EPA-660/3-73-003
Ecological Research Series
Organic Nutrient Factors
Effecting Algal Growths
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
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was consciously planned to foster technology
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2. Environmental Protection Technology
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This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
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assessed for their long- and short-term
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fate of pollutants and their effects. This work
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For sale by the Superintendent of Documents, U.S. QoTemment Printing Office, Washington, D.C. 20402 - Price $2,80
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EPA-660/3-73-003
July 1973
ORGANIC NUTRIENT FACTORS EFFECTING ALGAL GROWTHS
By
Nicholas L. Clesceri
with contributions by: Gerald C. McDonald
Inder Jit Kumar
William J. Green
Project 16010 DHN
Program Element 1B1031
Project Officer
Thomas E. Maloney
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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Review Notice
This report has been reviewed by the Environmental Pro-
tection Agency and approved for publication. Approval
does not signify that the contents necessarily reflect
the views and policies of the Environmental Protection
Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for
use.
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ABSTRACT
Effects of wastewater organic fractions on the growth rate
of Selenastrum capricornutum and Anabaena flos-aquae were
investigated. Effluent from a conventional activated
sludge facility was membrane filtered, freeze-dried, and
gel fractionated. Apparent molecular weights (AW) were
assigned to the appropriate fractions. These and organic
carbon data showed 6970 of the effluent organics had an AMW
less than 700.
Absorbancies and regression analyses within algal ex-
ponential growth phases demonstrated the control growth
rate for Selenastrum was 0.43 and for Anabaena was 0.34.
Selenastrum growth rates were monitored using Lake George
water as the diluent for the media employed. An inhibi-
tion in growth occurred. Halving the nitrogen concentra-
tion in modified Gorham's had no significant effect on
growth rate.
In concentrating organics from natural water (Lake George
and Saratoga Lake), raw sewage, and sewage effluent, thin
film evaporation was preferred when using natural waters
whereas freeze-drying was advantageous when working with
sewage samples. Also, the soluble organic component in
municipal wastewater was characterized and the effect of
chemical-physical treatment on it has been shown.
This report was submitted in fulfillment of Project Number
16010DHN, under the sponsorship of the Office of Research
and Monitoring, Environmental Protection Agency.
111
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CONTENTS
Section
I Conclusions 1
II Recommendations 3
III Introduction, Objectives and
Technical Review 5
IV Procedures and Results 33
Physical and Chemical Investigations
Biological Investigations
V Discussions 105
VI Acknowledgements 123
VII References 125
VIII Appendices 135
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FIGURES
No. Page
1 PARTIAL SCHEMATIC STRUCTURE OF SEPHADEX
DEXTRAN GEL, AFTER DETERMANN (20) 27
2 EXPERIMENTAL PROCEDURE FLOW DIAGRAM 34
3 ELUTION DIAGRAM RESULTING FROM THE FRAC-
TIONATION OF CONCENTRATED EASTERN-WESTERN
TREATMENT PLANT EFFLUENT ON SEPHADEX G-10 41
4 ELUTION DIAGRAM RESULTING FROM THE FRAC-
TIONATION OF CONCENTRATED COXSACKIE PLANT
EFFLUENT ON SEPHADEX G-10 42
5 SCHEMATIC FLOW DIAGRAM OF WATER POLLUTION
CONTRON FACILITY, BATAVIA, NEW YORK 43
6 ELUTION DIAGRAMS OF THE FRACTIONATION OF
UNCONCENTRATED AND CONCENTRATED SAMPLES OF
RAW DOMESTIC SEWAGE FROM ELNORA, NEW YORK,
ON SEPHADEX G-15 49
7 GEL CHROMATOGRAPHY APPARATUS 50
8 ELUTION DIAGRAM OF THE FRACTIONATION OF
CONCENTRATED EASTERN-WESTERN TREATMENT
PLANT EFFLUENT ON BIO-GEL P2 56
9 GEL CHROMATOGRAPHY OF STANDARD SOLUTIONS
1 AND 2 ON SEPHADEX G-10 59
10 GRAPHICAL REPRESENTATION OF THE RELATION-
SHIP BETWEEN MOLECULAR WEIGHT AND ELUTION
VOLUME FOR SEPHADEX G-10 60
11 GEL CHROMATOGRAPHY OF STANDARD EGG ALBUMIN
SOLUTION ON SEPHADEX G-25 AND SEPHADEX G-50 61
12 GRAPHICAL REPRESENTATION OF THE RELATION-
SHIP BETWEEN MOLECULAR WEIGHT AND ELUTION
VOLUME FOR SEPHADEX G-25 62
v
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No. Page
13 ELUTION DIAGRAMS OF THE FRACTIONATION OF
CONCENTRATED BATAVIA EFFLUENT ON SEPHADEX
G-10. RUN 1 THROUGH RUN 6 64
14 ELUTION DIAGRAMS RESULTING FROM THE FRAC-
TIONATION OF CONCENTRATED COMPOSITE FRONTAL
PEAK G-10-I ON SEPHADEX G-25 72
15 ELUTION DIAGRAMS RESULTING FROM THE FRAC-
TIONATION OF CONCENTRATED COMPOSITE FRONTAL
PEAK G-25-I ON SEPHADEX G-50 76
16 CONTINUOUSLY STIRRED BATCH CULTURE APPARATUS 82
17 GRAPHICAL REPRESENTATION OF THE CORRELATION
BETWEEN ABSORBANCES AND SELECTED GROWTH
PARAMETERS FOR SELENASTRUM CAPRICORNUTUM AND
ANABAENA FLOS-AQUAE 90
18 SELENASTRUM CAPRICORNUTUM GROWTH CURVES 93
19 GRAPHICAL REPRESENTATION OF THE RELATIONSHIP
BETWEEN THE SELENASTRUM CAPRICORNUTUM GROWTH
RATE AND VARYING ORGANIC CARBON CONCENTRA-
TION OF THE CONCENTRATED EFFLUENT AND FRAC-
TION G-50-I 95
20 ANABAENA FLOS-AQUAE GROWTH CURVES 100
21 GRAPHICAL REPRESENTATION OF THE RELATIONSHIP
BETWEEN ANABAENA FLOS-AQUAE GROWTH RATE AND
VARYING CARBON CONCENTRATION EFFLUENT 103
APPENDIX A
No.
1 CHROMATOGRAM OF STANDARD SOLUTION ON
SEPHADEX G-15 COLUMN WITH PHOSPHATE BUFFER 158
2 CALIBRATION CURVE FOR SEPHADEX G-15 COLUMN 159
VI
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No. Page
3 CHROMATOGRAM OF SARATOGA LAKE WATER THIN 164
FILM CONCENTRATE
4 CHROMATOGRAM OF SARATOGA LAKE WATER FREEZE- 165
DRIED CONCENTRATE
5 PERCENTAGE CURVES FOR CHROMATOGRAMS OF 167
SARATOGA LAKE WATER
6 CHROMATOGRAM OF CLIFTON KNOLLS EFFLUENT 171
FREEZE-DRIED CONCENTRATE
7 CHROMATOGRAM OF CLIFTON KNOLLS EFFLUENT 172
THIN FILM CONCENTRATE
8 PERCENTAGE CURVES FOR CHROMATOGRAMS OF 175
CLIFTON KNOLLS EFFLUENT
9 CALIBRATION CURVE FOR SEPHADEX G-15 COLUMN 179
10 CHROMATOGRAM OF LAKE GEORGE WATER THIN FILM 180
CONCENTRATE
11 CHROMATOGRAM OF LAKE GEORGE WATER FREEZE- 181
DRIED CONCENTRATE
12 PERCENTAGE CURVES FOR CHROMATOGRAMS OF LAKE 184
GEORGE WATER
13 CHROMATOGRAM OF ELNORA RAW SEWAGE THIN FILM 186
CONCENTRATE
14 CHROMATOGRAM OF ELNORA RAW SEWAGE FREEZE- 187
DRIED CONCENTRATE
15 PERCENTAGE CURVES FOR CHROMATOGRAMS OF 189
ELNORA RAW SEWAGE
vii
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APPENDIX B
No. Page
1 SCHEMATIC OF AN INFRARED CARBONACEOUS 234
ANALYZER
2 SCHEMATIC OF UPFLOW GEL COLUMN 241
3 SCHEMATIC OF DOWNFLOW GEL COLUMN 242
4 EXPERIMENTAL PROCEDURE USED DURING 246
LABORATORY CHEMICAL TREATMENT STUDIES
5 EXPERIMENTAL PROCEDURE USED DURING CHEMICAL- 247
PHYSICAL TREATMENT STUDIES
6 EXPERIMENTAL PROCEDURE USED DURING ANALYSIS 249
OF UNCONCENTRATED SAMPLES AND VALIDATION OF
FREEZE-DRYING
7 STANDARD CURVE FOR APPARENT MOLECULAR WEIGHT 253
DETERMINATIONS
8 CHROMATOGRAMS OF UNTREATED AND LIME TREATED 255
EASTERN-WESTERN SEWAGE CONCENTRATES SAMPLES
OF 6/6/69
9 CHROMATOGRAM OF EASTERN-WESTERN SEWAGE SAMPLE 258
OF 6/30/69
10 CHROMATOGRAMS OF UNTREATED AND LIME TREATED 260
WATERFORD SEWAGE
11 CHROMATOGRAMS OF INFLUENT AND CHEMICAL- 264
PHYSICAL EFFLUENT - NEW YORK STATE PILOT
PLANT SAMPLE OF 9/26/69
12 CHROMATOGRAMS OF CONCENTRATED INFLUENT 267
CHEMICAL TREATMENT EFFLUENT AND CARBON COLUMN
EFFLUENT - NEW YORK STATE PILOT PLANT SAMPLES
OF 10/10/69
viii
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APPENDIX B
No. Page
13 CHROMATOGRAMS OF CONCENTRATED INFLUENT, 269
CHEMICAL TREATMENT EFFLUENT AND CARBON
COLUMN EFFLUENT - NEW ROCHELLE PILOT PLANT
14 CHROMATOGRAMS OF UNCONCENTRATED AND CON- 272
CENTRATED ELNORA SEWAGE
15 CHROMATOGRAMS OF UNCONCENTRATED AND CON- 273
CENTRATED CHEMICALLY TREATED ELNORA SEWAGE
16 CHROMATOGRAMS OF UNTREATED, LIME TREATED 275
AND SODIUM HYDROXIDE TREATED ELNORA SEWAGE
APENDIX C
No.
1 SCHEMATIC OF NEW YORK STATE PILOT PLANT 294
2 SCHEMATIC DIAGRAM OF TOTAL ORGANIC CARBON 299
ANALYZER
3 SCHEMATIC DIAGRAM OF MSA MODEL 200 INFRARED 300
ANALYZER
4 CONTINUOUSLY STIRRED BATCH CULTURE APPARATUS 302
IX
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TABLES
No* Page
1 Elements Required by Green Plants 12
2 Vitamin Requirements of Algae as Reported 14
in the Literature, after Droop (22)
3 Total Organic Carbon Concentration of 40
Membrane Filtered Wastewaters
4 Selected Operational Data for Batavia 45
Wastewater Treatment Plant, 1968
5 Chemical Analysis of Membrane Filtered 46
Batavia Effluent, Collected 1/22/69
6 Comparison of Recovery of Organic Carbon, 47
Thin Film Rotary Evaporation vs Freeze-
drying
7 Properties of Dextran Gels Sephadex, after 51
Determann (20)
8 Concentrations of Organic Compounds in 58
Sephadex G-10 Column Standards
9 Organic Carbon Content of G-10 Fractions 70
10 Organic Carbon Content of G-25 Fractions 74
11 Organic Carbon Content of G-50 Fractions 75
12 Selected Parameters for Wastewater Fractions 78
13 Percent of Original Effluent Organic Carbon 80
Content in Fractions
14 1/10 Gorham's Medium 84
15 Basic ASM Medium 85
x
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No.
16 Effect of Fractions on the Growth Rate of 91
Selenastrum capricornutum
17 Effect of Organic Fractions in Reduced Con- 94
centrations on the Growth Rate of
Selenastrum capricornutum
18 Selenastrum capricornutum Growth Rates 97
Produced by Fraction Combinations
19 Effect of Organic Fractions on the Growth 99
Rate of Anabaena flos-aquae
20 Effect of Organic Fractions in Reduced Con- 101
centrations on the Growth Rate of Anabaena
flos-aquae
21 Ana/baena flos-aquae Growth Rates Produced by 102
Fraction Combination
APPENDIX A
No.
1 Concentration and Organic Recovery Data 161
Saratoga Lake Water, Sample No. 1
2 Concentration and Organic Recovery Data 163
Saratoga Lake Water, Sample No. 2
3 Chromatographic Data for Saratoga Lake Water, 166
Sample No. 1
4 Concentration and Organic Recovery Data 170
Clifton Knolls Effluent
5 Chromatographic Data for Clifton Knolls 173
Effluent
6 Concentration and Organic Recovery Data 176
Lake George Water, Sample No. 1
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APPENDIX A
No.
7 Concentration and Organic Recovery Data 178
Lake George Water, Sample No. 2
8 Chromatographic Data for Lake George Water, 182
Sample No. 2
9 Concentration and Organic Recovery Data Raw 185
Sewage, Elnora
10 Chromatographic Data for Raw Sewage, Elnora 190
APPENDIX B
No.
1 Classification ,of Sewage Fractions as 218
Presented by Rudolfs and Balmat
2 Typical Strength Distributions of Organic 219
and Nitrogenous Matter in Sewage Fractions
as Percent of Total Parameter
3 Organic Constituents of the Soluble Fraction 221
as Determined by Painter & Viney
4 Elution Characteristics of Standards 252
5 Treatment and Soluble Organic Carbon 254
Recovery Data - Laboratory Chemical Treat-
ment Studies
6 Effect of Chemical Treatment on Eastern- 257
Western Sewage Sample of 6/6/69
7 Apparent Molecular Weight Distribution of 259
Eastern-Western Sewage Sample of 6/30/69
8 Effect of Chemical Treatment on Waterford 261
Sewage
Xll
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APPENDIX B
No.
9 Treatment and Soluble Organic Carbon 263
Recovery Data, Chemical-physical Treat-
ment Studies
10 Effect of Chemical-physical Treatment on 265
New York State Pilot Plant Samples of
9/26/69
11 Effect of Chemical-physical Treatment on 268
New York State Pilot Plant Samples of
10/10/69
12 Chemical Treatment and Soluble Organic Carbon 271
Recovery Data - Elnora Sewage
13 Effect of Concentration by Freeze-drying on 274
Untreated and Lime Treated Elnora Sewage
Samples
14 Effect of Chemical Treatment on Elnora 276
Sewage Analyzed Unconcentrated
xiii
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SECTION I
CONCLUSIONS
The work undertaken and described has proven that in-
creased algal growth has been exhibited upon addition of
organic wastewater fractions to cultures of selected
algal species. Based upon these data, it is expected
that such effects may also occur in the natural regime.
Several other conclusions resulted from this study.
1. The organic constituents of secondary effluents are
in the main of low molecular weight.
2. The removal of nitrogen and phosphorus from treatment
facility effluents may not completely obviate the pos-
sibility of algal proliferation in the receiving water-
course.
3. The siting of pollution control facilities and the
treatment schemes to be provided should be carefully in-
vestigated, especially in those areas where minimal dilu-
tion may be expected.
4. Increased removal of organic carbon should be mandated
in facilities proposed to discharge to low flow streams
and essentially stagnant bodies of water.
5. An inhibition in the growth of Selenastrum capri-
cornutum occurred when Lake George water (soft water
oligotrophic lake) was used as the diluent for the growth
media.
6. Nitrogen concentration could be lowered in modified
tenth Gorham's medium without altering the growth rate of
Selenastrum capricornutum.
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SECTION II
RECOMMENDATIONS
Based on analysis of the results and experience gained,
the following recommendations are made:
1. Further investigation should be undertaken to assess
fully whether or not the Batavia effluent is unique in
its growth rate enhancement potential.
2. It should be determined if other forms of treatment,
e§» physico-chemical, produce effluents with such growth
enhancement potentials.
3. The gel chromatography technique should be further
investigated to determine whether such a tool may produce
wastewater characterizations with applicability to treat-
ment plant operation and effectiveness.
4. Further studies should be undertaken to ascertain the
exact nature of those factors, within the fractions,
responsible for the growth rate response noted.
5. Further studies involving the ascertainment of the
effects of wastewater fractions on ultimate growth should
be undertaken.
6. Carbon 14 uptake studies should be included in any
further undertakings. The use of such technique will
allow for an assessment of effects on photosynthetic
activity.
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SECTION III
INTRODUCTION, OBJECTIVES AND TECHNICAL REVIEW
For many years man has been aware of the natural process
of aging taking place in the earth's lakes. This natural
process in the limit leads to degradation of the resource
and a concomitant unfitness for use. This awareness has
today become acutely heightened as a result of the ac-
celeration of this natural aging process through the
activities of man.
With the onset of the Industrial Revolution, man in his
quest for progress and power began to destroy the environ-
ment, its land, air and water resources. Those activities
which accompany civilization and progress, namely in-
creased and unchecked waste discharge, increased utiliza-
tion of land and fertilizers to meet an ever-expanding
requirement for food and the rerouting and damming of
streams have led specifically to a rapid acceleration of
the destruction of the water resource (66, 85, 89, 90).
Eutrophication has become the shibboleth of the day.
Skulberg's definition is adopted here as exemplifying the
true interpretation; "the process of eutropication is
primarily caused by the supply of plant nutrients to the
aquatic environment even though other factors are in-
volved, such as morphometric relations and the degree of
biological utilization of the nutrient substances. The
manifestation of eutrophication is the increasing of
primary productivity; in extreme cases, the water bloom
phenomenon developes and in stagnant water oxygen supply
becomes depleted" (85).
The investigation of the effects of man-made wastes,
perhapes the primal cause of the eutrophic phenomenon has
received minimal examination to date and thus requires
specific and intensive effort. The complexities of such
investigations are compounded by the very nature of dis-
charged wastes; they "contain everything" in the realm of
nutrients (52).
The nutrient constituents of the aquatic environment
responsible for these eutrophic phenomena, the main cat-
egories of interest being the macronutrients, nitrogen
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and phosphorus, the trace element micronutrients vanadium,
molybdenium, etc and in several cases the accessory growth
factors cyanocobalamin, biotin and thiamine, have been the
subject of studies which have led to the development of
perceptible results in some cases, and in other, founda-
tions for speculation and hypotheses requiring further in-
vestigative scrutiny (77, 91).
The endeavors undertaken and documented in this investiga-
tion involve a hitherto neglected but specific aspect of
algal metabolism, namely, the effect of wastewater organic
compounds on the growth of algae. To attain this end, the
organic component of the effluent from a modern well-
operated conventional activated sludge system was separated
into fractions according to molecular size by gel permeation
chromatography. The fractions were then examined as to
their possible effect on algal growth by standard culture
comparison techniques. The results of the chromatography
and the biological investigations are presented in the
following chapters.
An examination and comparison of the effectiveness of
thin film evaporation and freeze-drying for concentrating
organics from natural and wastewaters was also determined
(Appendix A). Both methods were studied as to their rela-
tive polymerization or hydrolysis effects and their re-
covery efficiency so far as organic carbon was concerned.
In addition, the soluble organic component in municipal
wastewater has been characterized and the effect of
chemical-physical treatment has been shown (Appendix B).
Nutrient Factors in Algal Growth
1. The Natural and Synthetic Environment
No question has been raised as to the efficacy of nu-
trients for sustaining and being requisite for the growth
of algae. The only query yet to be answered fully is
what concentrations of specific nutrients are required
for sustained growth of a nuisance algal bloom in a
particular situation.
The investigations into the limiting requirements of nu-
trient factors have been carried on extensively in lab-
oratory experiments; the use of which has at times been
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assailed as an unnatural system with no possible validity
for extrapolation to the natural realm. However, Provasoli
has stated the case for laboratory cultures rather suc-
cinctly, "It is often said that laboratory conditions are
wholly artificial and that laboratory experiments at best
vaguely approximate natural conditions. But the ecological
arena is populated by the products of the continuous chal-
lenge of nature to the potentialities of the organisms...
In fact, the challenges provided by nature are as strenuous
to the organisms as are those inflicted on them by extreme
conditions in exploratory laboratory experiments" (77).
The obvious analogy that an in vitro requirement or its
physiological equivalent should of necessity be found in
vivo has been borne out in extensive investigations. The
requirements for all nutrient species save for nitrogen
and phosphorus have been noted as being approximately the
same in vitro and in vivo. The needs for nitrogen and
phosphorus though are 10 to 100 times greater in vitro.
If one realizes that the bloom may be concentrated by
wind and density differences in the resource the algae
may well have derived their required nutrients from an
extremely large volume of water thus negating the need
for the existence of high concentrations of these elements
in the natural regime.
2. Eutrophication Case Studies
That the production of eutrophic conditions in the aquatic
environment has been seriously accelerated by the discharge
of waste is without question. Several monographs relating
the changes for the worse caused by unchecked discharges
document the definite implication of waste discharges as
the primal causative factor in accelerated eutrophication
(18, 23, 35, 68, 89).
Among the products of the eutrophic phenomenon are (1) dense
mats of algae which render the water resource aesthetically
unattractive to those seeking recreational opportunities,
(2) loss of the economic value of lake shore property,
(3) clogging of water treatment filters, (4) the production
of odors and tastes, (5) discoloration of the water, and
in the limit, (6) anoxic conditions and their coincident
problems.
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Since Hasler's fine description of the problem in 1947
(44), the literature has been replete with the examples
of man's enhancement of the degradation of the aquatic
environment. Lakes Washington, Green, Zoar, Tahoe, Clear,
and even the Great Lakes, have been examined and found to
be suffering from the general malaise symptomatic of
eutrophication (7, 25, 42, 89). Neither has the estuarine
environment escaped notice (83). The list of victims does
not diminish as more notice is taken of the problem.
3. Macronutrient Requirements
As the body of information has grown regarding the eutrophic
phenomenon, the causative agents have begun to receive
scrutiny. Nitrogen and phosphorus have been implicated as
the key limiting factor in the growth of algae in the
natural environment (2, 24, 39, 48, 49, 50, 52, 56, 66, 77,
82, 89). This conclusion is logical if one considers the
important part these elements play in bioenergetics and the
synthesis of protein and their minimal levels of concentra-
tion in the environment. It should be noted that the pre-
ponderant attention focused on these elements has in the
main resulted from the knowledge that their removal from
discharges could be effected and that, for example, the
environmental carbon concentration would never be completely
subject to human control.
Phosphorus requirements. Although phosphorus, as noted
previously, is an important macronutrient and has been
regarded as perhaps the key element in the nutritional
pattern of algae, contradictions developed by several in-
vestigators and the variability of results as to the
exact nature of the limiting concentration are evident.
Tucker in his study of Michigan lakes found that the
minimal concentrations of phosphorus available were not
limiting to the growth of algae (91). Gerloff and Skoog
(39) and Goldman (42) in their studies of eutrophic lakes
found that phosphorus was not the limiting nutrient. The
concentrations of phosphorus claimed to be limiting have
exhibited striking disparity. Ketchum (48) indicates that
in his experiments with Nitzschia closteriuro, a phosphorus
concentration of 17 ug/1 was limiting to cell division
and the rate of assimilation of phosphorus. Rhode (80)
has developed much information on the required concentration
8
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of phosphorus. He discovered that 0.2 mg/1 of phosphorus
as phosphorus was limiting for Ankistrodesmus falcatus
and that the growth of Scenedesmus quadricauda increased
as the phosphorus concentration was raised to 1.0 mg/1.
However, he found that the addition of only 2 ug/1 of
phosphorus to "Bacteria filtered" Lake Erken water con-
taining 10 ug/1 of phosphorus produced full growth of
Asterionella formosa while artificial medium containing
9 to 18 ug/1 allowed for no growth of the organism.
Based on these anomalous data, Rhode proposed the
existence of a phosphorus sparing factor in the natural
regime; that is one of several factors which principally
change the phosphate dependence of the organism and thus
facilitate its use of and coincident proliferation with
low phosphorus concentration. Sawyer (82), as a result
of his work on Wisconsin Lakes, has posited a concentration
of 0.015 mg/1 as a minimal requirement for the occurrence
of nuisance blooms, since this concentration occurring at
the time of the Spring turnover did not allow for the
development of nuisance blooms and thus was indicative of
a well-behaved lake. The data of Shapiro and Ribiero (83)
led them to conclude that in the estuarine environment
they studied, phosphorus was definitely not the limiting
factor for the growth of blue-green algae in their experi-
ments.
V
Also worthy of note are those instances of inhibition of
algal growth by phosphorus. Chu (12) found phosphorus
concentrations in excess of 8.9 to 17.8 mg/1 inhibitory
to the growth of selected algae. Rhode (80) in his in-
vestigations with Asterionella formosa found that, al-
though 0.002 mg/1 added to filtered Lake Erken water
produced maximum growth, an increase to a level of 0.05
mg/1 was inhibitory. He also ascertained that a concen-
tration of 5 ug/1 was inhibitory to Dinobryon and Uroglena.
Thus one can see that the phosphorus situation is a mosaic
of alternating limitation, non-limitation, variability of
limiting concentrations and finally inhibition. The re-
sults lead to the conclusion that phosphorus in many in-
stances is limiting, the concentration range is low and
narrow and organism dependent, and that the environmental
requirements seem to be orders of magnitude lower than
those resulting from culture experiments. The phosphorus
sparing factor of Rhode noted previously has been posited
9
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as one explanation of this phenomenon and Luxury uptake as
another.
Nitrogen requirements. Nitrogen, as is the case with phos-
phorus, has been alternately implicated and acquitted in
the specific investigations for a limiting factor in the
growth of algae. The literature is replete with the
results of investigations on the limiting nature of
nitrogen (2, 24, 36, 38, 39, 49).
Gerloff and Skoog (39) found that nitrogen was the "primary
limiting factor" for the growth of Microcystis aeruginosa
in several Wisconsin lakes. Kratz and Myers (49) concluded
from their data that nitrogen metabolism was the process
determining the maximum growth rate for the algal species
studied, Anabaena variabilis, Anabaena cylindrica, Anacystis
nidulans, and Nostoc muscorum. Dugdale and Goering (24)
discovered in their study of diatom blooms in the Sargasso
Sea that nitrogen was the key limiting factor, while on the
other hand, Goldman found little evidence for a nitrogen
or even a phosphorus limitation in his studies on Clear
Lake (42). Also Shapiro and Ribiero (83) on the Potomac
Estuary found nitrogen to be non-limiting to the growth of
blue-green algae, a function they infer of the nitrogen
fixation capability of such algae. But such a conclusion
may not be extrapolated to all blue-green algae as Fogg (33)
has noted the ability of fixation has been discovered in
only 21 species belonging to 8 genera. Even though the
capability exists, it has been shown that atmospheric ni-
trogen is not generally as efficient as ammonia or nitrate
in satisfying the metabolic requirements for nitrogen in
blue-green algae. Kratz and Myers (49) showed that nitrogen
fixation alone would only support 7570 of the growth of
Nostoc muscorum produced when nitrate was provided and used
as the source of nitrogen.
As with phosphorus, investigations have led to the con-
clusion that nitrogen in certain concentrations can be
inhibitory to the growth of selected algae. Ketchum in
his excellent review article (48) has noted that Zobell
found the upper limits of nitrogen concentrations for
the growth of Nitzschia closterium to be 0.7 tng/1 (NH3),
70 mg/1 (N02) and 560 mg/1 (N03), all as nitrogen. In
contrast to these data, Chu (12) found concentrations of
10
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nitrogen in the form of an ammonium salt in excess of 5.3
mg/1 as nitrogen to be inhibitory to Staurastrum para-
doxum; and 17 mg/1 N when a nitrate source is used to be
inhibitory to Asterionella gracillima.
The limiting concentration of nitrogen thus varies from
species to species within a wide range of concentrations.
Again we are presented with a mosaic of alternating lim-
itation, non-limitation and inhibition dependent on species
and resource investigated.
4. Micronutrient Requirements
Inorganic micronutrients. The elements required by algae
for proper growth and development are many. Eyster has
noted that 20 elements may be required in the biochemistry
of green plants and has presented them in tabular form (26).
The data are reproduced herewith as Table 1.
As can be seen from the tabulation, the sole difference
between macro and micro nutrients rests on the relative
concentrations of each set required for sustained growth
of algae.
Eyster has noted that the trace elements are commonly em-
ployed as metal constituents of enzyme systems which enter
into biological reactions (26). Provasoli (77) has noted
in his review that molybdenum, copper, manganese, vanadium
and cobalt were demonstrated to be essential to the growth
of Chlorella, Scenedesmus and Anabaena and infers from such
that the requirements for these elements hold for all species
of algae. The need of nitrogen fixation for trace elements,
such as boron, calcium and molybdenum has been pointed out
by Eyster (26).
Since the quantitative requirements of algae for these
trace elements are minimal the in vitro determination of
limiting concentrations is fraught with difficulty. Two
major problems may cast a cloud on the attempts at the
determination of limiting concentrations. The lack of
purity of the nutrient salts used in medium makeup thus
leading to trace element contamination, and the seeming
dependency of trace element requirements on medium com-
position (92). Confusion as to the limiting concentration
11
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Table 1. Elements Required by Green Plants
Macromitrients Micrgnutrients
(IP-2 - IP"4 M) (IP"3 M and Less)
C, H, 0, N Fe, Mn, Cu, Zn
P, S, K, Mg Mo, V, B
Ca* Cl, Co, Si
Na**
* Except for algae where it is a micronutrient
** For blue-green algae
12
-------
may also develop as a result of the similarity of symptoms
produced by a lack of several elements. Notwithstanding
the above, the requirements for trace elements are absolute
but the limiting concentrations of the elements have not
been definitely fixed. It is hoped that the implementation
of new purifying techniques in the production of the nu-
trient salts will have a salutory effect on attempts at
the determination of a quantitative requirement.
Organic micronutrients. Noteworthy compounds also required
in raicroquantities for the growth of some algal species are
the organic growth factors, vitamin B-^, thiamine and biotin
(22, 32, 77). The interest in the presence of algal forms
of a requirement for these items developed out of the real-
ization that the early cultivation of algae in the laboratory
relied on the placement of soil extracts of unknown organic
composition in the medium. Provasoli considers the pattern
of vitamin needs stereotyped as only the above factors either
singly or in combination seem to be required by algae. Droop
has prepared a tabulation of the percentages of strains in
each division requiring these factors (22). Reproduced in
Table 2 are the data of Droop.
Vitamin 6^2 i-s required by more species than either thiamine
or biotin (77). This contention is borne out by Droop (22)
who posits that the species requiring the growth factors,
807o require vitamin B^. Provasoli has shown that the
sensitivity, depending on the organism involved, is 0.1 ng/1
to 5 ng/1 of vitamin "B12 (77). Sensitivity, having only
empirical value, is defined as the smallest amount of the
vitamin giving measurably increased growth as compared with
a control.
The determination of the concentration of vitamins required
in the test medium or environment is complicated by the
low levels and in the case of B^2 by the fact that many
species are able to employ analogues of 6^2 and more specif-
ically several blue-green species and Chlorella have been
found to be able to synthesize B^2 in a deficient medium
(22, 50, 56, 77). Kratz and Myers state that numerous at-
tempts to elicit a growth response from selected blue-green
algae, Anabaena variabilis, Anacystis nidulans and Nostoc
muscorum, by the addition of vitamins to a culture medium
have proved fruitless (49). Droop (21) reviewing the
13
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Table 2. Vitamin Requirements of Algae as Reported
in the Literature, After Droop (22).
Strains Requiring
Division
Chlorophyta
Euglenophyta
Chryptophyta
Pyrrophyta
Chrysophyta
Bacillariophyta
Phaeophyta
Rhodophyta
Cyanophyta
Totals
Non- Only
Total Auxotrophs Auxotrophs Bi 9
47
10
11
17
13
54
1
1
25
179
22
10
11
17
12
21
0
1
1
95
25
0
0
0
1
33
1
0
24
84
10
0
2
12
2
11
0
1
1
39
Bl2
Only and
Thiamine Thiamine
7
1
1
0
1
6
0
0
0
16
5
9
8
0
6
4
0
0
0
32
Thiamine
and
Biotin
0
0
0
1
1
0
0
0
0
2
B;L2
Thiamine
and
Biotin
0
0
0
4
2
0
0
0
0
6
-------
sensitivity of data for various species and the lowest
level of B^2 detected in the sea (0.1 ng/1) proceeds to
allow as that such a concentration could afford a crop of
25 million Skeletenoma, equal to the highest number ever
recorded in nature. From this he supposes that B-^ is
always in excess in the sea. Daisley, however, disputes
this contention as he feels that the growth rate and
not yield is the more important measurement as a certain
rate of growth is required to overcome losses due to
predation and sinking (16).
5. Growth Substances
For the last thirty years, literature has been produced
regarding the effect of organic growth substances on the
growth of algae. Growth substances being differentiable
from the growth factors in that the latter may be con-
sidered essential to the growth of certain species, that
is, be an absolute requirement; while the former produce
increased growth rates and yield but their absence from an
otherwise full complement medium does not inhibit growth.
The major studies to date have involved the determination
of possible stimulatory effects by auxins, plant growth
hormones of non-specific nutritional value. The first
report of the effect of these compounds on algae was
produced in 1937 by Yin (95). He found that the addition
of 10 mg/1 of 3-indoleacetic acid (CJ_Q Hg 02N) produced
a 207o increase in the mean diameter of Chlorella vulgaris
but did not measurably increase the growth rate. At the
same time Leonian and Lilly (55) reporting on their studies
of the effects of heteroauxin (3-indoleacetic acid) on
Chlorella pyrenoidosa, C_. minata, Cystococcus cohaerans,
Oocystis naegelii and Scenedesmus flavescens stated that
insofar as the species they investigated were concerned
heteroauxin was inhibitory to growth rather than stimulatory.
In 1938 Pratt (73) in studying auxin effects on Chlorella
vulgaris produced evidence to show that such compounds stim-
ulated the rate of multiplication of the organism. In his
experiments with 3-indoleacetic acid, 3-indolebutyric acid
and 3-indolepropionic acid he was able to show that con-
centrations of 50 mg/1 of each of the compounds placed
individually in the medium greatly increased cell multipli-
cation and that lesser concentrations in the order of 10
mg/1 produced smaller but very clear increases :'* the rate
15
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of multiplication of the organisms. The effectiveness of
the three compounds was, however, not the same. 3-indole-
acetic acid gave the greatest increase, almost twentyfold,
while the 3-indolebutyric acid produced the least increase.
Pratt was unable to detect any change in the mean diameter
of the cells. Coincident with these experiments he also
tested the effect of various compounds, among them potassium
acetate, glucose, acetic acid, allanotoin, and glycine, in
the same concentration, based on carbon content, as would
be added to the medium by 50 mg/1 of 3-indoleacetic acid.
These compounds produced an increase in cell multiplication,
with the greatest increase caused by the addition of glucose;
however, the increases in no way equaled those demonstrated
after the addition of the 3-indoleacetic acid. Brannon and
Bartsch (9) also investigated the effects of auxins on
algal growth. Based upon their experimental work with
Chlorella vulgaris they found that auxins had a salutary
effect on the 12 day yield of the test organism. The in-
crease in cell number over the control ranged from a low
of 73.170 produced with a concentration 6.7 ppm of indole-
propionic acid to 261.4% with phenylacetic acid in a concen-
tration of 33.3 ppm. Intermediate increases were noted for
other auxins, eg, 119.0% increase for 10.0 ppm of 3-indole-
acetic acid, 166.57o for 6.7 ppm of indolebutyric acid and
172.4% for 33.3 ppm of naphthalenacetic acid. They also
looked into the effect of such compounds on Coccomyxa
simplex and Mesotaenium caldariorum. The data regarding
these species are minimal but it is stated in the discus-
sion portion of their paper that "suitable concentrations
of naphthalenacetic, indoleacetic, indolebutyric, indole-
propionic and phenylacetic acids applied in sugarfree
inorganic culture media stimulated reproduction in Chlo-
rella vulgaris, and Coccomyxa simplex, but not in
Mesotaenium caldariorum." It should be noted for further
reference that the substances tested were dissolved in
ethanol before addition to the culture media. Brannon and
Sell (10) in 1945 found that concentrations of 10 ppm and
20 ppm of 3-indoleacetic acid stimulated the growth of
Chlorella pyrenoidosa and produced greater than a fourfold
increase in dry weight after thirty days. Meanwhile Manon
(58) discovered that a concentration of 10 ppm of 3-indole-
acetic acid was inhibitory to the growth of Chlorella
vulgaris and that a concentration of 50 ppm was definitely
toxic. He attributed the disparity between his results and
those of Brannon and Bartsch (9) to the possible use of
16
-------
different strains of the organism, due to the poor taxonom-
ical situation with Chlorella existing at that time, and
to possible differences arising out of a variation in the
purity of the acid used in the two experimental procedures.
Conrad and Saltman (15) in their treatise on growth sub-
stances note that Sven Algeus in 1946 performed extensive
experimental work on the effects of 3-indoleacetic acid,
3-indolebutyric acid, 3-indolepropionic acid, naphtha-
lenacetic acid and phenylacetic acid on cultures of
Chlorella vulgaris and several species of Scenedesmus.
It is reported that Algeus found that a concentration of
0.1 mg/1 of 3-indoleacetic acid exhibited a threefold in-
crease in cell number for Scenedesmus obliquus and that
the other compounds produced measurable but not as pro-
found effects. Further tests with the 3-indoleacetic acid
and cultures of S. acuminatus, S. dimorphus, and S. quad-
ricauda either produced no increase or inhibited growth.
In his work with Chlorella vulgaris, Algeus found that one
strain of the species responded with a threefold increase
in cell number at a concentration of 1.0 mg/1 of 3-indole-
acetic acid, while the other strain evidenced the effects of
several inhibition at this concentration. The foregoing
data lend credence to the first contention of Manos cited
above.
Williams in 1949 (93) found that the use of 3-indoleacetic
acid produced an increase in the wet weight of disks cut
from the brown alga Laminaria agardhii; but the article
reporting his work shows no replicate data and it is noted
therein that almost half of the treated samples disinte-
grated at concentrations ranging from 0.1 mg/1 to 10 mg/1.
Jacobs (46) reported that 3-indoleacetic acid in a concen-
tration of 100 mg/1 had a stimulatory effect on rhizoid
formation in Bryopsis plumosa. He found that no rhizoids
were formed in the controls. Davidson (17) shortly there-
after reported that fronds of Ascophyllum nodosum and
Fucus eyanescens responded to concentrations of 3-indole-
acetic acid from 10'* M (175 ng/1) to 10~4 M (17.5 mg/1)
with 10"5 M (1.75 mg/1) giving a 57.1% increase in apical
growth. A 10"^ M (0.0186 mg/1) concentration of naphtha-
lenacetic acid produced this maximum growth, but concentra-
tions in excess of 10"6 M (0.186 mg/1) proved to be
inhibitory. Corresponding concentrations of 3-indolebutyric
17
-------
acid also proved inhibitory. In the case of Fucus evanescens
growth was promoted by 3-indoleacetic acid in concentrations
from 10~8 to 10~^ M and by 10~^ M concentrations of naphtha-
lenacetic acid, and 3-indolebutyric acid. It should be
noted that, as with the earlier work on the effect of auxins,
the auxins used here were dissolved in an alcohol solution.
Provasoli (75, 76), studying the effect of auxins on the greei
alga Ulva lactua, concluded that the initiation of germlings
and elongation of the filaments were brought about by the
combination of kinetin and 3-indoleacetic acid in concen-
trations of 0.10 mg/1 and 0.05 mg/1 respectively.
The effect of auxins on the growth of diatoms has been
minimally investigated. Bentley (8), in experiments with
the marine diatom Skeletonema costatum, found that 3-indole-
acetic acid affected chain length and cell multiplication.
Skeletonema under natural conditions in the sea exists as a
long chain organism with the chains very often containing 50
or more cells. In a synthetic medium, it was discovered that
the organism was essentially unicellular with a few chains
of 2 to 3 cells evident. Optimum concentrations of 3-indole-
acetic acid, 10 ng/1 to 100 ng/1 resulted in an,improvement
of chain length, an average of 8 cells per chain after treat-
ment, and a doubling of cell number.
Miller and Fogg (65) in their study of Monodus subterraneous
elicited evidence that 3-indoleacetic acid had no effect
whatsoever on the growth of the organism. In their wide-
ranging study they also noted that several organic substrates^
including glucose, tryptone and the growth factors thiamine
and vitamin 6^2 also had no effect on the growth of the
organism; however, it was found that several of the sub-
strates tested were able to serve as sources of either phos-
phorus, eg, sodium glycerophosphate, or nitrogen, eg, gluta-
mine, urea and succinamide.
In 1958, Bach and Fellig (6) and Street et al (87) reported
on their findings that ethanol led to a stimulation of the
growth of Chlorella vulgaris. Street and his colleagues
discovered that the addition of an ethanolic solution of
3-indoleacetic acid produced a 160% increase in cell number,
while an aqueous solution of the same compound in the same
concentration only allowed for an 117o increase. Bach and
18
-------
Fellig (6) at the same time reported that while small concen-
trations of ethanol in the culture medium led to a twofold
increase in cell number, when the same experiments were con-
ducted on a shaking table the stimulatory effect disappeared.
This result led them to postulate that carbon was limiting
in the earlier experiments on auxin effects and that the
agitation of the cultures would have, in removing the limi-
tation, negated the requirement for carbon and thus reduced
measurably the stimulatory auxin effects noted. Their further
experiments with auxins bear out this contention as they were
unable to find any stimulatory effect. Although the above
work casts doubt on the validity of the conclusions drawn
from the early work of Brannon et al, the fact remains that
the work of Algeus, Bentley and Conrad and Saltman (14),
wherein the use of alcohol was scrupulously avoided and no
carbon limitation was evident, has not been refuted.
Prakash and Rashid (72) in studying the effects of humic
substances on algal growth found that humic acid fractions
obtained by gel permeation chromotography exerted a stim-
ulatory effect on the growth of Gonyaulax tamarensis. The
effects noted were: increasing yield, growth rate and
14C uptake. They attribute these effects at concentrations
of 2 ug/1 to 35 ug/1 to the fact that these small amounts
may act as stimulants and may be involved in cellular met-
abolic processes. They refer to other experimental work
which may indicate that the humic substances act as
sensitizing agents enhancing the permeability of the cell
membrane and consequently, increasing the uptake of nu-
trients from the surrounding medium.
6. Carbon
Recently a controversy has developed over the true nature
of the key limiting factor for algal reproduction in the
natural environment. Kuentzel (51) has proposed that the
key limiting factor is carbon. He reasons that carbon as
C09 is the major nutrient, since nuisance blooms of algae
are always associated with the discharge of excessive
amounts of organic matter to the resource. He infers from
these postulates that the organic matter acted upon by the
bacteria produce the carbon necessary and that the large
amounts of carbon required for the production of such blooms
19
-------
cannot be made available from the atmosphere or from HC03
dissolved in the water. He supposes a "perfect" symbiosis
between the bacteria and the blooming algae and states that
before a bloom of algae may occur a bloom of bacteria must
have taken place.
The fact that carbon is a major nutrient required by the
algae and that the unchecked discharge of carbonaceous
material could be the source of such a carbon requirement
is not in dispute. What has been seriously questioned is
his contention that the concentration of phosphorus in the
aquatic environment is already sufficient to support massive
blooms of algae and that any steps taken to require the re-
duction of the amount of phosphorus discharged to the envi-
ronment without at the same time taking out more of the
carbonaceous material would be self-defeating. Shapiro
(84) and Kuentzel (54) have exchanged statements regarding
their personal and professional interpretations of the
facts relating to phosphorus concentrations in the eco-
system and their suitability for algal growth. Shapiro
has stated that the phosphorus requirements of the growing
algal cell are essential and well known and that a con-
centration in the environment suitable to provide such
amounts is a sine qua non. The fact that low concentra-
tions are always found in the water during a bloom he
attributes to previous phosphorus uptake by the algae.
Kuentzel on the other hand argues that algae can produce
numbers in excess of the stoichiometrically determined
requirements for the cells and that only a minimum amount
of phosphorus is required for a bloom. This amount al-
ready being present, he feels that the removal of phos-
phorus from discharges is a wasted effort. What must be
taken into account, however, is the rapid turnover of
phosphorus in the aquatic environment, a matter of 3 to
5 minutes according to Rigler (79), the ability of algal
species to utilize organic phosphorus (12, 80) and the
species dependency of the required phosphorus concentration
in the environment. It would seem from the data previously
presented that perhaps in certain situations the attainment
of phosphorus concentrations in the natural realm allowing
for no algal proliferation might well be impossible.
Saunders in his article on the interrelationship between
phytoplankton and dissolved organic matter (81) has noted
20
-------
that the activity of the organic matter, although com-
prising only a very minor portion of the total number of
factors affecting algae, may be very significant when in-
tegrated into the total picture. He ascribes to organic
matter four possible functions relating to algal growth:
(a) utilization as an energy source or provision of an
essential nutrient required in the building of cellular
material, (b) action as an accessory growth factor vitally
essential to survival of the species, (c) toxicity leading
to inhibition or complete cessation of growth and finally
(d) the organic compounds may act as a chalating agent
forming complexes with trace metals in the environment and
thus produce beneficial or detrimental results depending
on the circumstances involved.
Since in the process of evolutionary development, algal
forms have arisen which cover the range from obligate
autotrophy to auxotrophy and finally, in some species
under certain conditions heterotrophy with all the shadings
in between evident, it would not be amiss to prepose that
some of the organic compounds extant in a wastewater ef-
fluent could affect the growth of algae in the natural
environment. As a specific example, one can note the oc-
currence in treated wastewater effluents of vitamin B^2
previously noted as a required accessory growth factor
for many algal species.
Concentration and Separation
1. Freeze-drying
The use of the freeze-drying technique to preserve and
concentrate biological samples has been in use for at
least fifty years. But it was the requirements for
plasma and dried penicillin during World War II which
brought the technique to fruition.
Freeze-drying, simply stated, is the removal of water
from a frozen sample via sublimation in vacuo. The beauty
of the method as a means of preservation is related to
the fact that the material is locked into the ice matrix
during prefreezing and remains in position during the
dehydration, thus affording no opportunity for interaction.
21
-------
Theory. The major portion of the following discussion is
based on two articles by Merryman which in combination
present an excellent presentation of the factors involved
in the freeze-drying technique (61, 62).
As pointed out by Merryman, the freeze-drying process in-
volves the harmonious integration of three distinct factors:
(a) the provision of heat to provide the energy required
for sublimation of the ice, (b) the transfer of the water
vapor sublimed through the specimen already dried, and
(c) the removal of water vapor from the specimen surface.
Assuming that the water vapor is removed, the function of
the introduced heat and its interrelationship with the
sublimation procedure, can easily be explained. The sample
after being frozen is composed of ice crystals rigidly
confined in a crystal lattice with the solute locked within
this matrix. The water molecules within the lattice are
in constant violent motion and it is statistically prob-
able that a molecule may through its violent motion over-
come the energy barrier and escape from the ice crystal.
If heat is provided to the system, most commonly by con-
duction, the probability of escape is increased. When
the water molecule escapes to the more unrestricted vapor
state it takes with it the latent heat of sublimation as
energy. At -25° to -30°C Greaves has calculated that this
is 672 calories/gram (43).
Since the escaping molecule has removed with it an amount
of energy equal to the heat of sublimation, the temperature
of the remaining ice mass will drop. If heat is added to
the system an equilibrium will be set up so that in the end
the energy removed by the subliming water molecules will
just balance that amount added by the external heat source.
The foregoing has been presented on the basis that all water
vapor and its coincident energy is removed from the system.
Such is not, in practice, the true case.
As the ice sublimes, the atmosphere above the drying sur-
face becomes suffused with water vapor. As this concentra-
tion of vapor increases, the probability of a return to the
ice crystal becomes greater. If the return rate increases,
the temperature of the sample will increase, positing a
22
-------
fixed heat input, as the returning molecule will bring with
it the energy it originally removed. In the limit, the
specimen will melt, thus the second and most important con-
sideration is the prompt removal of the water vapor from
the surface of the dried specimen.
The only force driving water vapor across the dried specimen
is the concentration gradient. The vapor produced at the
drying surface must diffuse through the already dried speci-
men to the specimen surface. To achieve this effect, the
equipment in use has been designed to achieve almost zero
vapor concentration at the specimen surface. For without
this condition, the rate of diffusion through the dried
specimen being low (a shell of dried material 1 mm thick
reduces the drying rate by a factor of 1,000) the sample
would eventually cease sublimating and melt. A zero vapor
concentration at the specimen surface is achieved by having
the vapor condense on a surface and thus be effectively
removed from the system. To achieve this end the mean free
path must be large, ideally equal to the distance between
specimen surface and the condenser. This is the sole
purpose of the vacuum system to enlarge the water vapor
mean free path by removing almost all gas molecules from
the area around the specimen surface and condenser and
also from the interstices of the dried sample.
From the foregoing it can be seen that the controlling
factor for freeze-drying is the rate of diffusion of the
sublimed water vapor and the sole motive power for this
transfer of water vapor from the drying boundary to the
condenser surface is the concentration gradient.
2. Thin Film Evaporation
The idea of conducting evaporation under vacuum was first
conceived by Howard, who invented a vacuum pan in 1812 (98).
By using a jet condenser and an air pump he reached a boil-
ing point of 60°C. The study of evaporation under artifi-
cially reduced pressure led to the development of multiple
effect evaporators. The importance of moving the liquid
itself relative to the heating surface was realized early
in the Nineteenth Century. In 1830 Dubrufaut described an
ideal evaporator for sugar liquors in the following words:
23
-------
"Concentrate the juice as rapidly as possible
so as to avoid any alteration; give the liquid
to be evaporated great speed, and, if possible,
accelerate the movement of the liquor so as to
evaporate in the form of a thin film."
The concept of thin film, however, took a practical shape
only towards the close of the Nineteenth Century in the
construction of the Lillie evaporator, which was patented
in 1890 (98, 99). There have been a number of improvements
since then in which this concept has been usefully employed.
The laboratory scale rotary thin film evaporator is a de-
velopment of the late forties. Partridge (100) has described
such an apparatus for use in the concentration of small
quantities of liquid at low temperatures under partial vac-
uum. The present-day rotary thin film evaporator is only
a slight modification of the Partridge apparatus (evaporator).
It is surprising to note that although this apparatus is
used in almost every laboratory for concentrating small
amounts of liquids, there is practically no literature
evaluating its performance.
Theory. The process of thin film evaporation can be divided
into four basic steps: 1) introduction of heat to supply
the energy necessary for evaporation, 2) formation of thin
film for efficient heat transfer, 3) evaporation of water
from the thin film vacuum, and 4) removal of water vapor with
the help of a water aspirator or a vacuum pump.
The process is similar to distillation. Applying the prin-
ciple of differential distillation, Fredette (101) arrived
at a simple mathematical relationship for calculating the
recovery of organic materials dissolved in water. The re-
lationship is expressed as:
xo
In(n0/ni) = XL dx/ (cx-x) = (l/(c-l)) (ln(xo/xL))
where
n0 and n-^ are the initial and final moles of the
solvent
XQ and x are the initial and final mole fraction
of the solute^in the liquid.
c is a constant and its value is dependent on the vapor liquid
equilibrium curve for the system. Fredette (101) assumed this
24
-------
carve to be a straight line while arriving at the above
relationship. In this case c is the slope of the straight
line.
The above relationship can be used to find the recovery of
known compounds in the solvent. Fredette (101) obtained a
very good correlation between the experimental and theoret-
ical values based on the above relationship.
The investigations related to these concepts within this
report are discussed in Appendix A.
3. Gel Permeation Chroma tography
The separation of substances differing in molecular weight
by passing a solution containing these substances through
a bed of gel grains is termed gel permeation chromatography
or more simply gel filtration. Many materials have served
as a source of polymer gels. Among these are agar, starch,
polyacrylamide and polyvinyl alcohol (53). The most widely
used material has become dextran first described for use as
a gel by Porath and Flodin in 1959 (70).
Sephadex dextran gels. Dextran is a high polymer carbo-
hydrate produced during the growth of Leuconostoc
mesenteroides on sucrose. The soluble polysaccharide
developed consists exclusively of glucose and contains a
preponderence ( > 90%) of «<- 1,6 glycosidic linkages.
Since it contains 3 hydroxyl groups per glucose unit, it
is water soluble.
The reaction occurring upon the mixture of an alkaline
dextran solution and epichlorhydrin proceeds exothermally
to form polymer chains cross- linked with glyceryl ether
bonds. The reaction described first by Flodin (31) in his
dissertation and reproduced by Determann in his monograph
(20) is as follows:
Dex-OH + qH2-CH-CH2-Cl — > Dex-0-CH2-CH(OH)-CH2-Cl
Dex-0-CH2-CH(OH)-CH2-Cl + NaOH^Dex-O-CI^-CH-C^ + NaCl + H20
25
-------
Dex-0-CH2-CH-CH2 + OH-Dex > Dex-0-CH2-CH(OH)-CH2-0-Dex
As can be seen from the above equations, the dextran poly-
mers are cross-linked as a result of the reaction of epi-
chlorhydrin with two hydroxyl groups on two different
polymer chains. Gels of vastly different swelling prop-
erties may be produced depending on the concentration and
molecular weight of the dextran polymer and the concentra-
tion of epichlorhydrin. This swelling property directly
affects the size of the accessible regions within the gel
structure and thus allows for the production of gels capable
of separation over a wide range of molecular weights. The
structure of a portion of the Sephadex dextran gel is as
depicted in Figure 1.
From experimentation it has been found that the swelling
properties of the gel increase with decreasing concentra-
tions and molecular weight of dextran and decreasing concen-
trations of epichlorhydrin.
Theoretical aspect £[. Several numerical constants useful in
the interpretation of gel filtration data have been advanced.
These constants have been developed from experimentation
with the gels in a columnar format. The setup is used almost
exclusively in gel filtration work. The volumes existing
in the gel column play an integral part in the determination
of these parameters. The total volume of a gel bed (Vt) may
be thought of as being composed of three separate volumetric
entities:
V,. = V + V. + V (1)
t m i o v '
Where V is the volume of the bed taken up by the gel matrix,
V^ is the inner volume, that volume taken up by the eluent
inside the gel grains and V0 the void volume, that portion
of the volume outside the gel grains.
The measurement of Vm and V. are quite difficult but have
been calculated by the manufacturer for the various Sephadex
gels and may be used in the development of the column param-
eters.
26
-------
GLYCERYL
ETHER
BRIDGE
H C—0 —CH2
° vA
Fig. l PARTIAL SCHEMATIC STRUCTURE OF SEPHADEX
OEXTRAN GEL, AFTER DETERMANN .
27
-------
Determann (20) has a presentation of the development of one
such parameter. K^ the fraction of the inner volume avail-
able to the solute molecule was originally proposed by
Wheaton and Baumann. The formula for this parameter is:
Ve = V0 + (Kd x Vt) (2)
Since the elution volume (V ) which is the volume of eluent
passed through the column prior to the appearance of the
molecule under study in highest concentration is a known
factor the K^ may be calculated as follows:
Kd - Ve - VQ (3)
A compound completely excluded from the inner volume would
have a K of 0 while a compound with a K^ of 1 would have
the fulldinner volume of the gel available to it. Inter-
mediate values of K^ would indicate compounds with only a
portion of the inner gel volume available.
This parameter is independent of the geometry and packing
density of the column and thus is useful for comparing
work done with the same gel by different investigators. The
sole drawback to the use of this constant resides in the
uncertainty of the measurement of V. .
To overcome this uncertainty, Laurent and Killander (53)
developed the concept of a Kav constant. The difference
between this constant and K rests in the fact that the
total volume of the gel phase (Vj. - V0 = V. -t- V ) instead
^ *-' J_ 10
of the inner volume (Vj) is used in the calculations. The
elution volume of a substance may then be found by:
V=V+K • (V - V }
ve o av ^ t o'
and K_.T would be calculated from:
av
(5)
- v0
As with K, this constant is independent of column geometry
and packing density. Its main advantage is based on its
28
-------
calculation using clearly defined and easily obtained
volumetric parameters.
Models for gel filtration. Gel chromatography having de-
veloped out of a few accidental observations was at first
without a theoretical basis. Flodin in his investigations
(30, 31) clearly outlined the prime factors essential to
separations by gel chromatography: The elution volume of
a molecule is independent of flow rate and sample concen-
tration and is largely determined by its molecular weight.
The analysis of experimental data collected over the past
years has served to substantiate these contentions.
The previous discussion regarding the constants K
-------
Kd = k (1 - 2r)3 (6)
R
Employing proportionality factors for r and R he was able
to arrive at the final format of:
Kd1/3 = kL - k2 x M (7)
where M represents molecular weight. This basic equation
has been checked extensively against the experimental data
and found to be valid in most cases.
Subsequently, Squires (86) assuming that the inner volume
was composed of cones, cylinders and cracks developed the
following experssion:
= KI - K?M1/3 (8)
0
The expression has been found to be valid for a number of
proteins fractionated on Sephadex gels but has not repre-
sented the experimental data equally well in all cases.
Laurent and Killander (53) following the basic premise of
earlier investigators that the position of a molecule in
the elution chromatogram is a function of the volume avail-
able to it have proposed another model for the gel structure.
They posit what they consider to be the "simplest model,"
by assuming that the dextran chains are infinitely long,
straight rigid rods distributed at random throughout the
gel. Using this model, they are then able to employ the
formula of Ogston (67) to calculate the available volume
for a spherical particle in such a system,
Kav " e~fl"L(r + R)2 (9)
In the above expression L is the concentration of rods in
the gel expressed as cm of rod per cm3, r is the radius of
the spherical particle and R the radius of the rod. From
data provided in the literature and in their experiments
they calculated the K using the void volume, total volume
and elution volume for various proteins and dextran frac-
tions. The experimental data showed that the model hypoth-
esized is able to satisfactorily explain gel filtration on
the basis of a steric exclusion of solute molecules from
the gel phase.
30
-------
Restricted diffusion. Ackers (1) has proposed a mechanism
of gel filtration on the basis of a decreased rate of dif-
fusion into the gel structure for large molecules. It is
assumed in this explanation that the diffusion rate was
restricted due to steric effects and friction in the gel
pores. But this explanation encounters difficulty when one
considers that the position at which a substance is eluted
does not vary appreciably with the rate of eluent flow.
This indicates that the exclusion principle and not re-
stricted diffusion is the operative mechanism as the flow
rate would have an effect on the elution position if re-
stricted diffusion was the mechanism (20, 53).
Summary. It may be well to reiterate the salient points
regarding gel filtration namely, (1) the elution volume of
a substance is dependent on its molecular size, (2) the
elution volume does not vary appreciably with the rate of
eluent flow up to the technically realizable maximum, (3)
the elution volume is not temperature or concentration de-
pendent and (4) the elution volume is directly proportional
to the log of the molecular weight of the substance under
study (3, 4, 20). These findings based on the analysis of
experimental data led to the conclusion that in the majority
of cases the operating principle in gel filtration is based
on the exclusion of a substance from the gel interior due
to steric factors.
31
-------
SECTION IV
PROCEDURES AND RESULTS
Physical and Chemical Investigations
1. Sampling, Preliminary Treatment and Concentration
The relative merits of a grab sample versus a composite
sampling procedure for the collection of the effluent to be
fractionated and subsequently used in the biological anal-
yses were thoroughly investigated.
Due to the obvious lack of a clearcut advantage for a short
term composite over a grab sample and the unfavorable eco-
nomic and time demands required for a long term composite,
it was decided that all samples of effluent would be grab
samples. At all times samples of effluent were collected
prior to chlorination.
It was realized that in a study of this nature, care must
be taken to maintain the integrity of the sample under in-
vestigation. To accomplish this end the following proce-
dural steps were devised and implemented.
Samples were collected and stored in previous acid-washed
18 liter polyethylene containers and returned immediately
to the laboratory. The time of travel involved no more
than 5 hours. Upon arrival an alinquot of the wastewater
was taken for total organic carbon analysis using a Beckman
carbonaceous analyzer. Immediately thereupon preliminary
treatment was undertaken. In rare instances, the time of
delivery to the laboratory did not allow for the immediate
implementation of preliminary treatment. In these cases
the wastewater sample was placed in a cold room maintained
at a constant temperature of 4°C until preliminary treatment
was undertaken on the succeeding day.
The flow diagram shown in Figure 2 depicts the total treatment
scheme proposed for use in the study. To render the effluent
amenable for fractionation and subsequent biological analyses,
some means had to be obtained to remove the particulate matter
extant in the wastewater.
33
-------
EFFLUEN
MEMBRANE
FILTRATION
(0.45 )
ff
CONCENTRATION
FREEZE
DRYING
CONTINUOUS
I—frlCENTRIFUGATION
CONCENTRATION
THIN RLM
ROTARY EVAPORATION
FRACT1QNATION
SEPHADEX
G-K)
FRONT
FRACTIONATION
SEPHADEX
FRONT
FRACTIQNATIQN
SEPHADEX
G-5O
AUGAL
CULTURE
FLASKS
Fig. 2 EXPERIMENTAL PROCEDURE FLOW DIAGRAM
-------
Continuous centrifugation. The first method investigated
for the separation of participates was continuous centrif-
ugation. The equipment already available at the laboratory
was a Lourdes continuous centrifuge model CFR-2. Several
samples of wastewater were collected to assess the advis-
ability of this method of preliminary preparation. It was
found during the operation that the sample temperature after
passage through the system increased appreciably to a point
where it was higher than ambient. It was felt that such a
temperature increase might have a deleterious effect on
sample quality. This factor, coupled with the low rate of
flow, average 5 1/hr, and the need for constant attention,
induced a search for a more reliable and quicker means of
separation.
Membrane filtration. In place of the above method a large
membrane filter apparatus, 142 mm diameter, was employed
and the sample filtered through a 0.45 u mean pore size
membrane filter. The system was notably successful as it
afforded ease and simplicity of operation, coupled with
a high rate of separation.
The sample to be filtered was discharged into an 18 liter
container that had been previously cleaned and acid-washed
with hot dilute HCl (1:10 dilution). The container, equipped
with an orifice at its base, was placed in an elevated posi-
tion to provide sufficient head to fill the filter apparatus
prior to the actual commencement of filtration. A water
aspirator was used for vacuum filtration of the sample.
Subsequent to the filling of the collection flask and sooner
if the level of particulates in the sample required it, the
membrane filter was changed and the process repeated until
all of the wastewater had been filtered. The contents of
the collection flask were discharged into an acid-washed
18 liter container, which when filled was placed in the cold
room. The minimum rate of filtration experienced during the
investigation was 20 liters per hour. After the effluent
sample had been filtered, an aliquot of the filtrate was re-
moved for measurement of organic carbon.
After separation of the particulates ( ^ 0.45 u) from the
soluble organic matter, a concentration step was employed.
This step was taken to insure that the chroma to graphic
35
-------
column effluents would contain organic carbon in concen-
tration readily detectable with the Beckman carbonaceous
analyzer. It has been found that with a 2.5 x 100 cm
column, the minimum sample organic carbon concentration
that will allow detection of the column effluent fraction
carbon concentration by the analyzer is approximately 100
mg/1.
An additional benefit is also enjoyed due to the use of a
concentration procedure. If the sample organic concentra-
tion is increased, the number of passes through the gel
column required to produce fraction volumes sufficient to
be usable in the subsequent biological analyses are pro-
portionately reduced.
Thin film rotary evaporation. At first thin film rotary
evaporation procedures were attempted. The experimental
apparatus included a Calab thin film rotary evaporator and
a 500 ml cold finger condenser filled with ethylene glycol.
The ethylene glycol was maintained at or below 10°C with
the aid of a Forma cold finger.
This concentration procedure proved very troublesome. The
apparatus, as constructed, required continuous monitoring
to insure proper operation, especially the constant sample
delivery accessory. When left unattended, the system was
highly erratic and on one occasion an overnight concentra-
tion attempt resulted in complete filling of the apparatus,
including the vacuum pump, with sample wastewater.
In addition to the operational difficulties, it was found
that it was necessary to raise the temperature of the waste-
water sample to approximately 34 C to insure reasonable
evaporation rates. As with the continuous centrifugation
step, it was felt that this elevated temperature would have
a deleterious effect on the sample composition, and that,
under the circumstances, another more reliable and less
troublesome concentration procedure should be sought.
Freeze-drying. The final concentration procedure investigated
was freeze-drying. A Virtis 10 liter large port freeze-dryer
was obtained, and fitted during its manufacture with 16 3/4"
inlet ports to allow a greater rate of sublimation. The
apparatus is claimed by the manufacturer to be capable of
concentrating 8 liters during a single run. However, it
36
-------
was found that for best operation 5 liters was the optimum
load that could be imposed on the system at any one time.
Thus, the maximum rate of concentration using the above
system was 5 liters per day.
The freeze-drying procedure followed firstly involved the
transfer of the filtered wastewater to ten 1200 ml freeze-
drying flasks. A measured 500 ml volume of the filtered
wastewater was placed into each freeze-drying flask. The
sample flasks were then sequentially shell frozen by rapid
manual spinning in a dry ice acetone bath (-61°C), and then
placed on the 3/4" ports of the concentration apparatus.
A McLeod gauge was integrated into the system to give an
overall value for the vacuum attained during its operation.
Using a high volume Welch Due-Seal vacuum pump, a vacuum of
less than 10 microns mercury was maintained throughout the
freeze-drying operation, except during the one minute equil-
ibration period following the application of a flask to a
freeze-dryer port. During this equilibration period a
maximum vacuum reading of 100 microns mercury was noted. At
no time were sample flasks placed on the apparatus less than
one minute apart. This step was taken to insure that sudden
overloads were not imposed on the system.
Initially, severe difficulty was encountered in the operation
of the system. The flasks were geared to be used with either
Pyrex glass connectors or stainless steel connectors, both
items of thin wall construction. The initial samples were
concentrated in flasks using glass connectors of thick Pyrex
glass and with the Virtis filters in place in the flask caps.
With this setup in use, a melt-back phenonmenon occurred,
essentially ruining the concentration procedure being under-
taken. Thereupon, it was decided that the use of the filters
in the flask cap would be foregone, and a flask connector of
larger inside diameter used to connect the flask to the freeze-
dryer port. These two steps completely corrected all diffi-
culties experienced with the system.
It is surmised that the melt-back phenomenon had been occur-
ring due to constriction of the water vapor mean free path
resulting from the use of a flask connector with a smaller
inside diameter than recommended by the Virtis Company. With-
out the escape of the water vapor and its concomitant heat of
sublimation, the temperature of the sample rose, leading even-
tually to melt-back.
37
-------
Once the 500 ml samples had been reduced to approximately
50 to 75 ml, another 500 ml of the wastewater was added to
each flask and the composite again shell frozen in the dry
ice acetone bath and introduced onto the freeze-dryer. This
sequence was repeated until the total volume of effluent
collected, save for a minor amount retained for use as a
flask wash, had been concentrated.
After the concentration had been completed the concentrate
was filtered through a 0.45 micron membrane filter. The
flasks were washed with small volumes of unconcentrated
effluent and this wash also introduced onto the membrane
filter. The filtrate obtained was stored in the cold room
in a Pyrex reagent bottle. The solids remaining on the
membrane filter was acidified with 0.1 N HCl and the result-
ing organic material also stored in a Pyrex container and
placed in the cold room.
Upon completion of this step the organic carbon concentrations
of the filtrate and the acid wash were measured. Combining
these data with the known volumes of both portions, the total
organic carbon content of the concentrate was calculated.
Using this figure, and the organic carbon content of the
original sample, a recovery factor was determined.
Criteria of selection. It was considered to be of prime im-
portance that the effluent selected for fractionation and
subsequent biological analysis be truly representative of
secondarily treated wastewaters. To this end, two criteria
were posited as guidelines for the selection of the wastewater
sampling site. These criteria were: (a) low effluent organic
carbon content, thus presupposing an efficiently operated
wastewater treatment plant, and (b) a wastewater treatment
facility utilizing a form of treatment applicable for use in
presently planned and future large scale regional facilities.
Effluents investigated. In line with the above guidelines,
three facilities were investigated as possible sources of
effluent.
Eastern-Western wastewater treatment facility. The
Eastern-Western facility located in Albany County, New York
was the first plant investigated as a possible source of the
required effluent. This facility of the Town of Colonie,
New York serves approximately 80 families and utilizes
38
-------
extended aeration as the treatment method. The proximity
of the site to the laboratory was a consideration in its
favor, but the organic carbon concentration in the effluent
and a critical appraisal of the operation of the plant and
the form of treatment employed raised doubts as to its suit-
ability for use in the study.
The total organic carbon concentration of the sample collect-
ed from this facility is noted in Table 3. This high concen-
tration, 21 mg/1, was not in keeping with the requirements
imposed by the first criterion and also gave pause in that
it was indicative of possible operational difficulties. Al-
though a preliminary chromatographic analysis was performed,
Figure 3, the facility, due to its noncorapliance with either
of the criteria set forth above, was deleted from the list
of possible sources of effluent.
Coxsackie Reformatory wastewater treatment facility.
The second effluent investigated for possible use in the
study was that produced by the trickling filter plant lo-
cated at the Coxsackie Correctional Facility, Coxsackie,
New York. The carbon concentration of the original sample
collected, 11 mg/1, gave hope that the facility would produce
the type of effluent required. Critical examination, however,
proved otherwise. Subsequent samples proved to be high in
organic carbon concentration and heavily loaded with partic-
ulates, which led to much difficulty in filtration.
However, the above was not the sole reason for discontinuance
of the use of this effluent. It was felt also that its lack
of accord with the second criterion regarding applicability
for large scale systems should militate against its use in
the study. Thus, as with the extended aeration facility,
the Coxsackie installation was removed from contention. As
noted previously for the extended aeration facility, prelim-
inary chromatographic work was performed on the effluent and
such chromatographic data are presented in Figure 4.
Batavia water pollution control plant. The final plant
investigated as a possible source for the effluent for the
study was the 2.5 mgd conventional activated sludge facility
located at Batavia, New York. Figure 5 represents a schematic
diagram of the treatment facility.
39
-------
Table 3. Total Organic Carbon Cencentration of
Membrane Filtered Wastewaters
Total Organic
Facility Date Sampled Carbon mg/1
Eastern-We stern 9-30-68 21
Coxsackie 10-18-68 11
Batavia 12-14-68 8
1-22-69 7
3-17-69 9
40
-------
80 P
GEL-
SPL-
SPL VOL -• 5ml TOC -580mq/l
COLUMN PARAMETERS I
2.5cm x 95cm; 60rnl/hr.
SEPHADEX G -10
CONCENTRATED
EXTENDED AERATION
EFFLUENT
35 4O 45 50 55 60 65 70 75 80 85 90
FRACTION NUMBER
Fig. 3 ELUTION DIAGRAM RESULTING FROM THE FRACTIOMATION OF
CONCENTRATED EASTERN-WESTERN TREATMENT PLANT EFFLUENT
ON SEPHADEX G-IO.
-------
30r
o
o
20
GEL - SEPHADEX G-IO
SPL - CONCENTRATED TRICKLING
FILTER EFFLUENT
SPL VOL- 5ml TOC-280mg/l
COJJMN PARAMETERS!
2.5cm x 95cm; 60ml/hr.
(AMW. > 700)
35 40 45 50 55 60 65 70 75 80 85
FRACTION NUMBER
90
Fig. 4 Eu/TION DIAGRAM RE8ULTINO FROM THE FRACTIONATION OF
CONCCNTRATEO COXSACKIE TREATMENT PLANT EFFLUENT
ON SEPHADCX G - IO.
-------
U)
THICKENER . OVERFLOW
VACtlUM FILTRATION
AND DISPOSAL
KEY
SEWAGE FLOW
SLUDGE FLOW
DESIGN POPULATION 25,000
AVG. FLOW! 25 mgd
MAX. FLOW: 026fi*4
LWASTE
MIXED UQUOR
SCREENING ft
COMMMUnON
u
3
k.
Z
AERA
TANK
TION
NO.I
AERA
TANK
TION
N02
Fig. 5
SCHEMATIC FLOW DIAGRAM OF WATER POLLUTION
CONTROL FACILITY, BATAVIA, NEW YORK.
-------
Noted in Table 3 are carbon concentrations of three rep-
resentative samples collected from this facility. As can
be seen from the data presented therein, the total organic
carbon concentrations of the effluents fully satisfied the
requirements of criterion a. In addition, as was not the
case with Eastern-Western or the Coxsackie Reformatory
facility, operational data were available for this facility.
Table 4 represents monthly averages of the BOD and suspended
solids loadings and removals effected by the plant during
the year 1968. From a review of these data, it is quite
readily descernible that the facility was well operated and
did produce an acceptable effluent.
This information, coupled with the fact that conventional
activated slude treatment is well suited to wide-scale usage
on a regional basis as required by the second criterion,
prompted the selection of this facility as the sampling
source. The chemical characteristics of the 1-22-69 effluent
used in the study are shown in Table 5.
Freeze-drying. Depicted in Table 6 are data regarding re-
covery of total organic carbon resulting from both thin film
rotary evaporation methods and freeze-drying concentration.
The data contained therein clearly indicate that the freeze-
drying method produced a higher recovery of organic carbon
in the soluble portion. No data were garnered as to the
carbon content of the acid wash for the thin film system so
that a comparison on an overall recovery basis is not pos-
sible; but based upon the data relative to the recovery of
the soluble organic component, however, it is obvious that
freeze-drying was the more efficacious of the two methods
in this particular instance.
No question has ever been raised as to the efficacy of freeze
drying in regard to retaining the basic character of a sample
concentrated by this method. However, to test the validity
of this basic assumption with particular reference to waste-
water, near the conclusion of the study an untreated sample
of wastewater from Clifton Knolls, New York was concentrated
by the freeze-drying method and both the unconcentrated and
concentrated portions subjected to fractionation on Sephadex
G-15. The total organic carbon concentrations of the frac-
tions resulting from the concentrated sample were measured
with the Beckman Model 315 carbonaceous analyzer and those
produced by the unconcentrated sample were measured by the
44
-------
Table 4. Selected Operational Data for Batavia "Wastewater
Treatment Plant, 1968
Average
Flow
Month mgd
January
February
March
April
May
June
July
August
September
October
November
December
Year Avera
2.4
2.5
3.3
2.3
3.3
2.7
2.3
1.9
1.5
1.3
2.6
2.6
ge
Average
Influent
Suspended
Solids
Ibs/day
4760
3670
5182
3670
6262
4644
3064
3418
3315
3007
4530
4321
Average
Influent
BOD
Ibs/day
5627
3936
5447
4899
7370
7383
6777
4900
3293
2566
5240
4313
Percent
Suspended Percent
Solids BOD
Removed Removed
90
89
88
84
83
87
91
88
88
90
87
87
88
88
89
88
86
84
85
89
85
89
89
88
84
87
45
-------
Table 5. Chemical Analysis of Membrane Filtered Batavia
Effluent, Collected 1-22-69
Constituent Concentration*
Cl 200.00
Total Alkalinity as CaC03 327.00
NH4 as N 8.00
Organic N as N 12.00
N02 as N 0.20
N03 as N 1.20
Fe (total) 0.41
Total PC-4 as P04 7.06
Orthophosphate as PCv,. 6.95
304 116.00
Ca as CaC03 360.00
Mg as CaC03 40.00
Na 116.00
K 7.00
Organic Carbon 7.00
pH 7.90
* all expressed in mg/1 except for pH
The above analyses with exception of Organic Carbon were
performed by the Division of Laboratories and Research
New York State Health Department.
46
-------
Effluent
Table 6. Comparison of Recovery of Organic Carbon, Thin
Film Rotary Evaporation vs Freeze-drying
Total Percent
Organic Total Organic Recovery Total Organic
Concen- Carbon Carbon in Soluble Carbon in Percent
tration In Original Soluble Organic Organic Acid Soluble Recovery
Method Sample (mg) Component (mg) Component Component Total
Eastern-
Western
STP
Coxsackie
Reforma-
tory
STP
Batavia
STP
1-22-69
Batavia
STP
3-17-69
Batavia
STP
12-14-68
Thin- 84 57
Film
Rotary
Evapora-
tion
Thin- 33 20.1
Film
Rotary
Evapora-
tion
Freeze- 213.5 190
drying
Freeze- 360 284
drying
Freeze- 42 38
drying
68
61
89.0 25 100
79 14 83
92 2.5 97
-------
MSA LIRA Model 20 low level carbon analyzer. The resulting
chromatograms are depicted in Figure 6. The data developed
clearly indicate that the concentration of a wastewater
sample by the freeze-drying method neither affects the basic
character of the sample, nor leads to selective carbon losses
in that the respective peaks are changed only in height, rep-
resentative solely of a change in concentration.
Based on the above information, it was concluded that the
concentrated Batavia effluent and the resulting chromato-
graphic fractions were in all aspects truly representative
of the organic matter extant in the unconcentrated effluent.
Additional data on this and other concentration procedures
are found in Appendix 1.
2. Fractionation
The original method of choice for the fractionation of the
organic matter in the concentrated wastewater was the sepa-
ration of organic from inorganic components in the concen-
trate via ion exchange resins follows by fractionation of
the organic components by gel permeation chromatography.
Preliminary experimentation using the ion exchange procedure
and an unconcentrated wastewater proved that the method would
be totally unsatisfactory. Although the removal of the in-
organic fraction was successful, a major portion, greater
than 50%, of the effluent organics was lost due to adsorption
onto the resin.
Therefore the ion exchange step originally posited was elim-
ated and the fractionation procedure narrowed to gel perme-
ation chromatography alone. The chromatography setup used
is shown in Figure 7. Standard chromatographic columns 2.4
x 100 cm, fitted with upflow adaptors to optimize the reso-
lution of the fractionation procedure were employed (Pharmacia
Fine Chemicals) . This apparatus was operated in the cold roofl
At first it was felt that possibly Bio-Gel, a cross-linked
polyacrylamide manufactured by Bio-Rad Corporation of
Richmond, California, would be satisfactory for use as the
gel in the procedure. However, investigation showed that
Bio-Gel would be unsatisfactory. Attention was then focused
on the Sephadex gels manufactured by Pharmacia Fine Chemicals,
The Sephadex gels finally used in the investigation were G-10,
G-25, and G-50. Data relative to these gels may be found in
Table 7.
48
-------
GEL - SEPHADEX G-15
I
o
o
I
s
o
10
SPL- R/SW DOMESTIC SEW3E
(NOT CONCENTRATED)
ELNORA, N.Y
SPL VOL-IOml TOC-62mg/!
TT(AMW300)
I(AMW>I500)
MW 180)
ETCAMW.
-------
Ul
o
ELUANT RESERVOIRS
SAMPLE
RESERVOIR
G-IO
COLU
3 WAY
STOPCOCK
M,
EFFLUENT
5-ml
SIPHON
y
FRACTION
COLLECTOR
COLUfc
ZCOL
G+50
JMN
s
Fig. 7 GEL CHROMATOORAPHY APPARATUS.
-------
Table 7
L/l
After Determann (20)
Approximate Separation Range
Type
G-10
G-15
G-25, coarse
G-25, medium
G-25, fine
G-50, coarse
G-50, medium
G-50, fine
G-75
G-100
G-150
G-200
Particle Water
Size Regain Gel Bed
(dry; in u) (ml/g) (ml/g)
40-120
40-120
100-300
50-150
20-80
100-300
50-150
20-80
40-120
40-120
40-120
40-120
1.0 + 0.1 2-3
1.5 + 0.1 2.5-3.5
2.5 + 0.2 4-6
it it ti
ti n it
5.0 ± 0.3 9-11
It !1 11
11 11 11
7.5 + 0.5 12-15
10.0 + 1.0 15-20
15.0 + 1.5 20-30
20.0 + 2.0 30-40
Peptides and
Globular Proteins
up to 700
up to 1,500
1,000 5,000
it ti
it n
1,000 30,000
u it
ti tt
3,000 70,000
4,000 150,000
5,000 400,000
5,000 800,000
Dextran
Fractions
up to 700
up to 1,500
100 5,000
ti n
it ii
50 10,000
n n
it n
1,000 50,000
1,000 100,000
1,000 150,000
1,000 200,000
-------
Column packing. To assure that the chromatographic analyses
would be successful, the columns were packed with the utmost
care. The gel was swelled for a minimum of 24 hours in the
cold room and the column filled, prior to packing, with glass
distilled water. This water had been stored in the cold room
for the same length of time as the swelling gel. The outlet
of the column was shut off and a funnel fitted with a rubber
stopper was placed into the top of the column and this filled
partly with the glass distilled water.
In one pass sufficient gel to fill the column was introduced
into the funnel and continuously stirred while the gel set-
tled into the column. After a bed height of approximately
5 cm had been established, the outlet of the column was
opened slightly to enable passage of water out of the column
and thus increase the rate of sedimentation of the gel into
the column. After the final bed height had been established,
the outlet was shut off and the funnel removed. The column
above the bed was allowed to remain filled with water to
assure that the top of the bed did not go dry.
The upflow adaptor to be inserted in the top of the column
was connected to the eluent reservoir bottle and the res-
ervoir outlet opened. In this way water was continuously
allowed to flow through the adaptor. The adaptor was then
gently introduced into the top of the column through the
head of water above the column bed with care, so as to assure
that no air bubbles were trapped beneath or on the side of
the adaptor gasket. When the adaptor reached the top of
the bed the gasket control was tightened securely and the
column top closed.
The column was then inverted. This inversion step was de-
vised because previous experience in packing columns had
shown that if the eluent reservoir connection was made to
the bottom adaptor, the gel bed would rise off the column
base. By inverting the column, this difficulty was avoided.
After the connections had been secured the eluent was allowed
to run through the column for 48 hours prior to any sample
application. The eluent used throughout the study was glass
distilled water.
Sample application. As shown in Figure 7 the eluent and
sample reservoirs were connected to a three-way stopcock,
which connection allowed the addition of either sample,
52
-------
up to 10 ml, or eluent. Prior to the actual fractionation
of a sample, approximately 2 to 3 ml of the sample was run
into the column, followed with a bed volume of eluent, which
was run to waste. This initial step was taken to assure (1)
that mixing of dissimilar samples did not occur in the sample
feed line leading to the three-way stopcock, and (2) that
the sample volume registered by the pipette used as the sample
reservoir truly represented the volume applied to the column.
The sample to be fractionated was drawn up into a 10 ml syringe,
and then discharged through a Swinnex membrane filter (0.45 u)
into the 10 ml pipette which served as the sample reservoir.
The stopcock was then adjusted so that the sample could flow
on to the column bed. After sample introduction had been
completed, the stopcock was switched to the eluent position.
Careful checks were made to insure that no air bubbles were
present in the feed line leading to the column.
The column effluent was collected in 5 ml portions, using a
5 ml siphon and an LKB Radirac fraction collector. Several
positions were available for the placement of the eluent
reservoir bottle so that varying flow rates could be obtained.
This eluent reservoir placement was especially critical for
the more loosely cross linked gels, G-25 and G-50.
Column standardization. It has been demonstrated that over
a wide range of elution volume of an organic compound, that
volume in which the maximum concentration of the compound is
obtained, is directly proportional to the log of the molec-
ular weight of the compound (3, 4, 20, 31, 71).
To determine the apparent molecular weight of the fractions
produced by the application of an unknown sample onto a
Sephadex column, the column must first be standardized. The
standards used for G-10 with an upper exclusion limit of
molecular weight 700 were solutions containing glucose (mo-
lecular weight 180), sucrose (molecular weight 342), raffinose
(molecular weight 594.5) and egg albumin (molecular weight
45,000).
Blue dextran, consisting of a dyed dextran with a molecular
weight of approximately 2 million was also used to standardize
the G-10. This dextran was used specifically to determine
the degree of band broadening experienced during the passage
of a sample through the columns.
53
-------
The standard solutions were applied to the G-10 column and
the organic carbon concentrations of the 5 ml fractions
analyzed with the Beckman carbonaceous analyzer. A plot
was then developed of milligrams per liter carbon versus
fraction number, with each fraction number of maximum con-
centration of each of the organic compounds in the standard
solution taken as the elution volume of that organic com-
pound. Based on these data a plot of fraction number versus
log molecular weight was subsequently produced, thus pro-
viding a standard curve for the G-10 column. Using the
elution volumes of the fractions of an unknown sample, one
could then enter the curve and deduce an apparent molecular
weight.
For the G-25 and G-50 standardizations, the standard solution
contained egg albumin (molecular weight 45,000). The points
chosen to develop the standard curves for the G-25 and G-50
columns were the theoretical end of the bed calculated from
data provided by the Sephadex manufacturers and the elution
volume of the egg albumin, which denoted the void volume of
the column.
Carbon analysis. The organic carbon in each of the 5 ml frac-
tions was determined using the Beckman carbonaceous analyzer.
A 20 microliter sample of the fraction was removed with a
syringe and discharged into the effluent port of the analyzer.
The sample then passed into a catalytic combustion chamber
maintained at a temperature of 950°C. In this catalytic
combustion chamber the carbonaceous portion of the sample
is oxidized in the presence of a cobalt catalyst and pure
oxygen carrier gas. This gas then carries the carbon diox-
ide produced from the combustion of the carbonaceous material
and the water vapor out of the furnace. The water vapor is
condensed in a trap and the carbon dioxide oxygen mixture re-
maining is then swept into the infrared analyzer.
The range of the instrument was calibrated with oxalic acid
(H2C204 • 2H20). For a range between 0 mg/1 and 100 mg/1 C,
the oxalic acid solution was made up initially in a concen-
tration of 96 mg/1 C, by dissolving 500 rag of the compound
in distilled water and making the solution up to 1 liter
volume. Portions of this solution were then diluted to pro-
duce standards containing 48 mg/1 C and 24 mg/1 C. At tha
commencement of each set of analyses using the carbon ana-
lyzer, the standards were injected and the results noted.
54
-------
By judicious adjustment of the gain, the instrument can be
made to read out the concentrations of carbon existing in
the respective standards, and respond linearly in the ranee
0-100 mg/1.
Fraction compositing. To secure a sufficient volume of frac-
tions for the subsequent biological analyses, the wastewater
sample concentrate under investigation was fractionated
several times on Sephadex G-10 and the similar resulting
fractions composited. The fractions comprising the front
from G-10 (molecular weight greater than 700), after the
compositing step, were reconcentrated by freeze-drying and
this concentration introduced onto the G-25 column.
The same procedure as followed with the G-10 column was also
employed in compositing the similar fractions developed dur-
ing the G-25 chromatographic analysis. The front from the
G-25 column (molecular weight greater than 5,000) was recon-
centrated and introduced onto the G-50 column, and the frac-
tion appearing in the G-50 effluent composited.
The composite fractions were stored in the cold room in acid-
washed serum bottles provided with syringe caps. Prior to
storage, the fractions were analyzed for pH, conductivity and
organic carbon concentration.
3. Chromatographic Data
Results with Bio-Gel P-2. The first attempts at fractionation
were made with Bio-Gel P-2. Several difficulties, however, •
were encountered with the gel leading to its disuse after a
short experimental procedure. It was first found that when
blue dextran was applied to the gel, the color group of the
dextran was adsorbed onto the gel material and no amount of
eluent passed through the column would remove it from the
gel bed. Subsequently a sample of concentrated effluent from
the Eastern-Western facility was applied to the column. The
resulting chromatographic data shown in Figure 8 showed only
one peak approximately at the middle of the bed volume. This
can be compared with the Sephadex G-10 chromatogram of the
same effluent shown in Figure 3. No attempt was made to
investigate the reasons for the failure of Bio-Gel P-2 to
fractionate the wastewater organic components or to allow
blue dextran to pass through without adsorption of the dex-
tran color group. These occurrences provided the impetus
55
-------
80
o»
O
t
O
O
z
g
o:
O
Ui
60
40
20
GEL- BIO-GEL P2
SPL - CONCENTRATED
EXTENDED AERATION
EFFLUENT
SPL VOL- 5ml TOC- 580mg/l
COLUMN PARAMETERS:
2.5cm x 90cm
35 4O 45 50 55 60 65 70 75 80 85
FRACTION NUMBER
Fig. 8 ELUTION DIAGRAM OF THE FRACTIONATION OF
CONCENTRATED EASTERN - WESTERN TREATMENT
PLANT EFFLUENT ON BlO-GEL P2.
-------
for a switch to the Sephadex gels.
Standardization of Sephadex feels.
Sephadex G-10. The standard solution used with Sephadex
G-10 was split into two portions. . .one containing egg albu-
min, raffinose and glucose and the other sucrose. The reason
for this split was that originally a standard solution had
been made up with all four compounds and the closeness of
raffinose, glucose and sucrose in molecular weight did not
allow for the resolution of these three compounds into sep-
arate and distinct peaks. The concentrations of these com-
ponents in the standards used for the Sephadex G-10 gel
column are given in Table 8. The chromatograms resulting
from the application of standards 1 and 2 to the G-10 column
are shown in Figure 9.
Based on the data presented therein, the elution volumes of
the four standards and their respective molecular weights
were used to produce a plot of elution volume versus log
molecular weight for this specific column. A regression
analysis was employed to obtain a line of best fit for the
data. Using this plot, shown in Figure 10, one would then
be able to assign an apparent molecular weight to the or-
ganic fractions produced upon passage of an unknown sample
through the G-10 gel.
Sephadex G-25 and G-50 standardizations. Both G-25, with
an upper exclusion limit of 5,000, and G-50, with an upper
exclusion limit of 30,000, were standardized with a solution
of egg albumin in glass distilled water at a concentration
of 250 mg/1. The chromatograms resulting from the applica-
tion of this standard to the G-25 and G-50 columns are shown
in Figure 11.
Using the elution volume of the egg albumin and the theoret-
ical end of the bed calculated from data provided by the
manufacturer, the graphical representation of elution volumne
versus log molecular weight was developed for the G-25 column.
This standard curve is shown in Figure 12.
G-10 Chromatographic data. The 1-22-69 Batavia concentrate
containing 380 mg/1 of total organic carbon was concentrated
by the freeze-drying method to a total organic carbon concen-
tration of 1500 mg/1, with a resulting percentage recovery
of 97%.
57
-------
Table 8. Concentrations of Organic Compounds in
Sephadex G-10 Column Standards
Compound
Egg Albumin
Raffinose
Glucose
Sucrose
Standard
Number
1
1
1
2
Concentrat ion
mg/1
250
687
623
625
Molecular
Weight
45,000
594.5
180
342
58
-------
GLUCOSE
g 10-
GEL-SEPHADEX G-IO
SPL-EGG ALBUMIN (250mg/0
RAFF!NOSE«687mg/l)
GUUCOSE(623 mg/1 )
SPL VOL- 10ml
COLUMN PARAMETERS:
25cm x 85cm; 48ml/hr.
SUCROSE
50 60
FRACTION NUMBER
GEL- SEPHADEX G-IO
SPL- SUCROSE(625mg/l)
SPL VOL- 10
COLUMN PARAMETERS:
25 cm x 85cm ;45ml/hr
40 50 60
FRACTION NUMBER
70
80
Fig. 9 GEL CHROMATOORAPHY OF STANDARD SOLUTIONS
I AND 2 ON SEPHADEX G-IO.
59
-------
7Or
65
60
55
oc
u.
45
40
100
GEL- SEPHADEX 6-10
VOID VOLUME-175ml
2.5cm x 85cm
JLUCOSE
{SUCROSE
.RAFFINOSE
1
I
i
i
200
300 400 500 700 1000
MOLECULAR WEIGHT
Fig. 10 GRAPHICAL REPRESENTATION OF THE RELATIONSHIP
BETWEEN MOLECULAR WEttHT AND ELUTION VOLUME
FOR SEPHADEX G-IO.
60
-------
50
00
o
o
o
tr
o
E
o
o
<
o
20
10
60'
50-
40
30
20
K>
+
GEL- SEPHADEX G-25
SPL- EGG ALBUMIN
(250mg/l)
SPL VOL- 5ml
COLUMN PARAMETERS
2.5cm x 92cm§ I20ml/hr.
OOOOd
1
30
40 50 60 70
FRACTION NUMBER
80 90
GEL- SEPHADEX 6-50
SPL - EGG ALBUMIN
(250mg/l)
SPL VOL - 10ml
COLUMN PARAMETERS
2.5cm x 90cm} I80ml/hr.
30 40 50 60 70
FRACTION NUMBER
80 90
Fig. 11 6EL CHROMATOORAPHY OF STANDARD EOO ALBUMIN
SOLUTION ON SEPHADEX G-25 AND SEPHADEC G-50.
61
-------
OOr
90
80-
70
60
50
4O
CALCULATED
,BED VOLUME
1000
GEL-SEPHADEX 6-25
2.5cm x 92cm
VOID
VOLUME
j 1 1—I—
MOLECULAR
5000
WEIGHT
OOOO
Fig. 12 GRAPHICAL REPRESENTATION OF THE RELATIONSHIP BETWEEN
MOLECULAR WEIGHT AND ELUTION VOLUME FOR SEPHADEX G~25
62
-------
The resulting concentrate was applied to the G-10 column
in 10 ml passes. The graphs shown in Figure 13 represent
the chromatograms produced as a result of the six runs on
G-10 with the Batavia concentrate and are noted therein as
Runs 1 through 6. The apparent molecular weights listed
thereon are derived from the standard G-10 curve. Those
organic fractions in the lower portions of the chromatogram
have not been assigned a molecular weight as this area of
a G-10 elution profile contain fractions with an indefinable
molecular weight (20).
The data in Table 9 show the total organic carbon content in
milligrams of each of the G-10 fractions for all six runs
performed. The determination of the percentage of original
effluent organic carbon that each fraction represented was
based on these data. To ascertain the percentage of applied
carbon represented by the various G-10 fractions, the values
of carbon applied and total organic carbon content in each
of the fractions are totaled and the percentages calculated
from these totals. For example, the total organic carbon
applied to the G-10 column in the six runs was 90 rag and of
that amount 27.9 tng was found in the frontal peak, and thus
the G-10 frontal peak, based on these data, was calculated
to represent 31% of the organic carbon applied to the column.
Since no change had occurred in the character of the original
sample through the concentration and chromatographic opera-
tions, this percentage should hold as a valid measure of the
proportion of the G-10 frontal peak in the unconcentrated
membrane filtered effluent. This fact has great significance,
particularly with regard to the calculation of the percent of
the effluent organic carbon content represented by the various
fractions.
The fractions produced during the six runs on G-10 were com-
posited in acid-washed serum bottles and stored in the cold
room. The G-10 frontal peak was kept for further fraction-
ation on Sephadex G-25 and the remainder of the peaks for
biostimulation studies.
G-25 chromatographic data. The G-10 frontal peaks appearing
in maximum concentration at fraction 35 noted as peak G-10-I
were, subsequent to compositing, examined for total organic
carbon concentration and reconcentrated by freeze-drying to
63
-------
300r
2KA.M.W 250)
GEL-SEPHADEX G-IO
SPL-SECONDARY
(CONCENTRATED BY
FREEZE DRYING)
BATAVIA, NY
SPLVOL-IOml 7OC-l500mg/l
COLUMN' PARAMETERS:
2.5cm x 85cm ; 50ml/hr.
I(A.MW>700)
JEA (A.M.W. NOT DEFINED)
50 55 60 65 TO
FRACTION NUMBER
Fig. 13a EumON DIAGRAM OF THE FRACTIONATION OF CONCENTRATED
BATAVIA EFFLUENT ON SEPHADEX G-IO, RUN I.
-------
cr.
Ln
300
TKAM.W 260)
GEL - SEPHADEX G-IO
SPL- SECONDARY EFFLUENT
(CONCENTRATED BY
FREEZE DRYING)
BATAVIA, NY
SPL VOL- 10ml TOG-1500 mg/l
COLUMN PARAMETERS:
2.5cm x 85cm ;50ml/hr
mA(AMW NOT DEFINED)
50 55 60 65 70
FRACTION NUMBER
Fig. 13b EumON WAORAM OF THE FRACTIONATION OF CONCENTRATED
BATAVIA EFFLUENT ON SEPHADEX G-IO, RUN 2.
-------
IHAMW. 250)
GEL- SEPHADEX G-IO
SPL- SECONDARY EFFLUENT
(CONCENTRATED BY
FREEZE DRYING)
BATAVIA, N.Y.
SPLVOL-IOml TOC-l50Omg/l
COLUMN PARAMETERS:
25cm x 85cm ; 50mg/l
I(AMW>700)
JUACAMW. NOT DERNED)
50 55
FRACTION
60 65
NUMBER
Fig. 13c ELUTION OUORAM OF THE FRACT.ONAT.ON OF CONCENTRATED
BATAVIA EFFUUENT ON SEPHADEX G-IO, RUN 3.
-------
,1(AMW >700)
260 h .- 3I(AMW 250)
GEL-SEPHADEX 6-10
SPL-SECONDARY EFFLUENT
(CONCENTRATED BY
FREEZE DRYING)
BATAVIA, N.Y
SPL VOL- 10ml TOC-1500mg/l
COLUMN PARAMETERS:
2.5cm x 85cm ; 50ml/hr
HrA(A.M.W NOT DEFINED)
50 55 60 65 70
FRACTION NUMBER
Fig. 13d EOJTJON DIAGRAM OF THE FRACT1ONATION OF CONCENTRATED
BATAVIA EFFLUENT ON SEPHADEX G-IO, RUN 4.
-------
300r
00
TKAM.W 250)
GEL-SEPHADEX G-IO
SPL-SE3CONDARY EFFLUENT
«X)NCENTRATED BY
FREEZE DRYING)
BATAVJA, N.Y
SPL VOL - 10ml TOCH500mg/l
COLUMN PARAMETERS:
2.5cm x 85cm ; 50ml/hr.
I (AM.W. » 700)
TEAfAMW NOT DEFINED)
50 55 60 65
FRACTION NUMBER
ISC
70 75 80 85
Fig. I3e ELUTJON DIAGRAM OF THE FRACTIONATIOW OF CONCENTRATED
BATAVIA EFFLUENT ON SEPHADEX G-IO, RUN 5.
-------
vD
300
260
220
^
6 180
z
8 140
a:
3 100
ICAAAW 700)
o
<
e>
oc
o
60
20
TJ(AMW 250)
30
GEL - SEPHADEX G-IO
SPL - SECONDARY EFFLUENT
(CONCENTRATED BY
FREEZE DRYING)
BATAVIA, NY
SPL VOL - 10 mI TOC -1500 mg/l
COLUMN PARAMETERS:
2.5cm 85cm \50m\/\vr.
NOT DERNED)
35 40
45
50 55 60 65 70
FRACTION NUMBER
75
80 85
Fig. I3f ELUTION DIAGRAM OF THE FRACTIONATION OF CONCENTRATED
BATAVIA EFFLUENT ON SEPHADEX G-IO, RUN 6,
-------
Table 9. Organic Carbon Content of G-IO Fractions
Organic
Carbon
Run
1
2
3
4
5
6
Total
Applied
(mg)
15
15
15
15
15
15_
90
.0
.0
.0
.0
.0
.0
^^^^
.0
4
4
4
5
4
4
•i^
27
I
.3
.3
.5
.1
.9
.8
^^M^
.9
4
5
5
5
5
5
•^H
31
Organic Carbon
of Fractions
II
.1
.6
.4
.6
.2
il
.5
Ilia
1
1
1
1
1
1
•M
9
.4
.5
.6
.7
.8
.7
M^BB
.7
Content
(mg)
Illb
0.66
0.73
0.64
0.63
0.77
0.65
4.08
0
0
0
0
0
Q
2
IV
.38
.43
.27
.32
.33
.36
^^••^•B
.09
70
-------
assure measurable carbon concentrations upon fractionation
on the G-25 column.
This concentrate, containing 720 mg/1 organic carbon, was
applied to the G-25 column in two passes of 10 ml each.
The resulting chromatographs are shown in Figure 14 as
Runs 1 and 2. The frontal peak noted as G-25-I, commenced
its appearance at fraction number 40, the same position
noted for the initial appearance of the egg albumin standard
during the column standardization procedure; and, the second
peak denoted as G-25-II appeared in highest concentration
at fraction number 80, very close to the calculated bed
volume of the column. Using the G-25 standard curve appar-
ent molecular weights of greater than 5,000 and 1,000 were
assigned to peaks G-25-I and G-25-II respectively. The
data in Table 10 show the total organic carbon content in
milligrams of each of the G-25 fractions. Following the
procedure used with the G-10 fractions, the determination
of the percentage of original effluent organic carbon con-
tent was based upon these data.
Subsequent to the completion of fractionation, these G-25
fractions were stored in anticipation of further experi-
mental procedures.
G-50 chromatographic data. The total organic carbon concen-
tration of the composite G-25-I was found to be 64 mg/1.
129 ml of this fraction was then reconcentrated by freeze-
drying and the final volume made up to 25 ml with glass
distilled water. The total organic carbon concentration of
this final volume was found to be 330 mg/1, indicating a
recovery factor of 100%.
This G-25-I concentrate was then applied to the G-50 column
in four passes. Although the column had been successfully
standardized, the data developed from this particular set^
of chromatographic experiments showed some dissimilarity in
the shape of the elution profile produced during each one
of the passes. The G-50 column was then repacked and re-
standardized and the effluent from each of the four G-50
runs was composited. This composite was reconcentrated by
freeze-drying and the final volume was made up to 10 ml.
The total organic carbon concentration was found to be
380 mg/1.
71
-------
ro
KAMW. > 5000)
GEL-SEPHADEX G-25
SPL- COMPOSITE FRONTAL
PEAK G-IO I
SPL VOL- 10 ml TOC-720 mg/l
COLUMN PARAMETERS:
2.5cm x92cmil2Oml/hr.
I (AMW 1000)
35 40 45 50
55 60 65 70
FRACTION NUMBER
75
80 85
Fig. 14a ELUTION DIAGRAM RESULTING FROM
CONCENTRATED COMPOSITE FRONTAL
SEPHADEX G-25, RUN I.
THE FRACTIONATION OF
PEAK G-IO-I ON
-------
00
KAMW > 5000)
GEL- SEPHADEX G -25
SPL- COMPOSITE FRONTAL
PEAK G-IO I
SPL VOL- 10ml TOC-720mg/l
COLUMN PARAMETERS:
2.5cm x 92cm j!20ml/hr
XICAMW 1000)
55 60 65 70
FRACTION NUMBER
Fig. I4b ELUTION DIAGRAM RESULTINO FROM THE FRACTIONATION OF
CONCENTRATED COMPOSITE FRONTAL PEAK G~IO-I ON
SEPHADEX G-25, RUN 2.
-------
Table 10. Organic Carbon Content of G-25 Fractions
Organic Organic Carbon Content
Carbon Applied of Fractions
Run (ing) (mg)
Front II
1 7.2 4.9 1.9
2 7.2 5.5 1.5
Total 14.4 10.4 3.4
74
-------
This secondary concentrate was then applied to the G-50
column in two passes of 4 ml and 5 ml each. The chromato-
grams produced upon fractionation of the G-50 column were
similar and the peak in each case based on its point of
appearance in the respective chromatograms adjudged to be
a frontal peak and assigned a molecular weight of greater
than 30,000. The G-50 chromatographs, produced as a result
of these aforementioned final passes, are shown in Figure 15
Table 11 depicts data regarding the total organic carbon con-
tent of the applied concentrate and the fraction produced by
the G-50 column. As with the G-10 and G-25 data, these data
were used to determine the percent of original effluent or-
ganic carbon content represented by the G-50-I fraction.
Table 11. Organic Carbon Content of G-50 Fractions
Organic Carbon Organic Carbon Content
Applied of Fractions
Run (mg) (mg)
1 1.5 1.2
2 1.9 1.5
Total 3.4 2.7
Analyses of fractions. Data relative to the fractions ob-
tained from the G-10, G-25 and G-50 fractionations are shown
in Table 12. The data presented therein include total or-
ganic carbon concentration, pH, conductivity and apparent
molecular weight. As evidenced from the conductivity data,
the G-10 column succeeded in separating the great majority
of the wastewater inorganic constituents from the frontal
peak, the greatest percentage of these inorganic constituents
appearing in fraction G-10-IIla.
Conductivity determinations of the composite fractions were
made to allow an interpretation of biological effects pro-
duced by the fractions in light of inorganic constituent
concentrations. If the fraction did exhibit an effect, and
its conductivity was low, then the interpretation of this
effect as arising from an organic causative factor would be
75
-------
KAMW. > 30pOO)
j.
J.
GEL-SEPHADEX G-50
SPL - COMPOSITE FRONTAL
PEAK G-25 I
SPL VOL- 4mJ TOG -380 mg/l
COLUMN PARAMETERS:
2.5 cm x 90 cm ; 05 ml/he
5O 55 6O
FRACTION
65 70
NUMBER
75
80
85
90
Fig. I5a ELUTION DIAGRAM RESULTING FROM THE FRACTIONATION OF
CONCENTRATED COMPO8TTE FRONTAt PEAK G~25 I ON
SEPHADEX G-5O, RUN I.
-------
40r HAMW > 30,000)
GEL - SEPHADEX 6-5O
SPL - COMPOSITE FRONTAL
PEAK 6-25 I
SPL VOL-5m! TOC-380mg/l
COLUMN PARAMETERS:
£5cm x90cm ;B5ml/hr.
55 60 65 70
FRACTION NUMBER
75
80 85
90
Fig. 15b EutflON CMA9RAM RESULTING FROM THE FRACTIONATtON OF
COMPOSITE FRONTAL PEAK G~25 I ON SEPHADEX G~50, RUN
2.
-------
Table 12. Selected Parameters for Wastewater Fractions
Total Organic
Carbon
Concentration
Conductivity
Apparent
Molecular
Fraction
G-10-I
G-10-II
G-10 Ilia
G-10 Illb
G-10- IV
G-25-I
G-25-II
G-50-I
mg/1
120
100
60
30
17
64
39
23
PH
5.6
8.2
7.1
7.7
6.3
7.1
6.4
6.4
umhos/cm
850
2,400
20,000
380
34
56
5.7
8.5
Weight
>700
250
*
*
*
>5,000
1,000
> 30, 000
* indefinable
78
-------
appropriate. The organic carbon concentrations of the
composite fractions were determined for use in the bio-
logical portion of the study. With these data in hand,
and an approximation of the percentage of original effluent
organic carbon content that each of the fractions repre-
sented, the volume of fraction required to be placed in a
culture flask to match a set effluent dilution level would
be easily obtainable.
Infrared analysis. Consideration was given to infrared
analysis of the fractions as an aid in the identification of
the organic compounds extant therein. One attempt was made
to obtain the infrared spectrum of an organic fraction.
The fraction chosen for investigation was G-10-II. One ml
of the chosen fraction was placed in a test tube, the tube
fitted into a port on the freeze-drying apparatus, and the
sample concentrated. Subsequently the dry sample was taken
up with 10 ml of spectral quality chloroform and analyzed
using a Beckman IR 20 spectrophotometer. No spectrum was
obtained.
A generalization regarding infrared analysis requires that
the unknown represent 10% by weight of the sample solution.
Such a high percentage, even if only 1 ml of chloroform was
used, would require more fraction volume than that obtained
in the fractionations already performed, thus leaving no
sample for the culture experiments. Based on the above, all
further attempts at infrared analysis were deleted from the
study.
Percentage of original effluent represented by fractions.
As noted previously, calculations were performed to adduce the
percentage of organic carbon applied to each column that the
respective fractions represented. Using the postulate that
concentration of freeze-drying and passage through a gel col-
umn allowed for no change in the basic character of the organic
constituents in the concentrated effluent, one would then
simply calculate the percentage of original effluent organic
carbon content represented by such fractions. The above data
are presented in Table 13.
In addition, this table also contains data relative to the
organic carbon concentrations of each fraction in the original
79
-------
Table 13. Percent of Original Effluent Organic Carbon
Content in Fractions
Percent of
Organic Carbon
Gel Fraction Applied
G-10
G-25
G-50
I
II
Ilia
Illb
IV
I
II
I
31
35
11
5
2
72
24
80
Percent of
Original
Effluent
Organic Carbon
Content
31
35
11
5
2
22
7
18
Organic Carbon
Concentration
in Original
Effluent
(mg/1)
2.4
0.8
0.4
0.2
0.5
1.3
80
-------
effluent. These data were based on an effluent carbon con-
centration of 7 mg/1. For example, as shown in Table 13,
fraction G-10-II is shown to represent 35% of the original
effluent organic carbon concentration. Thus, if such con-
centration was 7 mg/1, fraction G-10-II would be found in
the original effluent to have a concentration of 2.4 mg/1.
Biological Investigations
1. Materials and Procedures
To carry out the extensive biological investigations planned,
it was decided that one room in the North Hall Laboratory
would be converted into an algal culture room. To achieve
this end, the windows of the room were covered with raasonite
to exclude extraneous light and a Carrier air conditioner
installed to maintain the temperature of the room at 23 +
A continuously stirred batch culture apparatus noted in
Figure 16 was constructed, using pressed board for the
structure and 6 Dayton 1/100 horsepower motors for motive
power. Six spots were provided on each one of the culture
apparatus for the placement of algal culture flasks. A
portion of a rubber stopper (size 7) was cut out to provide
space for the placement of a 2 by 8 cm magnet. The magnet
and stopper were then formed into a unit by wrapping the
combination with ordinary uninsulated copper wire. The
stopper was placed on the shaft of the motor so that as the
shaft revolved, the magnet would also revolve. A teflon
coated 4 cm magnet was put in each culture flask and the
interaction of the magnets kept the cultures continuously
stirred.
Sixteen of the continuously stirred batch culture apparatus
were built, providing facilities for 96 individual algal
culture experiments.
Suspended above the culture apparatus were banks of 40 watt
fluorescent bulbs. These banks were suspended from bracing
and assured that the irradiant setting for the algal culture
experiments could, at will, be raised or lowered. The set-
tings used in these experiments were 550-foot candles for
Selenastrum capricornutum (85, 86) and 150-foot candles for
Anabaena flos-aquae and Microcystis aeruginosa.
81
-------
oo
to
' x
1/100 HP
MOTOR
7
I
r
X
ELECT Rl
STRIP
1
C I
[LET
CULTURE APPARATUS
Fig. 16 CONTINUOUSLY STIRRED BATCH CULTURE APPARATUS.
-------
The algae selected for use in this study were those proposed
for use in the provisional algal assay procedure; Microcystis
aeruginosa obtained from the laboratories of Dr. George P.
Fitzgerald at the University of Wisconsin; and Selenastrum
capricornutum and Anabaena flos-aquae obtained from the
Pacific Northwest Laboratory, Federal Water Quality Adminis-
tration, United States Department of Interior at Corvallis,
Oregon.
The media used in the investigation were: 1/10 Gorham's medium,
constituents and concentrations shown in Table 14 (45), and
the basic ASM medium of the provisional algal assay procedure
(47). The constituents and concentrations of basic ASM are
as noted in Table 15. Basic ASM medium is the ASM medium of
McLachlan and Gorham (60) with a reduction in the concentra-
tion of K2HP04 from 17.4 mg/1 to 3.48 mg/1, a reduction in
the Na2EDTA concentration from 7.4 mg/1 to 1.0 mg/1 and the
addition of 50 mg/1 of sodium carbonate.
Stock solutions of both media were made up as noted in the
respective tables. Prior to a run of experiments, aliquots
of the stock solutions sufficient to make up 18 liters of
medium were discharged into 5 liters of glass distilled water
and sufficient glass distilled water added to make up 18
liters. This bottle of medium was then stored in the cold
room and used during the experimental procedure. The life
of a bottle of medium at no time exceeded 3 weeks. Directly
prior to experimentation, Erlenmeyer flasks containing ali-
quots of the medium were stoppered, autoclaved with the teflon
coated magnet in place and stored in the culture room.
During the initial phases of the experimental procedures, only
1/10 Gorham's was used as the medium. It was discovered, how-
ever, that in using this medium reproducible growth rates for
the cultures under investigation were not obtainable. This
same effect was noted by Dr. Fitzgerald (29). It has been
posited that the decrease in carbonate concentration seriously
reduced the buffering capacity of the medium, thus allowing
for wide and erratic pH changes which in the limit led to non-
reproducible growth of the algae.
At this point, a switch was made to the basic ASM medium, in
which the algae exhibited a reproducible growth rate subse-
quent to several inoculations.
83
-------
Table 14. 1/10 Gorham's Medium
Compound
Stock For 1 Liter of
Solution Solution Use
(g/1) (ml)
Final
Concentrat ion
in Medium (mg/1)
NaN03
K2HP04
CaCl2-2H20
Na2C03
MgS04*7H20
NaSi03-9H20
Ferric Citrate
Citric Acid
Nao • EDTA
49.60
3.90
3.60
2.00
7.50
5.80
0.60
0.60
0.10
1
1
1
1
1
1
1
1
1
49.6
3.9
3.6
2.0
7.5
5.8
0.6
0.6
0.1
Glass Distilled Water - Dilute to 1 Liter.
84
-------
Table 15. Basic ASM Medium
Compound
NaN03
K2HP04
CaCl2'2H20
Na2C03
MgS04-7H20
MgCl2
FeCl3
Na0 * EDTA
Stock For 1 Liter of
Solution Solution Use
(g/1) (ml)
8.50
0.348
1.47
5.00
4.90
1.90
0.032
0.100
10
10
10
10
10
10
10
10
Final
Concentration
in Medium (mg/1)
85.0
3.48
14.7
50.0
49.0
19.0
0.32
1.0
Glass Distilled Water - Dilute to 1 Liter.
85
-------
At the commencement of the investigation, the algae were
cultured in 500 ml aliquots of the medium contained in 1
liter flasks. Subsequently it was determined that, to ar-
rive at even a minimal concentration of the organic waste-
water fractions in a 500 ml aliquot of medium, it would
require inordinately large volumes of effluent fractions.
Thus to reduce the volume of fractions required, the cul-
ture volume was reduced. At this juncture, the cultures
were transferred and grown in 100 ml portions of Basic ASM
medium contained in 250 ml Erlenmeyer flasks.
Upon transference to the new chosen conditions, the cultures
did not immediately respond and several weeks passed before
a representative and a reproducible growth rate could be
obtained. Microcystis aeruginosa never recovered from the
initial shock of the transfer. It was found that Microcystis,
under the posited conditions, would only grow if the culture
was left completely unstirred. A decision was made at this
time that the study would be carried forward using Anabaena
flos-aquae alone as a representative of blue-green algae
and Selenastrum capricornutum as a representative of the
green algae.
Throughout the investigation, the cultures were transferred
at set times after initial inoculation. Selenastrum capri-
cornutum being transferred from a culture flask that had
been allowed to proliferate for 4 or 5 days and Anabaena
flos-aquae from a flask growing for 6 to 7 days. The vol-
ume of inoculum used at all times was 1 ml. During the
culture experimentation involving the organic fractions of
the Batavia effluent, selected flasks were marked and the
desired volume of organic fraction, based on previous cal-
culations, was drawn up from the serum bottle into a syringe,
and the contents of the syringe discharged directly into the
culture flask. Immediately thereupon, the flask was inocu-
lated along with controls and placed on the continuously
stirred batch culture apparatus.
The growth of the cultures under investigation was followed,
using spectrophotometric techniques. The chosen instrument
was the Beckman DU-2 spectrophometer. The culture to be
measured was treated as follows: a sample of the culture was
removed from the culture flask with a 10 ml sterile disposable
pipette and introduced into a 5 cm spectrophotometric cell,
which had been previously acid-washed and set aside specifi-
cally for use with only one species of algae. The 5 cm cell
86
-------
was then placed in the DU-2 and the absorbance of the culture
measured at 750 nm (94) . After the measurement of the absor-
bance, the contents of the 5 cm cell were reintroduced into
the culture flask and the flask returned to the continuously
stirred batch culture apparatus.
Although absorbance measurements are widely used as reflec-
tions of the growth of a culture, cell counts are also
performed in order to develop a correlation between the
absorbance measured and the cell concentration of the sample.
Selenastrum and Microcystis were counted with a brightline
haemocytometer, using the red corpuscle method.
Since Anabaena flos-aquae is not a unicellular algae it did
not lend itself readily to the cell counting method and thus
a correlation between dry weight and absorbance was developed.
After an absorbance had been measured 25 ml to 50 ml of the
culture was removed from the flask and membrane filtered
using a 0.45 u membrane filter and a 47 mm filter holder
apparatus. Prior to the discharge of the culture onto the
membrane filter, the filter was washed with glass distilled
water. After completion of the filtration of the Anabaena
flos-aquae, the filters were removed from the filter holder,
placed in petri dishes and then in a constant temperature
oven at 70°C for 24 hours. Subsequent to this drying opera-
tion, the filters were allowed to cool for one hour and were
weighed. The difference between this weight and original
weight of the filter was interpreted as the dry weight of
the algae. Dry weight measurements of a culture with a
given absorbance were performed in triplicate and the indi-
vidual weights in the series averaged after correction based
on changes in the weight of a control filter. In both cases
of cell count or dry weight determinations, sufficient
observations were made so as to produce a statistically
reliable correlation between the chosen parameter and
absorbance.
For any given culture, whether spiked with a fraction or a
control, a regression analysis was performed using those
points determining the log phase of growth. Using this re-
gression technique a growth rate was developed, which then
allowed for a comparison of the growth characteristics of
the cultures.
87
-------
In addition, these investigations attempted to ascertain
the effect of various concentrations of nitrogen and phos-
phorus on the growth rate of Selenastrum capricornutum.
The media employed were as described in Tables 14 and 15
with one minor alteration in the modified Gorham's medium.
The concentration of Na^COo was increased to 50 mg/1 as an
aid to pH control. Conditions of illumination and tempera-
ture were as noted previously.
For these experiments, the culture method employed bubbling
tubes (ie, 25 x 200 mm optically matched pyrex test tubes
filled with 25 mm of growth medium which was continuously
aerated and mixed by passing water saturated air through a
section of glass tubing that is vertically placed to the
bottom of the culture tube). The growth of the culture
was followed using a Bausch and Lomb Spectronic 20 at 750
nra and a one inch cell path.
2. Biological Results
Methodological considerations. Subsequent to the conclusion
of the chromatographic portion of the study and the coincident
garnering of the organic fractions, biological investigations
were undertaken. As noted in the section on experimental pro-
cedure the test organisms to be studied, Selenastrum capri-
cornutum and Anabaena flos-aquae, were cultured under the
posited conditions both as controls and in culture vessels
spiked with selected organic fractions or combinations thereof,
It was decided at the outset that the first sets of experi-
ments would involve addition of the fractions in such amounts
as to produce a final concentration of the selected fractions
in the culture vessel approximating that as was found to
exist in the filtered effluent (Table 13). Since it was felt
that an effect not being had at this maximum level would in
all probability not exist at a reduced concentration, adherend
to this procedure would immediately allow one to note if and
what fractions would produce a change in growth rate when adde
to the culture.
The response to the organisms to this fraction addition was,
as noted previously, to be followed and ascertained by com-
parison and the growth rates developed with those found for
the control cultures. The growth rate for an individual
culture was determined by employment of a linear regression
88
-------
technique on those points of the growth curve representing
the log phase of growth. For each set of experiments the
individual growth rates found were then averaged to produce
the mean growth rates presented herein.
To adjudge the validity of the mean growth rates obtained,
such data were subjected to statistical analysis using
Student's T distribution for small sampling theory. Based
on this technique the 95% confidence interval for each mean
growth rate was determined. These data then afforded a
measure of significance of the difference in growth rates
obtained with the controls and treated cultures.
Since the growth rates were to be determined by measurement
of culture absorbance at 750 nm, it was decided that some
correlation between absorbance at such wavelength and a chosen
paramater of growth should be provided. Presented in Figure
17 are graphical presentations of such correlations. In the
case of Selenastrum capricornutum this parameter was cell
concentration and for Anabaena flos-aquae dry weight was
determined in accordance with the method outlined in the
experimental procedures section.
Effect of the organic fractions on the growth of
Selenastrum capricornutum.
Maximum levels of organic fractions. Presented in Table
16 are data relative to the effect produced on Selenastrum
capricornutum when exposed to the concentrated effluent and
its organic fractions in the culture vessels. Noted in this
table are the concentrations of the fractions and the growth
rates found under the varying test conditions. Also shown
therein are the 95% confidence intervals.
As can be seen from such data, three of the organic fractions,
G-10-IIIb, G-25-II, and G-50-I, and the concentrated effluent
caused significant responses in Selenastrum capricornuturn.
The largest changes in growth rate were produced with the
addition of fraction G-50-I and the concentrated effluent,
with minimal changes in growth rate demonstrated upon addition
of the apparently low molecular weight fractions G-10-IIIb
and G-25-II. The data also indicate the response found was
selective in that no correlation between the increase in
89
-------
4000r
3000
rt
-§2000
1000
DU-2-5cm CELL
75Onm
COEFF OF CORR- 0.968
0.1 0.2 03
ABSORBANCE
0.4
o>
80
60
40
20-
ANABAENA FU»-AQUAE
DU-2-5cmCELL
750nm
CXEFF OF CX)RR-0.935
0.1
0.3
0.4
Fig. 17 GRAPHICAL REPRESENTATION OF THE CORRELATION BETWEEN
ABSORBANCES AND SELECTED GROWTH PARAMETERS FOR
SELENASTRUM CAPRICORNUTUM AND ANABAENA FUOS-AQUAE.
90
-------
Table 16. Effect of Fractions on the Growth
Rate of Selenastrum capricornutum
Concentration of Growth
Fraction mg/1 Rate
Fraction Organic Carbon KIQ (day~
Control
G-10-II
G-10-IIIa
G-10-IIIb
G-10-IV
G-25-II
G-50-I
2.0
0.6
0.3
0.2
0.4
1.3
0.43
0.43
0.42
0.49
0.43
0.50
0.72
Confidence
) Interval
0.42
0.41
0.40.
0.48
0.39
0.49
0.70
- 0.44
- 0.45
- 0.44
- 0.50
- 0.47
- 0.51
- 0.74
Concentrated
Effluent
7.0
0.96
0.89 - 1.03
91
-------
growth rate and the organic carbon concentration of the
fractions in the culture flask is evident. In point of
fact within the fraction series itself that fraction having
the highest organic carbon concentration in the culture
vessel produced no effect whatsoever.
Displayed in Figure 18 are growth curves one representative
of the Selenastrum capricornutum control and the other of
the culture with concentrated effluent added. These two
curves represent the culture extremes encountered in the
studies with Selenastrum capricornutum. Growth curves for
the other fractions were found to lie in the area bounded
by these curves.
Reduced levels of organic fraction. The data presented
in the foregoing section represent the effect produced upon
addition of the fractions and the concentrate at the postu-
lated maximum concentration. It was decided that the effect
of reduced concentrations should be investigated. The ex-
periments in this section were narrowed so that only those
fractions showing a growth enhancement at such maximum con-
centration were investigated. Table 17 contains data rela-
tive to the effect on growth rate when the organic fractions
and the concentrate are present in the culture flask in re-
duced concentrations. In the case of fractions G-10-IIIb
and G-25-II, the biostimulatory effect previously noted for
these fractions is removed completely upon reduction to
approximately 1/2 of the concentration originally eliciting ,
a response.
In the case of fraction G-50-I and concentrated effluent, the
initially high concentration used allowed for investigation
of the effects of these entities at two reduced levels. The
levels chosen were 1/2 and 1/10 of the initial concentration.
As can be seen from the data, a reduction to the 1/2 level
still afforded a growth rate higher than that found for the
control. However, further reduction in their respective con-
centrations to levels 1/10 of the initial removed all vestiges
of growth rate enhancement.
Figure 19 graphically represents the relationship between the
growth rate attained versus the percentage of full complement
organic carbon concentration for fraction G-50-I and the con-
centrated effluent.
92
-------
LOO-
O.9O
o.eo-
0.70-
O80-
0.80
O.4O
030
UJ 0.20
o
I
0.10
O.O9
008
O.07
OO6
O06
0.04
0.03
CAPRICORMUTUM
MEDIUM BASIC ASM (100ml)
LIGHT-550 FT CANDLES
° CONCENTRATED EFFLUENT (7mg/0
•CONTROL
40
80
120
I6O
200
TIME
Fig. 18 SELENASTRUM CAPRICORNUTUM GROWTH CURVES.
93
-------
Table 17. . Effect of Organic Fractions in Reduced
Concentra t ions
on the Growth Rate of
Selenastrum capricornutum
Fraction
G-10-IIIb
G-25-II
G-50-I
Concentrate
Concentration of
Fraction tng/1
Organic Carbon
0.2
0.2
0.7
0.1
3.5
0.7
Growth
Rate 95% Confidence
Km (day"1) Interval
0.43 0.39 - 0.47
0.45 0.40 - 0.50
0.63 0.60 - 0.66
0.43 0.40 - 0.46
0.86 0.79 - 0.93
0.43 0.40 - 0.46
94
-------
SELENASTRUU CAPRJCQRUUTUU
o CONCENTRATED EFFULJENT
FULL COMPLEMENT* 7mg/l
• 6-501
FULL COMPLEMENT* 1.3mg/l
100
PERCENT OF FULL COMPLEMENT
Fig. 19 GRAPHICAL REPRESENTATION OF THE RELATIONSHIP BETWEEN THE
SELENASTRUM CAPRICORNUTUII GROWTH RATE AND VARYING ORGANIC
CARBON CONCENTRATION OF THE CONCENTRATED EFFLUENT AND
FRACTION G-50 I.
95
-------
Effect of fraction combinations. Since the effects pro-
duced by fractions G-10-IIIb and G-25-II at the maximum con-
centration of 0.3 mg/1 and 0.4 mg/1 were small; and in that
fraction G-50-I produced an effect not matching that of the
concentrated effluent it was decided that combinations of
the organic fractions should be tested to ascertain if and
to what extent synergism played a part. The combinations
chosen are as noted in Table 18.
The data displayed in Table 18 clearly show that the first
combination used that of fraction G-10-IIIb plus G-25-II
produced a growth rate equal to that had when the fractions
were introduced individually into the culture vessel. It
should be noted that in these combination experiments the
concentrations of the individual fractions were as noted
in Table 16.
The second combination involved fractions G-10-IIIb, G-25-II
and G-50-I and the data from that run is also presented in
Table 18. A synergistic effect seems to be evident in that
the growth rate is substantially higher (0.85) than that
produced by fraction G-50-I alone (0.72) and is very close
to the range of the K-^Q produced by the concentrated effluent.
The effect of phosphorous was also investigated. In exam-
ining the organic fractions it was found that the highest
concentration of total phosphorous existed in the concen-
trated effluent. The concentration extant in such concen-
trate was 1.5 mg/1 as P while no discernible phosphorous
concentration could be found in the several organic fractions.
It was decided, therefore, to test the effect of the phos-
phorous contained in such concentrate on the Selenastrum
capricornutum culture. In adding the volume of concentrate
necessary to produce the maximum organic carbon concentration
it was found that 3 micrograms of total phosphorous as P was
added to the culture vessel under investigation. A sufficient
volume of stock phosphorous solution was thus employed to add
3 micrograms to a set of Selenastrum capricornutum cultures.
Such procedure involved the addition of 50 microliters of a
348 mg/1 K2HP04 solution. The KIQ after addition of this
minimal amount of phosphorous was discovered to be 0.50 +
0.02, the same growth rate obtained with the addition of
fractions G-10-IIIb and G-25-II individually or in combina-
tion.
96
-------
Table 18. Selenastrum capricornutum Growth Rates
Produced by Fraction Combinations
Growth 95%
Fraction Rate Confidence
Combination Km (day ) Interval
G-10-IIIb + G-25-II 0.49 0.46 - 0.52
G-10-IIIb + G-25-II 0.85 0.82 - 0.88
+ G-50-I
97
-------
Effect of the organic fractions on the growth of Anabaena
flos-aquae. The same format followed in the experiments
with Selenastrum capricornutum was used in the tests con-
ducted with Anabaena flos-aquae. The sole difference, as
noted in the previously set forth experimental procedure,
involved a reduction in the level of illumination from
550 foot-candles to 150 foot-candles.
Maximum level of organic fractions. Outlined in Table
19 are the data relative to the effect of the organic frac-
tions in the postulated maximum concentrations on the growth
rate of Anabaena flos-aquae.
Those items eliciting a response from the organism were as
displayed: G-50-I and the concentrated effluent. In con-
trast with the data developed during the experiments with
Selenastrum capricornutum the low molecular weight fractions
had no effect on the organism.
At first it was thought that perhaps an increased growth rate
was being had when the fractions G-10-IIIa and G-25-II were
used in the Anabaena cultures; however, a test of the signif-
icance of these data at the 95% confidence level indicated
otherwise. As with the Selenastrum experiments, the concen-
trated effluent was discovered to have produced the most
dramatic increase in growth rate with fraction G-50-I pro-
ducing a lesser but strong effect.
It should be noted that the effects produced by these items
in no way matched levels of increase determined with Sele-
nastrum capricornutum, indicating that the sensitivity of
Anabaena flos-aquae for the causative factors within the
fraction G-50-I and the concentrated effluent was not as
pronounced as that displayed by Selenastrum capricornutum.
Shown in Figure 20 are typical growth curves for an Anabaena
flos-aquae culture both under control conditions and with
the concentrated effluent added to the culture. These were
the extreme conditions encountered in this set of experiments.
Reduced levels of organic fractions. Table 20 contains
data regarding the growth rate responses experienced when
fraction G-50-I and the concentrated effluent were applied
at reduced levels to the cultures. The chosen reductions
were as used previously 1/2 and 1/10 of maximum concentration.
98
-------
Table 19. Effect of Organic Fractions on the Growth
Rate of Anabaena flos -aquae
Fraction
Control
G-10-II
G-10-lIIa
G-10-IIIb
G-10-IV
G-25-II
G-50-I
Concentration of
Fraction mg/1
Organic Carbon
2.0
0.6
0.3
0.2
0.4
1.3
Mean Growth
Rate
Km (day'1)
0.34
0.34
0.38
0.33
0.34
0.37
0.41
957o Confidence
Interval
0.32 - 0.36
0.30 - 0.38
0.34 - 0.42
0.29 - 0.37
0.29 - 0.39
0.33 - 0.41
0.38 - 0.44
Concentrated
Effluent
7.0
0.54
0.50 - 0.58
99
-------
I
O.9OJ-
Q8O-
0.70-
QJBO-
QftO-
04O-
030-
020
0.10-
009
008
0.07
0.06
0.05
004-
OJ03
ANABAEW FLOS-AQUAE
MEDIUM-BASIC ASMdOOml)
UGHT- 150 FT CANDLES
o CONCENTRATED EFFLUENT(7 mg/|
• CONTROL
40
80
120
160
eoo
£40
TIME
Fig. 20 ANABAENA FLOS-AQUAE GROWTH CURVES
100
-------
Table 20. Effect of Organic Fractions in Reduced
Concentrations on the Growth Rate of
Anabaena flos-aquae
Concentration of Mean Growth
Fraction mg/1 Rate 95% Confidence
Fraction Organic Carbon Ki r> (day"1) Interval
G-50-I 0.7 0.37 0.33 - 0.41
0.1 0.33 0.30 - 0.36
Concentrated
Effluent 3.5 0.41 0.39 - 0.43
0.7 0.33 0.31 - 0.35
101
-------
A reduction of 1/2 in the concentration of G-50-I extant in
the culture flask removes all vestiges of growth rate en-
hancement. In opposition to this situation the concentrated
effluent concentration had to be reduced to a level 1/10 of
the original concentration before such occurrence would be
had. At the 1/2 level the effluent had an effect equal to
that produced by the G-50-I fraction in full complement.
Figure 21 is a graphical representation of the relationship
between K^Q and the percentage of full complement organic
carbon concentration for the concentrated effluent.
Effect of fraction combination. Since the data prior
to statistical analysis indicated that G-10-IIIa and G-25-II
could possibly have produced a response from Anabaena flos-
aquae it was decided to investigate the effect of fraction
combinations on Anabaena flos-aquae. The data so developed
are as shown in Table 21.
Table 21. Anabaena flos-aquae Growth Rates
Produced by Fraction Combination
Mean Growth
Fraction Rate 9570 Confidence
Combination KXQ (day"1) Interval
G-10-IIIa + G-25-II
+ G-50-I 0.41 0.39 - 0.43
G-10-IIIa + G-25-II 0.36 0.34 - 0.38
The data presented clearly show that no interactive effects
whatsoever were found. The KIQ produced by the combination
of G-10-IIIa, G-25-II and G-50-I was the same as that ex-
hibited by the culture when G-50-I alone was introduced.
The result of the G-10-IIIa, G-25-II combination reinforces
the findings ascertained with these fractions individually,
namely, that they had no significant effect on the growth
rate of Anabaena flos-aquae.
As was done during the Selenastrum capricornutum experiments,
the effect of the phosphorous added with the concentrated
effluent was also investigated. The growth rate observed in
this experiment was 0.33 + 0.03. In contrast with the effect
produced by this minimal addition of phosphorous upon the
102
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0.60
050
i
040
030
ANABAENA FLOS-AQUAE
CONCENTRATED EFFLUENT
FULL COMPLEMENT=7 mg/l O.G
50
PERCENT OF FULL COMPLEMENT
100
Fig. 21 GRAPHICAL REPRESENTATION or THE RELATIONSHIP
BETWEEN ANABAENA FLOS-AQUAE GROWTH RATE AND
VARYINQ CARBON CONCENTRATION OF THE CONCEN-
TRATED EFFLUENT.
103
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growth rate of Selenastrum capricornutum, no effect whatso-
ever was adduced upon addition of phosphorous to the
Anabaena flos-aquae culture.
104
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SECTION V
DISCUSSION
Preliminary Methodology
The data developed during the initial phases of this study
bear considerable note. Reference is specifically made to
the use of freeze-drying as a concentration technique and
the employment of gel chromatography to separate out into
fractions the organic compounds extant in the test effluent.
Reviews of the literature for data relative to applications
of the above techiques have elicited minimum evidence that
such experimentations have been undertaken in the environ-
mental fields. With reference to freeze-drying as a concen-
tration method, the sole reference discovered is that of
Painter and Viney (69) who employed freeze-drying in a study
of the composition of domestic sewage. Gel chromatography
experiments have been performed but most of the effort to
date has centered around the fractionation of the organic
material in natural waters, eg, the work of Gjessing and
Lee (40). However, one report has recently been produced
regarding the fractionation of raw wastewater and treated
effluents using gel chromatography. This work was performed
by Zunkerman and Molof in conjunction with their investiga-
tions of physical chemical treatment (97).
1. Freeze-drying
The results heretofore adduced, and more particularly noted
in Table 6, clearly indicate that the freeze-drying technique
was for these experiments superior to thin film rotary evap-
oration as a means of concentration. Although as noted
previously the total percentage recovery was not obtained
for thin film rotary evaporation, a comparison of the per-
centage recovery based on the soluble organic component shows
that the freeze-drying technique afforded a substantial in-
crease in percentage recovery for such component. When
cognizance is taken of ths possible problems encountered due
to the effect of heat and coincident biological degradation
on the sample to be concentrated, the freeze-drying technique
again seems superior to any technique employing the addition
of heat to the sample. Another point of interest lies in the
lack of interaction among the many and varied components in
105
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the sample when freeze-drying is employed. This lack of
interaction is due to the fact that the components within
a sample are locked within the ice matrix and thus are not
afforded an opportunity for interaction. Such is not the
case with the thin film method.
Any question as to the efficacy of freeze-drying is dis-
pelled upon examination of the data developed from the
fractionation of the unconcentrated and concentrated water
samples. The elution diagrams displayed in Figure 6 show
that the technique produced a concentrate which is the
equivalent of the original effluent sample and as a result
selective losses of organic material were not evidenced.
When taken in concert, these four points namely, (a) the
high degree of recovery obtained, (b) the minimization of
any changes in the sample due to temperature effects, (c)
the oviation of selective carbon losses, and (d) the lack
of any interaction between sample components allow that
this form of concentration is most effective. A strong,
but secondary point in favor of this method, is the ease
and simplicity with which the concentration may be carried
out.
2. Gel Chromatography
It is indeed unfortunate that this process long the partic-
ular province of the biochemist has not been more extensively
employed in the environmental field. It would seem that the
technique would be most applicable to the characterization
of wastewaters, both raw and as treated effluents. The data
produced herein leads to some important considerations re-
garding the basic character of biologically treated wastewaters
Under the given conditions of analysis, it is evident that
biologically treated wastewaters, whether they are effluents
of activated sludge systems, trickling filters or extended
aeration processes, contain organic compounds which are in
the main of low molecular weight. The chromatographic data
displayed hereinbefore show that 69% of the effluent organic
carbon of the conventional activated sludge system at Batavia,
New York was made up of organic compounds with an apparent
molecular weight less than 700. Further analysis showed that
18% of the original sample organic carbon content has an
apparent molecular weight in excess of 30,000. With respect
106
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to trickling filter and extended aeration effluents, it has
been shown that the percentages of organic material with an
apparent molecular weight less than 700 are 72 and 71 re-
spectively.
The above results coincide with those arrived at by other
investigators in their studies of the composition of waste-
waters. For example, Dean et al (19) have found through
their experiments that the low molecular weight fractions
represented approximately 70% of the organic carbon. In
another study, Bunch et al (11) indicated that low molec-
ular weight compounds make up 60% of the effluent organic
carbon. However, data not consonant with the above has
been presented by Zuckerman and Molof (97). They employed
gel chromatography as a means of assessing the validity of
their proposed hydrolysis reaction in the physical chemical
treatment scheme. Based upon their data, they have averred
that the effluent from an activated sludge system would only
contain organic compounds with a molecular weight greater
than 1200. This contention does not seem to be correct in
light of the data developed herein. Their results it should
be noted were developed from analysis of an unconcentrated
sample with the chromatographic effluent continuously moni-
tored for COD.
Growth Rate Response of the Algae
Significant effects on the growth rate of the test organisms
under investigation were evident upon addition of certain
fractions and the concentrated effluent to the culture ves-
sels. As noted previously growth rate enhancement was dis-
covered upon addition of fractions G-10-IIIb, G-25-II and
G-50-I and the concentrated effluent to the Selenastrum
capricornutum cultures. Enhancement was also noted when
the Anabaena flos-aquae cultures were spiked with fraction
G-50-I and the concentrated effluent.
A review of the data in Table 12 allows for a preliminary
assessment of the causative agency for such effects. Noted
in such table are data relative to the conductivity of each
of the chromatographic fractions. The conductivity of the
active fractions ranged from a low of 5.7 umhos/cm for frac-
tion G-25-II to a high of 380 for fraction G-10-IIIb with
the high molecular weight fraction G-50-I having a conduc-
tivity of 8.5. In light of this information it could
107
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definitely be assumed that any effects being had with frac-
tions G-25-II and G-50-I were due solely to an organic
agency. In the case of fraction G-10-IIIb such a clearcut
determination is not possible. The high conductivity evi-
denced by this particular fraction may be an indication that
the increased growth rate is the result of inorganic factors
or possibly organic ions. One possible explanation for such
an effect could be the provision of a trace element or ele-
ments unavailable from the Basic ASM medium.
The results generated by addition of the fractions both
singly and in combination bear note. Fraction G-50-I in
the case of Selenastrum capricornutum produced the most
dramatic and wide ranging effects. As can be seen from
Figure 19 the effect produced by this fraction closely
paralleled that found with use of the concentrated effluent.
In point of fact the combination of G-50-I with G-25-II and
G-10-IIIb produced a growth rate almost equal to that de-
veloped by the concentrated effluent. Based on these data
it would not be amiss to assume that the major portion of
the growth rate enhancement found upon testing of the con-
centrated effluent was due in the main to the action of the
several fractions. Anabaena flos-aquae was in no way sim-
ilarly affected. Response was developed only with the
concentrated effluent and fraction G-50-I and the effects
were not as strong as those exhibited with the Selenastrum
capricornutum culture. The percentage increase in growth
rate was much smaller for Anabaena for both fraction G-50-I
and the concentrations of fraction G-50-I.
The concentrations of the fractions used in this study is a
point of interest. Reference is specifically made to the use
of zero dilution of the effluent as an upper limit. Such a
selection, it is felt, is perfectly acceptable in light of
the existence of wastewater treatment facilities on streams
and water resources incapable of providing more than minimum
dilution of the effluent. It would seem that the data de-
veloped indicate a pressing need for the reassessment of
siting procedures, and treatment method selection. The need
for such reassessment is reinforced when one considers that
growth rate response was evident upon usage of reduced con-
centrations of fraction G-50-I and the concentrated effluent.
The present tack taken by the regulatory agencies it is felt
will lead in the near future to a mandate for the removal of
phosphorus in treatment facilities, as phosphorus has been
108
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popularly indicated as the prime causative agent for in-
creased algal growth in the aquatic environment. Based
on the data herein presented, however, it would seem pos-
sible that in certain situations serious algal problems
would remain notwithstanding the removal of phosphorus
from the effluent.
Recent work conducted at Lake Tahoe by Middlebrooks, et al
(63, 64) has indicated, as have previous investigations,
that the biostimulatory effects of secondary effluents are
considerable. The investigations conducted closely paral-
leled the techniques followed in this study. The main
points of comparison are the employment of the batch culture
technique, the use of growth rate as an indication of re-
sponse and the use of an organism of the genera Selena strum,
namely, Selenastrum gracile. The experiments undertaken
showed that the addition of secondary effluent to Lake Tahoe
water produced marked enhancement of the growth rate of the
organism. In addition, it was noted that the effects could
in part be the result of the addition of biostimulatory
agents in the effluent such as organic material, vitamin
^12 > etc> as the growth rate responses were in some cases
higher than could be accounted for solely on the basis of
the amount of nitrogen and phosphorus in the sample. At
this juncture it would be well to discuss the nature of the
factors effecting these results.
1. Carbon
The first point to be discussed is the relationship of this
enhancement of growth rate to the concentration of carbon
in the fractions and concentrated effluent. As shown in
Tables 16 and 19 the full fraction concentration expressed
as organic carbon ranged from a low of 0.2 mg/1 for G-10-IV
to a high of 2.0 mg/1 for G-10-II. It could be assumed that
the effect produced upon addition of the stimulatory fractions
was due to bacterial degradation of the organic carbon, the
release of such carbon into the system and its subsequent use
by the algal species.
However, to accept such a premise requires that several im-
portant assumptions be made. The first of these being that
Basic ASM medium is carbon limited, which is definitely not
the case. The second relates to the biodegradability of
organic compounds. For example, one would have to assume
109
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that organic compounds of molecular weight greater than
30,000 were more subject to biodegradation than those ex-
isting in fractions G-10-11 which had an apparent molec-
ular weight of 250. Such an assumption it is posited is
not supportable. Also if one was to assume equal oppor-
tunities for bacterial attack then one would be forced to
accept the proposition that approximately equal concen-
trations of carbon qua carbon, 2.0 mg/1 for fraction G-10-11
versus 1.3 mg/1 for fraction G-50-I had produced vastly
different growth rate responses.
2. Phosphorus and Nitrogen
The cursory investigation undertaken in this research with
phosphorus clearly indicates that if at all phosphorus
played a minor role in the increased growth rate response
of the organisms. As far as the individual fractions are
concerned phosphorus could have played no part whatsoever
since when tested for total phosphate no measurable con-
centration was discovered in any of these fractions.
In the case of the concentrated effluent it was of course
noted that the addition of phosphorus in the same amount
as would be added by this concentrate afforded an increase
in the growth rate of Selena strum capricornutum. This
increase growth rate, 0.50, however, does not approach that
produced by fraction G-50-I, 0.72, and is minimal when com-
pared with that produced by the concentrated effluent, 0.96.
Anabaena flos-aquae was totally unaffected by the addition
of phosphorus.
In the case of nitrogen the analysis is not as straight for-
ward as that noted previously for phosphorus. Investigation
of the nitrogen content of the chromatographic fractions
proved to be impossible due to the small amounts of such
fractions available for all experiments; thus, the part
played by nitrogen must be examined indirectly.
In the case of the high molecular weight fraction G-50-I, data
developed in previous investigations (59) indicate that the
achievement of a growth rate response equal to that produced
by this fraction (0.72) would require the addition of 0.6 tng
of nitrogen to the culture. Such an amount of nitrogen could
not be made available from the organic compounds contained
within fraction G-50-I. The conductivity exhibited by this
110
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fraction would immediately remove any consideration of an
inorganic nitrogen source. Thus it would seem that the
increased growth rate response caused by fraction G-50-1
must be the result of a factor other than nitrogen.
The situation regarding the part played by nitrogen in the
effects exhibited by fractions G-25-II and G-10-IIIb is
unclear. Fraction G-10-IIIb possessed a conductivity of
such magnitude as not to allow one to discount the possi-
bility that inorganic nitrogen compounds in such fraction
were responsible for the increased growth rate of Sele-
na strum capr icjornut urn. In addition the organic compounds
extant in the fraction may also have served as a source of
nitrogen for the organism. The low conductivity of fraction
G-25-II does not permit an interpretation of its effects as
having been produced as a result of the presence of an inor-
ganic nitrogenous compound in said fraction. However, the
same may not be said for an organic nitrogen factor.
Since Anabaena flos-aquae was solely affected by fraction
G-50-1 it would seem that the general import of the discus-
sion noted previously would hold. In addition since it has
been noted that this organism is capable of nitrogen fixa-
tion it is surmised that its susceptibility to variations
in nitrogen concentration would not be as marked as that
shown by Selenastrum capricornutum.
3. Other Possible Causative Factors
Several possible alternative explanations may also be ad-
vanced for the effects exhibited in this study. First
among these would be an accounting for such effects based
on the provision of a requisite vitamin or a growth sub-
stance such as 3-indoleacetic acid. Both of these items
it has been pointed out by Painter and Viney (69) have
been discovered to be constituents of wastewaters. It
would seem, however, on the basis of molecular weight con-
siderations that the only such substance with a match to
the fractions obtained would be vitamin B^2- The apparent
molecular weight of fraction G-25-II would present a range
for the presence of said substance. However, it should be
noted that a vitamin requirement has yet to be established
for Selenastrum capricornutum.
Ill
-------
A possible explanation with application to all the active
fractions would be chelation; but an assumption such as
this would require one to discount the effectiveness of
the Na2'EDTA already present in the Basic ASM medium.
A second consideration involves a parallel to that explana-
tion proposed by Prakash and Rashid (73) for the effect of
humic substances on marine algal growth. This explanation
supposes that a factor or factors within the fractions act
as sensitizing agents for cellular transport. It is surmised
that such agents act to enhance the permeability of the algal
cell membrane and thus increase the uptake of required nu-
trients from the basal medium.
Final consideration may be given to the possible addition of
trace metals in the active fractions as biostimulatory agents.
However, it would seem in light of the extremely low conduc-
tivities exhibited by fractions G-25-II and G-50-I, that only
fraction G-10-IIIb would be capable of assuming this role. In
addition it would not be amiss to assume that the sum total
of the trace element requirements for both algal species had
been satisfied by trace element impurities in the stock
chemicals used to make up the basal medium.
4. Summary
The main thrust of the study hereinbefore delineated has been
the garnering of organic wastewater fractions and the ascer-
tainment of their effect on algal growth. This work has
substantially been based on kenetic considerations. A fruit-
ful field of investigation would be the assessment of ultimate
growth effects. Such investigation would be of possible
assistance in offering an explanation of the effects noted
herein. The explanations now proffered for such effects are
suppositive in nature. The true assessment of the causative
factors involved must await an expansion of the conventional
wisdom.
As mentioned, the effect of various concentrations of phos-
phorus and nitrogen on the growth rate of Selenastrum
capricornutum was also investigated. The experimentation
consisted in obtaining growth rates of the test alga in
Basic ASM and modified tenth Gorham's media, both of which
used glass distilled water and/or 0.45 u membrane-filtered
Lake George water (an oligotrophic soft water lake in the
112
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eastern Adirondack Mountains) as dilution water. The
rationale for using glass distilled water and Lake George
water was to allow interpretation of the adequacy of the
synthetic media for algal culturing. Once these growth
rates were established, the concentrations of nitrogen and
phosphorus in the two media were fractionally reduced to
one-half and one-quarter of the full amount and the growth
rate at each concentration level ascertained.
For the following illustrations, the ordinate indicates the
extent of growth attained in 24 hours on a relative basis
with Basic ASM medium made up with glass distilled water
the norm for comparison since this medium supported the
greatest rate of growth. During the experimentation only
the component under study was altered from its concentration
in the original medium. As illustrated in Figure 22, the
effect of nitrogen reductions on the growth rate is less
severe than fractional reductions in the phosphorus concen-
trations. The growth rate for Selenastrum capricornutum in
Basic ASM is 1.32. In noting the effect of nitrogen reduc-
tion, it can be seen that a 50% reduction in nitrogen con-
centration produced a growth rate of 0.96 or a one-third
reduction in cell concentration over the 24 hour period.
This is contrasted with a reduction in K rate to 0.74 when
the nitrogen is further reduced from 0.5 nitrogen to 0.25
nitrogen. The overall change in cell concentration produced
by a decrease in nitrogen concentration from the full nitro-
gen level to one-fourth was 4070.
The influence of changes in phosphorus concentration is also
illustrated in Figure 22. A reduction in cell concentration
of 407o was affected by a 50% reduction in phosphorus level.
A further reduction in the phosphorus level to one-fourth of
the full medium phosphorus level produced an overall reduction
in cell concentration of 4570. The experiments indicated in
Figure 22 and the following figures are illustrative of the
nutrient limitation exhibited by the decreased concentrations
of the required elements, nitrogen and phosphorus, with the
organism being consistently more sensitive to reductions in
phosphorus concentration than nitrogen concentration.
In Figure 23 the data were obtained by using the Basic ASM
medium with Lake George water substituted for glass distilled
water. The striking result evident in these data is the
113
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12
TIME (hrs)
24
Fig. 22 EFFECT OF VARYING NUTRIENT CONCENTRATION
IN ASM(DD) FOR SELENASTRUM CAPRICORNUTUM.
114
-------
1.0
75
o
HI
I
-50
25
FULL N&P-
12 18
TIME (hrs.)
24
Fig. 23
EFFECT OF VARYING NUTRIENT CONCENTRATION
IN ASM(LG) FOR SELENASTRUM CAPRICORNUTUM
115
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substantial reduction in growth rate for the full complement
of nitrogen and phosphorus from 1.32 with glass distilled
water as diluent to a value of 0.98 with Lake George water
as diluent. The substitution of Lake George water as diluent
for the Basic ASM medium thus produces a 29% reduction in
cell mass production. A similar effect was noted in the
growth rate of Selenastrum capricornutum in modified tenth
Gorham's medium due to the Lake George water diluent. It
appears that Lake George water contains some factor inhibi-
tory to the growth of Selenastrum capricornutum with greater
inhibition prevalent in the Basic ASM medium. The growth
rates produced by fractional reductions in phosphorus are
comparable to those obtained with these same reductions with
glass distilled water. However, the effect of a reduction
in nitrogen concentration to the one-half level was more pro-
nounced with Lake George water as diluent than with glass
distilled water as diluent. The K rate for the one-half ni-
trogen level in glass distilled water was 0.96, whereas the
K value for Lake George water was 0.72, a 20% decrease in
cell concentration due to Lake George water.
The basal medium for experimentation illustrated in Figure 24
was modified tenth Gorham's medium with glass distilled water
as diluent. It can be seen that the growth rate for the full
complement of nutrients is 1.15 which is considerably higher
than the value obtained for Basic ASM with Lake George water
and modified tenth Gorham's with Lake George water. The
difference noted in K rate for a reduction of nitrogen to the
one-half level, that is, a value of 1.03 and a concomitant 25%
reduction in cell concentration, was not as pronounced as the
reductions noted for a similar decrease in nitrogen level with
Basic ASM, using either glass distilled water or Lake George
water. When the nitrogen was fractionally reduced to one-fifth
its original level there was drastic reduction in the growth
rate to a value of 0.40 and a 50% reduction in cell concentra-
tion. This indicated that for unimpeded growth a value at
least one-half of the full nitrogen concentration is required.
For a reduction in phosphorus concentration to one-half the
level originally posited a growth rate of 0.60 was obtained as
compared to 1.15 for the full complement of phosphorus. This
reduction in growth rate represents a decrease of 40% in cell
concentration. As noted previously, a reduction to one-half
phosphorus level in Basic ASM also produces a 40% change in
cell concentration. A predictable result occurred since both
116
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1.0
.75
.50
.25
FULL N6P
1/2 N
1/2 P
1/5 N
12 18
TIME (hrs.)
24
Fig. 24
EFFECT OF VARYING NUTRIENT CONCENTRATION
IN MODIFIED .16 (DD) FOR SELENASTRUM CAPRICORNUTUM
117
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media have approximately the same phosphorus concentration
and the organism is phosphorus limited in both situations.
Based upon the data presented in Figures 22 and 24 and the
relative concentrations of nitrogen and phosphorus in both
media, a comparison can be made between the results of Basic
ASM with one-half nitrogen and modified tenth Gorham's medium.
The Basic ASM medium with one-half nitrogen contains 7 tng/1
and the tenth Gorham's medium contains 8 mg/1 and approximately
the same phosphorus concentration in both media. The K rate
obtained for the modified tenth Gorham's medium is 1.15 while
the K rate for Basic ASM with one-half nitrogen is 0.96. For
a 15% increase in nitrogen level, one obtains 20% greater cell
concentration in modified tenth Gorham's medium as opposed to
the Basic ASM medium with one-half nitrogen. These proportions
again hold when one compares Basic ASM with one-fourth nitrogen
and modified tenth Gorham's with one-half nitrogen. The re-
spective growth rates being 0.74 for the former and 1.03 for
the latter, again a 20% increase in cell concentration for a
15% increase in nitrogen level. As depicted in Figure 25
higher growth rates were obtained with glass distilled water
as diluent than with Lake George water as diluent in both
Basic ASM medium and modified tenth Gorham's medium. All
curves indicate a predictable effect on the growth rate with
increasing concentrations of phosphorus indicated, phosphorus
is limiting. However, the limitation is less pronounced with
Basic ASM using Lake George water as diluent. It appears
that the alga cannot utilize the increased phosphorus concen-
tration because of some inhibitory factor present in Lake
George water.
The data presented in Figure 26 are a summation of experimen-
tations with various nitrogen concentrations in modified
tenth Gorham's medium with glass distilled water or Lake
George water. The curve for modified tenth Gorham's medium
was glass distilled water indicates that nitrogen is present
in excess in the full medium. If interest were placed on the
minimization of nutrient storage and carry over, the nitrogen
level could be reduced in this medium to one-half the level
presently prescribed. For Basic ASM medium once again the
growth rates were higher with glass distilled water than with
Lake George water. Increasing concentrations of nitrogen
elicited higher growth rates and within the range of concen-
trations indicated nitrogen was limiting for Basic ASM medium.
118
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1.3
1.2
.6
I
I
I
0
I
.1 G (L6)
.1 G (OD)
ASMCDD)
ASM(LG)
I
.6
.7
0 .1 .2 .3 4 .5
PHOSPHORUS (mg P/l)
Fig. 25 EFFECT OF VARIOUS PHOSPHORUS CONCENTRATIONS
ON VALUES OF Ke FOR SELENASTRUM CAPRICORNUTUM
119
-------
1.3
1.2
.9
.6
0
Fig. 26
1
• .1 6 (DO)
• ASM (DD)
A ASM(LG)
I
10.5
35 7
NITROGEN (mg N/l)
EFFECT OF VARIOUS NITROGEN CONCENTRATIONS
ON VALUES OF Ke FOR SELENASTRUM CAPRCORNUTUM
14
120
-------
In summary it can be stated that there appears to be an
inhibition to the growth of the organism as indicated by
reduced growth rates when Lake George water is used as a
diluent for the basic components of either modified tenth
Gorham's medium or Basic ASM medium. To this point, further
particular investigation is now being carried on to ascertain
the extent and the causative factor of such inhibition. It
was also demonstrated that the concentration of nitrogen in
modified tenth Gorham's medium may be reduced to one-half
the posited level without any significant change in the
growth rate of Selenastrum capricornutum.
Note; Phosphorous was always tested in the form of phosphate
in this latter work.
121
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SECTION VI
ACKNOWLEDGEMENTS
The author and contributors to this final technical report
wish to express their gratitude for advice to Drs. Donald
B. Aulenbach and William W. Shuster of the Bio-Environmental
Engineering Division, Rensselaer Polytechnic Institute.
Particular appreciation is extended to the late Professor
Edward J. Kilcawley of this division for his expert guidance
in several phases of the investigation. Additionally, the
technical advice of Drs. Henry L. Ehrlich, Department of
Biology, RPI and Leo J. Hetling, Director of Research, New
York State Department of Environmental Conservation is also
appreciated. The valuable assistance of Dr. Richard C.
Spear, currently at the National Environmental Research
Center, U. S. Environmental Protection Agency, Edison, New
Jersey, in the initial phases of research and described
within Appendix A is also acknowledged.
Lastly, special gratitude is expressed to Dr. James J. Ferris,
Research Coordinator, Rensselaer Fresh Water Institute for
his assistance (technical and editorial) during the prepara-
tion of this final technical report.
ORGANIC NUTRIENT FACTORS EFFECTING ALGAL GROWTHS is the final
technical report for a three year - three phase investigation.
These studies were supported in part by the U. S. Environmental
Protection Agency, Project #16010 DHN. In addition to these
funds, support was also obtained from the New York State
Science and Technology Foundation and the New York State De-
partment of Health. One of the contributors (Gerald C.
McDonald) was a trainee of the United States Public Health
Service (USPHS Training Grants 2T1RH4-06 and 3T1RH4-0 651 (67)) .
A substantial portion of the information within this final
technical report was taken from theses submitted to Rensselaer
Polytechnic Institute by Gerald C. McDonald (Ph.D. Thesis- 1971),
Inder Jit Kumar (M. S. Thesis - 1971) and William J. Greene
(M. S. Thesis - 1972).
123
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SECTION VII
REFERENCES
1. Ackers, G.K., "Molecular Exclusion and Restricted
Diffusion Process in Molecular-Sieve Chromatography,"
Biochemistry, 3, pp 723-730 (1964).
2. Allen, M.B., "General Features of Algae Growth in
Sewage Oxidation Ponds," California State Water Pol-
lution Control Board, Sacramento, California, Public-
ation No. 13, pp 11-34 (1955).
3. Andrews, P., "Estimation of the Molecular Weight of
Proteins, by Sephadex Gel Filtration," Biochem. J_. ,
91, pp 222-233 (1964).
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134
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SECTION VIII
APPENDICES
Appendix A
Comparison of Thin Film Evaporation and Freeze-drying
Methods of Concentration of Organics in Water.
Section I:
Section II:
Conclusions
Recommendat ions
Section III: Introduction, History and
Technical Review
Section IV: Procedures and Investigations
Section V: Discussion
Section VI:
References
137
139
141
153
191
197
Section VII: Organic Carbon of Fractions from 201
Sephadex G-15 Column
Appendix B
The Effect of Chemical-Physical Treatment on the
Soluble Organic Component of Wastewater.
Section I:
Section II:
Conclusions
Recommendations
Section III: Introduction, History and
Technical Review
Section IV:
Section V:
Section VI:
Section VII:
Procedures
Results and Data Analysis
Discussion
References
211
213
215
237
251
279
285
135
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Appendix^C
Materials and Apparatus 291
136
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APPENDIX A
COMPARISON OF THIN FILM EVAPORATION
AND FREEZE-DRYING METHODS OF
CONCENTRATION OF ORGANICS IN WATER
SECTION I: CONCLUSIONS
1. Organic carbon recovery in freeze-drying is less than
that in thin film evaporation for all the samples.
2. Relative recovery in freeze-drying is better for sewage
samples than for water samples.
3. Less recovery in freeze-drying may be due to (a) absence
of flask filters, (b) suction of dry solute into the con-
denser, (c) use of a large number of flasks, and (d) high
vacuum.
4. The chromatograms obtained for thin film and freeze-dried
concentrates of the same sample contain an identical number
of peaks at about the same elution volume for each peak. This
indicates the presence of the same compounds in the two con-
centrates.
5. The cumulative percentage vs molecular weight curves for
the two chromatograms which show the percentage of compounds
greater than a certain molecular weight plot out very close
to each other for all the samples. The maximum difference
between the two curves is less than 2 to 3% at any section.
This small difference in the elution profile is believed to
be due to instrumental and other errors involved.
6. Any significant polymerization or hydrolysis in either of
the concentration techniques employed in this work has not
been observed.
7. Although recovery in freeze-drying is less than that in
thin film evaporation, the similarity of the chromatograms
suggest that there has been no selective removal of a com-
pound (or compounds) from the sample during freeze-drying.
8. Thin film evaporation requires about three times as much
time as freeze-drying for concentrating the same amount of
a sample.
137
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9. By providing a vacuum pump and a condenser instead of
a water aspirator, in thin film evaporation, the rate of
evaporation is increased by more than 200 percent.
10. Both methods have merits and demerits but thin film
evaporation would be better for concentrating natural waters
because of higher organic recovery and freeze-drying would
be better for concentrating sewage samples because there is
less chance of degradation at lower temperatures.
138
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SECTION II: RECOMMENDATIONS
Based on analysis of the results and experience gained,
the following recommendations are made:
1. The precipitate which remains undissolved in 100% HCl,
should be treated with dilute NaOH. This might dissolve
some of the organic compounds if any are present in the
precipitate.
2. The remaining precipitate should be analyzed on a Leco
furnace for organic carbon.
3. Future research should also examine the possibility of
carrying out thin film evaporation at reduced temperatures
and pressures.
4. A larger freeze-drying flask (say 2 liters) would reduce
the possible loss of organic carbon due to the use of a large
number of flasks.-
5. To be absolutely certain about the hydrolysis and poly-
merization effects of the two methods, the acid filtrate
should also be fractionated on the gel column after neutral-
izing with NaOH and reconcentrating it.
6. A smaller fraction volume, in gel chromatography ie 1.5
ml instead of 5 ml used in this work, may give better defined
elution profiles.
139
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SECTION III: INTRODUCTION, HISTORY AND TECHNICAL REVIEW
In recent years considerable research has been done in
developing techniques for the determination of organic
compounds present in very low concentrations in surface
waters. These trace organics are shown to be of signif-
icance as causes of taste and odor, color and epidemio-
logical effects (1). The basic methodology for the deter-
mination of organic compounds consists of concentrating
the water sample followed by analysis of constituents.
The main problem, however, is to increase the concentration
of organic compounds present, to levels detectable by the
instrumental means, without destroying them or altering
their chemical composition.
Various concentration methods have been employed to this
end, which include carbon adsorption followed by elution
with an organic solvent, freeze concentration, solvent
extraction, rotary thin film evaporation and freeze-drying.
While each of these methods has some advantages and dis-
advantages, the last two have been studied in some detail
in this investigation. Both methods provide a means of
concentration by direct removal of water from the sample
and do not involve the use of any adsorption material or
the addition of an organic solvent.
1. Trace Organics
The trace organics present in natural waters originate from
a variety of sources. According to Lysyj et al (2) human
activity is the prime source of organic contaminants intro-
duced into waterways, lakes, reservoirs and other water
resources. These contaminants may consist of both biodegrad-
able and refractory materials. Sproul (3) in his work on
the origin of organics has indicated four general areas from
which they originate. These are (1) substances resulting
from microbial action, (2) industrial wastes, (3) domestic
wastes, and (4) natural runoff. Specific compounds iden-
tified from surface waters, as listed by Spicher (4) include:
Pesticides, such as DDT, DDD, methoxychlor, endrin,
dieldrin, aldrin, 2,4-D and lindane.
141
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Detergents, primarily alkylbenzene sulfonates.
Hydrocarbons, including benzopyrene, chrysene, anthra-
cene, phenanthrene, fluoranthene, 10,11 benzofluoranthene,
gasoline, kerosene and other fuels.
Other aliphatics and aromatic compounds, including
diphenyl ether, pyridine, lutidine, naphthalene, petralin,
chloro-ethyl ether, acetophenone, toluene, xylene, ortho-
nitrochlorobenzene, ortho-dichlorobenzene, proteins, poly-
saccharides, tannins and lignins, phenolic compounds and
their chlorine derivatives, substituted nitrobenzenes,
picolines, fatty acids, nitriles, aromatic ethers, aldehydes,
ketones, esters and organic acids.
2. Effects of Trace Organics
Taste and odor problems associated with trace organics are
well known. Further, these may interfere with coagulation,
foul ion exchange resins (5) and increase activated carbon
demand (6) thereby adding to the overall cost of water
purification.
Organic contaminants may also add or change the color of the
stream, exert excessive BOD, taint fish flesh and upset the
microbial purification cycle (7). In industry, where high
quality water is required, these trace organics contaminate
the product and/or may result in increased production cost
and periodic plant shut downs.
3. Detection, Concentration Method
Analytical tests for the detection of organic matter in water
and wastewater samples are outlined in the twelfth edition of
Standard Methods. These include tests of volatile matter,
chemical oxygen demand, biochemical oxygen demand, grease and
oil, phenol, alkylbenzene sulfonate and threshold odor. In-
frared carbon analysis and gas chromatography are often used
to measure the concentration of organic compounds and to iden-
tify them. Both of these instruments are rapid and sensitive
and yield reliable results for organic concentrations in
excess of one mg/1. For lower concentrations of trace or-
ganics, as is the case with surface waters, the sample must
be concentrated prior to instrumental analysis.
142
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Concentration methods commonly employed are carbon adsorp-
tion, solvent extraction, freeze concentration, distillation,
ultrafiltration, air drying and reverse osmosis. The other
concentration techniques as described by Kamtnerer (8) are
dialysis, coprecipitation and ion exchange. All of the
above methods of concentration are still being evaluated.
The carbon adsorption filter evolved by Braus et al (9) is
the most widely used method for the concentration of trace
organics, but this technique has been shown to have serious
disadvantages. Certain compounds may be irreversibly ad-
sorbed or altered on the carbon. According to Hoak (10) the
adsorbed organics tend to oxidize on the carbon surface, lead-
ing to identification of compounds not originally present.
The recovery is also pH dependent, having reduced efficiencies
at pH 8.5 or higher (11). Baker (12) has shown that in the
carbon filter, selective adsorption and desorption may occur
and the organic constituents may undergo chemical and bio-
logical modifications.
In solvent extraction the organic compounds are extracted by
treating the sample with a solvent, which preferentially dis-
solves the organic compounds in the sample. This method
eliminates the adsorption-desorption problems but it does
require the addition of organic solvents and frequently the
addition of chemicals like NaCl to reduce the solubility of
organic compounds sought.
Ultrafiltration is still in its development stage. The rate
of concentration is very slow and equipment is quite costly.
Molecules smaller than the membrane pore size pass through
it and are lost. The tightest membrane presently available
can retain compounds of molecular weight 500 or higher. The
concentrates obtained by this method usually require further
concentration by other means.
Distillation is not usually employed for concentration of
trace organics as it can result in the loss of volatile
organic matter and destruction of heat labile compounds.
In freeze concentration the effectiveness decreases with in-
creased dissolved inorganic content. The efficiency depends
on a number of factors including rate of cooling, stirring
and composition of the original sample (1).
143
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There are two other concentration methods in addition to
the ones discussed above. These are rotary thin film
evaporation and freeze-drying. Both methods are simple
and are used in laboratories in a variety of applications.
Their effectiveness in concentrating trace organics in
natural or wastewater has never been evaluated. Concen-
tration is effected by the direct removal of water from
the sample. The two methods differ mainly in the temper-
ature at which the water vapor is removed. Freeze-dry ing
is generally considered better than thin film evaporation
primarily because it is carried at lower temperatures,
offering little chance of any degradation of the organic
compounds.
4. Objective, Scope and Significance
An evaluation of the literature available on the concentra-
tion of trace organics in natural and wastewater indicated
that (a) the present knowledge on the effectiveness of thin
film evaporation or freeze-drying for concentration is
limited and inadequate and (b) research is needed to provide
basic information as to the relative effect of the two
methods regarding hydrolysis or polymerization of organic
compounds. It was the purpose of the study to determine
which of the two methods is better when used for the specific
purpose of concentrating natural and wastewaters. Both
methods of concentration have been studied as to their rel-
ative polymerization or hydrolysis effects and their recovery
efficiency so far as organic carbon is concerned.
Water samples were concentrated from four different sources.
The sources included a high-quality natural water from an
oligotrophic lake (Lake George, New York) to the extreme of
raw domestic sewage. The water samples were first filtered
through membrane filters of 0.45 u pore size. Concentration
was carried out by both methods simultaneously. The concen-
trates were then put on Sephadex G-15 columns for fractiona-
tion into different molecular weights (sizes). These fractions
were analyzed for organic carbon content on a total carbon
analyzer. The two chromatograms thus obtained for each sample
have been compared, on a relative basis, for any possible poly-
merization or hydrolysis reaction.
The control of pollution hinges on our capacity to accurately
identify the pollutants. Obviously it is impossible to treat
144
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a compound effectively unless its nature is known. Thus,
any pollution abatement program has to take into consid-
eration the nature of the compounds present. The obvious
approach to the problem of water quality is to identify
specific impurities, to develop methods of accurately
measuring their presence and concentration in water, and to
develop ways and means of removing or lessening their adverse
effects. Since some organics may be present in very minute
quantity, often below the lower limit of detectability of
presently available analytical techniques, these organics
have to be concentrated. The method of concentration has
to be such that it does not eliminate, alter, or damage the
compounds or their distribution in the water sample. This
evaluation of the concentration techniques selected will help,
to some extent, in the accurate identification of trace or-
ganics.
5. Thin Film Evaporation, Freeze-drying and Gel Permeation
Chromatography
Thin film evaporation. The history and theory of these pro-
cesses have been described previously. Discussions of these
techniques and review of their concepts can be found within
the main body of this text, pp 31-38.
Freeze-drying. Freeze-drying is normally a vacuum drying
process in which the water is sublimated from the frozen
state. Carman (18) defines it as a process of distillation
since a vapor is formed and condensed. Essentially it con-
sists of freezing the sample to be dried or concentrated,
keeping it under sufficient vacuum and withdrawing the water
vapor which is formed due to evaporation of frozen sample
under vacuum.
The process of freeze-drying was first described by Shackell
(19) in 1909 for drying biological products. In his process
the material was first thoroughly frozen in a salt-ice mixture,
The frozen material was then dried in vacuo in a desiccator
with sulfuric acid as desiccant. In 1921 Swift (20) described
a similar technique for drying bacteria from the frozen state,
which became standard for some years in many laboratories.
However, there was no major improvement in the process till
1931 when Reichel, et al (21) developed a practical procedure
for the rapid freezing and rapid dehydration under vacuum of
145
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serum in bulk. The serum is shell frozen at -78°C and is
connected to a condenser contained in a dry ice-acetone bath
(-78°C). The system is also connected to a vacuum source.
Rapid sublimation of water j.n vacuo from its surface keeps
the serum in the frozen state. Further improvements in this
method have only been concerned with engineering details
The use of freeze-drying for the concentration of water and
wastewater for analytical purposes has not been extensive.
McDonald (22) used it to concentrate sewage and has reported
a recovery of 83-100%. McDonald, et al (23) found that chro-
matograms from the gel filtration of unconcentrated raw
sewage and raw sewage concentrated by freeze-drying looked
identical, showing no alteration of the compounds when con-
centrated by this method.
It is interesting to note that the technique of freeze-drying
owes its invention and subsequent development to the science
of biology. Its use of drying foods was first suggested by
Flosdorf and Mudd (24) in 1935. Food technologists are pres-
ently working on the development of accelerated freeze-drying
methods by the use of microwaves, radiant heat energy, fluid-
ized beds, etc. All these methods aim at heating the frozen
product without thawing. However, the rate of dehydration
is not a major consideration in concentrating liquids for
analytical purposes, where the main aim is not to damage or
alter the compounds even slightly.
Gel permeation chromatography. The birth of column chroma -
tography is due mainly to.the Russian biologist Tswett (25)
who in 1906 separated plant pigment with a calcium carbonate
column. The technique passed almost unnoticed until 1931.
Adsorption chromatography became standard procedure during
the late thirties. Partition chromatography was introduced
by Martin and Synge (26) in 1941 which made the separation
of hydrophilic compounds like amino acids, polysacchrides and
proteins possible for the first time. This technique involves
partition of solutes between a liquid and a gas phase. One
of these phases is held immobile by an adsorbent material as
silica gel in the column.
During the first decade of its development, only liquid chro-
matography was employed. Following the introduction of gas
chromatography by Martin and James (27) in 1952, which affords
greater selectivity and resolution, liquid chromatography
received a major setback. Gas chromatography, on the other
146
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hand, had its own limitations and drawbacks, which included
difficulties of measurement of polar substances, macromole-
cules and preparative isolation of separated fractions.
Extensive research conducted in this field has diminished
these limitations to a great extent. The method, however,
is comparatively very complicated and expensive.
Interest in liquid chromatography was revived with the devel-
opment of gel filtration, which offered a simple separation
method and allowed both the treatment of macromolecules.
Separated fractions can readily be used for preparative work
which is a major advantage of this technique.
Gel chromatography has a relatively short history. The
technique was in its infancy only ten years ago. The intro-
duction of Sephadex Gel by Porath and Flodin (28) has con-
tributed much to its rapid development. Sephadex is a
modified dextran, obtained as a reaction product of soluble
alkaline dextran and epichlorohydrin. The polymer chains
are cross linked by glycerine ether bonds. Other gels avail-
able, besides Sephadex, include acrylic gel, polystyrene gel,
aerogel, agarose and biogel. Proper pore size, absence of
ionizing groups, and, in a given solvent, low affinity for
the substances to be separated are the main considerations
in the selection of a gel for a particular separation problem.
To establish diffusion equilibrium quickly, the gel particles
should be as small as possible. Low flow resistance and
chemical stability are other desirable properties (29).
Gel forming particles are allowed to swell in the solvent.
The amount of swelling depends on the structure of the gel
substance and that of the imbibing solvent (30). As a mix-
ture of molecules of various sizes is applied to a gel column,
the molecules larger than the largest pores of the swollen
gel pass through the mobile liquid phase outside the gel par-
ticles because they cannot penetrate into the pores. Smaller
molecules are retarded due to the temporary diffusion into
the stationary gel phase. Molecules penetrate the network
to an extent which, in most cases, is determined by their
molecular dimensions and the degree of cross linkage of the
gel. The smallest molecules which penetrate the deepest are
the last to be eluted. In the gel column, therefore, the
larger molecules move faster than the smaller ones and the
components of the mixture are eluted in the order of decreasing
molecular sizes. According to Porath and Flodin (28) the gel
147
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is automatically regenerated after each complete operation.
The use of gel filtration in the field of fractionating
organic compounds in natural and wastewater has not been
much. Gjessing and Lee (33) employed this technique to
separate several samples of river water and concluded
that Sephadex columns can fractionate natural water organic
matter into a number of distinct fractions. Gel filtration
was also employed by Lundblad and Breggard (38) for the
separation of low molecular weight components in urine.
Zuckerman (39) separated components of several treated and
untreated sewage samples on Sephadex columns.
Relatively short time required for separation, the complete-
ness of the separation, good reproducibility, and simplicity
of the analytical procedure are some of the major advantages
in fractionating the organic matter in natural and wastewater
by gel filtration.
Theory of freeze-drying. Carman (18) has discussed the theory
of freeze-drying in his paper entitled "Some Basic Principles
of Freeze Drying and Molecular Distillation." According to
this paper, freeze-drying is closely related to molecular
distillation. The conditions generally required for molecular
distillation are: a) a pressure above the liquid so low that
the vapor molecules which escape from the liquid do not col-
lide with each other or with air and are not reflected back,
and b) the boiling, which occurs when the pressure above the
liquid is less than the saturation vapor pressure, must be
suppressed (eg by freezing the liquid and keeping it frozen
during the freeze-drying process). In such cases the vapor
will only be formed by the escape of molecules from the
surface of the condensed phase.
From the vapor pressure, 7h , at equilibrium, Knudsen has shown
that the rate of collision, Rc, is given by:
Rc = Tt-l M in gms/cm /sec
v2 7>- RT
where
M = Molecular weight
R = Gas constant
T = Temperature
148
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If there is no reflection this will be equal to Wo which is
the absolute rate of evaporation
2 7>- RT
In the case of reflection instead of reentry
M (2)
RT
If the pressure of the vapor at the interface is P^ instead
of 7>-> then the rate of reentry =°^i P- / M
L N/2 7^ RT
rate of evaporation W = absolute rate of evaporation W
rate of reentry
W = W -«*c' Pi M
o c i s—^~—^T
Assuming^' = °< then W = W (1- P.^ (3)
When air is present and the vapor escapes to the condensing
surface by diffusion through the air layer, then
ln
where
P = Total pressure of air in the system
D = Interdiffusional coefficient for vapor and air
Tfc = Vapor pressure at the condensing surface
X = Thickness of air layer
D and T* are average values
From equations (3) and (4)
W - W0 (1 - P.) = MDP In (?£>£.) (5)
RTX P-PI
assuming the condensing surface is so cold that /^c = 0 and
149
-------
if P« 7h , Pi=2^ P
if P » 7>- , PjL^t ^ So
W M5P In /P ) /7)
WQ WQ RTX P- Tf
Depending on the conditions prevailing in the system, equation
(6) or (7) may be used to find the rate of evaporation in the
freeze-drying process. Usually the pressure in the system is
maintained at only a few microns and very few air molecules
are present as compared to vapor molecules. In this case
equation (6) is applied. It is seen that rate of evaporation
will be increased if the total pressure of air in the system
is decreased.
Theory of gel filtration. It has been shown that in gel
filtration, the molecules emerge from the column in the
order of decreasing molecular weights (sizes). This simple
fact has prompted many researchers to derive a relationship
between the molecular weight of a molecule and its elution
volume. Granath and Flodin (34) found alinear relationship
between the logarithm of the molecular weight and the ratio
of the elution volume to the total bed volume for a number
of dextrans. Whitaker (35) in his experiment on Sephadex
G-100, observed a linear relationship between the logarithm
of the molecular weight of a protein and the ratio of its
elution volume, Ve, to the void volume, V , of the column.
He also found that, within limits, the ra?io Ve/Vo was inde-
pendent of the solute concentration, sample size and dimen-
sions of the column, but it was found to be temperature
dependent for several proteins.
A quantitative expression to find the molecular weight from
elution volumes has been derived by Porath (36). Considering
the cavities in the swollen gel available for penetration of
the protein molecule of radius r, as conical with radius R,
he found that
kd = k(l- r ) (9)
R
where kd is the distribution coefficient
150
-------
3
As R is closely related to solvent regain S
R3 — (Sr -oC)
and r is proportional to the square root of molecular weight
M equation (9) may be written as
kd - k (1 - kx M1/2 _ ) 3 (10)
(Sr -«C ) 1/3
He also found a value of 1.64 for K for the Sephadex gels and
0.8 for oC • KI was found to be close to 0.012. Kd can be
found from the elution volume such as
^d = ~ if there is no adsorption.
vi
V^ is the inner volume and is approximately equal to solvent
regain.
Since all quantities except M in equation (5) are known or
can be found experimentally, M can easily be calculated.
Working along similar lines, Squire (37) has derived a
different equation. His treatment is based on the assump-
tion that the cavities available to the protein molecule
consist of a mixture of cones, cylinders and crevices. Ac-
cording to him
1/3 •*
= (1 + g (1 - M )) J (11)
where C corresponds to the molecular weight of the smallest
protein that cannot enter the gel and g is a constant repres
enting the geometric relationship between the three types of
cavities in a given gel. Equation (11) can also be written
as
Ml/3 = cl/3 (1 + g - (Ve)
g *o
Values of C1'3 and g for a particular gel column are
mined experimentally from a plot of (Ve/Vo)1/3 - 1 vs M
Once the values of C and g for a column are known, equation
(11) can be used to find the elution volume for a particular
compound of known molecular weight, or vice versa.
151
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SECTION IV: PROCEDURES AND INVESTIGATIONS
Methodology
1. Sample Collection and Storage
The sample in each case was collected in a 20-liter plastic
carboy which had been repeatedly and thoroughly cleaned and
washed. It was also rinsed two or three times with the
sample source. The carboy was filled with the sample, capped
and brought to the laboratory where it was placed in the
cold room. The cold room temperature is kept between 3° - 5 C.
The following samples have been taken:
Sample Type Location
Lake George Near the dock at
Water Smith Bay
Saratoga Lake Dock, 1.5 miles north 11/29/69
Water of Public Beach
Clifton Knolls At Treatment Plant 3/26/70
effluent after chlorination
Elnora At Treatment Plant 4/16/70
raw sewage Clifton Knolls
The sample was filtered through a membrane filter of 0.45 u
pore size and 142 mm diameter on the same day. The plastic
carboy in which the filtered sample was stored in the cold
room was also properly cleaned and rinsed.
Concentration of the sample was started simultaneously by
both methods. The thin film method took more time than
freeze-drying.
2. Procedure for Thin Film Evaporation
The motor is securely fixed to a ring stand such that the
teflon shaft is inclined at about 30° to the horizontal.
The evaporating dish is half filled with water and is placed
on the hot plate. The round bottom flask is then fixed to
153
-------
the tapered end of the shaft. Two liters of the water sample
was put in the feed bottle. About 500 ml sample was added
to the evaporating flask by means of the feed tube and the
system was connected to a water aspirator. When the water
started boiling in the flask, the motor was switched on to
rotate the flask. The thermostat was set to give the de-
sired temperature in the water bath. Water level in the
evaporating dish was maintained with the help of a constant
level device.
Sometimes, due to insufficient vacuum, the water in the flask:
did not boil when the motor was stopped. Usually it was the
ball joint that was the trouble spot and needed readjustment
and tightening.
A little grinding action at the ball joint sometimes sent
very small teflon grinding into the flask, which looked
like bacterial growth. When checked under a microscope,
its exact nature was revealed. Since the concentrate was
passed through a 0.45 u membrane filter before applying it
to the gel filtration column, or to the carbon analyzer, the
teflon particles did not affect the results in any way.
A continuous feed into the evaporating flask was also at-
tempted by adjusting the pinch clamp on the tygon tubing.
The rate of evaporation was so small that proper adjustment
of the pinch clamp could not be made. The feed was, there-
fore, let intermittently into the flask.
3. Freeze-drying
The center well of the freeze-dryer is filled to 1/3 its
depth with acetone. Dry ice is added to the acetone in
very small pieces one at a time so that the acetone does
not overflow the vessel as CC^ escapes when the acetone
is cooled. More dry ice is added when the bubbling stops.
All the quickseal valves are closed, oil level in the vacuum
pump is checked and the pump is started.
The sample to be concentrated is put in a freeze-drying flask
of 1.2 liter capacity. It is then put in a dry ice/acetone
bath and rotated continuously at about 80 rpm for "shell"
freezing of the sample. The flask is kept in a slanting
position while rotating.
After all the liquid in the flask is completely frozen, it
154
-------
is attached to one of the ports on the manifold. The quick-
seal valve is opened and a high vacuum is reestablished in
the system by the pump. It has been observed that when the
pressure is more than 100 u of Hg, it is difficult to main-
tain a sample in the frozen state during the drying operation.
Usually the pressure is kept less than 50 u of Hg.
Although the capacity of each freeze-drying flask is 1.2
liters, only about 500 ml of the sample can be concentrated
in a flask at a time. Sample volumes of more than 650 ml
showed a tendency of melting adjacent to the walls and the
bottom of the flask. This tendency was more pronounced at
the bottom of the flask. It appears that the ice thickness
in the flask becomes too great to conduct all the heat,
supplied by the atmosphere, to the evaporating surface. No
additional heat is supplied to the system but normally the
ambient room temperature combined with the condensation of
moisture in the room on the external surface of the flask
supplies just the right amount of heat to efficiently sub-
limate the ice content of the sample.
After all the flasks have been frozen, some of the acetone
is taken out of the center well and dry ice is added in its
place. For overnight operation an extender is placed on the
center well and filled with the dry ice. It is then covered.
At the completion of the operation, the quickseal valves are
turned only 1/4 turn clockwise to retain vacuum in the flasks
and the vacuum pump is stopped. The vacuum is released by
opening a valve which is not attached to any flask. The
flasks are then removed along with the quickseal valves and
the vacuum in each flask is released very slowly so as not
to disturb the powdered concentrate in the bottle. The
sample is usually not concentrated to complete dryness. If
the apparatus is not to be reused immediately, it is better
to release the vacuum in the flask after the ice has melted.
Although the ports were internally baffled, ice clogging was
noticed occasionally on a few ports which resulted in the
melting of the sample. The concentrated sample was filtered
through 0.45 u membrane filter. Acid filtrate is obtained
by dissolving the precipitate in 1:10 dilute HCl and filter-
ing it through the membrane filter.
155
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4. Procedure for Gel Chrotnatography
A calculated amount of Sephadez G-15 was swollen in double-
distilled water in the cold room for 24 hours. The column
was assembled vertically and a stoppered funnel was fixed
onto the upper end. A flow adaptor was fixed at the upper
end. A flow adaptor was fixed at the lower end and the
outlet was closed. The column was then filled with the
solvent. The swollen gel along with some solvent was put
into the funnel when a layer of about 15 to 20 cm of the
gel had settled, the outlet was opened and the solvent was
allowed to flow out slowly. More gel came into the column
and was packed gradually. The verticality of the column
was checked with a plumb bob. The eluent reservoir bottle
was connected to the top of the column and flow of buffer
was maintained for 36 hours so that the gel is properly
settled.
To apply a sample to the column, the eluent above the gel
packing was removed with the help of a 10 ml syringe. What-
ever was left was allowed to penetrate the gel by keeping
the outlet open. A measured amount of sample was then appliej
to the column. Flow rates through the column were maintained
by adjusting the position of the eluent reservoir. Five ml
fractions were collected with the help of an LKB fraction
collector, and were analyzed on the carbon analyzer for
organic carbon.
A chromatogram is obtained by plotting the organic carbon
(rag/1) as ordinate and elution volume or fraction number as
abcissa. The elution volume of a particular compound is the
effluent volume as measured from the application of the
sample to the elution of the component at maximum concentra-
tion.
5. Column Standardization
The gel column must be standardized before any sample is
applied to it for analysis. The Sephadex G-15 column was
standardized using a solution of blue dextran (a dextran
with a blue dye chemically bound to it - molecular weight
approximately 2 x 10°), raffinose (molecular weight 594.32),
maltose (molecular weight 360.32), glucose(molecular weight
180.16) and sodium chloride (molecular weight 58.44). The
156
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homologous compounds were selected as standards because they
eliminate the possibility of interference due to different
configurations of other molecules. Another reason is that
carbohydrates are also present in the samples to be analyzed.
The above compounds were dissolved in double-distilled water.
and the solution was filtered through 0.45 u membrane filter
before applying it to the column.
Phosphate buffer was used as eluent. It consisted of the
following compounds:
Potassium dihydrogen phosphate 225.7 mg/1
Dipotassium hydrogen phosphate 577.4 mg/1
Disodium hydrogen phosphate 886.8 mg/1
The pH of the buffer was 7.6.
Length of the gel column was 90.5 cm and its diameter was
2.5 cm. The rate of flow through the column was 50 ml/hr.
The effluent was collected in 5 ml fractions which were an-
alyzed on the carbon analyzer. The chromatogram obtained
is shown in Figure 1.
The molecular weight of the sample is plotted against its
elution volume on a semilog paper and a calibration curve
is obtained, as shown in Figure 2. The elution volume has
been shown to be a linear function of the logarithm of the
molecular weight over a considerable range.
6. Procedure for Organic Carbon Determination
The carbon analyzer was calibrated for the range 0 mg C/l to
100 mg C/l using oxalic acid (HO^CCOoH • H20 molecular
weight 126.07) standard solutions. These solutions were
prepared in carbon dioxide free, double-distilled water in
carbon concentrations of 2, 4, 10, 20, 40, 80 and 100 mg/1.
By use of the sensitivity switch the instrument was adjusted
to give the maximum reading when 40 ul of 100 mg C/l standard
solution was injected. Readings corresponding to each stan-
dard concentration were obtained at that gain control and a
calibration curve was obtained. The calibration curve was
not linear for this instrument.
Each 5 ml fraction was acidified to a pH of 2.0 with 2N HC1
and purged of C02 with nitrogen gas for five minutes at a
gas flow rate of about 500 cc/minute. Fourty ul of the
sample was then injected with a Hamilton SK 148, 50 ul
syringe for organic carbon analysis. The Beckman furnace
157
-------
Ui
OO
I
o
o
90
80
70
60
4O
30
2O
10
IV
30
tl III
4O 50 6O 70
FRACTION NUMBER
I.DEXTRAN BLUE
-------
1500
1000
500
200
K
(D
U
100
-I
D
U
UJ
o
50
20
10
35
DEXTRAN BLUE (mwt. 2xl(ft
RAFFINOSE (mwt. 594.32)
MALTOSE (m.wt. 360.32)
GLUCOSE tm. wt. 180.16)
SODIUM CHLORIDE (m.wt. 58.44)
COLUMN LENGTH
INSIDE DIAMETER
90.5 cm
2.5 cm
45 55 65 75
FRACTION NUMBER
85
95
Fig. 2 CALIBRATION CURVE FOR SEPHAOEX G-15 COLUMN
159
-------
had arrangements for measuring total as well as inorganic
carbon separately but the inorganic channel did not give
consistent readings. The above procedure of purging in-
organic carbon was, therefore, employed.
Results and Data Analysis
1. Saratoga Lake Water
Concentration. About 20 liters of Saratoga Lake water were
collected on November 24, 1969 from the east shore, about
1-1/2 miles from the south end of the lake. The temperature
of the water sample as collected was 12°C and its pH was 8.35.
The sample was filtered through a 0.45 u membrane filter.
Thin film evaporation was started with 2 liters of sample in
the feed bottle. More sample was added when the initial feed
had evaporated. A total of 5 liters was concentrated by this
method to 141 ml, a concentration factor of 35.5. Although
the sample was colorless, the concentration was slightly
colored and appeared to be somewhat viscous in the evaporating
flask. Therefore, further concentration was not carried out.
On filtering, however, the viscous appearance turned out only
to be due to a slight color effect. The concentrate was not
viscous.
The freeze-drying was started in 8 flasks. Five hundred ml
of the sample was put in each flask, shell-frozen in a
dry-ice/acetone bath and the flask was connected to the
manifold. The freeze-dryer was kept running at night but
the V-belt of the vacuum pump broke and the frozen samples
melted in the flasks. The freeze-drying was again started
after replacing the V-belt in the morning. The concentrated
contents of the 8 flasks were transferred into 2 flasks and
again freeze-dried. The final concentrate obtained was 62
ml (concentration factor of 64.5) and it was colorless.
Table 1 represents the concentration and recovery data for
this sample.
The organic carbon recovery in the freeze-drying method is
only 60% of that in the thin film evaporation. The recovery
has been calculated on the basis of total organic carbon
recovered in each method, which includes both the concentrate
and the acid filtrate.
Another sample of Saratoga Lake water was obtained and con-
centrated by both methods. A relative recovery of 7970 was
160
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TABLE 1
CONCENTRATION AND ORGANIC RECOVERY DATA
SARATOGA LAKE WATER
SAMPLE NO. 1
Concentration Data
Initial volume of the sample
Final volume (concentrate)
Volume of the acid filtrate
Concentration factor (based on
concentrate volume only)
Temperature of the water bath
Vacuum pressure
Organic Recovery Data
Organic carbon content
Concentrate
Acid filtrate
Total organic content of the
sample (as recovered)
Total organic carbon recovered
per liter of the sample
Percentage recovery (based on
10070 recovery in thin film
evap ora t ion)
Total including acid filtrate
Only concentrate
Thin Film
Evaporation
5 liters
141 ml
16 ml
35.5
34.5°C-37°C
40-60 mm
of Hg
100 tng/1
30 mg/1
14.580 rag
2.916 mg
100%
100%
Freeze-
drying
4 liters
62 ml
15 ml
64.5
20-50 u
of Hg
100 mg/1
52 mg/1
6.980 mg
1.745 mg
60%
55.5%
161
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obtained this time. The concentration data for this sample
are shown in Table 2. The concentration by freeze-drying
was carried out on a "Freeze-mobile" (The Virtis Company,
Inc.) and not on the bench model freeze-dryer as previously
described.
Chromatographic studies. The concentrates obtained by both
methods were applied to the gel column packed with Sephadex
G-15 to fractionate the organic compounds present. Phosphate
buffer was used as eluent. The fractions were analyzed on
the carbon analyzer. The readings for individual fractions
are shown in a later section, (pages 255-262). The chroma-
tograms obtained are shown in Figures 3 and 4, for the rotary
thin film and freeze-dried concentrates, respectively. The
chromatographic data are shown in Table 3.
The two chromatograms look very much alike, although there
are some minor differences. The peaks appear at the same
elution volume in both chromatograms and are identical in
number. This shows the presence of the same compounds in
both the concentrates and, therefore, no relative hydrolysis
or polymerization.
This, in fact, is an oversimplification of the statement
(analysis). The necessity to compare the two chromatograms
in some other way is obvious. It was decided to plot the
cumulative percentages of the compounds eluted with successive
fractions. As the elution volume is a linear function of the
logarithm of the molecular weight, the graph (Figure 5)
obtained gives, at a glance, the percentage of compounds in
the sample which are greater than a certain molecular weight.
In this way the comparison is made of the compounds in the
two concentrates as they are eluted from the gel column. For
example, the thin film concentrate has 1670 of the compounds
with apparent molecular weight greater than 1,500, whereas
the freeze-dried concentrate also has 1670; similarly for
apparent molecular weight greater than 350, the corresponding
figures are 85% and 83% for thin film and freeze-dried concen-
trates, respectively.
The two percentage curves (Figure 5) for the thin film concen-
trate and the freeze-dried concentrate plot out very close to
each other and differ not more than 2.5% at any section. This
small difference is hardly of any importance, considering the
162
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TABLE 2
CONCENTRATION AND ORGANIC RECOVERY DATA
SARATOGA LAKE WATER
SAMPLE NO. 2
Concentration Data
Initial volume of the sample
Final volume (concentrate)
Volume of the acid filtrate
Concentration factor (based
on concentrate volume only)
Temperature of the water bath
Vacuum pressure
Organic Recovery Data
Organic carbon content
Concentrate
Acid filtrate
Total organic content of the
sample (as recovered)
Total organic carbon recovered
per liter of the sample
Percentage recovery (based on
100% recovery in thin film
evaporation)
Total including acid
filtrate
Only concentrate
Thin Film
Evaporation
2.5 liters
16 ml
55 ml
156
33°C
40-60 mm
of Hg
651 mg/1
24 mg/1
11.736 mg
4.694 mg
Freeze-
drying
2.5 liters
17 ml
28 ml
147
20-50 u
of Hg
390 mg/1
95 mg/1
9.290 mg
3.716 mg
100%
100%
79.2%
61.75%
163
-------
GEL SEPHADEX G-15
SAMPLE VOL. 10 ml
TOC 100 mg/I
FRACTION VOL. 5ml EACH
90
Fig. 3 CHROMATOQRAM OF SARATOGA LAKE
THIN FILM CONCENTRATE
WATER
-------
CT*
Ul
10
la
S 6
§
o
<
30
GEL
SAMPLE VOL. 12 ml
TOC 100 mg/t
FRACTION VOL. 5ml EACH
G-15
50 60 70
FRACTION NUMBER
80
90
Fig. 4 CHHOMATOQIWM OF SARATOGA LAKE WATER
FREEZE OWED CONCENTRATE
-------
TABLE 3
CHROMATOGRAPHIC DATA FOR SARATOGA LAKE WATER
SAMPLE NO. 1
Thin Film Freeze-
Preliminary Data Evaporation drying
Volume of the sample applied 12 ml 10 ml
3 3
Volume of gel column 444 cm 444 cm
Sample volume as percentage
of column volume 2.70% 2.25%
Organic content of sample 100 mg/1 100 mg/1
Total organic carbon applied
to column 1.20 mg 1.0 mg
Rate of flow through the
column 44 ml/hr. 28 ml/hr
Molecular Weight
Distribution Data
Compounds greater than apparent
molecular weight 1500 in the
sample 16% 16%,
Compounds greater than apparent
molecular weight 1200 in the
sample 43% 43%
Compounds greater than apparent
molecular weight 700 in the
sample 60% 60%
Compounds greater than apparent
molecular weight 350 in the
sample 85% 83%
Compounds greater than apparent
molecular weight 180 in the
sample 93% 91%
166
-------
100
90
80
70
60
LJ
2
Ul
Q.
I
_I
o
50
40
30
20 -
10 -
THIN FILM CONCENTRATE o-
FREEZE DRED CONCENTRATE o-
Pig. 5
1500 1000 500 200 100
MOLECULAR WEIGHT
50
PERCENTAGE CURVES FOR CHROMATOORAMS
OF SARATOGA LAKE WATER
167
-------
instrumental and other errors involved. Any significant
hydrolysis or polymerization of the compounds while the
sample is concentrated by either of the two methods is,
therefore, ruled out.
As is seen from Figure 5, the compounds of molecular weight
less than 200 constitute only 10% of the sample and 60% of
the compounds are with molecular weight greater than 700.
2. Clifton Knolls Sewage Effluent
Concentration. The Clifton Knolls Sewage Treatment Plant
is located about fifteen miles northwest of Troy, New York.
The sewage is treated by the contact-stabilization process
and the effluent is chlorinated before it is discharged into
the ponds. The chlorinated effluent sample was collected in
a 20 liter plastic carboy on March 26, 1970. Twenty ml of
HgCl2 (stock solution with 40 gms HgC^/liter) was added to
it to eliminate the possibility of any biological degrada-
tion during storage. The temperature of the effluent sample
was 20°C and its pH was 7.2. The sample was filtered twice,
first through a coarse filter and then through 0.45 u
membrane filter.
The freeze-drying was started with 2.5 liters of the sample
in 7 flasks. After one day, the flasks were removed and
ice remaining in the flasks was allowed to melt. When 2.5
liters more of the sample were added, the concentration was
again started. The concentrate from all the flasks was
transferred into one flask and was finally concentrated to
32 ml (a concentration factor of 156.2). Acid filtrate
obtained by dissolving the precipitate in 1:10 HCl was 22 ml.
As the sample was concentrated too much, a large amount of
material had precipitated in the flask.
Five liters of the sample was simultaneously concentrated by
thin film evaporation. The problem of heavy deposition along
the walls of the evaporating flask (near the top) was encoun-
tered while concentrating this sample. Although some deposi-
tion is always found while concentrating sewage sample and
has also been reported in the literature, in this case the
addition of HgC^ had also contributed much to this deposi-
tion. The concentrate obtained was 91 ml (a concentration
factor of 54.9) and the acid filtrate was 20 ml.
168
-------
The organic carbon recovery in the freeze-drying method was
90.8% of that in the thin film evaporation. The concentra-
tion and organic recovery data are shown in Table 4. This
comparatively higher recovery in freeze-drying is partly
due to the poor performance of the thin film evaporator in
this particular case. The deposition along the walls was
extensive, sticky, and difficult to dissolve completely in
dilute (1:10) HCl. It is believed that some organic carbon
must have been left in the flask along with the deposition
which could not be dissolved even after rotating the flask
with dilute HCl for about an hour. If acid filtrates are
excluded the relative organic carbon recovery in freeze-
drying comes to only 76.8%.
The acid filtrate obtained from freeze-drying contains 29.6%
of the total organic carbon recovered. In thin film evapora-
tion, it is only 16.5%. Comparatively greater amounts of
organic carbon in acid filtrate from freeze-drying may partly
be due to a larger concentration factor in this method, which
is 156.2 as compared to 54.9 in thin film evaporation.
Chromatographic studies. A 10 ml aliquot of the freeze-dried
concentrate was put on the G-15 Sephadex column. Phosphate
buffer was used as eluent. The flow rate through the column
was 46 ml/hour. The 5 ml fractions as analyzed on the carbon
analyzer are shown in a later section. The chromatogram is
shown in Figure 6.
The thin film concentrate did not have sufficient organic
carbon in it and thus was further concentrated before apply-
ing it to the gel column. The volume of the sample applied
to the column was 5 ml. The chromatogram is shown in Figure
7. The individual readings for the 5 ml fractions as anal-
yzed on the carbon analyzer are shown in a later section.
The general chromatographic data for the two concentrates
are shown in Table 5.
Both the chromatograms have two major peaks at apparent molec-
ular weight - 1,500 and 290; and two small peaks at molecular
weight - 600 and 180. Another good-size peak appears at the
end of the elution. According to the calibration curve, the
molecular weight of the compound should be about 30. Possibly
the nature of the compound is such that it has stayed in the
gel for more than what is theoretically required according
169
-------
TABLE 4
CONCENTRATION AND ORGANIC RECOVERY DATA
CLIFTON KNOLLS EFFLUENT
Concentration Data
Initial volume of the sample
Final volume (concentrate)
Volume of the acid filtrate
Concentration factor (based
on concentrate volume only)
Temperature of the water bath
Vacuum pressure
Organic Recovery Data
Organic carbon content
Concentrate
Acid filtrate
Total organic carbon in the
sample (as recovered)
Total organic carbon recovered
per liter of the sample
Percentage recovery (based on
100% recovery in thin film
evaporation)
Total including acid
Concentrate only
Thin Film Freeze-
Evaporation drying
5 liters
91 ml
20 ml
54.9
32°C-35°C
40-60 mm
of Hg
5 liters
32 ml
22 ml
156.2
50-100 u
of Hg
126 mg/1 275 mg/1
114 mg/1 168 mg/1
13.768 mg 12.496 mg
2.754 mg 2.499 mg
100%
100%
90.8%
76.8%
170
-------
GEL
SAMPLE VOL.
TOC
FRACTION VOL.
SEPHADEX G-15
10 ml
275 mo/1
5ml EACH
Fig. 6
60 70
FRACTION NUMBER
OF CLIFTON KNOLLS
FREEZE DRIED CONCENTRATE
EFFLUEMT
-------
-J
to
GEL
SAMPLE VOL.
TOG
FRACTION VOL
SEPHADEX 6-15
5ml
720mg/l
5 ml EACH
Fig 7
60
FRACTION
CHROMATOQFUUK OF CLIFTOW KNOLLS
THIN FILW CONCENTRATE
EFFLUENT
-------
TABLE 5
CHROMATOGRAPHIC DATA FOR CLIFTON KNOLLS EFFLUENT
Thin Film Freeze-
Preliminary Data Evaporation Drying
Volume of the sample applied 5 ml 10 ml
3 3
Volume of gel column 444 cm 444 cm
Sample volume as percentage
of column volume 1.1370 2.2570
Organic content of sample 72Q mg/1 2?5 mg/1
Total organic carbon applied 3.6 mg 2.75 mg
Rate of flow through the
column 38 ml/hr 40 ml/hr
Molecular Weight
Distribution Data
Compounds greater than apparent
molecular weight 1,500 in the
sample 17% 18 ^
Compounds greater than apparent
molecular weight 1,100 in the
sample 32%
Compounds greater than apparent
molecular weight 500 in the
sample 4-3% 41%
Compounds greater than apparent
molecular weight 180 in the
sample 73/0 74/0
Compounds greater than apparent
molecular weight 100 in the
sample 86% 88>4
173
-------
to its molecular weight and it has come out approximately
at the end of the bed volume. No definite molecular weight
can, therefore, be assigned to this peak which accounts for
about 9% of the organic carbon present in the sample.
All the peaks appear at the same elution volume in both the
chromatograms. The percentage curves shown in Figure 8
plot out very close to each other, with the maximum differ-
ence at any section less than 3 percent, which is believed
to be due to the instrumental and other errors involved.
No relative polymerization or hydrolysis is, therefore,
observed by either concentration techniques.
Molecular weight distribution in the effluent sample, as
revealed by the chromatograms, is such that about 30% of
the soluble organic material is larger than molecular
weight 1,200 and about 28% are smaller than molecular weight
180.
3. Lake George Water
Concentration. Lake George water was the first to be con-
centrated.However, this was done only to gain experience
about the procedure to be adopted in the research. Five
liters of the sample were concentrated to 50 ml by thin
film evaporation and 4.5 liters were concentrated by freeze-
drying in 9 flasks to a final volume of 58 ml. The concen-
trates were not filtered nor was any acid filtrate obtained.
The organic carbon recovery in freeze-drying was found to
be only 65% of that of thin film evaporation (Table 6). This
was, however, thought to be mainly due to the discrepancies
inherent in the initial work, such as lack of experience,
improper attention, and less than optimum operating perfor-
mance of the apparatus.
Another sample was collected on September 5, 1970 from Smith
Bay at Lake George, New York, in a 20 liter carboy. The
temperature of the sample was 20.5 C and its pH was 7.7.
The sample was filtered and concentration was started simul-
taneously by the two methods. The rotary evaporator outlet
was also connected to the freeze-drying apparatus. The
vacuum hose which was previously connected to the water
aspirator was now connected to one of the ports of the
manifold. The temperature of the water bath was 33 C-35 C.
The water evaporating from the sample was being condensed
174
-------
too
90
80
70
60
50
40
UJ
30
20
10
THIN FILM CONCENTRATE
FREEZE DRIED CONCENTRATE
1500
Fig. 8 PERCENTAGE
1000 500 200
MOLECULAR WEIGHT
100
CURVES FOR CHROMATOQRAMS
CLIFTON KNOLLS EFFLUENT
o-
50
OF
175
-------
TABLE 6
CONCENTRATION AND ORGANIC RECOVERY DATA
LAKE GEORGE WATER
SAMPLE NO. 1
Concentration Data
Initial volume of the sample
Final volume
Concentration factor
Temperature of water bath
Vacuum pressure
Organic Recovery Data
Thin Film
Evaporation
5 liters
50 ml
100
35°C-37°C
40-60 mm
of Hg
Freeze-
drying
4.5 liters
58 ml
77.6
50-100 u
of Hg
Organic carbon content of the
concentrate
Total organic content in the
sample (as recovered)
Total organic carbon recovered
per liter of the sample
Percentage recovery (based on
100% recovery in thin film
evaporation)
207 mg/1
10.35 mg
2.07 mg
1007o
104 mg/1
6.03 mg
1.34 mg
64.8%
176
-------
into ice in the condenser. The increased temperature
gradient and high vacuum of the freeze-dryer resulted in
a very rapid concentration of the sample. Two liters were
evaporated in less than 16 hours. Such a quick rate of
evaporation was not visualized and complete drying of the
sample occurred. Five liters of the sample were freeze-
dried using 5 flasks (500 ml in each flask, 2 times). The
freeze-dried concentrate of this sample also became complete-
ly dry. The residues were dissolved with double-distilled
water (free of organics) and they were filtered through a
membrane filter (0.45 u) . The precipitate was dissolved
in dilute (1:10) HC1, as per usual procedure and organic
carbon in both the concentrate and the acid filtrate was
determined on the carbon analyzer. The results show an
organic carbon recovery of 87.9% in the freeze-drying
method as compared to the thin film evaporation. Concen-
tration data are shown in Table 7.
ChromatQgraphic studies. The flow in the gel column had
decreased considerably. The column was, therefore, unpacked
and the gel (Sephadex G-15) was washed thoroughly with
double-distilled water. The column was then packed with
this gel and was again standardized. The calibration curve
for the column is shown in Figure 9.
A 10 ml aliquot of each concentrate was applied to the G-15
gel column for separation of the compounds of different
molecular weights or sizes. Phosphate buffer was used as
eluent (flow rate was 54 ml/hr). The 5 ml fractions col-
lected were analyzed on the total carbon analyzer for organic
carbon only. The chromatogram for the thin film concentrate
is shown in Figure 10 and that for the freeze-dried concen-
trate is shown in Figure 11. The readings for individual
fractions are given in a later section. Table 8 shown the
chromatographic data for the two concentrates.
As in the case of other samples the two chromatograms look
alike. There are two major peaks at apparent molecular
weight 1,500 and 180 and three minor peaks at apparent mo-
lecular weight 600, 370 and 275. There is one very small
peak at the end of elution of the sample. All these peaks
(even the smallest one) occur in both the chromatograms and
essentially at the same elution volume for each peak. About
377o of the compounds are of apparent molecular weight 180
and compounds greater than molecular weight 1,500 constitute
about 15%.
177
-------
TABLE 7
CONCENTRATION AND ORGANIC RECOVERY DATA
LAKE GEORGE WATER
SAMPLE NO. 2
Concentration Data
Initial volume of the sample
Final volume (concentrate)
Volume of the acid filtrate
Concentration factor (based
on concentrate volume only)
Temperature of the water bath
Vacuum pressure
Organic Recovery Data
Organic carbon content
Concentrate
Acid filtrate
Total content in the sample
(as recovered)
Total organic carbon recovered
per liter of the sample
Percentage recovery (based on 10070
recovery in thin film evaporation)
Total including acid
Concentrate only
Thin Film Freeze-
Evaporation drying
2 liters
14.5 ml
13 ml
138
33°C-35°C
40-60 mm
of Hg
296
49
4.929 mg
2.465 mg
5 liters
20 ml
10 ml
250
20-50 u
of Hg
504
75
10.830 mg
2.166 mg
1007o
1007o
87.9%
93.77o
178
-------
1500 - o-^. DEXTRAN BLUE (m.wt.2xK?)
1000 f-
500 -
200
l-
i
100
50
20
10
RAFFINOSE (m.wt. 594.32)
MALTOSE (mwt. 360.32)
o \GLUC08E (m.\wt, 180.16)
SODIUM CHLORIDE (m.wt.58.44)
\
\
\
COLUMN LENGTH
INSIDE DIAMETER
86.5 cm
2.5 cm
30 40
50 60 70
FRACTION NUMBER
80 90
9 CALIBRATION CURVE FOR SEPHADEX 6*15 COLUMN
179
-------
00
o
.-30
I
20
8
o
iO
30
GEL
SAMPLE VOL.
TOC
FRACTION VOL.
SEPHADEX G-15
10 ml
296 mg/l
5ml EACH
50
60 70
FRACTION NUMBER
Fig. 10 CHROMATOGRAM OF LAKE GEOROE
THIN FILM CONCENTRATE
80
WATER
90
100
-------
00
GEL
SAMPLE VOL.
TOC
FRACTION VOL.
SEPHADEX G-IS
10 ml
504 rng/l
5 ml EACH
40
50
60
FRACTION
Fi8- ll CHROMATOGRAM OF LAKE
FREEZE
DRIED
70
NUMBER
GEORGE
CONCENTRATE
80
90
100
WATER
-------
TABLE 8
CHROMATOGRAPHIC DATA FOR LAKE GEORGE WATER
SAMPLE NO. 2
Preliminary Data
Volume of the sample applied
Volume of gel colume
Sample volume as percentage
of column volume
Organic content of sample
Total organic carbon applied
to column
Rate of flow through the
column
Molecular Weight
Distribution Data
Thin Film Freeze-
Evaporation drying
10 ml
2.96 mg
10 ml
425 cm3 425 cm3
2,35% 2.35%
296 mg/1 504 mg/1
5.04 mg
54 ml/hr 54 ml/hr
Compounds greater than apparent
molecular weight 1,350 in the
sample
Compounds greater than apparent
molecular weight 600 in the
sample
Compounds greater than apparent
molecular weight 250 in the
sample
Compounds greater than apparent
molecular weight 150 in the
sample
17%
36%
61%
92%
16%
35%
61%
93%
182
-------
The percentage curves for the two concentrates, shown in
Figure 12, plot out close to each other and do not differ
by more than 2% at any section. The difference is, however,
too minor to arrive at any definite conclusion. It is be-
lieved that instrumental and other errors involved are
responsible for this small difference. The possibility of
any relative degradation in this case also is, therefore,
not apparent.
4. Raw Sewage, Elnora
Concentration. About 5 liters of raw sewage was collected
on April 15, 1970 from the sewage treatment plant at Clifton
Knolls. The temperature of the sample was 21 C and the pH
was 7.7. Five ml of HgCl2 (stock solution with 40 grams of
HgCl2/liter) was added to the sample to eliminate microbial
activity during storage. The sample was filtered twice,
through a coarse filter and a 0.45 u membrane filter.
Five hundred ml of the sample was put in the 2 liter round
bottom flask and was concentrated to 53 ml by rotary thin
film evaporation. The temperature of the water bath was
maintained at 34 -35 C and the vacuum provided with the help
of a water aspirator was from 1 to 2 inches of Hg. A small
amount of deposit had formed on the flask walls as in the
case of Clifton Knolls effluent sample. The deposit was
dissolved in dilute HC1 at the time of obtaining acid fil-
trate. Concentration of another 500 ml of the sample was
carried out by freeze-drying using only one flask. Final
volume of the concentrate was 121 ml. The concentration
and organic recovery data for the sample are shown in Table 9.
The original organic content in the sample was quite high,
ie 62 mg/1. Therefore, the sample was concentrated only 4
to 9 times. The organic carbon recovery was 92.570 in the
case of thin film evaporation and 91.7% in the case of
freeze-drying. So, the recovery in freeze-drying for this
sample is 99.270 of that in thin film evaporation.
Chromatographic studies. A 10 ml aliquot of each concentrate
waTs applied to G-15 Sephadex column. Phosphate buffer was
used as eluent and its flow rate through the column was 48
ml/hr. Five ml fractions obtained were analyzed on the
carbon analyzer. Individual readings for each fraction are
shown in a later section. The chromatograms obtained are
shown in Figure 13 and 14.
183
-------
100
90
80
70
60
ui
i
tu
g 50
£ 40
S
3 30
20
10
THIN FILM CONCENTRATE a-
FREE2E DRIED CONCENTRATE o-
i i
I50O 1000 500 200 100 50
MOLECULAR WEIGHT
Fig* 12 PERCENTAGE CURVES FOR CHROMATOGRAMS
OF LAKE GEORGE WATER
184
-------
TABLE 9
CONCENTRATION AND ORGANIC RECOVERY DATA
RAW SEWAGE, ELNORA
Concentration Data
Initial volume of the sample
Final volume (concentrate)
Volume of acid filtrate
Concentration factor (based on
concentrate volume only)
Temperature of water bath
Vacuum pressure
Organic Recovery Data
Organic carbon content
original sample
concentrate
acid filtrate
Total organic content
in the sample
as recovered
Percentage recovery
Percentage recovery based on 100%
recovery in thin film evaporation
total including acid filtrate
concentrate only
Thin Film
Evaporation
500 ml
53 ml
14 ml
9.95
34°-35°C
40-60 mm
of Hg
62 mg/1
522 mg/1
68 mg/1
31.000 mg
28.618 mg
92.5%
Freeze-
drying
500 ml
121 ml
12 ml
4.1
20-50 u
of Hg
62 mg/1
230 mg/1
48 mg/1
31.000 mg
28.406 mg
91.7%
100%
100%
99.2%
100.6%
185
-------
00
ON
60
50
40
O
CQ
30
20
to
35
45
Fig. 13
GEL
SAMPLE VOL.
TOC
FRACTION VOL.
SEPHADEX 6-15
10 ml
522 mg/l
5ml EACH
55
65 75 85
FRACTION NUMBER
CHROMATOQRAM OF EO.NORA RAW
THIN FILM CONCENTRATE
95
105
SEWAGE
-------
00
0
35
GEU
SAMPLE VOL.
TOC
FRACTION VOL.
SEFMM3EX G-13
10 ml
230mg/l
5ml EACH
45
55
65 75
FRACTION NUMBER
85
Fig. 14 CHROMftTOGRAM
OF ELNORA raw
DRIED COWCENTTMirE
95
SEWAGE
-------
Two major peaks appear in both chromatograms at apparent
molecular weight 300 and 180. Another large peak appears
at the end of the bed volume. No molecular weight can be
assigned to this peak as it cannot be ascertained from the
calibration curve, being at the end of the bed volume. This
peak accounts for 23% of the organic matter in the sample.
Two minor peaks appear at apparent molecular weight 1,500
and 60.
All the five peaks appear at exactly the same elution volume
in both the chromatograms. The percentage curves for the
two concentrates (Figure 15) plot out very close to each
other with a maximum difference of less than 2% at any
section. Obviously, the polymerization or hydrolysis of any
compound during concentration by the two methods, has not
occurred. Eighty-five percent of the soluble organic com-
pounds are smaller than apparent molecular weight 400 and
less than 10% are larger than molecular weight 1,200. The
chromatographic data for the sample are shown in Table 10.
188
-------
100
90
80
70
60
UJ
I
UJ
o
K
UJ
0.
50
40
30
20
10 -
THN FILM CONCENTRATION
D——a a
FREEZE OWED CONCENTRATION o o o
1500 IOOO 500 200 100 5O
MOLECULAR WEIGHT
Pig. 15 PERCENTAGE CURVES FOR CHROMATOGRAMS
OF ELNORA RAW SEWAGE
189
-------
TABLE 10
CHROMATOGRAPHIC DATA FOR RAW SEWAGE, ELNORA
Thin Film Freeze-
Preliminary Data Evaporation drying
Volume of the sample applied 10 ml 10 ml
3 3
Volume of gel column 444 cm 444 cm
Sample volume as percentage
of column volume 2.25% 2.25%
Organic content of sample 522 rag/1 230 mg/1
Total organic carbon applied
to column 5.22 mg 2.30 mg
Rate of flow through the
column 48 ml/hr 48 ml/hr
Molecular Weight
Distribution Data
Compounds greater than apparent
molecular weight 1,500 in the
sample 2% 2%
Compounds greater than apparent
molecular weight 400 in the
sample 13% 12%
Compounds greater than apparent
molecular weight 200 in the
sample ' 43% 42%
Compounds greater than apparent
molecular weight 100 in the
sample 68% 70%
190
-------
SECTION V: DISCUSSION
Two lake samples and two sewage samples were concentrated
by thin film evaporation as well as by freeze-drying. In
all cases the concentration resulted in a solid and a liquid
phase. The liquid phase contained from 70 to 9870 of the
organic matter recovered. This conforms with the findings
of Gjessing and Lee (33) who observed that 60 to 95% of
the organics remained in solution on concentration. The
amount of organic matter trapped in the solid phase would
depend largely on the concentration factor and the nature
of the sample.
In both the sewage samples a deposit had formed on the flask
walls when these were concentrated by thin film evaporation.
There was more deposit in the case of the Clifton Knolls
sample because of a greater concentration factor, ie 55
instead of 9.5 in the raw sewage sample. Similarity of the
chromatograms obtained in this work, as discussed hereafter,
discount the possibility of any interference due to these
deposits on the results. This is in accord with Fredette
(17) who also observed that deposits on the flask walls
did not affect his results.
Gjessing and Lee (33) have observed that color of the samples
decreased upon concentration. This is contrary to what has
been noticed in this work. Although no attempt was made to
quantitatively measure the color before and after concentra-
tion, it was observed that the color of the Saratoga Lake
water sample and the raw sewage sample had increased on con-
centration. The increase in color was more pronounced in
the case of thin film evaporation than in the case of freeze-
drying. In general, no decrease in color was noticed upon
concentration of any of the samples.
The concentration and organic recovery data for all the samples
show that organic carbon recovery in freeze-drying was less
than that in thin film evaporation. Some of the reasons for
the lower recovery in freeze-drying may be, a) the absence of
flask filters, b) the possible suction of dry solute from the
freeze-drying flask, c) the use of increased number of flasks,
and d) the high vacuum.
Manufacturers of the freeze-drying apparatus recommend the use
191
-------
of filters on the flasks if the sample is to be dried com-
pletely. Filters do help to arrest the flow of solute
molecules to some extent. However, when these were used,
the frozen sample in the flasks started melting. Presum-
ably the resistance to flow of vapor molecules through the
filter is so much that the rate of evaporation is consid-
erably decreased. Heat supplied by the atmosphere to the
frozen sample is more than that taken up by the evaporating
molecules, therefore, the sample starts melting. The use
of these filters was, therefore, abandoned.
In the freeze-drying process the sample is usually shell
frozen. As the flask is revolved at about 80 rpm, "pure"
ice is formed along the walls of the flask. The solute
particles remain in solution and are frozen in the end in
the concentrated form. These are the first to be exposed
to the actions of freeze-drying. Therefore, an increasing
shell of dried material develops as the drying boundary
progresses into the sample. All water vapor produced by
sublimation at the drying boundary must pass by diffusion
through this barrier. It is possible that some of the
dried material is drawn into the condenser along with the
water vapor.
In thin film evaporation only one flask is used to evaporate
the whole sample. In freeze-drying, on the other hand, a
number of flasks are used depending upon the size of the
sample and capacity of the freeze-dryer. The concentrate
from these flasks is transferred into one or more flasks
which usually need further concentration. Although effort
is made to take out all the concentrate without diluting
it excessively with rinse water, some of the concentrate
may still be lost in the flasks. Moreover, the greater the
number of flasks used, the greater may be the amount of dry
solute drawn into the condenser.
Absolute pressure in the freeze-drying process is of the
order of a few microns, whereas in the thin film evaporation
it is in the range of 1-2 inches of Hg. A higher vacuum may
contribute to the removal of dry solute from the freeze-
drying flasks.
Many organics are only sparingly soluble in water and their
concentration could exceed the solubility limit, thereby
placing some of the compounds in the precipitate. These
might have been difficult to dissolve even in dilute HCl.
192
-------
Concentration achieved in freeze-drying was many times more
than that in thin film evaporation in all of the samples
except raw sewage sample. This might have contributed some-
thing towards the low recovery in the freeze-drying process.
It is also observed that relative recovery in freeze-drying
is better for sewage samples than for water samples. Pos-
sibly the recovery in freeze-drying is dependent, to some
extent, on the amount of organic matter present in the
samples. In low organic water samples comparatively larger
portions of dry solute may be lost into the condenser.
The main purpose of this research was to determine if any
polymerization of hydrolysis occurred in either of the
concentration techniques employed. Gel chromatography was
the only convenient way of assessing the relative poly-
merization or hydrolysis effects. This was carried out in
the cold room at 4°C.
The chromatograms obtained show a different molecular weight
distribution for each sample. While the Saratoga Lake water
sample showed a predominance of high molecular weight com-
pounds in it, the Lake George water sample contained a
balanced amount of high and low molecular weight compounds.
In the case of Saratoga Lake water, more than 607o of the
compounds were of molecular weight greater than 700. This
observation is largely in conformity with that of Gjessing
and Lee (33) who separated several river water samples of
gel columns. They also found a preponderance of large molec-
ular weight (700+) compounds in river waters. The Lake
George water which is oligotrophic and unpolluted, had 36%
of the compounds larger than molecular weight 600 and 37%
of the compounds were with molecular weight about 180.
Chromatograms for sewage samples indicate that the raw
sewage sample had 85% of the compounds smaller than molec-
ular weight 400. The effluent sample, on the other hand,
had only 42% of the compounds smaller than molecular weight
400. This tends to indicate that low molecular weight com-
pounds in raw sewage are comparatively easily removed by
biological treatment.
As freeze-drying is carried out at much lower temperatures,
one would expect some difference in the chromatograms for
193
-------
thin film and freeze-dried concentrates. Absence of a peak
in one of the chroma to grains would mean that the compound
which was to come out at the elution volume had either been
altered, polymerized, or hydrolyzed, or has been totally re-
moved by the means employed for concentration. No meaningful
difference in the two chromatograms has, however, been found
for the samples analyzed in this investigation.
In all cases the volume of the sample applied to the gel
column was between 1% and 3% of the bed volume and the rate
of flow through the column varied from 28 ml/hr to 54 ml/hr.
Two chromatograms obtained for the thin film and freeze-
dried concentrates of the same sample have an identical
number of peaks and at about the same elution volume, indi-
cating, therefore, the presence of the same compounds in
both concentrates. The percentage curves for the two
chromatograms which indicate the percentage of compounds
larger than a certain molecular weight, do not show a
difference of more than 2 to 370 for any molecular weight.
This Jiinor difference is considered to be due to instrumen-
tal and other errors involved. McDonald et al (22) have
reported that chromatograms for unconcentrated raw sewage
and raw sewage concentrated by freeze-drying looked identical,
showing no alteration of the compounds when concentrated by
freeze-drying. It is, therefore, concluded that no poly-
merization or hydrolysis occurred during concentration by
either method.
It appears that not many heat labile compounds were present
in the water and wastewater samples analyzed, and if these
were present, the heat supplied in the thin film evaporation
was not sufficient to effect any degradation of the compounds.
The Lake George water sample was also concentrated at 60 C by
thin film evaporation. The chromatogram obtained was iden-
tical to the one obtained for the sample concentrated at 35°C
showing, thereby, the absence of heat labile compounds.
It is also believed that highly volatile compounds, if present,
must have been removed nearly equally in both methods. The
possibility of selective removal of a compound in either
method is discounted because of the similarity of the two
chromatograms.
The main drawback in thin film evaporation is the excessive
194
-------
time required to concentrate the sample. On the average,
one liter of sample could be concentrated in 24 hours. The
rate of evaporation can, however, be increased drastically
if the apparatus is connected to a suitable vacuum pump
and condenser.
As stated above, the low recovery in the freeze-drying
method is mainly due to the suction of dry solute into the
condenser. The loss could be minimized with the help of
a suitable device such as an improved filter which would
exclude the dry solute matter without hindering the transfer
of vapor molecules. From the results of this research, how-
ever, it may be concluded that it would be appropriate to
use thin film evaporation for concentrating water samples
and freeze-drying for concentrating sewage samples.
Freeze-drying has the inherent advantage, over thin film
evaporation of being carried out at much lower temperatures,
which affords less chance for degradation of the sample. If
a sewage sample cannot be preserved (it was preserved in
this research with HgC^) due to the special nature of the
experiment, freeze-drying might be the only reasonable way
of concentrating the unpreserved sample.
The possibility of carrying out thin film evaporation at
reduced temperatures, ie less than 10°C, needs to be inves-
tigated. This would combine the advantages of both freeze-
drying and thin film evaporation, ie less chance for degra-
dation and greater organic carbon recovery, respectively.
Lowering the temperature is certain to reduce the rate of
evaporation, but connecting the apparatus to a vacuum pump
and a condenser may make up for most of the loss.
195
-------
SECTION VI: REFERENCES
1. Baker, R.A., "Microchemical Contaminants by Freeze
Concentration," Journal WPCF, 37, 8 pp 1164-1170
(1965). ~~
2. Ihor, L., Nelson, K.H., and Webb, S.R., "Analysis of
Multi-component - Organic Mixtures in Aqueous Media
by Pyrolysis," Water Research, 4, pp 157-163 (Feb.
1970).
3. Sproul, O.J., "Origin of Organic Chemical Pollutants
in Water Supplies," Doctoral Dissertation, Washington
University, St. Louis, Mo. (1962).
4. Spincher, R.G., "Potassium Permanganate Effects on
Organic Refractories from River Waters," Doctoral
Dissertation, Washington University, St. Louis, Mo.
(1963).
5. Ward, R.F., "Organic Fouling of Strongly Basic Anion
Exchange Resins," Doctoral Dissertation, Washington
University, St. Louis, Mo. (1964).
6. Dornbush, J.N., "Physiochemical Removal of Naturally
Occurring Soluble Trace Organics from Water with
Selected Reactants," Doctoral Dissertation, Washington
University, St. Louis, Mo. (1962).
7. Ryckman, D.W. , Irvin, J.W. , and Young, R.H.F., "Trace
Organics in Surface Waters," Journal WPCF, 39, 3, pp
458-469 (1967).
8. Katnmerer, P.A., "Concentration Methods for Organic Com-
pounds in Water," University of Wisconsin, Water Chemistry
Seminar.
9. Braus, H. , Middleton, F.M., and Walton, G.U., "Organic
Chemical Compounds in Raw and Filtered Surface Waters,"
Anal. Chem.,JZ3, pp 1160 (1951).
10. Hoak, R.D., "Recovery and Identification of Organics in
Water," Int. J. Air, Water Poll., 16, pp 521-538 (1962).
197
-------
11. Hyndshaw, A.Y., Langhlin, H.F., Colebaugh, B.C., and
Filicky, J.G., "Factors Influencing the Efficiency
of Activated Carbons," J. New Eng. Water Works Assoc. ,
66, p 36 (1952).
12. Baker, R.A., "Chromatographic Evaluation of Activated
Carbon," J. AWWA, 56, 1, p 92 (1964).
13. Von Lippman, Z., Ver. deut, Zuckerind, 62, pp 967-979
(1912).
14. Badger, W.L., "Heat Transfer and Evaporation," The
Chemical Catalog Company, Inc., New York, pp 103-
108 (1929).
15. Webre, A.L., and Robinson, C.S., "Evaporation," The
Chemical Catalog Company, Inc., New York, pp 424-
426 (1926).
16. Partridge, S.M., "Rotary Film Evaporator for Laboratory
Use," J. Sci. Inst., 28, pp 28-29 (1951).
17. Fredette, P.E., "An Evaluation of Vacuum Rotary Thin
Film Evaporation for the Concentration of Trace
Organics," M.S. Thesis, Rensselaer Polytechnic
Institute, Troy, New York (1969).
18. Carman, P.C., "Some Basic Principles of Freeze-Drying
and Molecular Distillation," Conference on Freeze-
Drying of Foods, National Academy of Sciences, Washington,
D.C., Quatermaster Food and Container Institute, Chicago,
111., pp 77-84 (1961).
19. Shackell, L.F., "An Improved Method of Desiccation, with
some Application to Biological Problems," Amer. J. Physio_L
24, p 325 (1909).
20. Swift, H.F., "Preservation of Stock Cultures of Bacteria
by Freezing and Drying," J. Exper. Med., 33, p 69 (1921).
21. Reichel, J., Masucci, P., McAlpine, K.L., and Boyer, J.
unpublished work, reference taken from No. 25.
22. McDonald, G.C., Green, W.J., Hardt, F.W., Spear, R.D.,
Washington, D.R., and Clesceri, N.L., "Apparent Molecular
198
-------
Weight Distributions in Raw and Treated Wastewaters,"
Paper read at Fifth Annual International Association
on Water Pollution Research, San Francisco. Calif.
(Sept. 1970).
23. McDonald, G.C., and Clesceri, N.L., "Organic Nutrient
Factors Effecting Algal Growth," FWPCA Project No.
16010DHN, Progress Report for Year Ending 31 August 1968.
24. Flosdorf, E.W., and Mudd, S., "Procedure and for Preser-
vation in Lyophile Form of Serum and Other Biological
Substances," J. Immunology, 29_, p 389 (1935).
25. Abbot, D., and Andrews, R.S., "An Introduction of Chro-
matography," Longman, Green and Co. Ltd., London W.I.
(1965).
26. Alteman, A.G., "Chrotnatography as an Analytical Tool,"
Annals N.Y. Academy of Sciences, Vol. 137, p 335 (1966).
27. James, A.T., and Martin, A.J.P., Biochem. J. 50, p 679
(1952). Reference taken from Jacob Tadmor, N.Y. Academy
of Sciences Annals, 137, pp 103-126 (1966).
28. Porath, J., and Flodin, P., "Gel Filtration: A Method
for Desalting and Group Separation," Nature, 183, p 1657
(1959).
29. Determann, H., "Gel Chromatography," Springer-Verlag,
New York Inc., p 12 (1968).
30. Tiselius, A., and Albertson, J.P.A., "Separation and
Fractionation of Macromolecules and Particles," Science,
141, p 13 (1963).
31. Instruction Manual, Model 915, Total Organic Carbon
Analyzer and 191860 Air Purification Unit, Beckman
Instruments, Inc., Fullerton, Calif. (1969).
32. Service Manual, Model 200 LIRA Infrared Analyzer, Mine
Safety Appliances Company, Pittsburgh, Pa.
33. Gjessing, E., and Lee, G.F., "Fractionation of Organic
Matter in Natural Waters on Sephadex Columns," Water
Research, 1, 8, pp 631-638 (1967).
199
-------
34. Granath, K.A., and Flodin, P., Makromol. Chero., 48,
p 160 (1961).
35. Whitaker, J.R., "Determination of Molecular Weights of
Proteins by Gel Filtration on Sephadex," Anal. Chem.,
!35, pp 1950-1953 (1963).
36. Porath, J., "Some Recently Developed Fractionation
Procedures and their Application to Peptide and
Protein Hormones," Pure and Applied Chem., ji, pp 233-
244 (1963).
37. Squire, P.G., "A Relationship Between the Molecular
Weights of Macromolecules and their Elution Volumes
Based on a Model for Sephadex Gel," Archives of Biochem.
and Biophysics, 107, pp 471-478 (1964).
38. Lundblad, A., and Breggard, I., "Gel Filtration of Low
Molecular Weight Carbohydrate Components of Normal
Urine," Biochem., Biophysics Acta.,57, pp 129-134 (1962).
39. Zuckerman, M.M., "Chemical Versus Biological Wastewater
Treatment in the Production of High Quality Reuse Water,"
Doctoral Dissertation, New York University (1968).
200
-------
SECTION VII:
Fraction
No.
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15
COLUMN
Saratoga Lake Water Concentrated
By Thin Film Evaporation
Sample Volume 5 ml
T.O.C. 100 tng/1
Organic Carbon
mg/1
1,
1,
0
,0
1.5
2.0
5.5
7.5
8.2
8.5
8.0
10.0
11.0
12.0
11.5
10.0
9.5
9.0
8.5
7.0
6.5
5.0
5.0
5.5
6.0
7.0
9.0
9.5
7.2
7.3
6.5
6.0
5.5
6.5
4.0
Fraction
No.
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Organic Carbon
mg/1
3.0
2.5
1.5
1.5
2.0
2.2
1.5
1.5
1.3
2.0
2.0
1.0
2.0
1.5
2.0
1.0
1.5
1.5
1.5
1.5
1.0
1.5
2.0
1.0
2.0
1.5
2.0
1.0
1.5
0.6
1.0
1.0
0.5
201
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Saratoga Lake Water Concentrated
By Freeze-Drying
Sample Volume 10 ml
T.O.C. 100 mg/1
Fraction Organic Carbon Fraction Organic Carbon
No. mg/1 No. mg/1
32 1.0 62 4.3
33 2.0 63 3.8
34 4.0 64 3.4
35 5.0 65 2.3
36 6.0 66 1.8
37 6.5 67 1.9
38 7.0 68 2.0
39 6.5 69 1.9
40 9.0 70 2.0
41 9.5 71 3.0
42 10.0 72 2.6
43 9.0 73 2.6
44 9.0 74 2.5
45 8.5 75 2.5
46 8.2 76 1.8
47 8.0 77 1.6
48 5.9 78 1.6
49 5.4 79 1.5
50 4.5 80 1.2
51 4.3 81 1.1
52 4.4 82 1.4
53 5.1 83 1.7
54 5.7 84 1.7
55 6.6 85 1.8
56 6.9 86 1.6
57 6.0 87 1.5
58 5.8 88 1.6
59 5.5 89 2.0
60 5.0 90 1.5
61 5.4 91 1.6
202
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Clifton Knolls Effluent Concentrated
By Thin Film Evaporation
Sample Volume 5 ml
T.O.C, 720 mg/1
Fraction
No.
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Organic Carbon
mg/1
1.0
1.0
1.5
3.0
7.5
16.0
23.0
28.0
28.0
27.5
29.0
25.0
20.0
17.0
12.0
10.0
8.0
9.0
9.5
8.5
10.0
9.0
9.5
10.0
11.0
15.0
18.5
20.0
22.0
27.5
31.0
32.0
27.0
Fraction
No.
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Organic Carbon
tng/1
22.0
18.0
20.0
22,0
22.0
20.0
16.0
11.0
8.0
6,0
5.5
6.0
6.5
6.0
5
5
.0
.0
5.5
6.5
6.6
6.7
6.5
6.5
8.0
10.0
11.0
11.5
10.0
9.0
4.0
3.5
1.7
1.5
1.0
203
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Clifton Knolls Effluent Concentrated
By Freeze-Drying
Sample Volume 10 ml
T.O.C. 275 mg/1
Fraction
No.
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Organic Carbon
mg/1
2.0
2.5
3.0
4.0
7.0
24.0
37.0
41.0
35.0
30.0
23.0
22.0
20.0
18.0
16.0
16.0
15.0
14.0
13.0
13.0
12.-5
13.0
,5
5
13.
13.
12.0
13.0
16.0
17.0
25.0
34.0
36.0
43.0
48.0
44.0
35.0
Fraction
No.
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
Organic Carbon
tng/1
22.0
18.0
21.0
22.0
21.5
20.5
16.0
12.0
10.0
9.0
8.0
7.0
6.0
6.0
6.0
6.0
5.0
5.0
6.0
6.5
7.0
9.0
11.0
13.0
11.0
10.5
9.0
7.0
4.0
3.0
2.0
1.5
1.5
1.0
1.5
204
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Lake George Water Concentrated
By Thin Film Evaporation
Sample Volume 5 ml
T.O.C. 296 mg/1
Fraction Organic Carbon
No. mg/1
30 1.5
31 1.5
32 1.5
33 3.0
34 14.5
35 21.0
36 20.0
37 14.0
38 9.0
39 8.0
40 9.0
41 10.5
42 10.0
43 11.0
44 11.5
45 12.0
46 10.5
47 10.0
48 8.5
49 8.0
50 7.5
51 8.0
52 9.5
53 11.0
54 12.0
55 13.0
56 12.0
57 11.5
58 12.5
59 13.0
60 12.0
61 9.5
62 8.0
63 13.0
Fraction
No.
64
65
66
67
68
69
70
71
72
73,
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Organic Carbon
mg/1
24.0
31.0
37.0
33.0
22.0
11.0
5.0
3.5
3.0
3.0
2.5
3.5
2.0
2.0
1.7
2.0
1.8
2.0
1.7
1.3
1.2
1.3
1.4
1.8
2.3
3.3
2.4
2.4
1.8
1.9
1.5
2.1
1.3
205
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Lake George Water Concentrated
By Freeze-Drying
Sample Volume 10 ml
T.O.C. 504 mg/1
Fraction Organic Carbon Fraction Organic Carbon
No. mg/1 No. mg/1
30 1.5 64 56.0
31 1.5 65 70.0
32 3.0 66 65.0
33 11.0 67 61.0
34 31.0 68 39.0
35 37.0 69 18.0
36 33.0 70 9.0
37 23.0 71 6.0
38 16.5 72 5.0
39 14.0 73 4.0
40 16.0 74 3.5
41 18.5 75 3.0
42 19.5 76 3.5
43 20.0 77 2.5
44 20.0 78 2.0
45 20.5 79 2.0
46 21.0 80 2.2
47 19.0 81 2.5
48 16.5 82 2.3
49 15.5 83 2.1
50 16.5 84 2.0
51 15.0 85 2.5
52 15.0 86 2.6
53 21.0 87 3.5
54 22.0 88 3.7
55 24.0 89 4.0
56 21.0 90 3.0
57 24.0 91 3.0
58 30.0 92 2.0
59 30.5 93 2.0
60 25.0 94 1.7
61 20.5 95 1.5
62 20.0 96 1.7
63 33.0
206
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Fraction
No.
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Elnora Raw Sewage Concentrated
By Thin Film Evaporation
Sample Volume 10 ml
T.O.C. 522 mg/1
Organic Carbon
mg/1
2,
3,
2.0
2.0
.0
.0
4.5
6.0
7.0
7.2
6.0
5.5
5.5
5
5
0
0
6.0
6.0
6.5
7.0
8.0
8.0
8.5
9.0
10.5
12.0
15.5
18.0
25.0
33.5
45.0
53.0
57.0
52.5
45.0
39.5
Fraction
No.
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
Organic Carbon
41.0
29.0
19.0
11.0
8.0
7.0
7.5
7.5
8.0
8.5
11.5
9.5
8.5
7.0
7.5
7.0
6.5
5.0
6.5
12.0
21.0
32.0
40.0
43.0
42.0
37.0
20
17
5
5
11.0
7
5
0
0
4.0
4.0
207
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
(Continued)
Fraction
No.
66
67
68
69
70
Organic Carbon
mg/1
32.0
39.0
49.5
55.5
51.5
Fraction
No.
104
105
' 106
107
108
Organic Carbon
mg/1 _
3.0
3.0
2.5
2.0
2.0
208
-------
ORGANIC CARBON OF FRACTIONS FROM SEPHADEX G-15 COLUMN
Elnora Raw Sewage Concentrated
By Freeze-Drying
Sample Volume 10 ml
T.O.C. 230 mg/1
Fraction Organic Carbon Fraction Organic Carbon
No. mg/1 No. mg/1
35 1.0 70 31.5
36 1.0 71 22.5
37 2.0 72 12.0
38 2.5 73 7.0
39 4.7 74 4.0
40 3.5 75 3.0
41 3.0 76 3.0
42 2.5 77 2.5
43 2.0 78 3.0
44 2.0 79 4.0
45 1.5 80 4.5
46 1.5 81 3.5
47 2.0 82 4.5
48 2.0 83 3.5
49 2.5 84 3.0
50 3.0 85 3.0
51 3.5 86 2.0
52 3.5 87 2.0
53 3.5 88 1.5
54 5.0 89 2.0
55 6.0 90 4.0
56 6.0 91 8.0
57 7.0 92 14.0
58 9.0 93 20.0
59 10.0 94 24.0
60 22.0 95 20.0
61 30.0 96 14.0
62 29.5 97 10.0
63 24.0 98 6.0
64 19.0 99 3.0
65 13.0 100 2.0
66 12.0 101 1.0
67 14.0 102 1.0
68 22.0 103 1.0
69 31.0 104 1.0
105 1.0
209
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APPENDIX B
THE EFFECT OF CHEMICAL-PHYSICAL TREATMENT ON
THE SOLUBLE ORGANIC COMPONENT OF WASTEWATER
SECTION I: CONCLUSIONS
The work described in this appendix shows that the soluble
organic components of various municipal wastewaters all
exhibit strikingly similar chromatographic characteristics.
Several other conclusions result from this study.
1. The constituents of the soluble organic component of
municipal wastewater are primarily of low apparent molec-
ular weight (less than 1,500).
2. High pH lime treatment has no dramatic effect on the
apparent molecular weight spectra of the soluble organic
component in municipal wastewater.
3. The minimal changes observed in the apparent molecular
weight spectra of municipal wastewater after high pH lime
treatment can be attributed to some unknown removal mech-
anism or a combination thereof.
4. The soluble organic component of wastewaters after
chemical treatment and activated carbon adsorption is
characterized by a predominance of low apparent molecular
weight constituents.
5. The freeze-drying technique is an excellent method of
concentrating wastewater soluble organics.
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SECTION II: RECOMMENDATIONS
Based on the analysis of the results and experience gained,
the following recommendations are made:
1. Further investigations should be made on the identifica-
tion of the constituents of the soluble organic component
in municipal wastewater.
2. Further investigations should be made on the applicabil-
ity of the gel chromatography technique in designing and
controlling wastewater treatment processes.
3. Further investigations should be made on the specificity
of activated carbon adsorbtion for soluble wastewater organics
in various apparent molecular weight ranges.
4. Investigations of various wastewater treatment schemes
aimed at optimizing removals of soluble wastewater organics
should be made.
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SECTION III: INTRODUCTION, HISTORY AND TECHNICAL REVIEW
Present conventional wastewater treatment processes are
effective in removing particulate organic matter but less
effective in removing soluble organic matter. The need
for higher quality effluents has spurred research directed
toward development of waste treatment processes which are
highly efficient in the removal of soluble organic matter.
Reports (6, 40, 42, 49) to date indicate that chemical
treatment followed by granular activated carbon adsorption
(chemical-physical treatment) is an effective advanced
wastewater treatment process and that the molecular size
of the soluble organic compounds in wastewater may effect
the efficiency of activated carbon adsorption (28, 50, 51).
Molof and Zuckerman (54, 55) contend that molecular size
is the primary factor affecting adsorption and that the
chemical-physical treatment process can be optimized via
high lime (CaOH2) dosages. The resulting high pH allegedly
breaks down large molecular weight organic compounds by
alkaline hydrolysis into their more adsorbable components.
In view of the possible affects of molecular size on adsorp-
tion, a study was undertaken to characterize the soluble
organic component of wastewater by molecular size and to
determine if lime treatment has an effect on that component.
A second objective was to obtain data to characterize the
soluble organic component after activated carbon adsorption.
Gel chromatography has been used in recent studies (15, 25)
to analyze the soluble organic wastewater component. This
analytical technique has been particularly suitable for this
study since it allows the separation or fractionation of a
mixture of water soluble molecules into their approximate
molecular sizes. The method also lends itself to the approx-
imate determination of molecular weights of the fractionated
compounds after appropriate calibration curves have been
developed. Characteristic molecular size spectra or chroma-
tograms can be obtained from samples taken at various points
in a treatment process and compared to determine if certain
molecular sizes are preferentially removed, broken down or
added to the original mixture of solute molecules.
215
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In view of the applicability of gel chromatography it was
decided to use this method and the supporting methods of
freeze-dry concentration and combustion infrared total
organic carbon determination to accomplish the aims of
this study.
The results were obtained in three parts as follows:
Analysis of untreated sewage samples and laboratory
lime treated sewage samples to characterize the soluble
organic component of sewage and to determine if lime
treatment had an effect on it.
Analysis of waste samples from independent chemical-
physical treatment pilot plants to further confirm data
obtained in Part I and to characterize the soluble organic
component after activated carbon adsorption.
Confirmation of the efficacy of the freeze-drying
technique and comparison of the effect, on the soluble
organic component, of sodium hydroxide as opposed to lime.
Definition and Organic Content^ of the Soluble
Component of Domestic Sewage
A knowledge of how the soluble organic component of sewage
is defined and information on its specific organic content
was necessary to insure proper techniques and to interpret
the data obtained.
1. Definition of the Soluble Organic Component
A classification system based on both size and settling char-
acteristics of sewage fractions was presented by Rudolfs and
Gehm (38) in 1939. This classification system consisted of
four fractions as follows:
Settleable Solids
Pseudo Colloidal Sols
True Colloidal Sols
Soluble Solids
The true colloidal fraction was defined as those solids
passing a 0.2 micron filter and retained on a ultra-filter
passing only true solutions.
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Rudolfs and Balmat (37) expanded and revised this earlier
work to reflect specific size ranges and investigated
methods of separating sewage into the various fractions.
The electron microscope was used in determining the size
ranges obtained by the separation methods investigated.
Table 1 summarizes the resulting classification system
and separation methods.
Heukelekian and Balmat (16) separated domestic sewage frac-
tions utilizing the classification system outlined in Table
1. Composite winter and summer samples were obtained from
a single sewage treatment facility. The samples were then
classified and the relative contribution of each fraction
to total solids and organic matter was determined. Hunter
and Heukelekian (17) reported on a similar study involving
the same sewage and procedures used by Heukelekian and
Balmat. The contributions of the various fractions to total
solids, organic matter and nitrogenous matter were presented.
Additionally, Rickert and Hunter (35) modified the separation
method by substituting centrifugation at 50,000 rpm for the
membrane filtration step. The electron microscope was again
used to check the particle size range obtained. The contri-
butions of total solids and organic matter of the fractions
were reported. Painter and Viney (31) separated and charac-
terized the fractions of English domestic sewage with respect
to organic matter and nitrogenous matter. The fractions were
obtained using separation methods comparable with previous
work. A summary of the general characteristics of the sewage
fractions as derived from the literature is presented in
Table 2. Based upon this literature a general similarity in
the contribution of each fraction to total organic and ni-
trogenous matter is indicated.
2. Organic Content of the Soluble Component
Very little data on specific identification of organic groups
or compounds could be located in the literature.
Hunter and Heukelekian (17) reported on the ether soluble
and ether insoluble matter in the soluble component. The
specific compounds identified represented a small part (less
than 10%) of the total soluble organic matter and only alkyl
benzene sulfonate, sugars, and amino acids were significant
contributors.
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00
Table 1. Classification of Sewage Fractions as
Presented by Rudolfs and Balmat
Fraction
Settleable Solids
Supra Colloidal Solids
Colloidal Solids
Size Range
greater than 100 microns
1 to 100 microns
1 micron to 1 millimicron
Separation Method
settling for 1 hour
continuous centrifuging at
14,000 rpm at 100 cc/hr
ultra filtration
Soluble Solids
less than 1 millimicron
ultra filtration
-------
Table 2, Typical Strength Distributions of Organic and Nitrogenous
K5
Matter in Sewage Fractions as Percent of Total Parameter
Heukelekian
Fraction Balmat (16)
Settleable 31.5
Supracol-
loidal 23.5
Colloidal 14
Soluble 31
* % Organic Matter % Nitrogenous Matter
Hunter Painter Hunter Painter
Heukelekian Rickert and Viney Heukelekian and Viney
(17) Hunter (35) (31) (17) (31)
30 24 34 23 23
19 18.5 22 34 20
10 9 15 11 20
41 48.5 29 22 37
* As determined by volatile suspended solids or organic carbon
-------
Painter and Viney (31) and Painter, Viney and Bywaters (32)
reported on a complete study of the soluble component. Of
particular significance to this study is the fact that these
workers used freeze-dry concentration and measured organics
as organic carbon. About 807» of the organic carbon in the
soluble fraction was accounted for. Carbohydrates and or-
ganic acids comprised 5870 of the soluble organic carbon;
amino acids and anionic surface active agents comprised
about 22%, of the soluble organic carbon. The predominant
sugars in solution were found to be glucose and sucrose.
Volatile acids identified were acetic, propionic, butyric
and valeric. Acetic acid was in the greatest concentration.
Twelve or more non-volatile acids were detected at low con-
centration. A summary of the data presented by Painter and
Viney is given in Table 3.
Zuckerman (54) has characterized the soluble organic fractions
of several domestic sewages by chromatographic molecular
weight. This work indicates that as much as 5070 of the sol-
uble organic fraction (as measured by chemical oxygen demand)
is comprised of organic compounds of about 1,200 in molecular
weight.
Chemical Treatment with Lime
Lime has been widely used in the chemical treatment of waste-
water for at least 100 years (33). Interest in lime treat-
ment is increasing in view of the need for advanced waste
treatment methods incorporating removal of phosphorus.
The mechanism of lime treatment traditionally has been attrib-
uted to the precipitation of carbonates from solution (36, 44),
Contradictions to this theory have arisen in the past twenty-
five years. Rudolfs (36) has shown that 95% of the turbidity
in sewage is removed before appreciable calcium is precipi-
tated from solution and that the addition of urine to sewage
acted as a coagulant aid by reducing the lime dosage at which
clarification took place. The effects of urine constituents
indicated strongly that phosphate precipitation was responsi-
ble for the effects observed.
Stones (44) has shown that decarbonated samples of sewage gave
the same degree of purification as the original sewage after
both had been treated with lime. Coagulation by positive
metallic sols was advanced as the possible mechanism of puri-
fication.
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Table 3. Organic Constituents of the Soluble Fraction
as determined by Painter and Viney (31)
Average Percent of Total
Organic Constituent Organic Carbon In Sewage
Carbohydrate 31.3
Volatile Acids 11.3
Non Volatile Acids 15.2
Free Amino Acids 3.1
Bound Amino Acids 7.6
Anionic Surface Active Agents 11.2
Total Organic Carbon Accounted for 79.7
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Bishop (5) has reported on the results of standard jar
tests on various wastewaters using a range of lime dosages.
Unfiltered and filtered (0.45 u membrane filters) super-
natants were analyzed for residual concentrations of total
phosphorus, calcium and magnesium. The results showed that
calcium concentrations increased only gradually with lime
dosage up to a pH of about 10.0 while total phosphorous de-
creased rapidly until precipitation was essentially complete
at a pH of about 10.0. This phenomenon was interpreted as
the precipitation of phosphorous as hydroxyapatite (Ca^OH
(P04).j). The phosphorous in the wastewater was being re-
moved from solution with the calcium being added producing
little net change in calcium concentration.
Mulbarger, Grossman and Dean (29) have summarized the pre-
cipitations occurring during lime clarification. These
reactions in the light of Bishop's report can be summarized
as follows for raw wastewater:
In the pH range up to about 10.0, lime reacts with
hydrolyzed orthophosphate to form insoluble hydroxyapatite.
In the pH range from 10.0 to about 11.0 lime reacts
with the carbonate alkalinity in the wastewater to form
insoluble calcium carbonate.
In the pH range from about 11.0 to 11.5 insoluble
magnesium hydroxide is formed.
Various authors have reported pollutant removals resulting
from lime treatment in terms of several wastewater parameters
including turbidity, biochemical oxygen demand, chemical
oxygen demand, total organic carbon and total phosphorous.
Rudolfs (36) has shown that turbidity decreases up to a pH
of about 9.5, remains constant between pH 9.5 to 10.5 and
then decreases again between pH 10.5 to 11.5. There was no
relation between increased clarification and the reduction
of carbonates, however, phosphates caused a shift downward
of the optinum pH values for clarification. Although not
specifically mentioned, the increased turbidity removals
reported above pH 10.5 may be due to magnesium hydroxide
precipitation (5).
Owen (30) has reported 5-day biochemical oxygen demand
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removals in a raw wastewater of 79% after treatment with
720 mg/1 of slaked lime. Buzzel and Sawyer (9) have re-
ported that lime treatment of raw wastewater can result
in 80% to 90% removal of total phosphorous and 50% to 70%
of the 5-day biochemical oxygen demand.
Williamson, Heit and Caiman (53) have reported chemical
oxygen demand removals in a secondary effluent, filtered
through Whatman No. 5 paper before treatment, of about
45%. Although a pH was not monitored, these data showed
a steady reduction of a chemical oxygen demand up to a
lime dosage of about 400 mg/1 followed by a rapid reduction
between lime dosages of 400 mg/1 to 500 mg/1.
Bishop (5) has reported significant increases in clarifica-
tion and total organic carbon removals in the treatment of
a secondary effluent at lime dosages sufficient to produce
a pH of 11.5. The increased treatment efficiency at the
higher pH level was attributed to the precipitation of
magnesium hydroxide which further clarified the wastewater
by the removal of colloidal material.
Zuckerman (54) has reported that the high pH associated with
massive lime treatment causes a breakdown of high molecular
weight organics into organics of smaller molecular weight.
Gel chromatography and descending paper chromatography were
the primary analytical techniques used in this study.
Samples of raw sewage from various sources were analyzed for
their chemical oxygen demand molecular weight distributions
and it was reported that raw wastewater contains soluble
organic material larger than 1,200 molecular weight and
smaller soluble organics of below 400 molecular weight.
Analysis of the wastewater after treatment with lime showed
evidence that the larger than 1,200 molecular weight material
is altered and that only soluble organic material of low
molecular weight remained. Partial removal by adsorption
onto chemical floe and alkaline hydrolysis of the remainder
into smaller organic molecules were advanced as the causes
of the altering of the larger than 1,200 molecular weight
material.
Sephadex G-15 dextran gel packed in a 2.5 cm by 45 cm chro-
matographic column was used extensively in the chemical
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treatment studies. The samples were filtered through a
0.45 membrane filter and 2 ml samples were then passed
through the gel using a phosphate buffered eluent. A
continuous low level chemical oxygen demand procedure was
used to monitor the chromatographic eluent. The procedure
used is reported to be capable of automated chemical oxygen
demand analysis in the 0 to 6 mg/1 range (54). Alkaline
hydrolysis was further studied using descending paper chro-
matography and pure compounds. Organic solutions of 40 gm/1
cellulose, 40 gm/1 starch and 5 gm/1 bovine serum albumin
were subjected to pH levels of 9.0, 10.0 and 11.0 using
calcium hydroxide and sodium hydroxide independently.
Contact times of 50 minutes at the adjusted pH were used.
The samples were then neutralized with 0.1 N sulfuric acid.
Twenty-five micro-liter samples were then analyzed by de-
scending paper chromatography. The results reported indi-
cated that all samples adjusted to a pH of 9.0, 10.0 and
11.0 except the starch sample at 9.0, had undergone hydrol-
ysis.
Several workers have suggested other mechanisms which may be
involved in organic pollutant removals during lime treatment
other than precipitation and coagulation.
Sorption has been suggested by both Rudolfs (36) and Zuckerman
(54) as a possible contributing removal mechanism. A recent
report by Brunner and Sproul (7) on virus inactivation during
phosphate precipitation lends support to the possible signif-
icance of sorption in the removal process.
Stumm and Morgan (45) have pointed out that many naturally
occurring colloidal and soluble impurities contain ionizable
functional groups that are known to form complexes with poly-
valent metal ions. Many such groups would carry negative
charges in the pH ranges encountered during lime treatment.
This suggests the possibility of chemical interactions be-
tween calcium ions and organics to promote the formation of
insoluble precipitates. LaMere and Smellie (20) have dem-
onstrated this concept by flocculating potato starch, which
contains phosphoryl monoester, with cations such as Ca++.
Removals of organic pollutants, during lime treatment, are
reported to be closely related to the precipitation of phos-
phorous and magnesium hydroxide (5, 36). Calcium carbonate
precipitation appears to play a lesser role (5, 36). Removals
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of both soluble and colloidal impurities may be accomplished
to a lesser extent through sorption and complexing (5, 20,
45, 54). Increased clarification appears to occur at lime
dosages sufficient to produce a pH of about 11.5 (5, 36).
Adsorption of Soluble Organic with Activated Carbon
A review of the literature to ascertain how activated carbon
adsorption takes place, what effect molecular size has and
what types of organics would be considered refractory and
thence appear in adsorber effluents was undertaken to aid
in interpretation of carbon column effluent data.
1. Structure of Carbon and Kinetics of Adsorption
Activated carbon's adsorption properties can be attributed
to its highly porous structure (24, 43). Each activated
carbon particle contains a vast network of variable size
pores. This great porosity provides a very large surface
area for adsorbing organic molecules. A relatively small
part of the surface area comprises the outside of the acti-
vated carbon particles.
Consideration of this structure indicates that the interior
surface of the activated carbon must be used for effective
adsorption and that adsorption of a solute molecule takes
place in three consecutive steps (50). If the incidental
adsorption on the exterior surface is neglected these steps
can be summarized as follows:
Transport of the adsorbate to the exterior surface of
the activated carbon.
Diffusion of the adsorbate into the interior surface area.
Adsorption of the adsorbate onto the interior surface area,
Observations of adsorption rate and effects of temperature, mo-
lecular size and configuration on adsorption rate indicate that
the rate controlling step in rapidly mixed non-flow systems
is diffusion of the adsorbate into the interior pores (28, 50).
Reports on continuous flow columnar systems suggest that ex-
ternal transport mechanisms are rate controlling (19, 52).
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2. Effect of Nature, Molecular Size and Configuration of
Solute on Adsorption
Weber and Morris (28, 50, 51) have reported on the effect
of molecular size and configuration of alkylbenzenesulfonates
on adsorption rate and capacity. The rate of adsorption for
a homologous series of ABS molecules was found to decrease
with increasing molecular weight when the amount adsorbed
per unit time was expressed in molar units. This effect
was much less pronounced when adsorption rate was expressed
in weight units.
Further experiments with alkylbenzenesulfonates (ABS) of the
same molecular weight but with different alkyl chain structure
indicated that those molecules having structures which permit-
ted coiling or compactness were adsorbed at a greater rate.
Investigations into the effect of molecular size on adsorption
capacity indicated that high molecular weight materials are
adsorbed to a greater extent than materials of lower molecular
weight. As stated in a previous section, the rate limiting
step in the rapidly mixed non-flow system used by Morris and
Weber was indicated to be interparticle diffusion. The de-
crease adsorption rate with increasing molecular size can then
be attributed to decreasing interparticle diffusion rates with
increasing molecule size of the ABS homologs. A more compact
molecule would adsorb faster in such a system for the same
reason. The increase in adsorption capacity with increasing
size can be attributed to the increased energy of adsorption
or partition factor of higher molecular weight compounds.
Weber and Keinath (19, 52) have reported that molecular size
is much less important to adsorption kinetics in fluidized
adsorbers in which external transport mechanisms are rate
limiting. The column adsorption systems reported on exhibited
a generally higher mass transfer rate with increasing molecular
size of the adsorbate.
The nature of the adsorbate must be taken into account when
considering adsorptive tendency. Weber and Morris1 report
(28, 50) makes it clear that organic compounds of much higher
molecular weight than the ABS series previously mentioned
adsorb much faster even in a rapidly mixed system.
3. Organics Resistant to Activated Carbon Adsorption
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There do not appear to be any conclusive reports available
on the identity of the trace organics remaining in waste-
water effluents after activated carbon adsorption, although
attempts have been made to identify these compounds (27).
Bishop et al (6) have pointed out that activated carbon
does a poor job of removing colloidal organics. Strongly
hydrophilic low molecular weight organics have also been
suggested as an adsorption resistant group (6, 27).
Among the specific soluble organic compounds which might
be expected in activated carbon adsorber effluents are
sugars, amino acids, urea, low molecular weight glycols,
hydroxy acids, sulfates and sulfonates (6, 27).
A review of the literature indicates that many factors can
effect adsorption on organic carbon. Large colloidal organics
which are limited by size in their ability to enter the
activated carbon pore structure and low molecular weight
hydrophilic compounds which tend to stay in the aqueous phase
would be expected to be resistant to adsorption and may be
present in activated carbon adsorber effluents.
The Hydrolysis-Adsorption Process
Zuckerman and Molof (54, 55) have proposed a chemical-physical
treatment process which is similar to those reported by others
(5, 40, 42, 49). This process would consist of the following
steps (55).
Solids separation by chemical coagulation with lime
plus elevation of pH to 11.5.
Reaction in a chamber to allow hydrolysis to occur.
Neutralization with acid or by recarbonation with COo-
Sand filtration
Activated carbon adsorption.
The reported effect of the high pH on molecules of large
molecular weight (hydrolysis) was mentioned in previous
sections. The hydrolysis step is the basic difference
between processes which have been used previously (5, 40,
42, 49). The alleged need for such a step is based on gel
227
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chromatography studies of raw wastewater, secondary bio-
logical effluents and activated carbon adsorption effluents
as well as on the interpretation by Zuckerman and Molof of
data on the effect of molecular size on adsorption for a
homologous series of alkylbenzenesulfonates presented by
Weber and Morris (50) for a rapidly mixed non-flow adsorp-
tion system.
A summary of the major conclusions of Zuckerman and Molof
(54, 55) reports are as follows:
About 2570 of the organic material, passing a 0.45 micron
filter, in raw wastewater is colloidal in nature.
The molecular weight distribution of the soluble organics
in raw wastewater, as determined by filtering through a 0.45
micron filter and gel chromatography analysis of the filtrate,
consists primarily of compounds of above 1,200 and below 400
in molecular weight.
The molecular weight distribution of the soluble organics
in biological activated sludge effluents, as determined by
filtering through a 0.45 micron filter and gel chromatography
analysis of the filtrate, consists largely of compounds above
1,200 in molecular weight indicating that biological treatment
preferentially removes low molecular weight organics.
Activated carbon adsorption processes are limited by inter-
particle diffusion causing the preferential removal of low
molecular weight compounds.
Lime clarification removes some of the high molecular
weight material in raw wastewater by adsorption onto chemical
floe and the remainder are broken down by alkaline hydrolysis
to more adsorbable compounds of below 400 in molecular weight.
The increased concentration of organics of below 400 in
molecular weight, after the hydrolysis step, causes an in-
creased partition factor between the liquid phase and the
solid adsorbent which results in increased activated carbon
adsorption capacity.
Weber (48) has commented in depth on the interpretations of
previous literature on adsorption rate and capacity and on
the experimental procedures reported by Zuckerman and Molof
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(54, 55). These comments were based to a large extent on
experimental data presented by Weber and co-workers in re-
ports which have been cited in the preceding section (28,
50, 51). A summary of the comments presented and literature
referenced are as follows:
Adsorption from solution is often enhanced by high mo-
lecular weight material (19, 50, 51, 52).
While it has been demonstrated that interparticle
diffusion is rate limiting in rapidly agitated batch adsorp-
tion systems, it has also been shown that external transport
mechanisms govern the column type adsorption systems general-
ly used in wastewater treatment (19, 50, 52). If molecular
weight has an effect on adsorption it would be expected to
be most apparent in the batch type systems and not in the
columnar type systems used in wastewater treatment.
The report by Weber and Morris (50), which included
data on the effect of molecular size and adsorption for a
homologous series of alkylbenzenesulphonates, clearly stated
that the detrimental effect on adsorption rate with increas-
ing molecular size was much less evident when the data was
expressed in weight and not molar units.
The approximate 2570 fraction of colloidal material in
the filtered samples used for gel chromatography analysis
could have significantly affected the gel chromatography
results.
Increases in activated carbon effluent quality, which
are attributed to a hydrolysis effect, could be associated
with increased removal of colloidal organic materials by
coagulation (5, 6).
The previously cited commentary (48) also contained the results
of experiments to determine if hydrolysis of organic matter
can be expected at pH 11.5 under otherwise ambient conditions
and if a hydrolysis effect on activated carbon adsorption
occurs. The activated carbon systems were rapidly mixed batch
systems which should show the hydrolysis effect most readily.
The results indicated that minor hydrolysis of proteins oc-
curred at a pH of above 10 after several hours of contact. The
results of coagulation experiments using sodium hydroxide,
calcium hydroxide, and ferric chloride indicated that calcium
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hydroxide clarification at pH 11.5 and ferric chloride
clarification at pH 6.7 produced effluents which were
essentially the same in quality and that sodium hydroxide
produced an effluent of much poorer quality after batch
activated carbon adsorption. The organic removals attrib-
utable to the clarification process were much higher when
calcium hydroxide or ferric chloride was used. The conclus-
ion drawn from these data was that effective coagulation
under less severe conditions produced the same effect on
adsorption as coagulation at high pH.
There does not appear to be any comparable data on the mo-
lecular weight distribution of raw filtered wastewater,
however, some data are available on the proportion of high
molecular weight materials in secondary biological effluents.
McDonald (25), using gel chromatography, has indicated that
70% to 80% of the soluble organic material in activated
sludge effluent is of below 600 in molecular weight. Bunch,
Earth and Ettinger (8), using semi-permeable membranes, have
attributed about 6070 of the organic material in various bio-
logical effluents to low molecular weight organics. These
two reports appear to be in disagreement with data presented
by Zuckerman and Molof (54, 55) indicating that activated
sludge effluents contain material primarily of above 1,200
in molecular weight.
1. Pilot Plant Studies of the Hydrolysis-Adsorption process
A pilot plant study of the hydrolysis-adsorption process has
been carried out by Ecolotech Research, Incorporated under
the sponsorship of the New York State Health Department (56) .
The pilot plant had a 10,000 gallon per day capacity and was
operated for a three month period at New Rochelle, New York
starting in September 1969. The process was used to treat
New Rochelle raw wastewater.
The pilot plant units consisted of an upflow solids separator
designed for a surface settling rate of 560 gallons per day
per square foot, a hydrolysis chamber designed for a deten-
tion time of about eighty minutes, a neutralization tank
utilizing sulfuric acid, a sand filter designed for a surface
loading of 3.2 gallons per minute per square foot and five
downflow adsorption columns, in series, totaling 20 feet of
activated carbon. The activated carbon columns were loaded
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to produce a superficial detention time of about 60 minutes.
The process sequence of unit operations was described in the
preceding section.
During the period of operation, the plant achieved total
average COD removals of 947, and soluble COD removals of
84%. Clarification pH was maintained above 10.5 except
during plant upsets. Drops in pH were accompanied by poorer
effluent quality which has been attributed by Ecolotech
workers to an incomplete breakdown of high molecular weight
materials. It should be pointed out again, however, that
when lime clarification is used, the efficiency of the clar-
ification process and the activated carbon column effluent
is related somewhat to pH and that turbidity which is not
removed by the clarification and filtration process would
not be expected to be removed efficiently by the activated
carbon adsorption process (5, 6) due to the inability of
colloidal sized particles to enter the pore network of the
activated carbon.
Chromatograms (56) obtained by direct measurement of both
the New Rochelle raw wastewater and the chemically treated
wastewater indicate to this writer that the majority of the
organic material in the raw wastewater was of less than
1,200 molecular weight and that there was no apparent in-
crease in concentration of the low molecular weight compo-
nents. The chromatography data obtained in New Rochelle
do not appear to reflect previous findings (54, 55) com-
pletely.
Gel Permeation Chromatography
Gel permeation chromatography is a method of separating or
fractionating a mixture of solutes by the size of the re-
spective solute molecules. The use of gel permeation
chromatography is receiving considerable attention in the
environmental science field as an analytical tool for
studying the soluble organic compounds in water and waste-
water. The description of these chromatographic processes
has been discussed at length earlier in this report.
1. Applications of Sephadex Gels in the Water and
Wastewater Field
Several workers have utilized Sephadex gels in the water
and wastewater field.
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McDonald (25) has used Sephadex G-10 through G-50 to frac-
tionate the organic compounds in an activated sludge efflu-
ent. About 30% of the organic carbon in the effluent was
attributed to organics with a molecular weight above 600.
Hardt (15) used Sephadex G-10 and other gels to demonstrate
a build-up of complex organics in a high solids biological
reactor operating on a glucose substrate. Zuckerman and
Molof (54, 55), as mentioned previously, have used Sephadex
G-15 extensively to demonstrate changes in soluble organics
after chemical-physical treatment. Smith (41) used Sephadex
G-25 to fractionate concentrated kraft and sulphite process
pulping wastes before and after coagulation with iron and
aluminum coagulants. The predominant effect of the coag-
ulants was the removal of high molecular weight organics
from the wastes. Collins, Webb, Didwani and Lueck (10)
used Sephadex G-50 to fractionate the organic components
of wood pulp bleach effluents. Gjessing and Lee (14)
fractionated concentrated samples of lake and stream water
using various Sephadex gels. Solutes with apparent molecular
weights from less than 700 to more than 200,000 were detected.
Gel permeation chromatography has been used with success to
separate soluble organic compounds from complex wastewater
mixtures. The molecular weights determined for such unknown
mixtures of organics must be considered as approximate or
apparent due to differences in molecular structure between
organic groups and due to the possibility of non-steric inter-
actions between the gel or solvent phases with some organic
compounds. However, the results of gel permeation chromatog-
raphy analysis should be very useful in determining the
character and changes in character of soluble wastewater
organics before and after treatment.
Concentration by Freeze-Drying
In investigations involving organic solutes whose chemical
and physical properties are poorly defined or where analysis
requires the measurement of very low concentrations, solute
concentration is often desirable. In portions of this study
a concentration step was deemed necessary in the preparation
of samples for gel permeation chromatography in order to
avoid measuring very low organics concentrated wastewater
filtrates.
232
-------
1. Alternative Concentration Methods, (Thin Film Evaporation
and Freeze-drying)
Thin film evaporation and freeze-drying (lyophilization) were
possible concentration methods which have been used in the
past to facilitate the study of low concentration soluble
organics in water and wastewater (17, 31, 41). These pro-
cedures and their applicabilities have been presented within
Appendix A and elsewhere.
Combustion-Infrared Method for Total Carbon Content
The use of gel permeation chromatography for the separation
of organic fractions requires a detector specific for orgari-
ics. The detection parameter used must facilitate rapid and
accurate determinations since many fractions must be analyzed
in each column run. An instrument for the rapid determination
of carbon would fulfill the need for such a detector.
1. Description of Method
A method for the rapid determination of total carbon has been
developed within the last decade (39, 47). The method in-
volves the rapid combustion of an aqueous sample in a stream
of oxygen passing through a heated combustion tube, followed
by measurement of the carbon dioxide produced. The carbon
dioxide measurement is made on the gas stream with an infrared
analyzer specifically sensitized for carbon dioxide. Several
commercial instruments are now available from various manu-
facturers. A schematic of a carbonaceous analyzer is shown
in Figure 1.
2. Function of the System Components
The functions of the components shown in Figure 1 are describ-
ed as follows (15, 47).
Combustion of sample. The sample is injected through the
sample port utilizing a nicrosyringe capable of measuring
small volumes. A continuous pure oxygen purge passes the
sample at a controlled rate into a heat resistant glass com-
bustion tube which is maintained at a temperature of 950 C.
A loosely packed plug of asbestos, impregnated with a cobalt
catalyst, located at the condenser end of the combustion tube
intercepts the vaporized sample. The carbon containing sub-
stances in the sample are completely oxidized to carbon
233
-------
NEEDLE
VALVE
CHECK
VA1VE
PRESSURE
REGULATOR
U>
OXYGEN
SOURCE
(02 FLOW RATE
MAINTAINED AT
IOO-I5O cc/Min.
AT Spsig)
COMBUSTION TUBE
MAINTAINED AT 95O* C
SAMPLE
INJECTION
PORT
ATMOSPHERIC
VENT
RECORDER
AMPLIFER/
CONTROL
CONDENSOR
CONDENSOR
OUTLET
t
INFRARED
ANALYSER
4_
]HOKE
MICRON
FILTER
Fig. 1 SCHEMATIC OF
INFRARED
CARBONACEOUS ANALYZER
-------
dioxide and water vapor at this point. The resulting gases
pass out of the combustion tube and through a condenser where
water vapor is removed.
Infrared analyzer. The carbon dioxide gas from the oxidation
of the sample is passed through a filter and into the anal-
yzer section of the instrument. The infrared analyzer sec-
tion consists of two distinct components or sides. One side
acts as a reference while the other side analyzes the sample
stream. Each side consists of an infrared energy source
which is blocked ten times per second by a two-segmented
chopper, an infrared energy absorption cell and a detector
cell. The adsorption cell on the reference side is filled
with nitrogen gas which absorbs little of the infrared energy
emitted by the reference source. The absorption cell on the
sample side is of the flow through type and is placed directly
in the sample train. The detector cells are filled with low
pressure carbon dioxide gas and are separated by a flexible
metal diaphragm.
Energy transformation. When a sample is being analyzed a
portion of the infrared energy emitted by the sample source
is absorbed by the carbon dioxide from the sample combustion
as it passes through the sample cell. Absorption of infrared
energy in the reference cell is negligible. The infrared
energy transmitted out of each cell is passed into the dia-
phragm separated detector cells by means of infrared trans-
mitting windows. The energy entering each detector heats
the carbon dioxide gas in the detector and causes a corre-
sponding increase in pressure. The gas in the reference
detector is heated more since there is a negligible absorp-
tion of infrared energy by the nitrogen gas in the reference
absorption cell. The pressure differential between the two
detector cells cause the separating diaphragm to flex in
relation to the energy increment between the two sides. The
pressure difference varies with the amount of carbon dioxide
in the sample stream and is translated electronically into
a direct read out of total carbon.
3. Determination of Organic Carbon
The results from a carbonaceous analyzer include both organic
carbon and any inorganic carbon in the sample unless the latter
is eliminated. Van Hall et al (46, 47) found that adjustment
of the sample pH to between 2 and 3, followed by purging with
235
-------
nitrogen gas for three to five minutes at a gas flow rate
of 300 to 400 ml per minute serves to eliminate inorganic
carbonates in the sample. The resulting reading reflects
total organic carbon only. The nitrogen purge method was
tested on various aqueous organic solutions including sugars,
urea, amino acids, phenols, proteins, starches, lignins and
cellulose as well as on various wastewaters (39, 47). No
loss of organic acids, methanol, or phenol occurs but com-
pounds that are both volatile and slightly soluble in water,
such as light hydrocarbons, are swept out of the sample.
Hardt (15) has confirmed the general method of Van Hall
et al (47) using prepared solutions of sodium carbonate. A
nitrogen flow rate of 500 ml per minute for five minutes,
after acidification of the samples, was used during the
purging procedure.
4. Applicability of the Carbonaceous Analyzer to Waste-
water Analysis
Schaffer et al (39) have reported on the application of the
carbon analyzer in wastewater analysis. The results of
analyses by the infrared method were compared with carbon
analyses by a wet-oxidation method. The suitability,
accuracy, and precision of the infrared method were verified.
Three recent studies, utilizing gel permeation chromatography
for soluble wastewater organics, have also indicated the
applicability of organic carbon by the infrared method, as
a detection parameter (15, 25, 41).
Organic carbon determinations by the combustion infrared
method have been shown to be accurate and applicable to the
analysis of wastewater organics. The usefulness of this
method for analyzing gel permeation chromatography eluents
has been recognized by several workers (13, 15, 25, 41).
236
-------
SECTION IV: PROCEDURES
Operational Procedures
1. Sample Preservation
Preliminary studies indicated that a reduction in organic
material occurred unless wastewater samples were preserved.
Refrigeration at 4°C or chemical preservation with mercuric
chloride have been used in past studies (17, 44) and were
found to be acceptable for this study.
All waste samples were routinely kept in the 4°C cold room
after arrival in the laboratory and all analyses were com-
pleted as soon as possible. In addition, all samples col-
lected with the exception of the Eastern-Western sewage
sample of June 30, 1969, were preserved, at the time of
collection, by adding 1 ml of a 40 g/1 mercuric chloride
solution to each liter of sample. The aforementioned
exception was packed in ice immediately in lieu of chemical
preservation.
All samples obtained from the New York State Health Depart-
ment, Research Unit, had been kept under refrigeration
continuously after collection with the exception of the
New Rochelle samples of October 19, 1969 which underwent
an approximate 5 hour transit time from New Rochelle to
Albany, New York, without preservation. These samples were
refrigerated upon arrival in Albany.
2. Definition of the Soluble Organic Fraction
The soluble organic fraction is defined in this study as that
fraction of wastewater organics which pass a 0.45 micron pore
size membrane filter (54, 55). Organic material, in all
cases, was measured as organic carbon. The organic carbon
content of a sample after membrane filtration is termed solu-
ble organic carbon in the text from this point on.
3. Jar Tests
One and one-half liter aliquots of selected waste samples
were chemically treated in the laboratory with either sodium
hydroxide (NaOH) or calcium hydroxide (Ca(OH)2). The se-
quential procedure used in the jar tests was as follows:
237
-------
Determination of initial pH and initial soluble organic
carbon.
Flash mix at 100 rpm while chemicals were added by
pipette, from stock solutions, over a one minute period.
Equilibration for one minute and determination of pH.
Reduction of paddle speed to 20 rpm and flocculation for
a 20 minute period.
Stop paddle and settle for a 30 minute period.
Membrane filter supernatant, neutralize filtrate with
sulphuric acid (112804) to a pH between 7.0 and 7.6 and
determine soluble organic carbon content of neutralized
filtrate.
Chemical dosages were computed from the volume and concen-
tration of chemical added to the 1.5 liter aliquot of waste.
4. Sample Concentration by Freeze-Drying
All samples to be concentrated were prepared by membrane
filtering (0.45 micron) and neutralization to a pH of 7.0
and 7.6 (if this had not been done previously). The se-
quential procedure used was as follows:
Shell freeze a 500 ml aliquot of the sample in an
acetone and dry ice mixture.
Fill freeze-dryer condenser with acetone and dry ice
and evacuate the system to a vacuum of 20 microns of mercury.
Place sample bottle on freeze-dryer port and open three
way valve to vacuum.
Freeze-dry sample for about 24 hours.
Remove sample bottle and add additional 250 ml to 400
ml aliquots of sample as necessary.
Refreeze and concentrate further.
Melt finished sample and membrane filter (0.45 micron)
liquid concentrates.
238
-------
Place filtered concentrates in tightly capped test
tubes and store at 4°C for future use.
Subsequent to the completion of the above procedure, recovery
of soluble organic carbon was calculated. Organic carbon
concentration in the filtered concentrate was measured and
combined with the volume of the concentrate to provide the
total amount of organic carbon in such concentrate. These
data, combined with the amount of organic carbon in the
original sample, allowed for the calculation of the recovery
factor.
Analytical Procedures
1. Organic Carbon Determination
Organic carbon determinations were made using either the
Beckman or the modified carbonaceous analyzer both of which
have been described previously.
The Beckman instrument was calibrated with 20 micro-liter
samples to produce a full scale deflection of the recorder
at 100 tng/1 of organic carbon.
The modified instrument was calibrated with 50 micro-liter
samples of oxalic acid solutions to produce a full scale
deflection of 10 mg/1 of organic carbon on low range and
with 40 micro-liter samples to produce a full scale de-
flection of 100 mg/1 of organic carbon on high range.
A Hamilton Model SK 148, 50 micro-liter graduated syringe
was used to inject all samples.
The calibrations of the Beckman instrument and the low range
of the modified instrument were linear. The calibration of
the modified instrument on high range was not quite linear
and organic carbon was determined from a calibration curve
of standard organic carbon versus recorder reading.
Inorganic carbon was eliminated by acidification of a 5 ml
aliquot of sample of pH 2.0 with 2N hydrochloric acid (HCl)
and purging for five minutes with nitrogen gas at 500 cc/min.
This procedure follows that recommended in the literature
(15, 46) and is termed the conventional method from this
point on. In instances where a sample contained more than
239
-------
100 mg/1 of organic carbon an appropriate dilution was made
with double distilled water.
2. Gel Permeation chromatography
Organic solutes in the soluble organic fraction of waste-
water concentrates were separated (fractionated) by molec-
ular size using gel permeation chromatography (see Figures
2 and 3).
The procedure used was as follows:
Preparation of Gel columns. The Sephadex G-15 gel used was
completely swollen in an excess of phosphate buffered eluent
for a minimum of 48 hours. The column was then packed to
a gel bed height of 91 cm for both upflow and downflow
modifications. The procedure used to pack the chromatograph
column was as follows:
The column, with an upflow adaptor installed in the bot
torn, was mounted vertically in the cold room on a metal frara
A funnel fitted with a rubber stopper was placed in the
top of the column and the column was completely filled with
phosphate buffered eluent.
The swollen gel suspension was placed in the funnel in
one pour and stirred constantly with a glass rod as the gel
particles settled to the bottom of the column.
The lower outlet of the column was opened to permit
eluent flow after a few centimeters of the gel bed has built
up in the bottom of the column.
When the gel bed was completely formed to a height of
91 cm, it was washed with several bed volumes of eluent,
using about the same flow rate that would be used for sample
elution (40 ml/hr).
The column was then capped for downflow or upflow opera
tion by either placing a nylon sample net over the top surfa
of the gel and screwing on a plastic cap connected to the
eluent reservoir (downflow) or by connecting an upflow adapt
to the eluent reservoir, installing it in the top of the
column and inverting the column so that the eluent entered
240
-------
10-ml
SAMPLE PIPET
o
o
0
ELUANT
THREE WAY VALVE
SAMPLE
ELUANT
RESERVOIR
EFFECTIVE
PRESSURE
DIFFERENCE
5 ml SIPHON
FRACTION
COLLECTOR
EFFLUENT
INFLUENT
UP-FLOW ADAPTER
2.5 x 100 cm
CHROMATOGRAPHY
COLUMN
GEL
Fig. 2 SCHEMATIC OF UPFLOW GEL COLUMN
241
-------
• •
e
EUJANT
=*=
ELUANT
RESERVOIR
EFFECTIVE
PRESSURE
DIFFERENCE
Li
5ml SIPHON
i
FRACTION
COLLECTOR
CAP
JO.
J
NYLON SAMPLE NE
2.5 x 100 cm
CHROMATOGRAPHY
COLUMN
GEL
UPFLOW
ADAPTER
EFFLUENT
Fig. 3 SCHEMATIC OF DOWNFLOW GEL COLUMN
242
-------
the column from the bottom (upflow).
The elevation of the eluent reservoir was adjusted to
produce a flow rate into the fraction collector of 40 ml/hr.
Standardization. The packed columns were standardized for
apparent molecular weight (a.m.w.) determinations by eluting
2 ml to 3 ml volumes of the standards and collecting each
fractionated standard in 5 ml aliquots with the fraction
collector.
Each fraction was analyzed for organic carbon or conductivity
in the case of standard No. 3. The fractions at which the
solutes in the standards were eluted in greatest concentra-
tion were taken as their elution fraction (11). The logarithm
of the molecular weight of the respective solute was then
plotted against its elution fraction to develop a standard
curve for apparent molecular weight determinations.
Sample application. Samples were applied either directly to
the top of the gel bed by pipette in the case of the downflow
column or automatically from a 10 ml sample pipette in the
case of the upflow column. The direct application procedure
required removal of all the eluent from the top of the gel
bed, applying and drawing the sample into the gel, two
successive, 5 ml applications of eluent and then refilling
the top of the column and continuously eluting the sample
via the eluent in the reservoir.
Fractionation and examination of waste samples. Samples of
filtered wastewaters and wastewater concentrates were placed
on the columns in volumes ranging from about 170 to 2% of the
gel bed volume (5 ml to 10 ml). Fractions equalling about
1157o of the gel bed volume (105-five ml fractions) were then
collected and organic carbon determinations were made on each
fraction. The conventional organic carbon determination pro-
cedure was used. No organic carbon appeared in any of the
fractions before fraction 35 in any of the samples analyzed.
Chromatograms were obtained for each sample fractionated by
plotting fraction number versus organic carbon and connecting
the plotted points with a smooth curve. The appearance of
definite peaks in the chromatogram signified the presence of
organic solutes, of approximately the same apparent molecular
size, in relatively high total concentration. The presence
243
-------
of a peak when fractionating a mixture of unknown organic
solutes does not necessarily signify the presence of a
single compound as was the case with standard elutions.
A lack of definite peaks signified the presence of organic
solutes over a range of molecular sizes in insufficient
concentration to produce distinct peaks.
The apparent molecular weight of a peak was determined from
the standard curve for the column and represents the approx-
imate average apparent molecular weight of the organic con-
tained in the peak. Molecular weight determinations are
termed apparent because unknown organic mixtures were being
analyzed and because of the references to non-steric behavior
cited previously (11, 23).
When it was desired to compare samples treated to different
degrees, the concentrates being compared were either diluted
to contain the same organic carbon content or the column
sample volumes were adjusted to produce the same result.
Chromatograms obtained in this manner could be compared
directly to determine relative changes in peaks.
Calculation of percentages of organic carbon in peaks was
made by computing the percentage of organic carbon recovered
in the respective peak according to the following relationshi
C° x N° X 100 = %
Ci x N!
where:
GO = Average Organic Carbon in Peak's Fractions (mg/1)
G! = Average Organic Carbon in all Fractions (mg/1)
NO = Number of Fractions in Peak
N! ~ Number of Fractions Analyzed
The percentage obtained was then multiplied by the soluble
organic carbon in the sample before concentration to obtain
the equivalent amount of soluble organic carbon in the
original sample. It will be shown that no significant
changes in the organics occurred during the concentration
process making the results of this calculation valid.
The recovery of organic carbon from the gel column was cal-
culated according to the following relation:
244
-------
Total Vol Eluted (ml) X Average Organic Carbon (mg/1) x ^00=7
Sample Vol (ml) X Sample Organic Carbon (mg/1)
Experimental Procedures
Actual methodologies were conducted for this aspect of these
investigations in four distinct experimental phases. These
phases are described below.
The preparation and standardization of the G-15 upflow gel
column was done according to McDonald (25). Moreover, a
considerable amount of preliminary experimentation with
various buffer systems and Sephadex gels, with various
fractionation ranges, occurred in order to better understand
the intricacies involved in this particular phase of the
study.
Laboratory chemical treatment studies were done to obtain
data on the molecular size distribution of the soluble or-
ganics in untreated wastewater and to determine if chemical
treatment with calcium hydroxide caused significant changes
in molecular size distributions.
The upflow column, freeze-dryer, Becktnan carbonaceous anal-
yzer and jar test procedure were used throughout. The
sequence of operational and analytical procedures followed
is shown in Figure 4.
The primary objective of the treatment studies via chemical-
physical means was to obtain data on the molecular size dis-
tributions of chemical-physical effluents. The upflow column,
freeze-dryer and Beckman carbonaceous analyzer were again used.
All samples analyzed were supplied by the New York State Health
Department, Research Unit. The sequence of operation and an-
alytical procedures used is shown in Figure 5.
Analysis of Unconcentrated Samples and Validation of Freeze-
Drying
The analysis of unconcentrated samples and validation of
freeze-drying done to obtain further data on the effects of
pH on molecular size distribution in sewage and to validate
the freeze-drying procedure. Both freeze-dryed and uncon-
centrated samples were analyzed. The gel column was modified
245
-------
COLLECT GRAB SAMPLE
OF RAW WASTEWATER
JAR TEST
CHEMICALLY TREAT CONTROL
WITH Ca(OH)2 NO CHEMICALS
<= —J
MEMBRANE FILTER
SUPERNATANT5
CONCENTRATE FILTRATES
BY FREEZE DRYING
t
MEMBRANE FILTER
CONCENTRATES
t
FRACTIONATE CONCENTRATES
ON SEPHAOEX 6-15
t
ANALYZE FRACTIONS AND
PLOT CHROMATOGRAMS
Fig' 4 EXPERIMENTAL PROCEDURE USED DURING
LABORATORY CHEMICAL TREATMENT STUDIES.
246
-------
OBTAN TREATED AND UNTREATED
SAMPLES FROM N.Y. s. HEALTH DEPARTMENT
MEMBRANE FILTER SAMPLES
CONCENTRATE SAMPLES BY
FREEZE DRYING
MEMBRANE FILTER CONCENTRATES
FRACTIONATE CONCENTRATES
ON SEPHADEX G-fi
ANALYZE FRACTIONS AND
PLOT CHROMATOGRAMS
Fig. 5 EXPERIMENTAL PROCEDURE USED DURING
CHEMICAL - PHYSICAL TREATMENT STUDIES
247
-------
for downflow operation at the start of this phase and the
modified carbonaceous analyzer (high and low range as
applicable) was used to analyze fractions. Only the Elnora
sewage sample was analyzed. The sequence of operational
and analytical procedures followed is shown in Figure 6.
248
-------
COLLECT GRAB
SAMPLE OF CLIFTON KNOLLS
SEWAGE
CHEMICALLY
TREAT WITH
NoOH
t
CHEMICALLY
TREAT WITH
Ca(OH)g
CONTROL
NO CHEMICALS
V
MEMBRANE
FILTER
SU PERN ATA NTS
UNCONCENTRATED ALIQUOTS
OF CONTROL, Co(OH)2 AND
No OH TREATED
CONCENTRATE PORTION
OF CONTROL -AND
Ca(OH)2
FRACTIONATE
SAMPLES
ANALYZE FRACTIONS
AND PLOT CHROMATOQRAMS
Fig. 6 EXPERIMENTAL PROCEDURES USED DURING
ANALYSIS OF UNCONCENTRATED SAMPLES AND
VALIDATION OF FREEZE DRYING.
249
-------
SECTION V: RESULTS AND DATA ANALYSIS
1. Gel Column Standardization
Several runs were made with standards for both the upflow
and downflow column modifications. The results were iden-
tical for both types. The elution characteristics of each
standard are tabulated in Table 4. The fraction in which
the standard appeared in maximum concentration was taken
as its elution fraction (11).
The standard curve developed from these data is shown in
Figure 7. Inspection of this figure reveals that the
standards were eluted in the order of decreasing molecular
weight and that the relationship of elution fraction versus
the logarithm of molecular weight was approximately linear.
Figure 7 was subsequently used to determine the apparent
molecular weights of the unknown organic compounds in the
samples analyzed by chromatography.
The calculated bed volumes of both column modifications were
446 ml or 89-5 ml fractions; therefore, organics eluted frac-
tion 89 are under non-steric influences (Kav^ 1) and cannot
be defined as to apparent molecular weight.
2. Laboratory Chemical Treatment Studies
Introduction. The laboratory chemical treatment studies were
conducted using the procedure shown in Figure 4. The Eastern-
Western sewage of June 6, 1969 and the Waterford sewage of
June 24, 1969 were analyzed before and after lime treatment
while the Eastern-Western sewage of June 30, 1969 was analyzed
untreated only. A summary of chemical treatment, organic
carbon and organic carbon recovery data is presented in Table
5. Chemical treatment was carried well beyond pH 11 and a
significant decrease in soluble organic carbon occurred in
all sewage samples treated with lime. Freeze-dryer recoveries
of soluble organic carbon averaged about 75% while recoveries
of soluble organic carbon from the column averaged about 94%
giving an overall recovery of about 7170.
Chromatographic Results - Eastern-Western Sewage. Chromato-
grams for the concentrates of the Eastern-Western sewage of
June 6, 1969 are shown in Figure 8. The untreated concentrate
251
-------
Table 4. Elution Characteristics of Standards Fractionated
on 2.5 cm x 91 cm G-15 Sephadex Gel Columns
Standard
Molecular Weight
Elution Fraction
Sodium Chloride
Glucose
Sucrose
Raffinose
Egg Albumin
Blue Dextran
59
180
342
594
45,000
About
2,000,000
82
68
62
54
39
39
252
-------
GEL : SEPHADEX G-15
ELUENT : 1C-2 M PHOSPHATE BUFFERED
DISTILLED WATER
FLOW RATE
BED DIMENSIONS
BED VOLUME
40ml/HR. (8.2 ml/MR/cm2)
2.5 cm x 91 cm
446ml 189 FRACTIONS)
ro
z
2 60
i-
o
I 50
40
30
90 tP5?_
80
70
SODIUM CHLORIDE M.W. - 59
EXCLUSION LIMIT
vGLUCOSE M.W. • 180
SUCROSE MW. * 342
»RAFFINOSE M.W. - 594
EGG ALBUMIN S BLUE DEXTRAN
• -"°M.W. >I500
J L
•55 J£Q"£56400 600 000 1500 OR GREATER
MOLECULAR WEIGHT
Fig. 7 STANDARD CURVE FOR APPARENT MOLECULAR WEWKT
-------
Table 5. Treatment and Carbon Recovery Data Laboratory
Chemical Treatment Studies
Sewage Sample
Eastern Western
6/6/69
Aliquot 1
Aliquot 2
Eastern Western
6/24/69
Water ford
6/24/69
Aliquot 1
Aliquot 2
Aliquot 3
1
Ca (OH) 2
Dosage
mg/1
None
600
None
PH
Attained
11.6
1
I
None 1
\
107 \ 9.6
267 1 11.2
Soluble Organic
Carbon mg/1
Before
Treatment
75
75
56
76
75
76
After
Creatment
75
65
56
Dryer
Recovery
% Org. C
78
72
85
j
76 I 80%
63 ! 68%
f
55 I 74%
Column
Recovery
% Org. C
95
90
107
j
92
94
93
-------
SPL-EASTERN-WESTERN
SEWAGE 6/6/69
(A.M.W 270)
IE(AMW.I7O)
SPL VOL-9ml8
SPL TOC-433mg/l
IE (A.MW UNDEFINED)
SPL- EASTERN -WESTERN
SEWAGE 6/6/69
TREATED TO pH 11.6
(600 mg/ 1 LIME.pH 11.6)
SPL VOL. -9 mis
SPL TOC -433mg/
45
55 65
FRACTION
75
NUMBER
85
95
105
Fig- 8
CHROMATOGRAMS OF UNTREATED AND UME TREATED EASTERN-WESTERN
SEWAGE CONCENTRATES SAMPLE OF 6/6/69.
255
-------
produced four distinct peaks with apparent molecular weights
as noted in the chromatogram. The great majority of the
soluble organic carbon was eluted well below the exclusion
limit of the gel (apparent molecular weight 1,500). Peak
IV was eluted after the total bed volume (Kav > 1) and can-
not be defined as to apparent molecular weight. The con-
centrate derived from the lime treated sewage produced a
similar chromatogram which exhibits the same peaks, however,
all but peak IV showed a decrease in magnitude.
Table 6 summarizes the percentages of recovered soluble or-
ganic carbon in each peak and the calculated soluble organic
carbon concentration of each peak in the organic unconcen-
trated sample. This table indicates that all peaks with the
exception of peak IV decreased with chemical treatment.
Only 8.7% of the soluble organic carbon in the untreated
sewage concentrate was within the exclusion peak ( > 1,500
apparent molecular weight) indicating a very low percentage
of high molecular weight soluble organics in the sewage.
Figure 9 and Table 7 present the data obtained from analysis
of the Eastern-Western sewage of June 30, 1969. The sewage
concentrate produced a chromatogram strikingly similar to
the previous sample. The same four peaks were present al-
though the relative magnitudes of the peaks were somewhat
different; only 3.6% of the soluble organic carbon in the
concentrate was within the exclusion peak indicating again
a very low percentage of high molecular weight soluble or-
ganics in the sewage.
Chromatographic results - Waterford sewage. Chromatograms
for the three concentrates of Waterford sewage are shown
in Figure 10. The untreated concentrate produced a chroma-
togram which was again strikingly similar to both of the
previous chromatograms for untreated Eastern-Western sewage.
The chromatograms of the concentrates obtained after treat-
ment with 107 mg/1 of lime and 267 mg/1 of lime indicate the
presence of all original peaks and little relative change.
Peak II increased slightly in magnitude and peak III de-
creased significantly. Peaks I and IV changed little.
Table 8 summarizes the percentages of recovered soluble or-
ganic carbon in each peak and the calculated soluble organic
carbon concentration of each peak in the original unconcen-
trated sample. This table shows that only 6.1% of the soluble
256
-------
Table 6. Effect of Chemical Treatment on Eastern-Western
Sewage of 6/6/69'
S3
Ln
Peak
I
II
III
IV
Apparent
Molecular
Weight
1,500
270
170
Undefined
Kav > 1
% Soluble Organic
Carbon in Peaks
Aliquot
1
8.7
38.6
23.6
18.3
Aliquot
2
5.3
36.4
17.2
18.5
Calculated Organic Carbon
In Original Sample mg/1
Aliquot
1
6.5
29.0
17.7
13.7
Aliquot
2
3.4
23.6
11.2
12.0
-------
40r
SPL- EASTERN-WESTERN
SEWAGE 6/30/69
CONCENTRATED BY
FREEZE DRYING
(NO TREATMENT)
SPL VOL
SPLTOC
10 mis
360 mg/l
1KA.M.W 270)
]Z(A.M.W UNDEFINED)
45
55 65 75
FRACTION NUMBER
85
95
IO5
Fig. 9 CHROMATOGRAM OF EASTERN-WESTERN SEWAGE SAMPLE OF
6/30/69.
258
-------
Table 7. Apparent Molecular Weight Distribution of
Eastern-Western Sewage of 6/30/69'
K3
Ui
Peak
I
II
III
IV
Apparent
Molecular
Weight
1,500
300
220
Undefined
Kav > 1
7o Recovered Organic
Carbon in Peak
3.6
45.1
9.2
20.1
Calculated Organic
Carbon in Original
Sample (mg/1)
2.0
25.2
5.2
11.3
-------
nr (AMW >70)
70
SPL - WATERFORD SEWAGE
CONCENTRATED BY
FREEZE DRYING -
ALIQUOT I
(NO TREATMENT)
SPL VOL- 10 mis
SPLTOC- 432 mg/I
EZKA.M.W. UNDEFINED)
45
55 65 75
FRACTION (5ml)
HIIAMW 170)
85
95
105
SPL - WATERFORD SEWAGE
CONCENTRATED BY
FREEZE DRYING -
ALIQUOT 2
(I07mg/l LIME, pH 9.6
SPL VOL- a4mis
SPLTOC- 516 mg/1
2SC (A.M.W. UNDEFINED)
65 75
FRACTION (5 ml)
3KA.M.W. 270)
iHIlAMW 170}
TREATED TO PHILS
SPL - WATERFORD SEWAGE
CONCENTRATED BY
FREEZE DRYING -
ALIQUOT 3
(267mg/l LIME, pH 11.2)
SPL VOL- 7.6 mis
SPLTOC- 570 mg/1
. UNDEFINED)
65 75
FRACTION (5ml)
10 CHROMATOGRAMS OF UNTREATED AND LJME TREATED WKTERFORD SEWAGE.
260
-------
Table 8. Effect of Chemical Treatment on Waterford Sewage
i Peak
\
I
II
III
IV
1
Apparent
Molecular
Weight
1,500
300
170
Undefined
Kav> 1
70 Recovered Soluble
Organic Carbon in Peak
Aliquot
1
6.1
29.8
39.6
11.9
Aliquot
2
4.7
37.2
26.8
12.9
Aliquot
3
5.0
43.7
19.3
13.2
Calculated Soluble Organic
Carbon in Original Sample (mg/1)
Aliquot
1
4.6
21.8
30.1
9.1
Aliquot
2
3.0
23.5
16.7
8.1
Aliquot
3
2.8
24.0
10.6
7.3
Aliquot 1 = No chemical treatment
Aliquot 2 = 107 mg/1 Ca(OH)2 to pH 9.6
Aliquot 3 = 267 mg/1 Ca(OH)2 to pH 11.2
-------
organic carbon in the untreated sewage concentrate was within
the exclusion peak indicating a very low percentage of high
molecular weight soluble organic in the sewage. Peaks I and
III decreased with chemical treatment; however, a lime dosage
of 267 mg/1 did not substantially change the effect brought
about with a 107 mg/1 lime dosage.
3. Chemical Physical Treatment Studies
Introduction. The New York State pilot plant samples anal-
yzed during this phase were composites which allowed direct
comparison of aliquots taken at different points on the
treatment process. The New Rochelle sample set was not
collected in relation to pilot plant detention times and
therefore no attempt is made to compare between the various
aliquots.
The chemical treatment and soluble organic carbon recovery
data for each sample set are presented in Table 9. No lime
dosage or pH data are available for the New Rochelle sample
set. The chemical treatment effluent has been neutralized
at the pilot plant site. The plant was normally operated
using a pH in excess of 10.5 (56). Analysis was made using
the procedure shown in Figure 4.
New York State pilot plant samples.
Sample set of 9/26/69. Chromatograms of the concentra-
ted influent and carbon column effluent for 9/26/69 are
shown in Figure 11. Both chroma to grams show five peaks and
both chromatograms closely resemble each other. The carbon
column effluent concentrate contained predominently low
apparent molecular weight material.
Table 10 presents data on percentages of soluble organic
carbon and soluble organic carbon concentrations in each
aliquot. These data are expressed in terms of apparent mo-
lecular weight ranges for ease of presentation. Percentage
removals for each range are also presented. About 80% of
the concentrated influent soluble organic carbon was under
1,000 in apparent molecular weight. The carbon column ef-
fluent concentrate contained about 70% of its soluble
organic carbon below 1,000 in apparent molecular weight. The
percentage removals in all ranges were quite good, however,
the above 1,000 apparent molecular weight material was removed
262
-------
Table 9. Treatment and Soluble Organic Carbon Recovery Data
Chemical Physical Treatment Studies
Sample
NYS Pilot Plant
9/26/69
Aliquot 1
Aliquot 2
NYS Pilot Plant
10/10/69
Aliquot 1
Aliquot 2
Aliquot 3
New Roche lie
Pilot Plant
Aliquot 1
Aliquot 2
Aliquot 3
Location In
Treatment Process
Untreated
Carbon Column
Effluent
Untreated
Chemically Treated
Carbon Column
Effluent
Untreated
Chemically Treated
Carbon Column
Effluent
Chemical
Dosages
Used
88.5 mg/1
Ca(OH)2
and
17.7 mg/1
FeClo
LO
-------
g 35
SPL - WATERFORD MIXTUR
OF 9/26/09
CONCENTRATED BY
FREEZE DRYING
(NO TREATMENT)
SPL VOL
SPLTOC
7.3 mis
658 mg/l
45
55
65 75
FRACTION (5ml)
95
35
SPL -
SPL VOL
SPLTOC
WATERFORD MIXTURl
OF 9/26/69
CONCENTRATED BY
FREEZE DRYING
(FINAL EFFLUENT)
3 mis
244 mg/I
45
55
65 75
FRACTION (5mO
85
95
105
Fig. 11 CHROMATOGRAMS OP INFLUENT AND CHEMICAL-
R^YSICAL EFFLUENT - NEW YORK STATE PILOT
PLANT SAMPLE OF 9/26/69.
264
-------
Table 10. Effect of Chemical-Physical Treatment on Waterford
Sewage-Paper Waste Mixture of 9/26/69
N3
o>
Ui
Apparent
Molecular
Weight
Range
Above 1000
600-1000
100-600
Less than
100
% Recovered Soluble
Organic Carbon
Untreated
18.7
14.8
53.4
13.2
Column
Effluent
27.8
8.4
50.2
13.6
Calculated Soluble
Organic Carbon in
Aliquot (tng/1)
Untreated
7.5
5.9
21.4
5.3
Column
Effluent
1.95
0.60
3.52
0.95
i
%
Soluble
Organic
Carbon
Removal
74
90
83
:
82
-------
to a slightly lesser degree.
Sample set of 10/10/69. Chromatograms of the concentra-
ted influent, chemical treatment effluent and carbon column
effluent for 10/10/69 are shown in Figure 12. The untreated
and chemically treated concentrates show five peaks while
the carbon column effluent concentrate shows four. It ap-
pears that peak II was nearly completely removed. The high
lime treatment had no noticeable effects.
Table 11 presents data on removals, percentages and concen-
trations of soluble organic carbon in various apparent mo-
lecular weight ranges. About 8770 of the soluble organic
carbon in the carbon column effluent is attributed to organ-
ics below 1,000 in apparent molecular weight. Removals of
soluble organic carbon were high except for the 100 to 600
apparent molecular weight range.
New Rochelle sample set. Chromatograms of the concentrated
influent, chemical treatment effluent and carbon column ef-
fluent from the New Rochelle pilot plant are shown in Figure
13. The chromatogram for New Rochelle influent sewage con-
centrate is very similar to previous sewage concentrate chro-
matograms and indicates a predominance of low apparent molec-
ular weight organics. The chromatographic results for the
chemical treatment effluent concentrate show nearly the same
characteristics and exhibit a low concentration of apparent
high molecular weight organics. The carbon column effluent
concentrate again exhibits a predominance of apparent low
molecular weight organics. The characteristic peak II (ap-
parent molecular weight 270) found in all other sewage con-
centrates is absent and is assumed to have been removed by
the activated carbon.
4. Analysis of Unconcentrated Samples and Validation of
the Freeze-drying Technique
Introduction. The Elnora, New York sewage was used exclusive-
ly during this phase. The experimental procedure used is shown
in Figure 6. The sample was split into three smaller 1.5
liter samples. The first was not treated, the second was
treated with lime to a pH of 11.6 and the third was treated
with sodium hydroxide to a pH of 11.6. The untreated and
lime treated samples were each further divided into two sep-
arate aliquots 3 and 4 (lime treated). Aliquots 2 and 3 were
266
-------
§ 20
o 10
g 35
45
SPL - WATERFORD MIXTURE
OF 10/10/69
CONCENTRATED BY
FREEZE DRYING
(UNTREATED)
SPL VOL - 9 mis
SPLTOC- 278 mg/l
•o-o-o.
O.
55 65 75
FRACTION (5ml)
85
95
105
SPL -
SPL VOL
SPLTOC
WATERFORD MIXTURE
OF 10/10/69
CONCENTRATED BY
FREEZE DRYING
(CHEMICALLY TREATED)
8.75 mis
276 mg/l
65 75
FRACTION (5ml)
85
95
IO5
~40r
20
10
35
O-O—O—Q-O
SPL -
m
SPL VOL -
SPL TOC -
WATERFORD MIXTURE
OF 10/10/69
CONCENTRATED BY
FREEZE DRYING
(FINAL EFFLUENT)
9 mis
189 mg/l
45
85
95
105
55 65 75
FRACTION (5 ml)
12 CHROMATOGRAMS OF CONCENTRATED INFUJENT, CHEMICAL TREATMENT EFFLUENT
AND CARBON COLUMN EFFLUENT- NEW "IDRK STATE PILOT PLANT SAMPLES
OF IO/IO/69.
267
-------
Table 11. Effect of Chemical-Physical Treatment on Waterford
Sewage-Paper Waste Mixture of 10/10/69
Apparent
Molecular
Weight
Range
Above
1,000
600-1,000
100-600
Less than
100
% Recovered Soluble Organic
Carbon in Range
Untreated
16.3
13.5
44.6
25.5
Chemical
Effluent
18.2
12.3
47.4
22.1
Carbon
Column
Effluent
12.9
5.8
71.5
9.9
Calculated Soluble Organic
Carbon in Aliquot
Untreated
6.4
5.3
17.4
10.0
Chemical
Effluent
7.3
4.9
19.0
8.8
Carbon
Column
Effluent
1.40
0.64
7.85
1.09
°/0 Soluble
Organic
Carbon
Removal
78
88
55
89
ro
ON
oo
-------
35
m
SPL - NEW ROCHELUE SEWAGE
CONCENTRATED BY
FREEZE DRYING
(UNTREATED)
SPL VOL- 10 mb
SPLTOC- 330mg/l
45
56
65 75
FRACTION 15 ml)
85
95
SPL - NEW .ROCHELLE SEWAGE
CONCENTRATED BY
FREEZE DRYING
(CHEMICALLY TREATED)
SPL VOL- 9.75 mte
SPLTOC- 338 mg/1
FRACTION (5ml)
SPL - NEW ROCHELLE SEWAGE
CONCENTRATED BY
FREEZE DRYING
(FINAL EFFLUENT)
SPL VOL- 9 mis
SPL TOC - 222 mg/l
Fig. 13
65 75
FRACTION (5ml)
CHROMATOGRAMS OF CONCENTRATED ^INFUJENT; CHEMICAL TREATMENT &FUIENT
AND CARBON COLUMN EFFLUENT - New >ORK ROCHEU£ PILOT PLANT.
269
-------
concentrated by freeze-drying. The sample which was treated
with sodiim hydroxide was used unconcentrated only and is
designated as aliquot 5. Ten ml samples of each aliquot were
fractionated and analyzed using either the high range (con-
centrated) or low range (unconcentrated) of the modified
carbonaceous analyzer described previously.
The chemical treatment and soluble organic carbon recovery
data are presented in Table 12. Lime treatment produced a
detectable but not a large drop in soluble organic carbon
while sodium hydroxide treatment did not. Lime treatment
produced excellent clarification while sodium hydroxide
yielded a turbid supernatant.
Validity of the freeze-drying technique. Chromatograms of
the concentrated and unconcentrated aliquots of untreated
and lime treated sewage are shown in Figures 14 and 15 re-
spectively. Elnora sewage samples. There is little dif-
ference between the chromatograms for concentrated and
unconcentrated samples.
Table 13 summarises the percentages of soluble organic car-
bon in each peak and the amount of carbon each peak repre-
sents in the aliquot. Both the percentages and soluble
organic carbon concentrations obtained from concentrate anal-
ysis agree very closely with the analysis of the unconcen-
trated aliquots. It is obvious that freeze-drying produced
a minimal change in chroma to graphic results. Peak IV in
each concentrate appears to have a slightly lower concentra-
tion than its unconcentrated counterpart. The other peaks
agree so closely that any difference could be ascribed to
experimental accuracy.
Lime versus sodium hydroxide treatment. Figure 16 depicts
chromatograms for the unconcentrated aliquots (aliquots 1,
3 and 5). Sodium hydroxide produced practically no effect
as evidenced by the similarity between the chromatograms for
the untreated and NaOH treated wastewater. Lime appears to
have caused a slight decrease in peaks I and IV.
Table 14 summarizes the percentages of recovered soluble
organic carbon in each peak and the corresponding organic
carbon concentration of the peak in each aliquot. Less
than 370 of the soluble organic carbon in the untreated ali-
quot was in the high apparent molecular weight category
(peak I> 1,500). Lime treatment produced a 507o decrease
270
-------
Table 12. Treatment and Carbon Recovery Data Elnora Sewage
Aliquot
i
i.
2
3
4
5
Chemical
Dosage
IN O Tic
INUIlc
321 mg/1
Ca(OH)2
321 mg/1
Ca(OH)2
NaOH
pH
Attained
11.6
11.6
11.6
Solubl*
Carboi
Before
Treatment
69
DZ
69
DZ.
62
62
62
2 Organic
i (mg/1)
After
Treatment
69
OZ
A?
DZ
60
60
62
Freeze
Dryer
Recovery
Unconc .
"7 A °/
f^fo
Unconc .
70%
Unconc .
Column
Recovery
% Org. C.
nc\"j
yy/o
f AQcy
lUo/o
95%
98%
103%
-------
I0r
8
SPL - ELNORA SEWAGE
UNCONCENTRATED
ALIQUOT I - UIMTREAT
SPL VOL - 10 mis
SPL TOG - 62 mg/l
1KA.M.W 270)
HI(AMW 170}
EHAMW UNDEFINED)
T-O
35 45 55 65 75 85
FRACTION NUMBER
95 105
50
.-= 40
SPL - ELNORA SEWAGE
CONCENTRATED BY
FREEZE DRYING
ALIQUOT 2-UNTREATEO
SPL VOL- lOmte
SPL TOC- 230 mg/1
270)
m(A.M.W 170)
EKAM.W UNDEFINED)
45
55 65 75 85
FRACTION NUMBER
95
105
Fig. 14 CHROMATOGRAMS OF UNTREATED AND CONCENTRATED ELNORA SEWAGE.
272
-------
10
3IIAMW 270) m(AMW )7Q)
SPL - ELNORA SEMWVGE
UNCONCENTRATED
ALIQUOT 3 -
TREATED TO pH 11.6
321 mg/l
SPL VOL- 10 mis
SPLTOC. - 60mg/l
]2(AMW. UNDEFINED)
35
45
55 65 75
FRACTION NUMBER
85
105
40
30 -
10
UKAMW 170)
ILIAMW 270)
I(AMW.>I500) /CONCENTRATED
SPL - ELNORA SEWAGE
CONCENTRATED BY
FREEZE DRYING
AUQUOT 4-
TREATED TO pH H.6-
321 mg/l C
SPL VOL- 10 mb
SPLTOC- 230 mg/l
3KAMW UNDEFINED)
45
55 65
FRACTION
85
105
75
NUMBER
Fig. 15 CHROMffTOGRAMS OF UWX3NCENTRATED AND CONCENTRATED CHEMICALLY
TREATED ELNORA SEWGE.
273
-------
Table 13. Effect of Concentration by Freeze Drying on Untreated and Lime Treated
Samples of Elnora Sewage
Peak
I
II
III
IV
Apparent
Molecular
Weight
1,500
270
170
Undefined
Kav> 1
% Recovered Soluble Organic
Carbon in Peak
Untreated
Unconc.
2.8
37.8
27.2
24.4
Cone .
2.3
37.3
31.2
18.0
Lime Treated
Unconc .
1.4
37.2
28.6
27.7
Cone.
1.4
35.5
30.0
24.2
Calculated Soluble Organic
Carbon in Original Sample tng/1
Untreated
Unconc .
1.7
23.4
16.9
15.1
Cone.
1.4
23.1
19.3
11.2
Lime Treated
Unconc .
0,8
22.3
17.2
16.6
Cone.
0.8
21.3
18.0
14.5
NJ
•sj
-------
EKAMW. 270)
m(AMW 170)
SPL - ELNORA SEWAGE
UNOONCENTRATED
AUQUOT I - NO TREATMENT
SPL VOL- 10 mis
SPL TOC- 62mg/l
DZ(A.M.W UNDEFINED)
55 65
FRACTION
75 85
NUMBER
K>
SPL - ELNORA SEWAGE
UNCONCENTRATED
ALIOUOT 5 - TREATED
TO pH 11.6-(NoOH)
SPL VOL- 10 mis
SPL TOC- 62mg/l
nI5OO)
TREATED TO
pHllfi(Co(OH)2)
(AMW. 170)
SPL - ELNORA SEWAGE
UNCONCENTRATED
ALIQUOT 3-TREATED
TO pH (I.6r32lmg/l
SPL VOL- 10mis
SPL TOC- 60 mg/l
I2(A.M.W. UNDEFINED)
o-oo-o-o-o-
45
105
55 65 75 85
FRACTION NUMBER
g- I6 < CHROMATOGRAMS OF UNTREATED, UME TREATED AND SODIUM HYDROXIDE
TREATED ELNORA SEWAGE.
275
-------
Table 14. Effect of Chemical Treatment on Unconcentrated Elnora Sewage
] Peak
I
II
j III
IV
Apparent
Molecular
Weight :
1,500
270
170
Undefined
Kav > 1
% Recovered Soluble Organic
Carbon in Peaks
Untreated
2.8
37.8
27.2
24.4
Lime
Treated
1.4
37.2
28.6
27.7
NaOH
Treated
2.3
39.0
27.3
26.1
Soluble Organic Carbon in
Sample mg/1
Untreated
1.7
23.4
16.9
15.1
Lime
Treated
0.8
22.3
17.2
16.6
NaOH
Treated
1.4
24.2
16.9
16.2
ON
-------
in peak I while sodium hydroxide produced little decrease.
These data coupled with the fact that lime treatment caused
a drop in soluble organic carbon indicate that a removal
mechanism and not pH caused the effect observed.
A summary of these results is presented at the end of the
following section (Section VI).
277
-------
SECTION VI: DISCUSSION
1. Possible Effects of Colloidal Organic Matter on
Chromatographic Results
The literature on sewage constituents defines the colloidal
fraction as particles between one nannometer and one micron
in size (16, 17, 35, 37). Roughly 10% of the organics in
sewage can be attributed to the colloidal organic fraction
and 40% to the soluble organic fraction (16, 17, 31, 35).
The filter used in this work (0.45 micron) was separating
in about the middle of the colloidal range; therefore,
small amounts of colloidal organics were probably present
in the samples analyzed. Based on the swollen size of the
gel particles, it can be assumed that at least some sub
0.45 micron colloids would pass through the gel bed. Col-
loids would be completely excluded from the gel and would
appear in the exclusion peak (peak I) as high apparent
molecular weight material. In view of the foregoing it
is highly probable that an unknown portion of the organics
in the exclusion peaks can be attributed to organic col-
loids.
2. Apparent Molecular Weight Distribution of Domestic
Sewage
The fact that all five untreated sewage samples analyzed all
exhibited the same four peaks indicates strongly that the
nature of the soluble organic fraction is similar between
sewages from different locations. This is further confirmed
by the fact that two independent studies (17, 31) have re-
sulted in the identification of many of the same soluble
organic constituents or organic groups (sugar, organic acids,
amino acids, etc) and by considering the origins of domestic
sewage. If commercial and industrial wastes are excluded
there is little or no variability in the sources that make
up sewage (human body wastes, ground garbage, wash water,
etc) as evidenced by the fact that such measurements as
solid content, BOD content, and many other sewage parameters
vary little between sewage sources and in fact have become
quite standardized.
This study indicates that high molecular weight substances,
as defined by the exclusion limit of Sephadex G-15 of 1,500,
comprise less than 10% of the soluble organics in sewage as
279
-------
measured by organic carbon. The only study comparable is
that reported by Zuckerman and Molof (54, 55) which indi-
cates a much higher proportion of high molecular weight
substances. The remaining literature (17, 31) is not
conclusive but does report many low molecular weight or-
ganic compounds in sewage.
The peak identified as peak IV in all sewage samples could
not be defined as to apparent molecular weight since the
organics in this peak were obviously under non-steric in-
fluences. In all probability this peak contained low
molecular weight organics since non-steric influences are
reported to increase with decreasing molecular size and
increased degree of cross-linking of the gel (4, 11).
Sephadex G-15 is a tightly cross-linked gel. One large
group that is known to be retarded by Sephadex gels is
the aromatic and heterocyclic organic compounds including
bile pigments, certain amino acids, indol and skatol (23).
Urea is also strongly retarded(23). All of the foregoing
organics could be contained in sewage.
The existence of four peaks in a sewage sample indicate
that only four soluble organic compounds are predominant.
It has been clearly shown that many soluble organic com-
pounds are present in sewage (17, 31). In the case of
peaks II, III and IV, each peak represents a group of
soluble organics with similar chromatographic characteris-
tics which, due to limitations in resolution of the gel
column, appear as one peak theoretically of approximately
the same molecular weight. Peak I would theoretically
include all compounds with a molecular weight of 1,500 or
above (4, 11).
3. Effect of Lime Treatment
High pH lime treatment produced no effect on the high ap-
parent molecular weight material in the Waterford sewage-
paper waste mixture (see Figure 12) and in no case did
lime treatment cause a dramatic shift in apparent molecular
weight distribution as reported by Zuckerman and Molof (54,
55). A consistent but variable reduction in the amounts of
high apparent molecular weight organics (peak I) in sewage
did occur with lime treatment. The cause of this phenomenon
is most probably a removal mechanism such as coagulation,
sorption or complexing and not a hydrolysis effect (54, 55) .
280
-------
This is indicated by the data in three ways:
Lime treatment of sewage was accompanied by a drop in
soluble organic carbon from the system.
High pH-sodium hydroxide treatment did not produce the
degree of reduction in peak I in Elnora sewage as high pH-
lime treatment.
A lime dosage sufficient to produce a pH of about 9.5
accomplished very nearly the same effect on peak I in
Waterford sewage as a lime dosage sufficient to produce a
pH in excess of 11.0. A pH of 9.5 at ambient temperature
and pressure is generally considered by biochemists as a
rather mild environment for hydrolysis of organics.
4. Apparent Molecular Weight Distribution of Chemical
Physical Effluents
All three chemical-physical effluents analyzed contained
both high and low apparent molecular weight material;
however, the low apparent molecular weight material was
predominant. Although organics in some molecular weight
ranges were removed more efficiently than others, there
was only one case where removals within a range were poor
(see Table 11). This predominance of low apparent molec-
ular weight material is supported by data indicating that
the soluble organics of the untreated wastes were also of
predominantly low apparent molecular weight. Further
confirmation is given by other workers (6, 27) who state
that low molecular weight hydrophilic organics would be
resistant to activated carbon adsorption. The data pres-
ented are in substantial disagreement with the reports of
Zuckerman and Molof (54, 55) that low molecular weight
soluble organics are preferentially absorbed by carbon
columns.
5. Effect of Freeze-drying on Chromatographic Results
The fact that freeze-drying did not change the chromato-
graphic characteristics of the organics in untreated and
lime-treated Elnora sewage indicates that the nature of the
organics was not changed. This is not surprising since
freeze-drying has been used effectively for many years in
the food and pharmaceutical industries as well as in
281
-------
medicine in handling labile organics (12).
Freeze-dryer recoveries were comparable to those reported
by other researches (25, 31) and no large selected losses
of organic carbon occurred as indicated by the chromato-
graphic results with Elnora sewage. The losses experienced
are attributed to mechanical losses during processing. This
is supported by the work of McDonald (25) whose data indi-
cate that about 90% of the loss in organic carbon in freeze-
drying wastewater can be accounted for by acid washing the
glassware and filters used in preparing concentrates.
6. Summary
The results can be summarized as follows:
Chromatograms of five samples of domestic sewage from
four separate locations all exhibited striking similarity
showing four characteristic peaks at apparent molecular
weights of greater than 1,500, 270, 170 and undefined. The
undefined peak appeared after the elution of one gel bed
volume and, therefore, has a partition coefficient (Kav) of
greater than one, indicating non-steric influences. The
vast majority of the soluble organic carbon in sewage is
attributed to soluble organics of less than 1,500 in ap-
parent molecular weight.
Dosing sewage with lime did not create any dramatic
effects on the chromatographic results. A partial but
consistent reduction of the small quantities of the high
apparent molecular weight peak (peak I) in sewage was
observed. In some cases variable reductions in low appar-
ent molecular weight peaks also were observed. Dosing a
mixture of Waterford sewage and paper waste with lime
created essentially no effect. In the case of Waterford
sewage, a relatively low lime dosage created essentially
the same effect as a relatively high dosage. In the case
of Elnora sewage, sodium hydroxide produced little effect
on peak I while lime produced a reduction in peak I; further-
more, lime produced a decrease in soluble organic carbon
during treatment in all cases. This indicates that the
effects observed were probably due to a removal mechanism.
Freeze-drying produced little effect on the chromato-
graphic results and all indications are that this technique
282
-------
is an excellent method of concentrating soluble organics in
wastewater.
Chromatograms of three final effluents after chemical
treatment and activated carbon adsorption indicate that the
soluble organics remaining after chemical-physical treatment
are primarily of low apparent molecular weight.
Soluble organic carbon removals in all molecular weight
ranges were generally high after chemical-physical treatment.
283
-------
SECTION VII: REFERENCES
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Proteins by Sephadex Gel Filtration," Biochemical
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2. Andrews, P., "The Gel Filtration Behavior of Proteins
Related to their Molecular Weights Over a Wide Range,"
Biochemical Journal, 96, pp 595-606 (1965).
3. Anon., "The Freeze Drying Method," The Virtis Company,
Inc. (1970).
4. Anon., "Sephadex-Gel Filtration in Theory and Practice,"
Pharmacia Fine Chemicals, Uppsala, Sweden.
5. Bishop, D.F., "Advanced Waste Treatment Research at the
FWPCA-DC Pilot Plant," Phosphate Removal Seminar, Albany,
New York, April 28-29 (1970).
6. Bishop, D.F., and Marshall, T.P., et al, "Studies on
Activated Carbon Treatment," Journal of the Water
Pollution Control Federation, 39, No. 2, pp 188-203
(February 1967).
7. Brunner, D.R. , and Sproul, O.T., "Virus Inactivation
During Phosphate Precipitation," Journal of the Sani-
tary Engineering Division, American Society of Civil
Engineers, SA 2, pp 365-378 (April 1970).
8. Bunch, R.L., Barth, E.F., and Ettinger, M.B., "Organic
Materials in Secondary Effluents," Journal of the Water
Pollution Control Federation, 33, No. 2, Part 2 (Oct.
1967).
9. Buzzel, J.C., and Sawyer, C.N., "Removal of Algal Nutri-
ents from Raw Wastewater with Lime," Journa 1 of the Water
Pollution Control Federation, 39, No. 10, Part 2 (Oct.
1967).
10. Collins, J.W. , Webb, A.A., Didwania, H.P., and Lueck, B.F. ,
"Components of Wood Pulp Bleach Effluents," Environmental
Science and Technology, Vol. 3, No. 4, pp 371-377 (April
1969).
285
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11. Determann, H., "Gel Chromatography," Springer-Verlag
New York, Inc. (1968).
12. Flosdorf, E.W. , "Freeze Drying," Reinhold Publishing
Corp., New York, pp 1-67 (1949).
13. Ghassemi, M., "Chemical Nature and Physical Properties
of Color Causing Organic Substances in Natural Waters
and a Study of their Coagulation with Aluminum (111)
and Iron (11) Salts," Doctoral Dissertation, University
of Washington (1967).
14. Gjessing, E., and Lee, G.F., "Fractionation of Organic
Matter in Natural Waters on Sephadex Columns," Environ-
mental Science and Technology, _!, No. 8, pp 631-638
(Aug. 1967).
15. Hardt, F.W., "Effluent Separation of a High Solids Mi-
crobial System by Membrane Filtration," M.S. Thesis,
Syracuse University (1969).
16. Heukelekian, H., and Balmat, J.L., "Chemical Composition
of the Particulate Fractions of Domestic Sewage," Sewage
and Industrial Wastes, 31, 4, pp 413-423 (April 1959).
17. Hunter, J.V., and Heukelekian, H., "Separation and Mate-
rials Balance of Solids Fractions in Sewage," Proceed-
ings of the Fifteenth Industrial Waste Conference,
Lafayette, Indiana, pp 150-157 (May 1960).
18. Hunter, J.V. , and Heukelekian, H., "The Composition of
Domestic Sewage Fractions," Journal of the Water Pol-
lution Control Federation, 39, 8, pp 1142-1163 (Aug.
1965).
19. Keinath, T.M., and Weber, W.J., "A Predictive Model for
the Design of Fluid Bed Adsorbers," Journal of the
Water Pollution Control Federation, 40, No. 5, pp 741-
765 (May 1968).
20. LaMer, V.K., and Smellie, R.H., "Flocculation, Subsidence
and Filtration of Phosphate Slimes," Journal of Colloid
Science, II, p 704 (1956).
286
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21. Laurent, T.C., and Killander, J., "A Theory of Gel Fil-
tration and its Experimental Verification," Journal of
Chromatography, 14, pp 317-330 (1964).
22. Loven, A.W., and Heather, C.H., "Activated Carbon in
Treating Industrial Wastes," 159th ACS Conference,
Houston, Texas, Feb. 22-27 (1970).
23. Marsden, N.V.B., "Solute Behavior in Tightly Cross-Linked
Dextran Gels," Annals New York Academy of Sciences, 125,
pp 428-457 (1965).
24. Masse, A.N., "Removal of Organics by Activated Carbon,"
Federal Water Pollution Control Administration, Cincin-
nati, Ohio (Aug. 1968).
25. McDonald, G.C., "Effect of Wastewater Organic Fractions
on the Growth of Selected Algae," Ph.D. Thesis, Rensselaer
Polytechnic Institute, Troy, New York (June 1971).
26. Meryman, H.T., "Principles and Practice of Freeze Drying,"
Annals of the New York Academy of Sciences, 85, p 501 (1960)
27. Morris, J.C., and Weber, W.J., "Adsorption of Biochemically
Resistant Materials from Solution - 2," Cincinnati, Ohio
(March 1966).
28. Morris, J.L., and Weber, W.J., "A Predictive Model for the
Design of Fluid Bed Adsorbers," Journal of the Water Pol-
lution Control Federation, 40, No. 5, pp 741-765 (May 1968).
29. Mulbarger, M.C., Grossman, E., and Dean, R.B., "Lime Clar-
ification, Recovery and Reuse," Federal Water Pollution
Control Administration, Cincinnati, Ohio (June 1968).
30. Owen, R., "Removal of Phosphorous from Sewage Plant Efflu-
ent with Lime," Sewage and Industrial Wastes, 25, No. 5,
pp 548-556 (May 1953).
31. Painter, H.A., and Viaey, M., "Composition of a Domestic
Sewage," Journal of Biochemical and Microbiological Tech-
nology and Engineering, !_, 2,"pp 143-162 (1959).
32. Painter, H.A., Viney, M., and Bywaters, A., "Composition
of Sewage and Sewage Effluents," Paper presented at a
!37
-------
meeting of the Metropolitan and Southern Branch, Institute
of Sewage Purification, London, England (December 1960).
33. Pearse, L., et al, "Chemical Treatment of Sewage,11 Sewage
Works Journal, _7, No. 6, pp 1001-1108 (Nov. 1935).
34. Porath, J., and Flodin, P., "Gel Filtration: A Method for
Desalting and Group Separation," Nature, 183, pp 1657-
1659 (1959).
35. Rickert, D.A., and Hunter, J.V., "Rapid Fractionation and
Materials Balance of Solids Fraction in Wastewater and
Wastewater Effluent," Journal of the Water Pollution Con-
trol Federation, ^9, 9, pp 1475-1486 (Sept. 1967).
36. Rudolfs, W., "Phosphates in Sewage and Sludge Treatment, II-
Effect on Coagulation, Clarification and Sludge Volume,"
Sewage Works Journal, 19, No. 2, pp 178-190 (March 1967).
37. Rudolfs, W., and Balmat, J.L., "Colloids in Sewage, I.
Separation of Sewage Colloids with the Aid of the Electron
Microscope," Sewage and Industrial Wastes, 31, 4, pp 413-
423 (April 1959).
38. Rudolfs, W., and Gehm, H.W., "Colloids in Sewage, and Sew-
age Treatment," Sewage Works Journal, II, 5, pp 727-737
(Sept. 1939).
39. Schaffer, R.B., Van Hall, C.E., and McDermott, G.N., et al,
"Application of a Carbon Analyzer in Waste Treatment,"
Journal of the Water Pollution Control Federation, 37, No.
11, pp 1545-1566 (Nov. 1965).
40. Slechta, A.F., and Gulp, G.L., "Water Reclamation Studies
at the South Tahoe Public Utility District," Journal of
the Water Pollution Control Federation, 39, No. 5, pp 787-
814 (May 1967).
41. Smith, S.E., "Coagulation of Pulping Wastes for the Re-
moval of Color," M.S. Thesis, University of Washington
(1967).
42. Smith, D.R., and Berger, H.F., "A Chemical-Physical Waste-
water Renovation Process for Kraft Pulp and Paper Wastes,"
Journal of the Water Pollution Control Federation, 40, No. -
288
-------
43. Snoeyink, V.L., and Weber, W.J., "The Surface Chemistry
of Activated Carbon," Environmental Science and Tech-
nology, _!, No. 3, pp 228-234 (March 1967).
44. Stones, T., "Studies in Lime Precipitation at Salford,"
Journal of the Institute of Sewage Purification, Part 4,
pp 395-400 (1954).
45. Stumtn, W., and Morgan, J.J., "Chemical Aspects of Coag-
ulation," Journal of the American Water Works Associ-
ation, 54, No. 8, pp 971-992 (Aug. 1962).
46. Van Hall, C.E., Earth, D., and Stenger, V.A., "Elimina-
tion of Carbonates from Aqeous Solutions Prior to Organic
Carbon Determination," Analytical Chemistry, 37, 68, pp
769-771 (1965).
47. Van Hall, C.E., Safranko, J., and Stenger, V.A. "Rapid
Combustion Method for the Determination of Organic Sub-
stances in Aqueous Solutions," Analytical Chemistry, 35,
3, pp 315-319 (1963).
48. Weber, W.J., "Discussion of Proposed Hydrolysis Adsorption
Process," Journal of the Water Pollution Control Federa-
tion, 42, No. 3, Part I, pp 456-463 (March 1970).
49. Weber, W.J., Hopkins, C.B., and Bloom, R., "Physicochem-
ical Treatment of Wastewater," Journal of the Water Pol-
lution Control Federation, 42, No. 1, pp 83-89 (Jan. 1970).
50. Weber, W.J., and Morris, J.C., "Kinetics of Adsorption on
Carbon from Solution," Journal of the Sanitary Engineering
Division, Proceedings American Society of Civil Engineers,
8£, SA 2, pp 31-58 (April 1963).
51. Weber, W.J., and Morris, J.C., "Equilibria and Capacities
for Adsorption on Carbon," Journal of the Sanitary Engineer-
ing Division, Proceedings American Society of Civil Engi-
neers, 90, SA 3, pp 79-107 (June 1964).
52. Weber, W.J., and Keinath, T.M., "Mass Transfer of Perdurable
Pollutants from Dilute Aqueous Solution in Fluidized Adsor-
bers ," jChej[ru£al_EjLi^^ 63, P 79,
(1967).
289
-------
53. Williamson, J.N., Heit, A.M., and Caiman, C., "Evaluation
of Various Adsorbents and Coagulents for Wastewater
Renovation,11 AW TR-12, United States Public Health Service,
Cincinnati, Ohio (June 1964).
54. Zuckertnan, M.M., "Chemical Versus Biological Wastewater
Treatment in the Production of High Quality Reuse Water,"
Doctoral Dissertation, New York University (Dec. 1968).
55. Zuckerman, M.M., and Molof, A.H., "High Quality Reuse Water
by Chemical-Physical Wastewater Treatment," Journal of the
Water Pollution Control Federation, 42, No. 3, pp 437-455
(March 1970).
56. Zuckerman, M.M., and Molof, A.H., "High Quality Reuse Water
from a Newly Developed Chemical-Physical Treatment Process,
Paper presented at Fifth International Conference on Water
Pollution Research, San Francisco, California (July 1970).
290
-------
APPENDIX C
MATERIALS AND APPARATUS
Materials
This section describes the types of permanent equipment and
expendable supplies employed in this overall investigation.
When appropriate, the specific phase of the study in which
the equipment was used is noted parenthetically.
The chemicals used throughout all phases of this experimental
work were reagent grade, unless stated otherwise.
The gels used in the experimental work were of the Sephadex
type, manufactured by Parmacia Fine Chemicals, Inc.,
Piscataway, New Jersey. The specific Sephadex gels employed
were as follows:
Sephadex G-10. With a particle size range of 40-120
microns and a fractionation range of 0-700 molecular weight
for peptides, globular proteins and dextran fractions.
Sephadex G-15. Sephadex G-15, cross-linked, dextran gel
was used to form the gel bed. The approximate fractionation
range of this gel extends up to 1,500 in molecular weight.
Compounds above 1,500 in molecular weight are excluded for
the gel.
Sephadex G-25 medium. With a particle size range of
50-150 microns,- fractionation range 1,000-5,000 molecular
weight for peptides and globular proteins.
Sephadex G-50 medium. With a particle size range of
50-150 microns, fractionation range 1,000-30,000 molecular
weight for peptides and globular proteins.
1. Calcium Hydroxide Suspensions (Appendix B)
The calcium hydroxide (Ca(OH)2) suspensions, used in the chem-
ical treatment studies, were made up in 10 g/1, 20 g/1, 30 g/1,
and 40 g/1 concentrations. These reagents were freshly pre-
pared before use.
291
-------
2. Sodium Hydroxide Solution (Appendix B)
A IN sodium hydroxide (NaOH) solution was used in the chem-
ical treatment studies. This reagent was freshly prepared
before use.
3. Sulphuric Acid Solutions (Appendix B)
0.05N, 0.10N and 0.50N sulphuric acid (H2S04) solutions were
used for pH adjustments.
4. Mercuric Chloride Solution (Appendix B)
A 40 g/1 mercuric chloride (HgCl2) solution was used to pre-
serve some waste samples (44).
5. Hydrochloric Acid Solution (Appendix B)
A 2N hydrochloric acid (HCl) solution was used in making
organic carbon determinations.
6. Oxalic Acid Solutions (Appendix B)
The oxalic acid (HoC^O^^I^O) solutions used to standardize
the carbonaceous analyzers were made up to contain 2 mg/1,
4 mg/1, 6 mg/1, 8 mg/1, 10 mg/1, 19 mg/1, 38 mg/1, 57 mg/1,
76 mg/1, 95 mg/1, of organic carbon.
7. Phosphate Buffered Eluent (Appendix B)
The phosphate buffered eluent used in gel permeation chrotna-
tography studies was prepared from a stock solution containing
22.57 g/1 of KH2P04, 57.74 g/1 of K2HP04 and 88.68 g/1 of
Na2 H PO, (54). Buffered eluent was prepared in four liter
batches as needed by diluting 40 ml of the stock solution to
4 liters with double-distilled water. The resulting buffer
had an ionic strength of 0.0185.
Gel standards (Appendix B) The gel column standards were made
up as follows:
Standard No. 1. 1 g/1 raffinose (C, nHooO.. .,, molecular
weight 594), 2.5 g/1 glicose (C6H (K, mileculaf weight 180),
1.0 g/1 egg albumin (molecular weight approximately 10,000).
An unknown portion of the egg albumin did not go into solution.
292
-------
Standard No. 2. 0.5 g/1 sucrose (C-ioHooOA, molecular
weight 342).
Standard No. 3. 5.85 g/1 sodium chloride (NaCl, molec-
ular weight 58.5).
Standard No. 4. Standard No. 4 consisted of blue dextran
2,000. Blue dextran 2,000 is a high molecular weight carbo-
hydrate (molecular weight approximately 2,000,000) with color
groups attached to it to permit visual observation of the
compound as it is eluted through the gel column. This stan-
dard was made up in sufficient strength to clearly see the
sample as it was eluted. The exact concentration of blue
dextran in the standard was not determined.
Wastewater samples (Appendix B) Wastewater samples were col-
lected from the following locations and used in laboratory
chemical treatment and studies.
Eastern-Western sewage. A four liter grab sample of
sewage was collected from the influent sewer to the Eastern-
Western sewage treatment plant at about 10 a.m. on June 6,
1969. A second one liter grab sample of this sewage was col-
lected at about 11 a.m. on June 30, 1969. The Eastern-Western
sewage treatment plant serves a small residential development
of about 50 homes. The sewage is entirely of domestic origin.
Waterford sewage. A 10 liter grab sample of sewage was
collected on June 24, 1969 at about 10 a.m. from a manhole
located near outfall number 3 of the Waterford, New York
sewer system. This system carries both sewage and storm
water. There are no known industrial waste discharges
tributary to the sewer line sampled.
Elnora, New York sewage. An 18 liter grab sample of
sewage was collected from the influent sewer of the Clifton
Knolls sewage treatment plant located in Elnora, New York
at about 11 a.m. on April 17, 1970. This treatment plant
serves a residential development of about 100 homes. The
sewage is entirely of domestic origin.
New York State pilot plant. Two separate sets of samples
were provided by the New York State Health Department Research
Unit for a chemical-physical pilot plant shown and described
in Figure 1. A 5070 mixture of Mohawk Paper Mill wastes and
293
-------
PROCEDURE
(I)
USED:
ADD 15 1/2 QAL. OF SO/SO WATERFORD
WASTE TO TANK I.
(2) ADO CHEMICALS AND RAPD MIX FOR 9 MM.
(3) FLOCCULATE FOR 15 TO 20 MM.
(4) SETTLE FOR 2 HOURS
(5) SIPHON SUPERNATENT TO TANK 2 AND MAINTAIN
SLOW MIXING.
(6) PUMP SUPERNATENT TO COLUMNS.
OPERATING DATA:
(I ) CARBON USED - FN.TRA8ORB 3OO
(2) COLUMN AREA - O.OO54 SO. FT
(3) SURFACE LOADING-1.5 GPM/SQ.FT
(4) SUPERFICIAL CONTACT TIME-60 MM.
(5) CARBON VOLUME O.O696 CU. FT.
(6) CARBON WEIGHT - 1.7 LBS.
BACKFLUSHING CONNECTIONS
fO
TRIABLE
MIXERS
I"DIA. GLASS COLUMNS
9 FT LONG
TTOON TUBMG
2O GAL. MiXNG ,
FUXXULATION AND
SETTUNG TANK
20 GAL. SUPERNATENT
HOLDING TANK
SIGMA MOTOR
PUMP
CAR
COLUMNS
CONNECTIONS
EFFLUENT
TANK
Fig. 1 SCHEMATIC OF NEW YORK STATE PILOT PLANT.
-------
Waterford, New York sewage was being treated in daily batches
by this plant.
The first sample set consisted of composite samples of the
raw waste mixture and final effluent of September 26, 1969.
The chemical dosages used on that day were 88.5 mg/1 of
calcium hydroxide (Ca(OH>2) and 17.7 mg/1 of ferric chloride
(FeCl3>. The pH of the chemically treated waste, before
neutralization, was 9.5.
The second set of samples consisted of composite samples of
the raw waste mixture, neutralized chemically treated effluent,
and final effluent of October 10, 1969. The chemical dosage
used on that day was 222 mg/1 of calcium hydroxide. The pH
of the waste before neutralization was 11.5.
New Rochelle pilot plant. One set of grab samples was
provided by the New York State Health Department, Research
Unit from the New Rochelle pilot plant which has been de-
scribed previously (56). The pilot plant was treating the
influent waste to the City of New Rochelle, New York sewage
treatment plant by the hydrolysis adsorption process which
was previously described. New Rochelle is a city of about
75,000 people and the sewage is presumed to contain some
commercial and industrial wastes in addition to domestic
wastes.
The sample set consisted of raw wastes, chemically treated
effluent and final effluent and was collected at about 4 p.m.
on October 19, 1969. The samples were not composited in
accordance with plant detention time.
Other materials used (Appendix A)
Bunsen burner
Drying oven
Test tubes
2,000 ml round bottom flask
1.2 liter freeze-drying flask
Rubber gloves
Hammer, tongs and safety glasses
Funnel
Tygon tubing and pinch clamps
Plumb bob
50 ul syringe
295
-------
10 ml syringe
Water aspirator
Vacuum hose
Hot plate
Thermometer to read from 0°C to 100°C
Evaporating dish
Membrane filters, 0.45 u pore size. (Millipore)
20 liter plastic carboys
McLeod vacuum gauge
Apparatus
1. Centrifugation and Filtration Equipment
Continuous centrifuge. Model CFR-2, 1,800 ml capacity,
12,000 rpm, manufactured by Lourdes Instrument Corporation.
Filtration apparatus. 147 mm filtration unit used with type
HAWP 0.45 u membrane filters. Millipore Corporation, Bedford,
Massachusetts.
2. Concentration Equipment
Virtis large port freeze-dryer. Model 10-200, ten liter
capacity. The standard model is equipped with eight 3/4"
ports and eight 1/2" ports; to increase the rate of sub-
limation the freeze-dryer was specifically manufactured with
sixteen 3/4" ports.
Eight liter dry ice capacity extender. Model 10-137E.
Virtis McLeod gauge. Model 10-224, range 5 microns-5 mm Hg.
Virtis filter seal freeze-drying flasks. 1,200 ml capacity,
Model F-128.
All of the above items were manufactured by the Virtis Company,
Inc., Gardiner, New York.
Calab thin film rotary evaporator. Model 200, equipped with
a 500 cl cold finger condenser and 2,000 ml boiling flask.
California Laboratory Equipment Company, Oakland, California.
Welch duo-seal vacuum pump. Model 1402, Skokie, Illinois.
296
-------
3. Chromatographic Equipment
Sephadex chromatographlc columns. 2.5 cm by 100 cm K25/100
fitted with upflow adapters, manufactured by Pharmacia Fine
Chemicals, Inc., Piscataway, New Jersey.
Fraction collector. Type 3402 B, LKB RadiRac LKB Produkter
AB, Stockholm, Sweden.
25 mm syringe filter holders. Equipped with type HAWP 0.45
micron filters, catalogue number SX0002500, Millipore Corpora-
tion.
Conductivity bridge. Model 31, manufactured by Yellow Springs
Instrument Corporation, Yellow Springs, Ohio.
Microliter syringejs. Model 705, 50 microliter capacity, and
Model 710, 100 microliter capacity. Hamilton Company,
Whittier, California.
pH meter. Corning Model 7 equipped with a temperature com-
pensating electrod.
Total organic carbon analyzer. The laboratory carbonaceous
analyzer is designed to measure and record the concentration
of small amounts (1 to 100 pptn carbon) of organic material
in a sample of water. The major instrumental components
include (1) a supply of oxygen, (2) a system for sample
introduction and transport, (3) a muffle furnace, (4) infra-
red analyzer, (5) amplifier and (6) a read out system (re-
corder) .
The new Beckman Model 915 Total Organic Carbon Analyzer used
in this work contains two separate but similar channels for
the total carbon and inorganic carbon analyses. In the total
carbon channel the combustion tube is packed with cobalt
oxide impregnated asbestos fiber and is enclosed in a high
temperature furnace (950°C). Both the organic and inorganic
carbonaceous material is oxidized to C02 in the presence of
high temperature, oxygen and the catalyst. In the inorganic
carbon channel the combustion tube contains quartz chips
wetted with 85?0 phosphoric acid and is heated by a low tem-
perature furnace (150°C). The acid liberates CO* and steam
from inorganic carbonates. The operating temperature is
sufficiently high for the desired reaction, but is substan-
tially below that required to oxidize the organic matter (31) .
297
-------
A condenser is provided after each combustion tube to remove
the condensed steam from the effluent which, then, flows to
a select valve. This valve is manually operated and is
selected for the particular channel in which the sample is
injected. The outlet from the select valve is connected to
a filter and then to the infrared analyzer which is sensitized
to carbon dioxide. A schematic flow diagram for the carbon
analyzer showing all the components is presented in Figure 2.
The infrared analyzer, Model 200 LIRA supplied by Mine Safety
Appliances, was used to measure the concentration of C02 pro-
duced when the sample is injected into the Beckman furnace
described on the previous page. It consists of two separate
infrared sources, a detector, an amplifier and a meter,
(Figure 3). Two similar helics of nichrome filaments are
heated to 1,200°F by the application of 5.7 volts A.C. The
infrared energy beams from these filaments travel through
parallel cells. One beam traverses the sample cell; the
other beam, the comparison cell. The emergent radiation is
directed to a single detector cell. The temperature and
pressure of the gas in the detector is accordingly increased
by adsorption of radiation (32).
A rotating interrupter alternately blocks the radiation enter-
ing the sample cell and the comparison cell. When the gas
to be analyzed is introduced into the sample cell, it absorbs
some infrared energy and thus reduces the radiation reaching
the detector from the sample cell. The two beams become
unequal. The detector gas expands and contracts as the beams
pass through it from comparison cell and sample cell, respec-
tively. The microphone membrane moves in response to the
pressure changes of the detector gas. The condenser micro-
phone capacity varies with the membrane movement, thereby,
generating an electrical signal proportional to the differ-
ence between the two beams which, in turn, corresponds to
the amount of carbon dioxide in the sample cell. This signal
is amplified and rectified so that it may be read on the LIRA
meter or an external recorder. A Beckman Ten-Inch Laboratory
Potentiometric Recorder, Model 1005, was used in this work.
The amplifier is tuned so that only variations in infrared
intensity occurring at the interrupting frequency produce
an output signal. When no C02 is passed through the sample
cell the beams are equal and the output is zero. Increased
298
-------
VO
VO
^{PRESSURE!
5 REGULATOR
GAUGE
SAMPLED-
dl
D
FLOWMETER
OXYOEN .SLEELJL
SYWNGE NEEDLE GUJDE
CHECK
VALVE
LDW TEMPERATURE COMBUSTION TUBE
ATMOSPHERE
SAMPLE SELECT MUVE
CHECK
EMPERATURE COMBLBTJoJ* TUbE
SYWNQE NFFTHF GUIDE
L
-•-CONDENSER
OUTLET
CONDENSER
TO INFRARED
ANALYZER
CONDENSER
Fig. 2 SCHEMATIC DIAGRAM OF TOTAL ORGANIC CARBON ANALYZER
-------
u>
o
o
ROTATING
INTEHMJPTER-
FUER
CELL-1
SAMPLE
IN ,-OUT
SOURCE
COMFARISON
CELL
9ENSmVITY VOLT3M3E-
OPTTCAL BENCH
ELECTRICAL CONTROLS
RECORDER
Fig. 3 SCHEMATIC DIAGRAM OF MSA MODEL 2OO INFRARED ANALYZER
-------
sample concentration may be simulated by upsetting the
optical balance of the analyzer. This is done by operat-
ing the sensitivity test switch which increases the
comparison beam voltage.
4. Biological Equipment
Microscope. Spencer Company, Buffalo, New York.
Syringes. Model 810L10N Luer-Lok tip disposable syringes,
10 ml capacity. Becton, Dickinson and Company, New York.
Continuously stirred batch culture apparatus. Constructed
and depicted on Figure 4.
Culture apparatus motors. 1/100 HP, 115V, 1,520 rpm, manu-
factured by Dayton Electric Manufacturing Company, Chicago,
Illinois.
Analytical balance. In the preparation of culture medium,
stock solutions and chromatographic standards, a Sartorius
200 g/0, 1 mg Selecta Rapid Balance was used. This balance
was also employed in making dry weight determinations of the
cultures. Sartorius Werke, Gottinger, Germany.
Cell enumeration. Bright line haemocytometer, American
Optical Corporation.
Absorbence measurements. Beckman Model DU-2 spectrophotometer,
A 5 cm cell was used in making the absorbence measurements.
Dry weight determinations. 47 mm Hydrosol filter holder and
type HAWP 0.45 micron filter, Millipore Corporation.
301
-------
8 i
to g
z
CULTURE APPARATUS
Fig. 4 CONTINUOUSLY STIRRED BATCH CULTURE APPARATUS.
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
1. Re? + BTo.
3. Accession
w
4. Title
5.
ort D,
Organic Nutrient Factors Effecting Algal Growf&s
a, P 'ormir Orgsr ation
7. Author(s)
Nicholas L. Clesceri
9. Organization
Rensselaer Polytechnic Institute, Troy, N.Y.;
Fresh Water Institute at Lake George (FWI)
10. Project K'fj.
16010 DHN
11. Contract'G::;,.'-* /.'
13. Type Repe. -nd
Perioa Co
12, ' Spomnrisg
IS. Supplementary Notes
Environmental Protection Agency report number,
EPA-660/3-73-003, July 1973.
16. Abstract
Effects of vastewater organic fractions on the growth rate of Selenastrum capri-
cornutum and Anabaena flos-aquae were investigated. Effluent from a conventional
activated sludge facility was membrane filtered, freeze-drled, and gel fractionated.
Apparent molecular weights (AMW) were assigned to the appropriate fractions. These
and organic carbon data showed 69% of the effluent organlcs had an AMW less than
700.
Absorbancies and regression analyses within algal exponential growth phases demon-
strated the control growth rate for Selenastrum was 0.43 and for Anabaena was 0.34.
Selenastrum growth rates were monitored using Lake George water as the diluent for
the media employed. An inhibition in growth occurred. Halving the nitrogen con-
centration in modified Gorham's had no significant effect on growth rate.
In concentrating organics from natural water (Lake George and Saratoga"Lake), raw
sewage, and sewage effluent, thin film evaporation was preferred when using natural
waters whereas freeze-drylng was advantageous when working with sewage samples.
Also, the soluble organic component in municipal wastewater was characterized and
the effect of chemical-physical treatment on it has been shown.
i7s. Descriptors *Abatement, *Pollution Abatement, Water quality control,
*Algae, Cyanophyta, Chlorophyta, *Analytical Techniques, Bioassay,,
chromatography, spectrophotometry, *Bioassay, water pollution sources,
BOD, *Chemical properties, waste identification, *Chemical wastes,
-organic wastes, *Freeze-drying, vacuum, *Nutrients, eutrophication,
*Qajganjjtc/:/ew4astes, water pollution sources, *Photosynthesis, carbon
fixation, *Physico chemical, water quality, *Tertiary treatment *Thin
films, vapor compression evaporation, *Wastes, municipal, waste ident-
ification, *Water management.
Troy, NY, Elnora, NY Lake George, NY, Albany, NY Coxsackie, NY
ire. cowRRFIM&Group V\At Q3D, 04, 05A, 05B, 05C, 05D, 05E, 05F, 05G
18. Availability
19. "S irity -ss.
(import)
3C. Secur.,',y C/3SE,
(r-ge) ...
2t. .of
Pages
22, P'ice
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
Abstractor \ Institution
WRS1C 102 (REV. JUNE I97U
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