WATER POLLUTION CONTROL RESEARCH SERIES 12040 DLQ 08/71
Slime Growth Evaluation of
Treated Pulp Mill Waste
U.S. ENVIRONMENTAL PROTECTION AGENCY
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of-pollution
in our Nation's waters. They provide a central source of
information on the research, development and demonstration
activities in the Environmental Protection Agency, through
inhouse research and grants and contracts with Federal, State,
and local agencies, research institutions, and industrial
organizations.
Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications Branch
(Water), Research Information Division, R&M, Environmental
Protection Agency, Washington, B.C. 20^60.
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SLIME GROWTH EVALUATION OF
TREATED PULP MILL WASTE
by
Department of Microbiology
Oregon State University
Corvallis, Oregon 97331
for the
ENVIRONMENTAL PROTECTION AGENCY
Project #12040 DLO
August, 1971
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 60 cents
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EPA Review Notice
This report has been reviewed by the Environmental Protection
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.
ii
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ABSTRACT
The main objectives were to evaluate the slime growth potential of
pulp mill wastes treated by various methods of biodegradation. Wastes
were tested both before and after secondary treatment in order to
determine the type of biodegradable material present in the influent,
determine the extent of fermentation during treatment, and the amount
of biodegraded fermentable compounds discharged in the effluent. These
studies were carried out in an effort to define total carbon, readily
fermentable carbon, and to design a reasonably accurate and sensitive
method for predicting adequate water quality presently measured by BOD.
The gas chromatographic analysis of monosacchardies along with total
carbon and continuous culture proved to be a very effective analytical
procedure. By the use of these techniques, it was possible to prove
beyond a doubt that Sphaerotilus natans would grow in the treated
effluent, that the residual sugars in the treated effluent could be
detected easily by gas chromatography, that weekly variations in
treatment were noted, and that differences between the two treatment
ponds could be easily observed. These variations could not be easily
observed by the usual BOD determinations.
These techniques make it possible to determine when bio-treatment is
adequate, thereby aiding in the design of proper aeration equipment,
better treatment pond design, and a means of monitoring rapidly changes
in influent composition.
Our findings indicate that pond II at the Lebanon treatment plant is
more efficient biologically than pond I, that when the influent flow
rate is high, the total solids and sugar concentrations are high,
and it is during these surges that sugars appear in the treated
effluent at levels which will easily support slime producing micro-
organisms in the outfall and receiving streams.
This report was submitted in fulfillment of Project No. 12040 DLQ
under the sponsorship of the Environmental Protection Agency and
Oregon State University.
iii
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CONTENTS
Section
I Conclusions
II Recommendations
III Introduction
IV Materials and Methods
V Results and Discussion
VI Acknowledgments
VII References
VIII Glossary
Page
1
3
5
9
17
49
51
53
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FIGURES
No. Page
1 Gas Chromatographic Analysis of Monosaccharides Present 28
in the Aeration Pond Influent
2 Gas Chromatographic Analysis of Monosaccharides Present 29
in the Aeration Pond Effluent After Treatment
3 Gas Chromatographic Analysis of Monosaccharides in the 30
Spent Liquor of Continuous Mixed Cultures with Filter
Sterile Aeration Pond Influent as Substrate
4 Gas Chromatographic Analysis of Monosaccharides in the 31
Spent Liquor of Continuous Mixed Cultures with Filter
Sterile Aeration !>ond Effluent as Substrate
5 Gas Chromatographic Analysis of Mill Waste Influent and 32
Effluents Collected on February 16, 1971
6 Continuous Culture Apparatus 34
7 BOD of Mill Waste Influent and Effluent 35
8 Gas Chromatographic Analyses Showing Range of Mono- 36
saccharide Concentration in Aeration Pond I Effluent
9 Gas Chromatographic Analyses Showing Range of Mono- 37
saccharide Concentration in Aeration Pond II Effluent
10 Daily Flow Rate of Mill Wastes 44
11 Total Carbon Concentrations in Filter Sterilized Mill 45
Waste Influent and Effluents
12 Suspended Solids Concentration in Mill Waste Influent 46
and Effluent Grab Samples
13 Estimated Total Monosaccharide Concentration in Mill 47
Waste Influent and Effluent Composite Samples
vi
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TABLES
No. Page
1 Thin Layer Carbohydrate Detection Solvents 12
2 Comparison of Media Used for Sphaerotilus Isolation 18
3 Comparison of CGT and \ Cytophaga Agar for Recovery of 19
Sphaerotilus from Natural Waters
4 Reproducibility of Plate Counts Using \ Cytophaga Agar 20
5 Growth Promoting Properties of Pulp Mill Wastes Assayed 23
by Continuous Culture
6 TLC Band Pattern of Aeration Pond Influent 26
7 Approximate Monosaccharide Concentrations in Aeration 38
Pond I Effluent, 1970-1971
8 Approximate Monosaccharide Concentrations in Aeration 39
Pond II Effluent, 1970-1971
9 Approximate Monosaccharide Concentrations in Aeration 40
Pond Influent, 1970-1971
10 pH Values of Aeration Pond Influent and Effluents Over 42
a Nine-Month Period
11 Temperature Values of Aeration Pond Influent and Effluents 43
Over a Nine-Month Period
vii
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SECTION I
CONCLUSIONS
1. Plate counts of Sphaerotilus as an indicator organism, could
be useful in establishing water quality criteria where organic
pollution is suspected.
2. Total growth yields of Sphaerotilus obtained from continuous
cultures might provide a suitable means for assessing the efficiency
of different treatment methods.
3. BOD determinations alone do not give an accurate picture of the
secondary treatment efficiency at the Lebanon mill.
4. Continuous culture studies combined with total carbon, and total
monosaccharide determinations were found useful in assessing adequate
bio-treatment.
5. Increased aeration of the Lebanon mill influent results in total
bio-oxidation of the readily fermentable compounds, thus reducing
or eliminating slime growth potential in the effluent.
6. Total carbon and total sugar analyses indicate that pond II is
more efficient than pond I for secondary treatment.
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SECTION II
RECOMMENDATIONS
Several possibilities exist which could improve secondary treatment,
thus reducing slime growth potential in the receiving stream at the
Crown Zellerbach mill in Lebanon, Oregon:
1. Increase aeration by increasing aerating capacity.
2. Change placement of aerators so that influent short circuiting
would be avoided.
3. Operate ponds in series rather than parallel, thus insuring minimal
chance for short circuiting. (The ponds were not operated in series
during the period of sugar determinations.)
4. Redesign the treatment facilities in order that retention times
could be adjusted to correspond with the BOD surges in the influent,
or conversely, so that the inflow rate is such that BOD levels
remain relatively constant.
5. Remove biomass from the effluent and thus remove future possible
nutrients for slime growth.
6. Utilize total carbon and monosaccharide analyses either in place
of, or in conjunction with BOD determinations of treatment efficiency.
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SECTION III
INTRODUCTION
Project Plan and Specific Aim; The introduction of sulfite waste
liquors into streams often leads to the development of unsightly
masses of biological slime dominated by Sphaerotilus. This organism
has the ability to extract sufficient nutrients from large volumes
of water containing very low levels of wastes. As a result, it has
developed in areas that are judged to be in excellent condition
with respect to dissolved oxygen and BOD. For example, it has been
shown (10) that Sphaerotilus is able to grow in sulfite waste liquors
at BOD levels as low as 0.06 ppm. These characteristics of Sphaerotilus
add to the difficulty of developing adequate waste treatment procedures
for controlling slime and in establishing suitable water quality
criteria.
Problems arising because of slime growths in streams have been
reviewed by many authors and will not be recapitulated here (4,5,7,9).
It is sufficient to note that masses of Sphaerotilus often interfere
with the aesthetic quality, uses of water and the ecology of a stream.
A number of studies have been carried out on the nutrition and phys-
iology of Sphaerotilus to determine the specific conditions that
promote its development. These studies have shown that it is capable
of growing on a wide variety of carbon and nitrogen sources under
diverse conditions. The major constituents in spent sulfite liquor
which promote the growth of Sphaerotilus include the hexose sugars
and acetic acid. The quality of the receiving water also plays a role
in the stimulation of Sphaerotilus in streams.
One approach for controlling the development of Sphaerotilus in
streams receiving pulp mill wastes has been to employ biological
methods for treating wastes before release into a receiving stream.
The activated sludge process has been found to be effective in reducing
the slime potential of the wastes although growth of Sphaerotilus is
not completely prevented by this treatment (2).
A study was made by personnel of Crown Zellerbach Corporation to im-
prove the effectiveness of waste treatment for controlling biological
growths in streams receiving pulp mill wastes. Facilities were pre-
pared at the Lebanon, Oregon plant of Crown Zellerbach Corporation
for a variety of treatment schedules and environmental parameters to
test the efficiency in the treatment of pulp mill wastes. Two aeration
basins were available for treating the wastes. The following conditions
were investigated by Crown Zellerbach.
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a. The optimum phosphorus concentration for waste treatment.
b. Differences in the effectiveness between series and parallel
operation of the aeration basins.
c. The effect of BOD load on BOD reduction.
d. The effect of retention time on BOD reduction.
e. The influence of treatment temperature on efficiency.
f. The effect of recirculating treated wastes on treatment
efficiency.
In addition to the above conditions, a variety of chemical analyses
were performed by personnel of Crown Zellerbach on samples taken from
inlets and outlets of the aeration basins to assess the effectiveness
of the various treatment. These analyses include determinations of
BOD, COD, dissolved oxygen, pH, total organic carbon, reducing sugars,
total nitrogen, ammonia nitrogen, and ortho-phosphate.
In addition to a chemical analysis performed by Crown Zellerbach on
the treated wastes, the Microbiology Department Water Laboratory at
O.S.U. evaluated the growth promoting properties of the wastes in terms
of slime production. The objectives were to provide the necessary
microbiological investigations to accompany and be closely coordinated
with those being carried out by the Central Research Division of Crown
Zellerbach Corporation.
The objectives as planned were designed to determine the effectiveness
of the presently used experimental treatments of pulp mill waste. The
investigation encompassed the following:
a. Assess the growth supporting properties of paper mill
waste water using a mixed culture (obtained from the
mill fermentation pond) and a pure culture of Sphaerotilus
natans. This includes both the influent and effluent.
b. Develop techniques for determining qualitatively and
quantitatively which type of compounds have been removed
and which have not.
c. Determine what effect engineering changes in the treatment
system have on the biological removal of fermentable
wastes.
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It was anticipated that this investigation would assist engineers
in designing a better and more economical treatment system for removing
biodegradable compounds, many of which cause pollution of Oregon streams
and the surrounding environment.
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SECTION IV
MATERIALS AND METHODS
Initially it was planned to assess the growth promoting potential
of the treated pulp mill wastes using two approaches. One of these
involved the use of six experimental streams available at the Lebanon
site of Crown Zellerbach Corporation. These streams were to be supplied
with fresh water which could be used in preparing various dilutions
of both treated and untreated wastes for study.
Slime production in the streams was to be assessed by several means.
These included a plate count procedure and a slide assay technique.
Attempts to correlate these approaches with the biomass approach
used by Crown Zellerbach personnel were planned.
Plate Count Procedure: One-fourth strength Cytophaga (1/4 CA) was
selected as the medium of choice for recovering Sphaerotilus from
water. This medium contained the following ingredients:
Tryptone 0.125%
Yeast extract 0.125%
Beef extract 0.005%
Sodium acetate 0.005%
Agar 1.5%
Distilled water was used to prepare the medium since it was found that,
at times, tap water was toxic to Sphaerotilus.
The plating procedure consisted of spreading a known quantity of water
on the surface of a 1/4 CA plate. Bent glass rods were used to dis-
tribute the water samples on the plate. To prevent pseudomonad and
motile eubacterial colonies from spreading over the surface of the
agar and interfering with Sphaerotilus growth, the plates were dried
at 37°C for 24 to 48 hours before inoculation. By use of a dry agar
surface it was possible to plate up to 0.5 ml of water directly. The
plates were incubated at 18 C.
Sphaerotilus colonies appeared on the plates after 48 to 72 hours
incubation. The organism was readily recognized on the plates by the
appearance of the colonies. Under 15x magnification, colonies of
Sphaerotilus on 1/4 CA were long and filamentous with a coarse texture
and were generally branched. A high degree of retractility was also
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apparent in the colonies. A colony with some similarity to Sphaerotilus
was produced on 1/4 CA by a Bacillus. It was possible, however, to
differentiate the two organisms by the fact that the Bacillus colony
exhibited filaments with a folded appearance (similar to switchbacks).
Also, the Bacillus colonies were less refractile than those formed
by Sphaerotilus. Questionable colonies could readily be identified by
examining wet mounts of colony material under phase contrast for the
presence or absence of sheaths.
Slide Technique; Clean glass slides were immersed into the stream
for a period of time and the number of Sphaerotilus filaments which
attach to the glass counted.
Another approach for monitoring slime production involved the use of
the continous culture procedure. This method consisted of the addition
of filter sterilized pulp mill wastes at a constant rate to a continous
culture vessel inoculated with Sphaerotilus, and a mixed culture ob-
tained directly from the treatment ponds. The continuous fermentations
were carried out in 500cc Bellco spin flasks (Bellco Glass Co., Vineland,
N.J.). The culture medium (influent, effluent or fortified substrate)
was fed into the flasks using a polystaltic pump (Buckler Model 2-6100,
1327 16th Street, Fort Lee, New Jersey, 07024).
All samples were adjusted to pH 7 and filter sterilized, several hours
after collection. The turn-over rate of medium in the culture vessels
was varied from 24 hrs to 7 days and air was usually supplied at a
constant rate of 2.0 CFH. The flasks were usually run simultaneously
as follows:
Continuous culture I: mixed culture from Pond II inoculated into sterile
influent.
Continuous culture II: a pure Sphaerotilus culture inoculated into
sterile influent.
Continuous culture III: a mixed culture from Pond II inoculated into
sterile effluent.
Collection and Treatment of Samples
Grab Samples: Weekly 15-20 liter grab samples were collected directly
from the influent and effluent flow at the secondary treatment ponds.
Time of collection, plant treatment, pH, and ambient pond temperature
were recorded. These samples were subjected to the following treatment:
a. Centrifuged on a Sharpless continuous centrifuge
(Model TIP) to remove the heavier particulate materials
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(within several hours following collection.)
b. Total suspended solids were determined by drying the
centrifugate wet solids at 105 C under low vacuum for
24-48 hours, and equating this on the basis of mgm/ml
(ppm).
c. When needed for continuous culture studies, the liquid
was filter-sterilized by using an industrial Sietz filter,
following centrifugation. This filter sterilized liquid,
influent or effluent, was used as substrate for the
growth of a mixed culture and of gphaerotilus natans
in continuous growth fermentation flasks.
d. 150 ml portions of the centrifuged samples were treated
with BaCl? until no more precitate formed. The precipitate
was removed by centrifugation and the supernatant
lyophilized. The dried samples were stored for later use
in thin layer chromatography.
e. The above treatment from steps 'a' through 'df was
also carried out on the test samples of influent and
effluent collected from the spent substrate following
continuous culture growth in the laboratory.
24-hr Composite Samples: After setting up refrigeration units in
November, 1970, one liter, cooled (2-9 C), 24-hr composite samples
based on flow rate of influent and effluent were collected at
weekly intervals. These samples were used to quantitate carbo-
hydrates, free volatile acids, alcohols, and total carbon present.
Thin Layer Chromatography
For thin layer chromatographs, carbohydrates were extracted from the
lyophilized solids with 95 percent ethyl alcohol. This decreased the
interference of lignosulfonates and wood pigments resulting in the
development of a better thin layer chromatogram. The alcohol extract
was applied to thin layers of Silica Gel G (30gm) impregnated with
the following solutions (60ml): 0.02M sodium borate buffer; 0.02 M
sodium acetate; 0.1 M sodium bisulfite; 0.02 M boric acid; water.
The solvent mixtures used to develop the chromatograms are shown in
Table 1. Visualization agents employed were mixtures of cone H-SO./
dichromate (charring), cone H SO,/anisaldehyde (specific color de-
velopment), cone H-SO,/napthoresorcinol (specific color development)
and anilin/diphenyfamine (specific color development).
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Table 1
Solvent mixtures used to develop thin layer chromatograms for the
detection of pentoses and hexoses from aeration pond influents and
effluents.
No
1
2
3
Solvent
Acetone
Mixture
- Water
Chloroform - Methanol
Benzene
- Acetic Acid
Composition (ml)
90:10
60:40
20:20:60
- Methanol
n-Butanol - Acetic Acid 85:5:15
- Water 70:15:30
60:30:10
Ethyl Acetate - Acetic Acid 60:15:15:10
- Methanol - Water
n-Propanol - Water 70:30
85:15
90:10
95:5
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Gas-Liquid Chromatography
1. Carbohydrates
a) Preparation of derivatives
i. Silylation (14): Approximately 50 mg of lyophilized
wasted material and 1.0 ml of Tri-Sil (trimethylsilyl -
pyrimidine mixture obtained from Pierce Chemical Co.,
P.O. Box 117, Rockford, 111.) were introduced into a
plastic stoppered vial and shaken vigorously for about
30 seconds. The mixture was allowed to stand for at
least 5 minutes; one or two microliter quantities
were then injected into the gas chromatograph.
Column consisted of 3% SE-30 ultraphase on chromosorb
W(HP) stainless steel 1/8 inch x 6 ft, (SE 30
General Electric Co.)
ii. Alditol Acetate Derivatives (modified method of
Albersheim e_t_ al) (1) : Ten mg of myo-inositol
(internal standard) were added to 50 ml of BaCl_
treated, centrifuged mill waste and the pH adjusted
to about pH 6. Approximately 0.15 gm of sodium
borohydride (NaBH,) was added to reduce the supars
to their respective alcohols. After 30 min of
reaction time at room temperature, the excess NaBH,
was decomposed by the dropwise addition of glacial
acetic acid until evolution of hydrogen gas ceased.
The resulting boric acid was removed by evaporation in
dryness or near dryness on a rotary evaporator "in
vacuo". Ten ml of methyl alcohol was added, the mixture
stirred and evaporated to dryness. This process was
repeated two more times. The mixture was dried in an
oven at 100 - 110°C for 10 minutes. Ten ml acetic
anhydride and about 0.5 gm sodium acetate was added
and the mixture refluxed at 140 for 20 min, then
cooled slightly, removed from condenser and evaporated
to dryness on a rotary evaporator. Five ml of dichloro-
methane were added, the mixture stirred, and centrifuged
at 11,000 - 12,000 rpm for 20 min. Dichloromethane was
decanted off into a test tube and evaporated to 1 ml.
One or two microliter quantities were injected into
the gas chromatopraph for analysis.
b). Preparation of Column (8). One hundred mp ethylene
glycol succinate (EGS) were dissolved in 25 ml of
chloroform and 100 mg ethylene glycol adipate (EGA)
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and 200 mg. XF 1150 were dissolved in 25 ml of acetone.
The above mixtures were poured together quickly, mixed
with 10 gm of gas chrom P, and stirred gently occa-
sionally. After 1/2 hr, it was poured into a fritted
funnel and excess liquid allowed to drain off until
approximately 1 drop/30 sec. The mixture was poured
into a large petri dish and dried. A 4' x 1/8" stainless
steel column was filled by vibration (about 6cc.) and
equilibrated in the GC for about 2-6 Hr at 180 C before
used. It was advisable to flow a low level of helium
gas through the column during equilibration (5 to 10 Ib
helium gas.) All analyses were made using a F & M
High Efficiency Gas Chromatograph (Model 402) with a
Honeywell Strip Chart Recorder (ElectroniK 16) and a
Hewlett-Packard 3370A Integrator. The column was run
isothermally at 180°C. Injection port and detector
temperatures were 215 C and 210 C respectively. The
approximate gas flow rates were as follows: helium
70 ml/min, hydrogen 40 ml/min, and air 230 ml/min. Two
yl was the standard volume injected.
2. Volatile Fatty Acids, Alcohols
Sample Preparation (3): Waste liquor in the amount of
300 ml was adjusted to pH 10 with 3N NaOH in order to
form the sodium salts of the volatile acids. The liquor
was evaporated to dryness and the salts dissolved with
1.6 N H SO . Any remaining solids were filtered out
with Whatman #1 filter paper. One or two microliter
samples were injected into the gas chromatograph. Waste
liquor samples could also be injected without treatment
directly into the gas chomatograph.
Preparation of column. A 6' x 1/8" stainless steel
column was filled by vibration with a packing mixture
prepared as follows: Dissolve 1.0 gm of neopentyl-
glycolsuccinate (NPGS) and 0.1 gm of concentrated
phosphoric acid in about 50 ml of chloroform (enough
to form a smooth slurry with Porapak Q). Once dissolved,
mix with 10 gm 80-100 mesh Porapak 0 and thoroughly
stir the slurry in a small beaker. Evaporate to dryness
and fill column by vibration. Equilibrate at 180°C
for 6 to 12 hrs before using the column. It was advisable
to flow a low level of an inert gas through column
during equilibration (5 to 10 Ibs. helium gas). Analyses
were run on the same GC system described before in
section Ib. Isothermal operation at 160 C was used.
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Injection port and detector temperatures were 198 C
and 195 C respectively. Gas flow rates were approximately
as follows: helium 70 ml/min, hydrogen 20 ml/min and
air 300 ml/min. The standard injection volume was 3 pi.
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SECTION V
RESULTS AND DISCUSSION
Biological Analyses
It was apparent from the outset of this study that the experimental
streams could not be used effectively to assess growth promoting
properties of the wastes unless a constant flow could be established,
and it was hoped that this could be achieved following the planned
modification of the flow regulators. However, the modification never
was completely successful. Initially, considerable effort was expended
in modifying and improving a plate count procedure for the enumeration
of Sphaerotilus in water. A tentative procedure had been developed but
after preliminary testing it was felt that the procedure could be
improved. A variety of media were tested for their ability to recover
Sphaerotilus from water. These included CGT agar as described by
Dondero (6) and various dilutions of Cytophaga agar.
In preliminary experiments a pure culture of Sphaerotilus was
added to stream water and plated on the test media. The results of
these studies are shown in Table 2. It can be seen that higher numbers
of Sphaerotilus were obtained on dilute Cytophaga agar than on the
richer media tested. On full strength Cytophaga agar and on CGT
medium large numbers of eubacterial colonies developed and it is
possible that these organisms inhibited the growth of Sphaerotilus.
The results presented in Table 2 also show that a greater number of
Sphaerotilus were recovered on 1/4 strength Cytophaga agar than on
1/10 strength Cytophaga agar. Furthermore, colonies on the 1/4
strength medium were somewhat larger and more easily counted.
Additional studies were carried out to test this medium for the
recovery of Sphaerotilus from the natural environment. Samples of
water were collected in sterile containers from the experimental
streams receiving pulp mill wastes and were plated on both 1/4 CA
and CGT agar. As shown in Table 3, 1/4 CA was more effective in
recovering Sphaerotilus from natural samples than CGT medium. These
findings confirm the earlier results.
The procedure described above appeared to be suitable for the
recovery and enumeration of Sphaerotilus from natural bodies of
water. Using this procedure it was possible to obtain reproducible
results in triplicate plating tests. As shown in Table 4, each of
the plates prepared from an individual water sample agrees closely
with regard to the number of Sphaerotilus colonies present. It would
be desirable to determine the reliability of this procedure by further
testing with natural waters and to attempt to correlate plate count
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Table 2
Comparison of Media for the Isolation of Sphaerotilus
Medium Number of Sphaerotilus/ml
CGT medium 150
Cytophaga agar 160
l< Cytophaga agar 600
1/10 Cytophaga agar 430
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Table 3
Comparison of CGT and \ Cytophaga Agar for Recovery of Sphaerotilus
from Natural Waters
Medium Number of Sphaerotilus/ml
CGY 0
Jj; Cytophaga agar 300
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Table 4
Results of Triplicate Plating Tests Using ^ Cytophaga Agar
Sample
1
2
3
1
78a
32
31
Plate
2
71
32
35
3
74
28
28
*J
Number of Sphaerotilus colonies per plate
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results with biomass data. It would seem that the number of free
floating Sphaerotilus filaments and swarm cells in a stream should
be proportional to the density of Sphaerotilus in that body of water.
Data from the experimental stream studies which were planned should
have provided information on this aspect. If the plate count procedure
should prove to be a reliable technique for assessing the level of
Sphaerotilus development in streams, it is possible that the procedure
would have application in establishing water quality criteria and
in monitoring the water quality of streams receiving pulp mill wastes
or other wastes which promote Sphaerotilus growth.
A second approach which was planned to assess slime production in
the experimental streams involved a slide technique. Clean glass
slides were immersed into the stream for a period of time and the
number of Sphaerotilus filaments which attached to the glass counted.
Preliminary tests were carried out with this procedure in the ex-
perimental streams which had large populations of attached Sphaerotilus.
It was found that more than a few hours of incubation in the stream
produced slides so heavily laden with Sphaerotilus and silt that
the filaments could not be counted. While the filaments attached to
slides incubated in the stream a short time could be enumerated, it
was felt that further attempts to standardize this procedure would
be of little value until the streams became operative. Hence, no
further experiments were carried out on this aspect of the study.
While the experimental stream facility could provide a worthwhile
approach for assessing slime production, it requires accurate control
of the waste added. Until it becomes possible to regulate the flow
of wastes into the experimental streams reliably, this approach is
of little value. For this reason no attempt was made to sample the
streams on a regular basis.
The third approach for monitoring slime production involved the use
of the continuous culture procedure. This method consisted of the
addition of pulp mill waste, treated and untreated, at a constant
rate to a continuous culture vessel inoculated with natural activated
sludge organisms or Sphaerotilus.
After a known amount of waste passed through the culture vessel, the
dry weight of the resulting growth was determined and an analysis
of the residual sugars made. Preliminary experiments were carried out
to standardize this procedure. Initially it was planned to use a
dilute CGT broth as a basal medium for these studies. This medium
proved to be unsatisfactory, however, since in some cases viability
of the culture was lost before the addition of waste was initiated.
In other cases the growth resulting was not filamentous and considerable
wash-out occurred. A number of other basal media were tested and it
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was found that 1/10 strength Cytophaga broth was the most satisfactory
since filamentous attached growth resulted.
The procedure used to assess slime production by the continuous
culture approach involved inoculating a culture vessel containing 500
ml of 1/10 strength Cytophaga broth with approximately 30 ml of a
broth culture of Sphaerotilus. Growth was allowed to occur in the
vessel for approximately 24 hours before the waste was introduced.
This allowed the reserve material of the cells and the nutrients in
the growth medium to be depleted before the addition of waste.
Following this initial incubation period, 1/10 strength Cytophaga
broth supplemented with a known volume of autoclaved waste was
allowed to flow through the culture vessel at a rate of approximately
150 ml per hour. Incubation was at 25° C. The growth occurring in
the vessel was collected, dried and weighed. While most of the growth
remained in the culture flask, a small amount washed out in the effluent.
For this reason the vessel effluent was collected in a container with
potassium cyanide or acidified sodium hypochlorite and the dry weight
of the cells determined. This was added to the cell yield obtained
in the culture vessel.
In carrying out the analyses on the pulp mill wastes it was necessary
to run a control culture vessel containing the basal medium alone.
The amount of growth obtained in the control was subtracted from the
cell yield obtained in the experimental flasks receiving the wastes.
Also the amount of solids passing through the vessel was determined
and subtracted from the cell yield. As a result, the cell yields
reported for the different wastes represents the quantity of Sphaerotilus
produced due to the nutrients in the wastes added.
Efforts to initiate the regular sampling of wastes was made the
latter part of June, 1969. Unfortunately, it was not possible to
obtain satisfactorily treated wastes at this time since difficulty
was being experienced with the secondary treatment facility due to
a malfunction in the aerators. Nevertheless, preliminary tests were
performed on samples taken on two occasions in June. The cell yields
obtained with these wastes are shown in Table 5. It is interesting
to note that in the samples examined on 30 June, the cell yield
from the secondary effluent was nearly the same as that obtained
with the primary effluent. This would suggest either that the
secondary influent waste sampled at this time was more dilute than
that generally entering the secondary treatment facility or that the
treatment was inefficient in removing nutrients which promote Sphaerotilus
growth.
Regular sampling was begun in August after measures had been taken
to correct the malfunctioning of the secondary treatment facility.
22
-------
Table 5
Growth Promoting Properties of Pulp Mill Wastes as Determined by
Continuous Flow Procedures
Sample Date
6/24/69
6/30/69
8/6/69
8/12/69
8/22/69
Type of Waste
Secondary influent
Secondary influent
Secondary effluent
Secondary influent
Secondary effluent
Secondary influent
Secondary effluent
Secondary influent
Secondary effluent
Milligrams dry weight
Sphaerotilus per liter
waste
96
77
75
113
53
245
120
146
69
23
-------
Both secondary influent and secondary effluent samples were analysed
at approximately weekly intervals. The results obtained with the samples
analyzed in August also are shown in Table 5. It can be seen that the
yield obtained with the secondary effluent was less than that obtained
with the secondary influent collected at the same time. However, this
does not necessarily represent the efficiency of the secondary treatment,
A comparison between the growth promoting properties of the secondary
influent and effluent wastes could be used to assess efficiency of
the treatment only if the chemical composition of the secondary influent
were relatively constant over an extended period of time. However,
this does not appear to be the case when one notes the differences
in the quantity of Sphaerotilus produced in the secondary influent
obtained at approximately weekly intervals. Efficiency of the secondary
treatment process could best be assessed with the continuous culture
method by comparing the cell yields obtained with samples of effluent
waste collected at regular intervals during successive treatment
schedules. The number of samples which should be analyzed before the
project was terminated was not sufficient to permit conclusions to
be made regarding the effectiveness of a given treatment process.
Nevertheless, this approach should provide a suitable means for
assessing the efficiency of different methods of treatment.
Experiments were also carried out to test the possibility of using
acidified wastes for analysis by the continuous culture procedure.
The use of acidified wastes would permit samples to be collected and
preserved for a period of time before analysis. The results obtained
in four tests using acidified wastes was considerably different from
those obtained with the corresponding fresh wastes. Hence acidification
does not seem to be a satisfactory method for preserving wastes for
subsequent analysis of their slime growth promoting properties. (The
above results are those reported by Dr. R. E. Pacha, initial contract
supervisor).
Chemical Analyses
As a means of identifying specific chemical factors which permit
growth of Sphaerotilus, thin layer chromatography (TLC) and gas
chromatography (GC) were used for determining the efficiency of
carbohydrate removal in both the treatment ponds and continuous
culture process.
Thin Layer Chromatogranhy
These studies were qualitative rather than quantitative and were
used in an effort to find a fast and efficient method of separating
and identifying the sugars present in the mill waste.
-------
The heavy trailing encountered during the first attempts to develop
thin layer chromatoprams could he minimized by precipitating the
sulfates in the waste liquor with barium and by reconstituting the
lyophilized solids with 95% ethyl alcohol. Although some sugars are
only slightly soluble in alcohol, it was felt that the quantity of
these sugars present in the waste liquor was sufficiently low to
assure their complete solution.
Best results were achieved when chromatograms were developed twice
in a solvent mixture of propanol and water (85:15, 70:30). As seen
in Table 6 six bands could be distinguished. Visualization of the
bands with ninhydrin showed a slight positive reaction from the origin
up to and including Band I. Bands II through V showed R values
established for the standard sugar mixture. Band III contained at
least four bands so close together that no individual R values
could be assigned. Although the color reactions of the standard
sugars agreed with those of most bands in the region of 5.9 - 10.0
cm from the origin, no definite identification was possible because
the R values were not consistent. Band VI, probably consisting of
low molecular weight substances, was found to travel on the solvent
front.
There appeared to be no detectable differences between chromatograms
developed from effluent samples when based on the mode of operation
of the aeration ponds (parallel vs. series operation). The cultures
grown on untreated influent developed rapid growth with heavy slime
formation. The mixed cultures which were grown on spent effluent
developed slowly over a period of several weeks with slime formation
along the inside wall of the culture vessel. The chromatograms,
except for Bands IV and V appeared identical to those of the fresh
influent.
Increasing the air supply to 2.0 CFH did not increase growth or
alter the chromatographic picture. The fact that all particulate
matter was removed from the effluent by centrifugation and filtration
before being used as a substrate may be a partial explanation.
Bacterial cells make up approximately 150 to 170 ppm of the total
solids. These may recycle and serve as an excellent source of nutrients
for Sphaerotilus and other microorganisms. When this biomass, which
may constitute as much as 30% of the BOD, is removed as is done in
the laboratory, the cultures lack sufficient nutrients for growth
and/or utilization of residual carbohydrates.
The observations made thus far suggest that the quality of the
paper mill effluent could be improved by more efficient aeration in
the treatment ponds and by removal of the bacterial mass from the
final effluent discharged.
25
-------
Table 6
Band pattern of aeration pond influent on Silica Gel G developed
twice in a solvent mixture of n-propanol and water (85:15,70:30).
Band No Distance from Origin Approx Rf
(cm) -
I 3.8 - 4.2 33
II 5.9 - 6.2 50
III 6.4 - 7.5 58
IV 8.0-8.2 67
V 9.3 - 10.0 78
VI 11.0-12.0 95
26
-------
Gas Chromatography
The silylation procedure proved to be exceedingly fast and re-
producible but for purposes of quantitation and even qualitative
evaluation so confusing as to be impractical. Since silylation
involves the conversion of hydroxy and polyhydroxy compounds into
trimethylsilyl ethers, there may be as many as four peaks for each
sugar due to anomerization in addition to the many peaks resulting
from the silylation of phenols, organic acids, and certain amines.
Consequently, the procedure was discontinued in favor of the relatively
new alditol acetate approach which yields only one peak for each
monosaccharide, thus making identification and quantitation feasible.
The results of the initial attempts are shown in Figure 1 and 2.
Figure 2 represents the sugar analysis of an untreated influent
sample entering aeration pond II and Figure 3 an effluent sample
following treatment in pond II. It should be noted that ponds I and
II were operating on a parallel schedule and the samples analyzed
were grab samples rather than 24 hr composites. Based on retention
times for standard sugars (Fig 5a) the carbohydrates in the influent
(Fig. 2) were found to correspond with the retention times of the
pentoses rhamnose, arabinose, and xylose, and the hexoses mannose,
galactose, and glucose. Unidentified are some low molecular weight
aldehydes or ketones which have a shorter retention time than shown
for the standard (Fig 5 b,c,d).
A comparison of the chromatographed influent and effluent indicates
that fermentable carbohydrates still remain following aeration during
7 days retention time. The six monosaccharides were usually present
in varying amounts in both the treated and untreated effluent. How-
ever, not enough nutrients remain in the treated effluent to initiate
or support growth of a suitable mixed flora. Nevertheless, over a
period of seven days or more, Sphaerotilus will become established
and continue to grow at a rate which becomes a problem at outfalls
from paper mill plants.
Initial results with continuous mixed cultures inoculated into
influent and effluent liquor are shown in Fig. 3 and 4 respectively.
Growth is luxuriant on influent liquor and, in particular, the
production of slime as Sphaerotilus. The microorganisms observed in
such a culture are typical of those observed in a healthy bio-oxidation
process. Controlled fermentation in the laboratory results in a more
complete bio-oxidation of the influent carbohydrates than treatment
in the aeration ponds.
Bacterial growth is limited when inoculated into filter sterile
27
-------
» I > I 01
r
a i "
Figure 1. Gas chromatographic analysis of pentoses
and hexoses present in the aeration pond influent.
28
-------
Figure 2. Gas chromatographic analysis of pentoses
and hexoses present in the aeration pond effluent after
treatment.
29
-------
llttSfiflOl
Figure 3. Gas chromatographic analysis of pentoses and
hexoses in the spent liquor of continuous mixed cultures
supplied with filter sterile aeration pond influent as
substrate (500 ml per 24 hours and 2.0 CFH of air).
30
-------
Figure 4. Gas chromatographic analysis of pentoses and hexoses
in the spent liquor of continuous mixed cultures supplied with fil-
ter sterile aeration pond effluent as substrate (500 ml per 24
hours and 2.0 CFH of air).
31
-------
I ' , '
-s*.r~.
I tittittrt It. IITI
Figure 5. Gas chromatographic analysis of mill waste
influent and effluents collected on February 16, 1971.
(a) standard mixture of the six main wood sugars and the
internal standard, myo-inositol, (b) influent; arrows left
to right represent rhamnose, arabinose, xylose, mannose,
galactose and glucose respectively, (c) effluent from
aeration pond I with six wood sugars designated by arrows,
(d) effluent from aeration pond II containing only pen-
toses; rhamnose, arabinose, and xylose as designated by
arrows.
32
-------
effluent. The negligible growth response is due to the fact that
nutrients which support luxuriant growth have been depleted during
the initial treatment process. However, Sphaerotilus will initiate
growth over a period of 7 to 10 days. The fact that Sphaerotilus growth
occurs at all supports the contention that bio-oxidation in the treatment
ponds is not complete. Although the effluent BOD (Fig. 7) was within
the established limits and did not fluctuate appreciably during the
period of this research, Sphaerotilus grew to the exclusion of all
other microflora in the effluent, as demonstrated both in the lab-
oratory and in the outfall of the plant.
Figure 6 shows the continuous laboratory culture method using
Spin Flasks adapted for continuous culture operation. The flask on
the right in the photograph shows waste that was initially inoculated
with a mixed activated sludge culture obtained from the Lebanon
treatment pond. The substrate is a weekly sample of treated
effluent. The turnover rate was approximately 24 hours. Although the
flask was initially inoculated with a mixed culture as indicated above
after a period of approximately 7 to 10 days Sphaerotilus becomes
the predominating organism showing a gradual increase in filament
mass over a period of a month. It would appear that there is sufficient
energy for growth of Sphaerotilus, but not enough for other microbial
species. Upon addition of a 0.1% glucose the microflora will revert
to mixed culture typical of an activated sludge system.
The flask on the left in the photograph is fed a substrate consisting
of the untreated weekly sample. The system was treated identically
to the one on the right. The microflora remain unchanged over the
several months of continuous culture. The substrates in both cases
were filter sterilized to minimize unwanted biological changes
during the test and to remove the biomass and other inert suspended
solids which could serve as additional nutrients in the presence of
the proper microbiological enzymes.
The GC analysis (Fig. 5,8,9, quantitated in Table 7,8,9) shows
the amount of carbohydrates remaining in the secondary effluent
which is sufficient to support the growth of Sphaerotilus. Weekly
variations and differences between the two treatment ponds were
observed in the residual monosaccharides (Fig. 13). Suggested
reasons for these variations will be discussed later.
Laboratory treatment of the untreated influent showed that the
sugars could be bio-oxidized below the level of detection by GC
analysis within 24 hours (Fig. 3). Thus it would appear that the
nitrogen, phosphorus, and other elemental and micronutrients were
present in sufficient quantities to insure complete bio-oxidation.
During the 8-month period that these tests were run, nitrogen in
33
-------
V
Figure 6. Continuous culture apparatus.
34
-------
600
500
ttOO
300
'200
100
<>-© INFLUENT
0--(3 EFFLUENT
DATE OF SAMPLE
Figure 7. BOD of mill waste influent and effluent.
+BOD data obtained,from daily log at Crown Zellerbach,
Lebanon, Oregon.
*Data not available for this date.
35
-------
a
Figure 8. Gas chromatographic analyses showing range
of monosaccharide concentration in aeration pond I ef-
fluent, (a) highest sugar content, (b) average sugar
content, and (c) least sugar content obtained during
study. Arrows in b and c represent respective wood sug-
ars labelled in a.
36
-------
Figure 9. Gas chromatographic analyses showing range
of monosaccharide concentration in aeration pond II ef-
fluent, (a) highest sugar content, (b) average sugar
content, and (c) least sugar content obtained during
study. Arrows in b and c represent respective wood sug-
ars labelled in a.
37
-------
CO
Table 7
Approximate monosaccharide concentrations in aeration pond I effluent, 1970-1971.
Date
11/24
12/1
12/8
12/15
12/22
1/5
1/19
1/26
2/2
2/9
2/16
2/23
3/2
3/9
*
RHAMNOSE
*
A
.0002
.0004
.0006
.0003
0
.0005
0
0
0
.0002
.0002
0
.0014
0
B
1
3
5
2
4
1
1
11
*
.7
.3
.0
.5
0
.2
0
0
0
.7
.7
0
.6
0
ARABINOSE
A
.0010
.0007
.0006
.0005
.0001
.0002
.0002
0
.0002
0
.0001
0
0
0
B
8.3
5.8
5.0
4.1
0.8
1.7
1.7
0
1.7
0
0.8
0
0
0
XYLOSE
A
.0008
.0004
.0009
.0014
.0008
.0019
.0001
.0006
.0001
0
.0011
.0028
.0007
.0004
B
6.7
3.3
7.5
11.6
6.7
15.8
0.8
5.0
0.8
0
9.2
23.2
5.8
3.3
MANNOSE
A
.0002
.0006
.0008
.0027
.0003
.0003
.0004
.0062
.0014
0
.0001
0
0
0
B
1.7
5.0
6.7
22.5
2.5
2.5
3.3
51.6
11.6
0
0.8
0
0
0
GALACTOSE
A
.0003
0
0
.0011
0
0
0
0
0
0
.0001
0
0
0
B
2.5
0
0
9.2
0
0
0
0
0
0
0.8
0
0
0
GLUCOSE
A
.0368
.0054
.0064
.0008
0
.0002
0
0
0
0
0
0 .
0
0
B
306.4
45.0
54.4
6.7
0
1.7
0
0
0
0
0
0
0
0
TOTAL SUGARS**
A
.0704
.1007
.010
.0099
.0045
.0527
.0028
.0080
.0028
.0021
.0017
.0032
.0029
.0048
B
586
838
84
82
37
438
23
66
23
17
14
26
24
40
.2
.5
.1
.4
.5
.8
.3
.6
.3
.5
.2
.6
.1
.0
***
55.8%
7.4%
93.4%
68.6%
26.6%
5.9%
24.8%
84.9%
60.5%
9.7%
93.6%
87.2%
72.1%
8.2%
grams per liter, approximately
B = pounds per million gallons, approximately
**Total sugars indicates the concentration of the six identified sugars plus other unidentified
monosaccharides observed on the gas chromatograph
***Percent monosaccharides identified
-------
Table 8
CO
VO
12/15
12/22
1/5
1/19
1/26
2/2
2/9
2/16
2/23
3/2
:imate monosaccharide
RHAMNOSE
* *
A B
0
.0009
.0004
0
.0004
0
0
0
.0005
.0003
.0001
0
7.5
3.3
0
3.3
0
0
0
4.2
2.5
0.8
concentrations in aeration
ARABINOSE
A B
.0001
.001
0
.0002
0
.0005
0
0
0
.0003
0
0.8
0.8
0
1.7
0
4.2
0
0
0
2.5
0
XYLOSE
A
0
.0002
0
.0001
0
.0005
.0006
.0001
.0002
.0003
.005
B
0
1.7
0
0.8
0
4.2
5.0
0.8
1.7
2.5
4.2
pond II effluent, 1970-1971.
MANNOSE
A B
0
.0004
.0005
.0012
.0020
0
.0003
.0016
0
0
0
0
3.3
4.2
10.0
16.6
0
2.5
13.3
0
0
0
GALACTOSE
A B
0
0
.0003
.0003
0
0
0
0
0
0
0
0
0
2.5
2.5
0
0
0
0
0
0
0
GLUCOSE
A B
.0305
.0070
.0022
0
0
0
0
0
0
0
0
254.0
58.3
18.3
0
0
0
0
0
0
0
0
TOTAL SUGARS**
A B
.0601
.0500
.0038
.0019
.0115
.0013
.0007
.0022
.0063
.0016
.0009
500.4
416.4
31.6
15.8
95.8
10.8
5.8
18.3
52.5
13.3
7.5
***
50.9%
17.1%
89.5%
94.9%
20.7%
77.7%
129.3%
77.0%
11.2%
56.3%
66.6%
0
.0001 0.8 .0001
0.8 .0016
3/9 .0003 2.5 .0001 0.8 .0033 27.5
*
A^» grams per liter, approximately
B = pounds per million gallons, approximately
**Total sugars indicates the concentration of the six identified sugars plus other unidentified
monosaccharides observed on the gas chromatograph
***Percent monosaccharides identified
13.3 243.6%
-------
Table 9
Approximate monosaccharide concentrations in aeration pond influent, 1970-1971.
Date
11/24
12/1
12/8 .
12/15
12/22
1/5 .
1/19
1/26
2/2
2/9
2/16
2/23
3/2
3/9
RHAMNOSE
* *
A B
.0004
0018
.0003
.0012
0003
0
0
0
0
0
0
.0003
.0009
3.3
15.0
2.5
10.0
2.5
0
0
0
0
0
0
2.5
7.5
ARABINOSE
A B
.0055 45.8
.0037 30.8
.0013 10.8
.0085 70.8
.0023 19.2
.0020 16.6
.0048 40.0
.0026 21.6
.0025 20.8
.0039 32.5
.0021 17.5
.0059 49.1
.0022 18.3
XYLOSE
A B
.0103
.0063
.0034
.0126
.0051
.0028
.0062
.0041
.0066
.0060
.0022
.0383
.0046
85.8
52.5
28.3
104.9
42.5
23.3
51.6
34.141
55.0
50.0
18.3
318.9
38.3
MANNOSE GALACTOSE
A BAB
.0174
.0155
.0074
.0347
.0032
.0015
.0073
.0096
.0033
.0123
.0025
.0005
.0051
144.9
129.1
61.6
288.9
26.6
12.5
60.8
79.9
27.5
102.4
20.8
4.2
42.467
.0058
.0072
.0028
.0120
.0021
.0019
.0030
.0018
.0016
.0077
.0017
.0017
.0024
48.3
60.0
23.3
99.9
17.5
15.8
25.0
15.0
13.3
64.1
14.2
14.2
20.0
GLUCOSE
A B
.0123
.0024
.0009
.0051
0
0
0
0
.0009
.0005
.0003
.0098
0
102.4
20.0
7.5
42.5
0
0
0
0
7.5
4.2
2.5
81.6
0
TOTAL SUGARS**
A B
.0673
.1321
.0591
.0799
.1550
.0132
.0231
.0190
.0196
.0373
.0099
.0574
.0165
560.4
1100.0
492.1
665.3
1290.7
109.9
192.4
158.2
163.2
310.6
82.4
478.0
137.4
***
76.8%
27.9%
27.2%
92.7%
8.3%
62.0%
92-2%
95.2%
76.0%
81.5%
88.9%
98.4%
92.1%
*
A^= grains per liter, approximately
B = pounds per million gallons, approximately
**Total sugars indicates the concentration of the six identified sugars plus other unidentified
monosaccharides observed on the gas chromatograph
***Percent monosaccharides identified
-------
the form of ammonia and phosphorus in the form of phosphoric acid
was added in excess daily. The pH of the influent was approximately
7.3. However, this could vary over a range of 4.3 to 9.5 (Table 10),
while the treated effluent was fairly constant at about 6.8 (Table
10). The daily inflow varied from 3.77QMGD to 6.17 MGD (Fig. 10).
The temperature varied from 13 C to 22 C over the same period
(Table 11). The retention time depending upon the inflow rate was
approximately seven days. Only trace amounts of acetic acid and
methyl and ethyl alcohols were detected, if at all, by GC analysis of
the secondary influent and effluents.
The factors which appeared to have the greatest effect on the
efficiency of the treatment system are the influent flow rate
(Fig. 10) and the rate of aeration. If the inflow rate (Fig. 10) is
compared with total carbon (Fig. 11), total suspended solids (Fig.
12) and total monosaccharides (Fig. 13), it can be observed that
inflow increases generally coincide with increases in suspended
solids, total carbon, and monosaccharide concentrations in both
the influent and effluent x^astes. The occurence of these marked
changes was not reflected in changes in the effluent BOD (Fig. 7).
This would indicate that reliance on BOD as the only indicator of
the efficiency of treatment could lead to an erroneous conclusion.
The present design parameters as well as the capacity are approx-
imately identical. The only difference between the two ponds is
the aeration systems. Treatment pond II was aerated by two 75 HP
aerators while pond I was aerated by six 25 HP units. The horsepower
input on both was identical. Thus the two 75 HP units appear to do
a more effective aeration job resulting in a better biodegradation.
Several factors may account for the incomplete oxidation observed.
The placement of the aerators may be such that not all the influent
waste is subjected to sufficient aeration due to short circuiting,
and appears in the effluent discharge. The aerators may not have
the capacity to properly aerate the intermittent surges which con-
tain an abnormally high content of biodegradable substances. A
properly designed pond could insure that the retention time of all
volumes and levels of waste solids was such that the carbohydrates
are completely bio-oxidized. This would probably involve a redesign
of the treatment ponds, increased aeration efficiency or capacity,
or a different placement of the aerators.
41
-------
Table 10
pH values of aeration pond influent and effluent over a 9-month period,
DATE AND
SAMPLE
July, 1970
Influent
Effluent
August
Influent
Effluent
September
Influent
Effluent
October
Influent
Effluent
November
Influent
Effluent
December
Influent
Effluent
January, 1971
Influent
Effluent
February
Influent
Effluent
March
Influent
Effluent
AVERAGE pH
7.7
7.0
7.6
6.6
5.2
6.9
8.4
6.6
7.2
6.8
7.9
6.4
7.2
5.9
7.2
6.3
7.6
6.4
LOW
6.6
6.7
7.2
5.8
4.3
6.5
7.6
6.1
6.5
6.1
7.0
5.9
7.1
5.9
6.8
5.8
7.1
5.9
RANGE
HIGH
8.8
7.4
8.0
7.3
9.5
7.1
9.6
6.9
7.6
7.2
9.5
7.2
7.4
5.9
8.3
6.8
8.2
6.9
42
-------
Table 11
Temperature values of aeration pond influent and effluent over a
9-month period.
DATE AND
SAMPLE
July, 1970
Influent
Effluent
August
Influent
Effluent
September
Influent
Effluent
October
Influent
Effluent
November
Influent
Effluent
December
Influent
Effluent
January, 1971
Influent
Effluent
February
Influent
Effluent
March
Influent
Effluent
AVERAGE
TEMPERATURE
°C
35
27
35
27
30
28
29
21
28
20
19
16
25
16
29
21
23
16
RANGE
LOW
35
27
33
26
21
19
29
18
23
18
16
14
24
16
25
19
19
16
HIGH
35
27
36
27
35
25
29
23
32
21
23
17
26
16
32
22
27
17
43
-------
6,0-
5.5
5.0-
4,5;-
4.0
3,5-
X S X X X X X X* X S
DjifB OF ^
Figure 10. Daily flow rate* of mill wastes
rate data obtained from daily log at Crown
Zellerbach, Lebanon, Oregon.
44
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6QO
550
500
i»50
too
I
350
300
250
©O INFLDBNT
QO EPFUIINT, Pond I
EFFLUENT, Pond II
rHl *» - *N>
\ X. N,
fsj i^- en-
DATS OF SAMPLE
Figure 11. Total carbon concentration in filter
sterilized mill waste influent.and effluents.
*Data not available for these dates.
45
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wot
140
120
100
60
S 20
5
.Q-*-Q" INFLUENT
'© EFFLUENT, Pond II
«3 *»>
'O* ,°Ch fl CM ^tf {*,
s v, v, v v. s;
DATE OP SAMPLE
Figure 12. Suspended solids concentration in mil]
waste influent and effluent grab samples.
*Data not available for these dates.
46
-------
,800
4600
SHOO
300
200
100
DATE OP
Figure 13. Estimated total monosaccharide concentra-
tion in mill waste influent and effluent composite samples.
*Data not available for these dates.
47
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SECTION VI
ACKNOWLEDGMENTS
We acknowledge with sincere thanks the assistance of Mr. Ken
Byington and the personnel at the Lebanon Crown Zellerbach paper
mill, Lebanon, Oregon, and also the personnel at the Central
Research Division of Crown Zellerbach, Camas, Washington.
With a special thanks, we acknowledge the support by the Environ-
mental Protection Agency and the assistance provided by Mr. Don
May and Dr. Kirk Willard at the Pacific Northwest Water Laboratory.
49
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SECTION VII
REFERENCES
1. Albersheiin, P., Nevins, D. J., English, P. D. and Karr, A.,
"A Method for the Analysis of Sugars in Plant Cell-Wall
Polysaccharides by Gas-Liquid Chromatography," Carbohydrate
Research, _5, pp. 340-345 (1967).
2. Amberg, H. R. and Cormack, J. F., "Factors Affecting Slime
Growth in the Lower Columbia River and Evaluation of Some
Possible Control Measures," Pulp and Paper Mag. Can., 61,
pp. 70-80 (1960).
3. Baumgardt, B. R., "Practical Observations on the Quantitative
Analysis of Free Volatile Fatty Acids (VFA) in Aqueous Solutions
by Gas-Liquid Chromatography," Dept. Bulletin I, Dept. of
Dairy Science, U. of Wisconsin, Madison, 12 pages (June, 1964).
4. Curtis, E. J. C., "Sewage Fungus: Its Nature and Effects,"
Water Research, _3, pp. 289-3li (1969).
5. Dondero, N. C., "Sphaerotilus, Its Nature and Economic Sig-
nificance," Advanced Agplied_ Microbiology^, 16, pp. 276-278 (1968).
6. Dondero, N. C., Phillips, R. A. and Heukelekian, H., "Isolation
and Preservation of Cultures of Sphaerotilus," Applied Micro-
biology, f, pp. 219-227 (1961).
7. Harrison, M. E. and Heukelekian, H., "Slime Infestation-Literature
Review," Sewage Ind. Wastes, 30, pp. 1278-1302 (1958).
8. Maglothin, P., Personal Communication, University of Colorado,
Dept. of Chemistry, Boulder, Colorado, September 30, 1970.
9. Molder, E. G., "Iron Bacteria, Particularly Those of the
Sphaerotilus-Leptothrix group and Industrial Problems." J_.
Applied Bacteriology, 27, pp. 151-173.
10. Scheuring, L. and Hohnl, G., "Sphaerotilus natans Sein Okologie
und Physiologic," Schriften ver. Zellstoff Papier-Chem Ing.,
2£, pp. 1-151 (1956).
11. Sweeley, C. C., Bentley, R., Makita, M., and Wells, W. W.,
"Gas-Liquid Chromatography of Trimethylsilyl Derivatives of
Sugars and Related Substances," J.A.C.S., JJ5, pp. 2497-2407 (1963)
51
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SECTION VIII
GLOSSARY
Sphaerotllus - A sheathed bacteria commonly found in large slime
masses in flowing waters where organic pollution exists.
BOD - Biochemical oxygen demand is the amount of oxygen utilized
during microbial degradation or oxidation of organic material. With
large amounts of organic material, waters may be totally depleted of
free oxygen due to this phenomenon.
COD - Chemical oxygen demand measures the oxygen equivalent of organic
matter which is susceptible to oxidation by a strong chemical oxidant.
Biodegradable - Substances able to be broken down by living organisms.
Influent - Mill wastes which flow into treatment ponds.
Effluent - Wastes liquors which flow out of treatment ponds.
Continuous culture - A system where substrate is continuously fed
into a culture vessel of a specific volume. Spent substrate and
cells flow out at the same rate, thus keeping the volume constant.
The vessel culture may be aerated, cooled or heated, pH adjusted,
dissolved oxygen measured, etc.
Grab sample - A sample collected at one time and from one location,
thus not truely representing over-all conditions.
Lyophilized - This term is synonymous with freeze-drying; a process
which depends on extreme cold and vacuum. Water is removed by passing
from the solid to the vapor state without melting.
Biomass - The living organisms which are found in a particular system.
R- value - This value represents the distance traveled by each com-
pound from the origin relative to the solvent front, or
distance from base line traveled by compound.
f distance from base line traveled by solvent
The R- value is characteristic of a particular compound measured under
specific conditions such as; solvent system, temperature, manner of
development, type of paper or layer, and the grain direction of the
paper or layer.
53
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TLC bands - During TLC, a compound will move a specific distance
from the origin forming a spot or band. This band is characteristic
of only that compound under the specific conditions of the run. Thus,
two distinct and sharp bands in TLC would represent two different
compounds.
Pentoses - Monosaccharides containing five carbon atoms.
Hexoses - Monosaccharides containing six carbon atoms.
Short circuiting - Influent bypassing aerators without adequate aeration
resulting in a partially treated discharge.
Retention time - The time from sample injection into a system, such
as in GC or aeration ponds, until the same sample leaves the system.
54
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1
Accession Number
w
5
« Subject Field & Group
05 AD
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Oregon State University , Department of Microbiology, Corvallis, Oregon
Title
Slime Growth Evaluation of Treated Pulp Mill Waste
10
Authors)
A. W. Anderson
G. A. Beierwaltes
16
Project Designation
120^0 DIft
21
Note
22
Citation
23
Descriptors (Starred First)
*Sphaerotilus,
*pulp wastes,
*waste treatment, fermentation, gas chromatography, slime
25
Identifiers (Starred First)
*slime growth potential, secondary treatment, gas chromatography, monosaccharides,
total carbon, continuous culture, aeration.
27
Abstract
The main objectives were to evaluate the slime growth potential of pulp mill
wastes treated by various methods of biodegradation. Wastes were tested both before and
after secondary treatment in order to determine the type of biodegradable material present
in the influent, determine the extent of fermentation during treatment, and the amount of
biodegraded fermentables discharged in the effluent. The studies were carried out in an
effort to define total carbon, readily fermentable carbon, and to design a reasonably
accurate and sensitive method for predicting adequate water quality presently measured
by BOD.
Gas chromatographic analysis of monosaccharides, total carbon, and continuous
culture proved to be effective analytical procedures. These techniques showed that
Sphaerotilus natans would grow in treated effluent, that residual sugars in the treated
effluent could be detected easily by GC, that total solids and sugar concentrations par-
allel influent flow rate, that during influent surges, effluent sugar concentrations are
high enough to support slime growth, that weekly variations were evident, and that dif-
ferences between the two treatment ponds could be observed. These variations could not be
easily observed by the usual BOD determinations. Thus, adequate biotreatment could be
determined by these three methods. (There are 11 references).
Abstractor
A. W. Anderson
Institution
Oregon State University
WR:I02 (REV. JULY 1969)
WRSI C
SEND. WITH COPY OF DOCUMENT, TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 20240
* SPO: 1970-389-930
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