PB85-226900
Anaerobic-Aerobic Treatment Process for the Removal of Priority
Pollutants
Vanderbilt University-
Nashville, Tennessee
Jun 85
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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EPA/600/2-85/077
June 1985
ANAEROBIC-AEROBIC TREATMENT PROCESS
FOR THE REMOVAL OF PRIORITY POLLUTANTS
by
Zahava Slonim, Li-Ta Lien,
W. Wesley Eckenfelder and John A. Roth
Vanderbilt University
Nashville, Tennessee 37235
Grant No. CR810229
Project Officer
Thomas Short
Subsurface Systems Branch
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA/600/2-85/077
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
ANAEROBIC-AEROBIC TREATMENT PROCESS FOR THE REMOVAL
OF PRIORITY POLLUTANTS
5. REPORT DATE
June 1985
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Zahava Slonim, Li-Ta Lien, Wesley Eckenfelder,
John Roth
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Vanderbilt University
Nashville, TN 37235
10. PROGRAM ELEMENT NO.
ABRD1A
Coop. Agr.
CR810229
12. SPONSORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Ada, OK 74820
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The removal of 4,6-dinitro-o-cresol (DNOC) was investigated using an anaerobic
recycle fluidized bed reactor as a pretreatment stage followed by an activated sludge
reactor.
The DNOC was completely converted during the anaerobic pretreatment stage for
influent DNOC concentrations as high as 600 mg/1. While complete conversion of DNOC
occurred during the anaerobic pretreatment stage, there was only 25% COD removal.
The subsequent aerobic activated sludge stage reduced the anaerobic stage effluent
COD by 80%, resulting in about 85% overall removal.
Batch tests established a range of DNOC loading rates for the anaerobic fluidized
bed. The batch tests also indicated that DNOC did not degrade in the absence of a
readily biodegradable co-substrate, and could not be used as a single carbon source
by the anaerobic bacteria. This investigation used sucrose as the co-substrate.
Anaerobic DNOC biodegradation was found to be a function of sucrose concentration.
Previous investigation of aerobic treatment of DNOC using conventional activated
sludge process showed that DNOC removal is less than 25% and the concentration of DNOC
that is tolerated by activated sludge microorganisms is only about 50-60 mg/1. The
present investigation demonstrated that anaerobic-aerobic treatment is an effective
treatment process for the removal of DNOC.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
1 ??
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (R«v. 4-77) PREVIOUS EDITION is OBSOLETE
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NOTICE
THIS DOCUMENT HAS BEEN REPRODUCED
FROM THE BEST COPY FURNISHED US BY
THE SPONSORING AGENCY. ALTHOUGH IT
IS RECOGNIZED THAT CERTAIN PORTIONS
ARE ILLEGIBLE, IT IS BEING RELEASED
IN THE INTEREST OF MAKING AVAILABLE
AS MUCH INFORMATION AS POSSIBLE.
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DISCLAIMER
The information in this document has been funded wholly or in part
by the United States Environmental Protection Agency under cooperative
agreement CR810229 to Vanderbilt University. It has been subjected to
the Agency's peer and administrative review and has been approved for
publication as an EPA document. Mention of trade names or commercial
products does not constitute endorsement or recommendations for use.
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FOREWORD
EPA is charged by Congress to protect the Nation's land, air, and water
systems. Under a mandate of national environmental laws focused on air and
water quality, solid waste management and the control of toxic substances,
pesticides, noise, and radiation, the Agency strives to formulate and imple-
ment actions which lead to a compatible balance between human activities and
the ability of natural systems to support and nurture life.
The Robert S. Kerr Environmental Research Laboratory is the Agency's
center of expertise for investigation of the soil and subsurface environment.
Personnel at the Laboratory are responsible for management of research pro-
grams to: (a) determine the fate, transport and transformatiqn rates of
pollutants in the soil, the unsaturated zone and the saturated zones of the
subsurface environment; (b) define the processes to be used in characterizing
the soil and subsurface environment as a receptor of pollutants; (c) develop
techniques for predicting the effect of pollutants on ground water, soil and
indigenous organisms; and (d) define and demonstrate the applicability and
limitations of using natural processes, indigenous to the soil and subsurface
environment, for the protection of this resource.
This project was undertaken to evaluate the use of anaerobic pretreatment
for combined industrial/municipal waste treatment systems that treat organic
priority pollutants with an aerobic activated sludge process. The results of this
study indicate that 4,6-dinitro-o-cresol, a priority pollutant, can be almost
completely removed by anaerobic pretreatment provided a readily biodegradable
co-substrate is present. This information should be useful to those responsible
for regulating, designing, and operating combined industrial/municipal waste
treatment systems.
Clinton W. Hall
Director
Robert S. Kerr Environmental
Research Laboratory
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ABSTRACT
The removal of 4,6-dinitro-o-cresol (DNOC), a phenolic priority pollutant,
and the removal kinetics were investigated using an anaerobic recycle fluidized
bed reactor as a pretreatment stage followed by activated sludge reactor as
the aerobic treatment stage.
The DNOC was completely converted during the anaerobic pretreatment stage
(anaerobic bed effluent DNOC concentration «1 mg/1), when the influent DNOC
concentrations were as high as 600 mg/1. It was also found that while 100%
conversion of DNOC occurred during the anaerobic pretreatment stage, there was
approximately 25% COD removal. The subsequent aerobic activated sludge stage
reduced the anaerobic stage effluent COD by approximately 80%, resulting in about
85% overal1 removal.
Batch test studies employing controlled feed to the anaerobic microorganisms
provided preliminary data that were used for establishing a range of DNOC loading
rate onto the anaerobic fluidized bed. The batch test studies also provided early
indications that DNOC was highly toxic to the anaerobic microorganisms in the
absence of a readily biodegradable co-substrate, and could not, therefore, be used
as a sole carbon source by the anaerobic bacteria. Sucrose was used as the
co-substrate in this investigation. The anaerobic DNOC biodegradation was found
to be a function of sucrose concentration.
Previous investigation of aerobic treatment of DNOC using conventional
activated sludge process showed that DNOC removal is less than 25% and the
concentration of DNOC that is tolerated by activated sludge microorganisms is only
about 50-60 mg/1. The results of the present investigation demonstrate that the
iv
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anaerobic-aerobic treatment process is an effective treatment process for the
removal of DNOC.
This report was submitted in fulfillment of grant no. CR810229-01-1 by
Vanderbilt University under the sponsorship of the U.S. Environmental Protection
Agency. This report covers the period June 2, 1982 to May 31, 1984 and work was
completed as of May 31, 1984.
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CONTENTS
Foreword i i i
Abstract iv
Figures viii
Tab! es ix
Abbreviations and Symbols xi
Acknowledgement xii
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. State-of-the-Art Review 6
5. Experimental 22
6. Results and Discussion 30
References 64
Appendices
A. Experimental Apparatuses and Procedures
Anaerobic Shaker Bottle Batch Test Apparatus
and Procedures 70
Anaerobic Continuously-Stirred Batch Test
Apparatus and Procedures 72
Pilot Plant Apparatus and Procedures 76
B. Anaerobic Continuously-Stirred Batch Tests Results 78
C. Methods Used in Analytical Procedures 107
Preceding page blank
vii
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FIGURES
Number Page
1. Schematic of pilot plant apparatus 25
2. NaCl tracer curve for the anaerobic column 26
3. A linearized NaCl tracer curve for the anaerobic
column reflecting a first-order mixing in the column 27
4. The distribution of biomass in the anaerobic column 28
5. The effect of sucrose concentration on the removal of
DNOC i n conti nuously-sti rred batch test 48
6. The effect of sucrose/DNOC concentration ratio on the
removal of DNOC in the anaerobic column, run 1 52
7. The effect of sucrose/DNOC concentration ratio on the
removal of DNOC in the anaerobic column, run 2 53
S. The effect of influent sucrose concentration on the
removal of COD by anaerobic column and activated
sludge, run 1 59
?. The effect of influent sucrose concentration on the
removal of COD by anaerobic column and activated
sludge, run 2 60
viii
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TABLES
Number Page
1. Physical and chemical properties of
4,6-dinitro-o-cresol (DNOC) 7
2. Summary of aquatic fate of DNOC 9
3. Biomass distribution in the fluidized bed 29
4. Anaerobic shaker bottle batch test results 31
5. Results of shaker bottle batch test using
filtered primary sludge as co-substrate 32
6. The effect of sucrose concentration on the
co-metabolism of DNOC in continuously-
stirred batch test 33
7. The performance of the anaerobic col umn 34-36
8. The performance of the activated sludge reactor 37-39
9. Removal of DNOC in the anaerobic column 41
10. The effect of hydraulic retention time (HRT)
on the anaerobic column performance 42
11. By-products of the degradation of DNOC and
sucrose as identified by GC/MS 43
12. The effect of sucrose concentrations on the
co-metabolism of DNOC in continuously-
stirred batch test, run 1 45
13. The effect of sucrose concentrations on the
co-metabolism of DNOC in continuously-
stirred batch test, run 2 46
14. The effect of sucrose concentrations on the
co-metabolism of DNOC in continuously-
stirred batch test, run 3 47
15. The effect of DNOC concentrations on the
co-metabolism of DNOC in continuously-
sti rred batch test 50
16. The effect of high DNOC concentrations on
the co-metabolism of DNOC in continuously-
stirred batch test 51
IX
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TABLES (Continued)
Number Page
17. The effect of anaerobic column hydraulic retention
time (HRT) on soluble COD removal by the anaerobic-
aerobic system 55
18. The effect of influent sucrose concentration on soluble
COD removal by the anaerobic-aerobic system 57
19. The effect of influent sucrose concentration on Microtox
toxicity through the anaerobic-aerobic system.,, . . 62
20. Microtox toxicity data for the anaerobic-aerobic system 53
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ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
AC
AS
ATP
COD
CODS
DNOC
HRT
ymhos/cm
mV
N.D.
ORP
Q
RHRT
SS
t
TOA
TOC
VSS
anaerobic column
activated sludge
adenosine triphosphate
chemical oxygen demand
soluble chemical oxygen demand
4,6-di ni tro-o-cresol
effective concentration of toxicant
causing 50% reduction in light output
in Microtox toxicity test
hydraulic retention time (based on
influent flow rate)
micromhos per centimeter
millivolts
not detected
oxidation-reduction potential
flow rate
recycle hydraulic retention time
suspended solids
time
total organic acids
total organic carbon (soluble)
volatile suspended solids
SYMBOLS
CH4
C02
HC03
NaHC03
Na2S04-H2S04
N2
(NH4)2HP04
N05
methane
carbon dioxide
-- bicarbonate ion
-- sodium bicarbonate
-- sodium sulfate-sulfuric acid solution
-- nitrogen
-- dibasic ammonium phosphate
-- nitrite ion
xi
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ACKNOWLEDGMENT
We wish to thank Dr. Donald H. Kampbell from the U.S. E.P.A. Robert
S. Kerr Environmental Research Laboratory, Ada, Oklahoma for performing
the GC/MS analysis.
xii
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SECTION 1
INTRODUCTION
4,6-Dinitro-o-cresol (DNOC), one of the U.S. EPA priority pollutants,
is a yellow crystalline solid derived from ortho-cresol. DNOC was
introduced in 1892, in its potassium salt form, as the active ingredient
of the pesticide "Antinonin", used for controlling the nun moth. It is
among the most toxic of all compounds used as herbicides. Currently, it
is used primarily as a blossom-thinning agent on fruit trees and as a
fungicide, insecticide, and miticide on fruit trees during the dormant
season. It also has limited use in the dye stuff industry and for other
minor industrial purposes.
DNOC has been detected in the following industrial wastewaters:
organics and plastics manufacturing industries, pesticides manufacturing
industry, rubber manufacturing industry, wood-preservatives industry,
pharmaceutical manufacturing industry, coal mining, foundries, and iron
and steel manufacturing industry (5,6).
Literature on the degradation of DNOC shows that it undergoes biode-
gradation by pure soil microorganisms cultures under aerobic conditions.
However, results from laboratory scale activated sludge studies (1) show
that the removal of DNOC in the activated sludge process is less than 25%
and the concentration of DNOC that is tolerated by activated sludge micro-
organisms is only about 50-60 mg/1. These results indicated that the bio-
logical treatment of DNOC by activated sludge process is unsatisfactory.
Data from physical and chemical treatment of DNOC (5,67,52) also show that
these treatment alternatives might be technologically or economically
1
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unfeasible. Very little information is available about the anaerobic de-
gradation of most organic compounds, but some data indicate that aromatic
nitro groups can be reduced to ami no groups in anaerobic conditions. It was
also shown that some nitro-aromatic compounds that can be degraded under
anaerobic conditions are not degraded under aerobic conditions. All these
facts encouraged the study on the fate of DNOC under anaerobic conditions.
Treatment of DNOC by single anaerobic process alone risks inactivating
the environment-sensitive methanogens, in which case the organic loading of
the pollutant will not be reduced. It is also known that the benzene nu-
cleus, which most likely may still exist if DNOC is just cometabolized to
another aromatic compound during the anaerobic process, will undergo ring
cleavage in the presence of oxygenase under aerobic conditions. Anaerobic
pretreatment-aerobic treatment scheme for treating wastes containing DNOC is,
thus, very promising in achieving the purposes of biodegrading the priority
pollutant, DNOC, as well as lowering the organic loading of the waste.
The research reported here describes a pilot scale study on the
anaerobic-aerobic biological degradation of 4,6-dinitro-o-cresol. It pro-
vides an assessment of the parameters controlling its biodegradation, and an
assessment of its biodegradation by-products and their toxicity.
2
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SECTION 2
CONCLUSIONS
A biological treatment system for the removal of 4,6-dinitro-o-cresol
(DNOC), a phenolic priority pollutant, has been designed and tested. The
system was comprised of an anaerobic fluidized bed reactor as a pretreatment
stage followed by an activated sludge reactor as the aerobic treatment stage.
The results of this investigation show that, in contrast to the failure of
the conventional activated sludge process to remove DNOC (removal of DNOC
in the activated sludge process is less than 25% and the inhibitory thres-
hold of DNOC by the activated sludge microorganism is 50-60 mg/1), DNOC was
completely converted during the anaerobic pretreatment stage. The anaerobic
bed effluent DNOC concentration was less than 1 mg/1, with a DNOC concen-
tration in the influent up to 600 mg/1. It was also found that, while 100%
conversion of DNOC occurred during the anaerobic pretreatment stage, there
was less than 25% removal of COD during that stage. In the activated sludge
process most of the COD was removed resulting in 80-90% overall COD removal.
GC/MS preliminary data indicate that the anaerobic pretreatment stage induced
an initial structural conversion in the DNOC molecule, causing it to become
more biodegradable by the aerobic microorganisms in the activated sludge
process.
The initial breakdown of DNOC in the anaerobic pretreatment stage was
due to co-metabolism. DNOC itself was toxic to the anaerobic microorganisms
if no other, readily biodegradable, co-substrate was available to them. DNOC
cannot be, therefore, used as a sole carbon source by the anaerobic bacteria.
Sucrose has been used as the co-substrate in this study. The data suggest
that there is a relationship between influent sucrose concentration and the
performance of the anaerobic pretreatment stage in the conversion of DNOC. A
3
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ratio of sucrose to DNOC of 2:1 or higher resulted in a 95-100% conversion of
DNOC in the anaerobic pretreatment stage. The anaerobic microorganisms failed
to co-metabolize DNOC when the sucrose to DNOC influent concentration ratio
was less than 2:1.
Toxicity test results show that effluent from the anaerobic pretreatment
stage was more toxic to Microtox luminescent bacteria than the influent
into the anaerobic pretreatment stage. This increase in toxicity was pro-
bably due to the accumulated anaerobic degradation by-products of sucrose
and DNOC. However, after undergoing the aerobic treatment stage, the effluent
from the activated sludge reactor was less toxic to the Microtox luminescent
bacteria.
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SECTION 3
RECOMMENDATIONS
1) It is recommended that additional investigations be performed to corre-
late effluent quality with influent loading changes and obtain more data
concerning the kinetics of the reaction that is taking place in the anae-
robic pretreatment process. This information would be used to test modi-
fications in the operational hydraulic parameters of the anaerobic pretreat-
ment process as well as the following aerobic treatment process in order to
optimize the overall performance of the system.
2) Alternative, more economical co-substrates other than sucrose should be
studied.
3) The performance of the anaerobic pretreatment process should be tested
at lower operating temperatures, such as room temperature, in order to reduce
energy requirements for operation.
4) It is recommended that this anaerobic-aerobic treatment process be applied
to other priority pollutants, which have been found resistant to aerobic treat-
ment, and their degradation-kinetics patterns be studied.
5) Overall treatment efficiency of DNOC and other aerobic-treatment-resistant
priority pollutants should be tested in a two-stage anaerobic system.
6) Alternative anaerobic processes should be evaluated.
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SECTION 4
STATE-OF-THE-ART REVIEW
4,6-DINITRO-O-CRESOL CHARACTERIZATION
DNOC has been used as a herbicide for several decades. It is
produced either by sulfonation of o-cresol followed by treatment with
nitric acid or by treatment of o-cresol in glacial acetic acid with nitric
acid at low temperature. Some important chemical and physical properties
of DNOC are shown in Table 1. Due to its extensive use as a very effective
herbicide, large numbers of investigations have been carried out to study
its herbicidal mode of action as well as that of other nitrophenolic com-
pounds. It has been established (58) that DNOC has an inhibitory effect on
the assimilatory processes of soil microorganisms through uncoupling of the
oxidative phosphorylation. Jensen (58,59), Jensen and Lautrup-Larsen (12),
and Gundersen and Jensen (7) showed that the toxicity of DNOC is markedly
pH dependent, namely an increase in the inhibitory effect of DNOC occurs
with increasing acidity. It is generally assumed that the increased toxicity
in low pH range is due to the greater permeability of the cytoplasm membrane
to the undissociated molecules whose concentration depends on the hydrogen
ion concentration. Since an increase in pH decreases the concentration of
the undissociated DNOC there is a decrease in DNOC toxicity. At a pH range
of 7.5-8.3, DNOC is the least toxic for most microorganisms (7,12). Van
Rensen et al. (69,70) showed that DNOC is a potent inhibitor of photosynthesis,
it strongly suppressed electron transport in isolated chloroplasts. Van
Rensen and Wim (65) further demonstrated that DNOC acts as a competitive in-
hibitor of HC03 binding in the photosynthetic electron transport in pea
(Pisun sativum) isolated chloroplasts. Vlassak and Heremans (63) showed that DNOC
inhibited soil nitrification. It also inhibited nitrogenase obtained from
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TABLE 1. PHYSICAL AND CHEMICAL PROPERTIES OF
4.6-DIN1TRO-ORTHO-CRESOL (DNOC)*
Molecular formula: CfiH2(CH3)OH(N02)2
Synonyms: 3,5-dinitro-o-cresol
3.5-d1nitro-2-hydroxytoluene "W2
4,6-dinitro-ortho-cresol
2,4-dinitro-6-methylphenol (DNOC)
CAS no. 534-52-1
2-methyl-4,6-dinitrophenol
Antinonin, Dekrysil, .Detal, Dinitrol, Ditrosol,
Effusan, Elegetol, Kill, KIV, Lipan, Prokarbol,
Selinon, Sinox
Molecular weight: 198.13
Appearance: yellow crystal
Melting point: 85.8°C
Vapor pressure: 0.000052 mm Hg at 20°C
Solubility: sparingly soluble in water
readily soluble in alkaline aqueous solutions,
ether, acetone, and alcohol (about 10%)
Log octanol/water partition coefficient: 2.85
pKa: 4.46
Henry's law constant: 1.4 x 10 atas-m3.mole~^
'Adapted from 2, 3, 4.
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Azotobacter vinelandii, the inhibition was competitive, using STP as a sub-
strate. Merenyuk and Timchenko (62) reported that an addition of 300 mg
DNOC per kg soil reduced the counts of nitrifying and cellulytic bacteria.
Bringmann and KUhn (13,14) showed that DNOC starts to inhibit cell
multiplication of Pseudomonas putida at the concentration of 16 mg/1, DNOC
also inhibits degradation of glucose by P. fluorescens and by E. coli at
30 and 100 mg/1, respectively.
Hivicky and Casida (71) in their work with mitochondria from various
mammals and insects,and Popov, Velikii and Parkhomets (64) in their work with
rat liver cells demonstrated that the inhibitory effect of DNOC is due to the
uncoupling of oxidative phosphorylation.
ENVIRONMENTAL FATE OF DNOC
Aquatic Fate
Table 2 summarizes the aquatic fate data of DNOC. This pollutant
will probably undergo slow photolytic destruction in ambient surface waters.
Volatilization is not thought to be an important transport process, but
DNOC might be strongly sorbed by clay minerals as indicated by its high
octanol/water partition coefficient of log P=2.85. There is a possibility
that DNOC could undergo hydrolysis while sorbed onto the clay structure.
Although the biotransformation of DNOC has been demonstrated, it is uncer-
tain whether biodegradation is an operational fate in ambient surface
waters (4).
Fate in Soil
Biotransformation and biodegradation occur in soil and are probably
the main processes of removal of DNOC from agricultural soils.
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TABLE 2. SUMMARY OF AQUATIC FATE OF DNOC (4)
Environmental
process
Photolysis
Oxidation
Hydrolysis
Summary statement
Slow photooxidation may be the main
aquatic fate pathway.
Displacement of the nitro-group by
hydroxyl radical may be possible.
Hydrolysis might occur during sorption
Confidence
of data
low
low
low
Volatilization
Sorption
Bioaccumulation
Biotransform-
tion/Bio-
degradation
on a clay surface; insufficient in-
formation to assess this pathway.
Probably not important.
This pollutant should be strongly
sorbed by clay minerals.
Importance is uncertain, due to
its high toxicity.
May be very slow in ambient surface
waters.
low
low
low
low
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BIODEGRADATION OF DNOC
Degradation of DNOC by Soil Microorganisms
The decomposition of DNOC in soil has been studied by numerous
investigators. Gunderson and Jensen (7) isolated from soil the bacteria
Arthrobacter simplex (then called Corynebacterium simplex) that uses DNOC
as nutrient in aerobic metabolism, with the formation of nitrite as meta-
bolite. They found that DNOC concentration of 200 mg/1 (at pH>7) appeared
to be non-toxic. Nitrite begins to appear in the cultures after 2 to 3 days
and approximately 70% of the DNOCnitrogen is recovered as nitrite after 6
to 7 days. Increasing the DNOC concentration to 500 mg/1 (at pH 8.0) re-
sulted in a prolonged lag-time for nitrite production, but after 14 days 50%
of the DNOC added was dissimilated. At 1,000 mg/1 inhibition was appreci-
able and at 2,000 mg/1 and 5,000 mg/1 no growth or degradation of the com-
pound took place. The authors suggested that the first degradation step of
DNOC is the removal of the para-nitro group and its conversion to nitrite.
They also suggested that the ring structure is subsequently ruptured, giving
rise to aliphatic compounds which are in turn assimilated by the organism.
Tewfik and Evans (8) isolated from soil a pseudomonad which meta-
bolized DNOC in pure cultures. The degradative pathway, as indicated by
chemical isolation of intermediates, sequential induction, washed cell
suspension experiments, and crude cell-free extracts, was shown to be as
fol1ows:
3,5-di ni tro-o-cresol>3-ami no-5-ni tro-o-cresol >3-methyl -5-ni tro-
catechol-^3-methyl-5-amino-catechol »2,3,5-trihydroxytoluene >
ring cleavage
This pathway is different in its initial steps from that employed by
Arthrobacter simplex, which, according to sequential induction evidence,
follows the following route:
10
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3,5-di ni tro-o-cresol »-3-methyl -5-ni tro-catechol >
2,3,5-trihydroxyto!uene »ri ng cleavage
The authors concluded that the presence or absence of a nitroreductase, a
different enzyme from nitrite reductase, is a determinant of the metabolic
route by which the nitrogen of nitro-compounds is made available to the
microorganisms.
Hamdi and Tewfik (9) showed that several species of Rhizobinum and
Azotobacter and Beijerinkia indica, all soil bacteria, can actively
degrade DNOC. They also determined that DNOC is reduced to
3-amino-5-nitro-o-cresol by Rhizobinum leguminosarum, in the early stages
of its degradation.
Chambers and Kabler (10) found that 100 mg/1 DNOC can be degraded by
phenol-adapted cultures, but not degraded by cultures adapted to other
tested phenolic derivatives. No percentage of removal was reported.
Tabak, Chambers and Kabler (11,57) extended the above investigation
and tested the degradation of 104 phenolic compounds and aromatic
hydrocarbons by phenol-adapted, aerobic soil bacteria. They observed loss
of color and accumulation of nitrite in enrichment cultures with several
aromatic nitro-compounds, including DNOC. Manometric experiments with the
phenol-adapted bacteria (mainly Pseudomonas and Flavobacterium) as seed,
showed only a slight oxygen uptake with 100 mg/1 DNOC in comparison with
the same concentration of o-cresol (7 percent in 3 hours) although loss of
color indicated 60 percent decomposition of DNOC in the same time. When
compared with the other 103 studied phenolic compounds, DNOC was classified
by the authors as "resistant to degradation" due to its low oxygen consumption
in Warburg tests, with phenol-adapted seed bacteria.
Jensen and Lautrup-Larsen (12) isolated from soils treated with 50-100
mg/1 DNOC, two groups of aerobic bacteria capable of metabolizing DNOC.
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Group I was comprised of seven strains of Arthrobacter-like organisms that
grew very slowly and feebly. When DNOC served as sole substrate, some 18
to 28 percent of the DNOC-nitrogen was assimilated in cell substance as com-
pared with 76-78 percent of the DNOC-nitrogen that was assimilated in cell
substance when glucose was introduced as an additional carbon source. Group
II was represented by two strains of Pseudomonads (possibly a species of
Agrobacterium) that grew rapidly; 17-19 percent DNOC-N was assimilated in
cell substance as cell-N and 46-47 percent was transformed into nitrite
ion when DNOC served as sole substrate. No other nitrogenous metabolite
other than nitrite was found. With glucose as a subsidiary carbon source,
the nitrite formation was repressed and approximately 90 percent of the
DNOC-N was found in the cell substance.
Wallnbefer et al. (66) investigated the transformation of DNOC by
Azotobacter sp. isolated from agricultural soil, they found that in the
absence of a nitrogen source, all strains of Azotobacter tested trans-
formed DNOC to 6-acetamido-2-methyl-4-nitrophenol. The transformation
was almost stoichiometric. In the presence of nitrogen source, bacterial
growth was markedly inhibited and no transformation of the DNOC was ob-
served. This demonstrates that there is a correlation between the re-
duction of DNOC in Azotobacter and the induction of the nitrogenase
enzyme system, which is represented by ammonium or nitrate compounds.
Jensen (60), Merenyuk et al. (62), and Hurle et al. (63) demonstrated
the importance of pretreatment and acclimation and showed that the rate of
decomposition of DNOC in the soil that had been treated with the same
compound was greater than the decomposition rate in a similar soil that
had not been pretreated.
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TREATABILITY AND TREATMENT ALTERNATIVES OF DNOC
Biological Treatment of DNOC
Tabak et al. (16) used a static-culture-flask biodegradation
screening test to determine the biodegradabil.ity of 96 organic priority
pollutants. They found that DNOC does not demonstrate significant
bio-oxidation even after 28 days of incubation period under enrichment-
culture conditions. The 5 and 10 mg/1 DNOC samples were only degraded
by 51 and 14%, respectively. Since the test was undertaken in favor-
able culture conditions, the actual removal performance in normal waste-
water treatment conditions is expected to be even poorer.
Fuentes (1) studied the fate of DNOC in bench-scale activated sludge
reactors using peptone and dextrose as main substrate to which acetone-
dissolved DNOC was added. He concluded that the main removal mechanism
for DNOC is biodegradation by microorganisms, whereas air stripping and
adsorption-on-floc do not contribute to the removal of DNOC in activated
sludge. Results of earlier short-term respirometer tests using unaccli-
mated sludge showed that DNOC becomes growth inhibiter within 20-30 mg/1,
and is toxic above 50 mg/1. For an influent DNOC concentration of less
than 30 mg/1, the removal efficiencies are between 8 and 23.5% for a sludge-
age-range of 3.5 to 9.5 days. The degradation of DNOC follows a zero-
order kinetic rate.
Additional data on the treatability of DNOC in activated sludge (5)
is given for synthetic wastewater as 58% removal and average achievable
concentration of 2.1 mg/1.
Physical and Chemical Treatments of DNOC
Sedimentation--
A DNOC concentration of 0.19 mg/1 was reported to be more than 95
percent removed from one coal mining effluent in a slurry pond (5).
13
-------
Vakulenko and Marchenko (67) reported that coagulation experiments done on
<4 mg/1 DNOC with 10-25 mg/1 Al2(804)3 indicated that the maximum degree of
removal was achieved at high concentrations of A^SO^.
Activated Carbon Adsorption--
Dobbs and Cohen (17) reported activated carbon adsorption data of
DNOC using Filtrasorb-300 granular activated carbon. The carbon dose
required to reduce DNOC concentration from 10 mg/1 to 1 mg/1 at pH 5.2 is
53 mg/1. Hutton (52) reported that DNOC of 0.011 mg/1 is more than 99
percent removed in a combined powdered activated carbon-activated sludge
treatment.
Steam Stripping
Treatability data for treating organic priority pollutants by steam
stripping has been reported by Hwang and Fahrenthold (18). Due to its low
activity coefficient (estimated as 308) and low vapor pressure, treatment
of DNOC by steam stripping is unsuitable. The removal as calculated by
the authors is less than 20% for steam stripping without reflux; even with
20 trays with reflux, the effluent DNOC concentration will still remain high.
ANAEROBIC DEGRADATION OF PHENOLIC AND NITROAROMATIC COMPOUNDS
Compared to the understanding of the aerobic degradation process,
relatively little is known about the anaerobic degradation of most organic
compounds. Published data of the anaerobic degradation of phenolic and
nitroaromatic compounds are limited, and sometimes conflicting on the
biodegradability of certain compounds.
Evans (47) reviewed the degradative mechanisms and routes of aromatic
compounds in anaerobic environments and summarized them into the following
three categories: a) photometabolism (by Athiorhodaceae), b) metabolism
through nitrate respiration (by Pseudomonas and Moraxella sp.)> and c)
14
-------
methanogenic fermentation by a consortium.
Healy and Young (48) studied the anaerobic degradation of 11 simple
aromatic lignin derivatives: vanillin, vanillic acid, ferulic acid,
cinnamic acid, benzoic acid, catechol, protocatechuic acid, phenol,
p-hydroxybenzoic acid, syringic acid, and syringaldehyde. They found that
all the tested compounds are biodegradable to methane and carbon dioxide
with different acclimation periods ranging from two days for syringic acid
to 21 days for catechol.
Hall as and Alexander (49) measured the transformation of mono and
dinitro aromatic compounds in sewage effluent maintained under aerobic or
anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic
acids, and 3- and 4-nitrotoluenes and much of the 1,2- and
1,3-dinitrobenzenes disappeared both in the presence and absence of
oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic
acid disappeared slowly, but no loss was evident in 28 days in aerated
sewage. Aromatic amines did not accumulate during the aerobic decompo-
sition of the mononitro compounds. They did appear in sewage incubated
aerobically with the dinitro compounds and anaerobically with all the
chemicals. Reduction of aromatic nitro group to amino group in anaerobic
environment is again confirmed. Further treatment of those amino by-
products shows that anaerobic by-products of mononitro compounds disappear
faster under aerobic than anaerobic conditions, whereas aerobic and
anaerobic by-products of dinitro compounds resist aerobic and anaerobic
microbial attack.
Recently, Boyd et al. (50) reported the anaerobic degradation of
phenol and the ortho, meta, and para isomers of chlorophenol,
methoxyphenol, and methylphenol (cresol), and nitrophenol in anaerobic
15
-------
sewage sludge. Eleven of the 12 monosubstituted phenols examined were
degraded in anaerobic digester sludge. The times required for complete
disappearance of phenol and the 12 monosubstituted phenols are given. The
presence of Cl and N02 groups, especially the meta- and para-substitutes,
inhibited methane generation. However, elimination or transformation of
these substituents was accompanied by increased gas production. No
obvious relationship between substituent position and susceptibility to
anaerobic degradation was observed. Even though the transformation
products of cresols were not observed directly, the authors state that the
reduction of aromatic N02 groups to NH2 in the anaerobic conditons almost
certainly occurred.
GENERAL INFORMATION ON BIODEGRADATION PROCESSES OF ORGANIC COMPOUNDS
Biodegradation includes several different microbial processes:
mineralization, detoxication, co-metabolism, activation, and defusing (20).
Mineralization is the conversion of an organic compound to inorganic
products. When an organic molecule is mineralized, the transformation is
usually the result of microbial action, and it is typically a growth-
linked process.
Detoxification refers to the conversion of a toxicant to innocuous
metabolite(s). Although mineralization is characteristically a
detoxication, many other kinds of detoxication are known.
Co-metabolism is the metabolism of a compound that the organisms are
not using as a nutrient. Co-metabolism does not result in mineralization;
hence, organic products remain.
Activation takes place when a nontoxic molecule is converted to one
that is toxic, or a molecule with low potency is made into a product of
greater toxicity to some species. Activation also may occur during
16
-------
co-metabolism.
Defusing, the conversion of a potentially hazardous substance into an
innocuous metabolite before the potential for harm is exerted, is so far
known only in pure laboratory cultures.
Some of these types of reactions take place among the phenoxy
herbicides. Thus, activation occurs when microorganisms convert
4-(2,4-dichlorophenoxy) butyric acid to 2,4-dichlorophenoxyacetic acid
(2,4-D), because the latter but not the former is phytotoxic. Similarly,
2,4-dichlorophenoxyethanol sulfate is activated to 2,4-D. In turn, 2,4-D
is detoxified as it is converted to 2,4-dichlorophenol but is mineralized
when it is converted to CC^. On the other hand, the removal of the
butyrate moiety of 4-(2,4-dichlorophnoxy) butyrate to yield
2,4-dichlorophenol is an example of defusing since the herbicide, 2,4-D is
not formed (20,21).
(METABOLISM
The phenomenon of co-metabolism, or sometimes called co-oxidation (22),
was first reported by Leadbetter and Foster (23) when they noted the
oxidation of (a) ethane to acetic acid and acetaldehyde, (b) propane to
propionic acid and acetone, and (c) n-butane to n-butyric acid and
2-butanone during growth of Pseudomonas methanica on methane, the only
hydrocarbon capable of supporting growth of the organisms.
Later, co-metabolism was defined by Foster (24) as follows:
"Nongrowth hydrocarbons are oxidized when present as co-substrates in a
medium in which one or more different hydrocarbons are furnished for
growth."
\
Because of the discovery of the phenomenon of co-metabolism, many
17
-------
compounds that were once considered recalcitrant may actually undergo
biodegradation. For example, the herbicide 2,4,5-trichlorophenoxyacetate
(2,4,5-T) has been regarded as a recalcitrant compound because of repeated
failure to isolate organisms capable of utilizing it as sole source of
carbon and energy for growth (25). However, later research (26) indicated
that 2,4,5-T could be oxidized by a co-metabolic process to the end product
of 3,5-dichlorocatechol, which could then be co-metabolized (27,28) by
another microbial species or completely metabolized (29).
The physiological basis for co-metabolism is not clear.
Microorganisms are unable to proliferate on compounds as sole carbon
source that they degrade by co-metabolism. The most likely hypothesis
involves enzyme specificity. Many enzymes present in microbial cells
catalyze reactions involving several different but chemically related
substrates. While the inital enzyme characteristically catalyses its
natural substrate to products that provide energy and a source of carbon
for the active species, some products of the enzyme reactions may not be
suitable for any other enzyme reaction in the culture and will subse-
quently accumulate (22,30).
Characterization and Identification of Co^metabolism
For organic compounds degraded by mineralizing conversion, an
increase in population density of species is generally paralleled by an
increase in degradation rate. Co-metabolism, however, is characterized by
the lack of this increase in degradation rate because the bacterial
species cannot use it for biosynthesis and have no growth benefits from
the presence of the compound (20,30).
Generally, proof of disappearance of substrate and/or accumulation of
end products and the inability of the microorganisms to utilize the
studied compound as its sole source of carbon and energy for growth are
18
-------
required to clearly demonstrate a co-metabolic process (32).
Enhancement of Co-metabolism--
Co-metabolism characteristically leads to a slow rate of products
formation if the number of cells bearing the responsible enzymes is small.
A number of techniques have been reported to enhance the rate of
co-metabolism.
Analog enrichment technique The effect of co-substrate was observed
by Broadbent and Norman (33), soil organic matter became a better source of
nutrient for the microbial soil population when readily decomposable
organic matter was added, the authors did not offer an explanation for
this phenomenon.
Horvath (34), studying the co-metabolism of 2,3,6-trichlorobenzoate,
demonstrated that microorganisms capable of a co-metabolic degradation of
organic pollutants can be enriched by application of biodegradable
analogues of the pollutant to the microbial ecosystem. Horvath (32)
indicated that the technique of "analogue enrichment" also accounts for
the enhanced rate of decomposition of organic matter described by
Broadbent and Norman (33).
Co-substrate enrichment technique Later, a structurally unre-
lated substance, glucose, was used as an enrichment agent for the study
of the co-metabolism of chlorobenzoates (35,36). The results showed that
employment of "co-substrate enrichment technique", considerably reduced the
time required for complete degradation of the studied chlorobenzoates.
Horvath (35) concludes that the addition of the co-substrate serves primarily
to increase the numbers of microorganisms capable of affecting an oxidation
of the halogenated compound and does not necessarily induce a specific
microbial population.
Glucose has been reported to be an effective co-substrate for
19
-------
decomposition of vanillin (37) and p-anisidine (38) by soil
microorganisms, alkyl benzene sulfonate by Pseudomanas sp. (39,40), and
diazinon by Arthrobacter sp. (41). The wide range of compounds for which
glucose serves as co-substrate supports the function of the co-substrate
proposed above by Horvath (35).
However, in some studies glucose was reported to be an inferior
co-substrate. In a study of the co-metabolism of cyclohexane by Beam and
Perry (42), the addition of hexadecane as co-substrate resulted in a
significantly higher rate of co-metabolic oxidation than with an equal
amount of glucose as co-substrate. The authors proposed that cyclohexane,
unlike compounds such as chlorobenzoates (35), may lack the capacity to
induce the requisite oxidative enzymes responsible for its degradation.
The addition of glucose, which is oxidized by pathways not involving an
oxygenase, thus did not help in inducing the oxygenase (43).
In another study on the effect of various co-substrates on the rate
of co-metabolic oxidation of malathion (o,o-dimethyl-S-(l,2-dicarbethoxy)
ethy phosphorodithionate) in soil (44), n-heptadecane was found to be the
most effective co-substrate. The addition of glucose, acetate and many
other co-substrates did not have an appreciable effect on the oxidation
rate. The same co-substrates were also tested for their effect on the
oxidation of DDT, lindane and dieldrin; however, no degradation of these
compounds was observed.
DiGeronimo, Nikaido and Alexander (45) compared structural analogues
and glucose as co-substrates and, in contrast to the finding by Horvath
(34,35,36), observed inappreciable degradations of chlorobenzoates, when
using the same techniques as Horvath except for inoculation with sewage
instead of pure cultures.
20
-------
Increased density of organisms technique -- Jacobson, O'Mara and
Alexander (46) demonstrated the co-metabolism of several herbicides in
sewage. The use of concentrated sewage microflora as seed greatly en-
hanced the rate of the co-metabolic conversion compared with the rate
of conversion with unconcentrated seed.
21
-------
SECTION 5
EXPERIMENTAL
To achieve the objectives of this investigation the experimental
study was divided into two main parts;
I. Anaerobic batch tests
II. Anaerobic-aerobic pilot plant applications.
I. ANAEROBIC BATCH TESTS
Two anaerobic batch testing techniques were used:
1. shaker bottle tests
2. continuously-stirred tests
Detailed information about these two testing techniques is given in
Appendix A.
The anaerobic batch test studies were designed to provide data on:
a. The biodegradability of DNOC in anaerobic conditions in the
presence of an additional carbon source (co-substrate).
b. The kinetics of the anaerobic biodegradation of DNOC.
c. The toxicity changes that result from the anaerobic
biodegradation of DNOC.
a. The Biodegradability of DNOC in Anaerobic Conditions in the Presence
of an Additional Carbon Source (co-substrate).
Early continuously-stirred batch tests results (see Section 6) indi-
cated that DNOC at a concentration of 100 mg/1 will not biodegrade in
anaerobic conditions unless an additional carbon source is provided. Sucrose
was chosen as the cosubstrate, since it is readily biodegradable. The
correlation between sucrose concentration and the biodegradation of DNOC
in anaerobic conditions was established. Filtered and unfiltered domestic
primary sludge were also tested as. possible, more economically feasible
22
-------
co-substrates.
b. The Kinetics of the Anaerobic Biodegradation of DNOC
Shaker bottle tests and continuously-stirred tests using varying
combinations of initial DNOC concentration and initial sucrose concen-
tration were performed. Samples were taken every 24 hours for the dura-
tion of the test, usually 7 days, and were analyzed for pH, oxidation-
reduction potential (ORP), suspended solids (SS), volatile suspended
solids (VSS), soluble chemical oxygen demand (CODs), soluble total or-
ganic carbon (TOC), and total organic acids (TOA). Samples were also
taken for determination of DNOC concentration, as well as sucrose
concentration (details are in Appendix C).
c. The Toxicity Changes that Result from the Anaerobic Biodegradation
of DNOC.
It was important to determine whether the anaerobic biodegradation of
DNOC resulted in by-product(s) having toxicity characteristics different
from the toxicity characteristics of the original compound. In order to
determine any toxicity changes due to the anaerobic biodegradation of
DNOC, daily samples were taken from the shaker bottle batch tests and from
the continuously-stirred batch tests and subjected to toxicity analysis
using the Beckman Microtox toxicity analyzer system. The method for
carrying out the toxicity analysis is outlined in Appendix C.
II. ANAEROBIC-AEROBIC PILOT PLANT APPLICATIONS
The anaerobic-aerobic pilot plant was set up in order to:
a. test the hypothesis that pretreatment of DNOC under anaerobic
conditions will result in bioconversion-products, or biodegradation-
products, which will be more readily biodegradable than DNOC in the
activated sludge process; namely, to test the impact of biological
anaerobic pretreatment of DNOC on the efficiency of the overall removal
23
-------
of DNOC in the activated sludge process.
b. provide data on the co-metabolism of DNOC as a function of the
concentration of an additional, more readily biodegradable, carbon source
(sucrose in this case) in the anaerobic pretreatment stage.
c. provide data on the toxicity changes that result from the
anaerobic pretreatment of DNOC.
d. provide data on some hydraulic design parameters for an optimized
performance of the anaerobic pretreatment filter column.
Pilot Plant Apparatus
The pilot plant consisted of one anaerobic upflow sand filter and one
activated sludge tank, as shown in Figure 1. The anaerobic upflow sand
filter consisted of a plexiglass column 8 feet high. Its lower section was
5 feet high and 3 inch ID and its upper section was 3 feet high and 4.25
inch ID. Prewashed sand, which was used as matrix for the attachment of
microorganisms, was 3.0 feet high in the lower section. When operating,
the bed expanded about 30% to a height of 4.0 feet. The temperature of the
column was maintained at 36.5°C by heating tapes surrounding the column.
The temperature was controlled by a Versa-therm proportional temperature con-
troller (Cole-Parmer Inst.) with a thermister sensor inserted into the column.
The anaerobic upflow sand filter column was operated as a flow through
system with a total internal flow rate maintained at about 3,000 I/day. Sodium
chloride tracer tests showed that the liquid phase was completely mixed
(Figures 2 and 3). However, the distribution of the biomass in the fluidized
bed was uneven (Figure 4). The microorganisms were predominately concentrated
»
in the upper section of the sand bed. The pattern of the biomass distribution
in the column bed resulted in a distinct dividing plane formed between the
sand particles in the lower section of the bed which were sparsely populated
with biomass, and the sand particles in the upper section of the bed which
24
-------
OFF GAS
ro
01
ANAEROBIC EFFLUENT
RECYCLE
ANAEROBIC
UPFLOW
COLUMN
FEED
TANK
RECYCLE
PUMP
ACTIVATED SLUDGE TANK
COMPRESSED
AIR
EFFLUENT
PUMP
Figure 1. Schematic of pilot plant apparatus.
-------
aooor
ro
en
HRTsSI min,
NoCI Recovery ss 105%
120
180
240 900 360
TIME ( min)
420 480 540 600
Figure 2. Sodium chloride (NaCI) tracer curve for the anaerobic column.
-------
.05F
10
o
£
E
» icr
»-
>
u
a
o
o
u
,o!
10
100
200 300
TIME (rein)
400
300
Figure 3. A linearized NaCl tracer curve for the anaerobic column
reflecting a first-order mixing in the column.
27
-------
22" ± 2"
26" 7 2"
ANAEROBIC
UPFLOW
COLUMN
B*WB
^M
B^M
.,r
-------
were densely populated with biomass. The biomass distribution in the
fluidized bed appears in Table 3.
TABLE 3. BIOMASS DISTRIBUTION IN THE FLUIDIZED BED
Sample Source VSS (mg/1)
Anaerobic bed upper section 10,700 - 11,300
Anaerobic bed lower section 1,940 - 3,050
Anaerobic column effluent 12 - 104
The activated sludge tank was the conventional 19 liters plexiglass
tank with a 14 liter aeration compartment and a 5 liter clarification com-
partment. Effluent from the anaerobic upflow column was pumped directly
into the activated sludge unit as feed.
The anaerobic filter column was started up by several inoculations
with seed from a local municipal sewage treatment plant sludge digester. It
was then operated during an acclimation period and throughout the testing
period as a flow through system. Loading concentrations of the different
components of the influent were carefully controlled. The anaerobic filter
column effluent and the activated sludge effluent were monitored, monitoring
details are in Appendix A.
29
-------
SECTION 6
RESULTS AND DISCUSSION
The results of the investigation are summarized as follows:
A) The removal of DNOC under anaerobic conditions;
B) The co-metabolism of DNOC under anaerobic conditions;
C) The removal of COD in the anaerobic-aerobic system;
D) The changes in toxicity due to anaerobic-aerobic treatment.
A) THE REMOVAL OF DNOC UNDER ANAEROBIC CONDITIONS
DNOC Removal in a Batch and a Flow-Through Anaerobic Reactor
Preliminary shaker bottle batch tests results (Table 4) in which
sucrose (3.0 g/1) was used as a co-substrate clearly indicated that
DNOC was degradable under anaerobic conditions. Over a period of 7 days,
the original DNOC concentration of 100 mg/1 was reduced to 15 mg/1. The
reduction in the soluble COD and TOC was only 16% and 22%, respectively,
which was coupled by an increase in TOA from 370 mg/1 at day 0 to 2,260
mg/1 at day 7.
Results (Table 5) from the shaker bottle batch tests in which filtered
primary sludge was used as a co-substrate also provided supporting evidence
that DNOC is anaerobically degradable. The DNOC concentration decreased
from 92 mg/1 in day 0 to 53 mg/1 in day 7.
Anaerobic continuously-stirred batch tests (Table 6) confirmed the
results of the anaerobic shake bottle tests. The original concentration
of 100 mg/1 of DNOC was reduced to 0.2 mg/1 over a period of 7 days in
the presence of 3.0 g/1 sucrose as a co-substrate. The decrease in
soluble COD was 11% over a period of 7 days while the TOA level increased
from 318 mg/1 at day 0 to 2,600 mg/1 at day 7.
DNOC was treated in the anaerobic-aerobic system (Tables 7 and 8)
30
-------
TABLE 4. ANAEROBIC SHAKER BOTTLE BATCH TEST RESULTS
Time
(days)
0
1
2
3
4
5
6
7
Sample*
S
D
S
D
S
0
S
0
S
D
S
0
S
D
S
D
PH
8.43
8.54
6.84
6.89
6.82
6.84
6.70
6.79
6.77
6.85
6.75
6.81
6.76
6.82
6.76
6.83
SS
(mg/1)
138
142
279
246
245
188
203
172
234
172
130
89.5
114
143
88.0
80.0
VSS
(mg/1)
64.0
69.0
197
153
177
123
145
115
156
114
111
68.5
93.0
86.5
73.5
56.5
TOA
(mg/1 )
341
371
2150
1950
2210
2230
2310
2160
2700
2180
2170
2600
2260
CODs
(mg/1)
3130
3340
2550
2810
2600
2780
2630
2790
2560
2710
2850
2980
3100
3240
2790
2820
TOC
(mg/1)
1720
1780
1430
1610
1390
1530
1490
1780
1360
1400
1660
1660
1750
1810
1320
1390
DNOC
(mg/1)
100
78.6
61.2
39.8
21.9
18.0
15.5
14.6
EC 50***
(5 m1n)
NTO**
10. IX
NTO
9.62%
NTO
17.6%
NTO
25.2%
40.9%
21.2%
9.97%
22.5%
56.4%
23.4%
33.5%
23.0%
EC 50
(15 m1n)
NTO
7.16%
NTO
7.82%
NTO
14.5%
NTO
23.4%
8.92%
14.5%
9.06%
17.8%
37.7%
15.7%
23.2%
18.2%
*S - Sucrose Only (3.0 g/1); D -
**No Toxlcity Observed
Sucrose (3.0 g/1) +
***ECRn 1s expressed in units of % of Initial sample
U50
mg/1)
concentration.
-------
TABLE 5. RESULTS OF SHAKER BOTTLE BATCH TEST USING FILTERED PRIMARY
SLUDGE AS CO-SUBSTRATE
Time
(days)
0
7
13
pH
7.3
7.2
7.3
SS
(mg/1)
130
195
145
VSS
(mg/1)
95
145
90
CODs
(mg/1)
579
630
674
Residual DNOC*
(mg/1)
92
53
50
^Initial DNOC concentration = 100 mg/1.
32
-------
TABLE 6. THE EFFECT OF SUCROSE CONCENTRATION ON THE CO-METABOLISM OF
DNOC IN CONTINUOUSLY-STIRRED BATCH TEST*
Sample
A
B
E
*Initial
Sample
A
B
E
Time
(days)
0
1/2
1
1 1/2
2
3
4
5
6
7
0
1/2
1
1 1/2
2
3
4
5
6
7
0
1/2
1
1 1/2
2
3
4
5
6
7
PH
8.0
7.8
6.8
6.9
6.9
6.9
6.9
7.0
7.0
7.0
7.9
7.9
6.9
6.9
6.9
6.9
6.9
6.9
6.9
6.9
7.9
7.9
7.9
7.9
7.9
7.9
7.9
7.9
7.9
7.9
SS
(mg/1)
74
101
237
208
192
196
144
103
62
116
54
77
185
176
157
158
157
132
140
147
51
56
64
74
55
62
53
48
54
93
VSS
(mg/1)
28
53
184
162
143
164
127
84
48
86
20
42
140
135
118
128
126
101
114
120
17
20
20
28
13
22
20
10
18
31
TOA
(mg/1)
278
297
2950
__
2670
__
2440
__
--
2460
318
309
2200
__
2680
__
2480
__
__
2600
320
274
446
--
280
228
220
CODs
(mg/1)
3440
3500
3130
3100
3170
3240
3040
3020
3020
3070
3590
3650
3340
3140
3140
3370
3210
3300
3180
3210
639
610
656
570
559
541
525
508
491
DNOC
(mg/1)
--
__
__
-_
-_
__
__
--
100
__
75.1
_-
46.5
16.9
4.4
0.8
0.5
0.2
100
__
--
__
95.0
--
._
101
98.7
102
Sucrose
(g/i)
3.0
0
--
0
0
0
0
0
0
3.0
0
--
0
0
0
0
0
0
3.0
--
0
--
0
0
0
0
0
0
Concentrations:
DNOC
(mg/1)
0
100
100
Sucrose NaHCO?
(g/D (g/i)
3
3
0
.0
.0
10.7
10.7
10.7
(NH4)?HP04
1.7
1.7
1.7
33
-------
TABLE 7. T!!E PERFORMANCE OF THE ANAEROBIC COLUMN
(A)
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Feed
DNOC
(mg/1)
10.0
20.0
30.0
50.0
75.0
100.
125
150
200
250
325
400
500
600
500
500
500
500
500
500
500
500
500
500
500
500
500
500
Cone.
Sucrose
(g/D
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
2.0
2.0
1.5
1.5
1.0
1.0
1.0
0.75
0.75
0.75
0.75
1.5
Influent
Q HRT*
(I/day) (days)
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
pH
7.0
7.0
7.0
6.9
6.4
6.4
6.4
6.3
6.3
6.3
6.3
6.4
6.4
Effluent
Influent Effluent
ORP Alkalinity SS VSS DNOC Sucrose COD TOC CODs TOC
(MV)(mg/l as CaC03)(mg/l){ing/l)(mg/l) (g/1) (mg/l)(mg/1) (ing/ 1) (ing/ 1)
-300
-290
-290
-290
-340
-280
-340
-310
-290
-160
-150
-120
0
3300
3300
3300
3400
3400
3000
2600
2000
1500
1500
1200
1100
N.D.
52 43 N.D.
110 86 N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
77 66 N.D.
N.D.
N.D.
100 82 N.D.
N.D.
110 93 N.D.
0.45
72 61 0.55
2.17
3.11
2.15
77 71 46.7
55.5
101
14 12 104
62.8
3200
3200
3200
3300
4070
4070
2950
2390
1820
1540
1540
2000 -
2350
2500 --
2800 --
1500 2960 1300
1500 2850 1220
1120 2170 705
882 1670 605
704 1260 585
590 1170 444
590 1240 494
Average gas
production
(I/day)
2.0
2.4
1.6
0.8
0.75
0.7
0.64
0.4
0.35
0.32
0.29
0.19
0.19
-------
TABLE 7. Cont.
OJ
en
Feed Cone.
No.
29
30
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
DNOC
(mg/1)
500
500
500
500
500
500
500
500
500
500
600
750
750
750
750
750
250
250
250
250
250
250
250
250
250
250
250
Sucrose
(9/1)
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
2.0
2.0
2.0
2.0
3.0
3.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Influent
Q HRT*
(I/day) (days)
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.8
4.8
6.0
6.0
7.5
7.5
10
10
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
2.5
2.5
2.0
2.0
1.6
1.6
1.2
1.2
Effluent
pH
6.3
6.3
6.2
6.2
6.2
6.5
6.2
6.1
6.1
6.1
6.1
Influent Effluent Average gas
ORP Alkalinity SS VSS DNOC Sucrose COD TOC CODs TOC production
(MV)(mg/1 as CaC03) (mg/1) (mg/1) (mg/1) (g/1) (mg/1) (mg/1) (mg/l)(mg/l) (I/day)
-70
-90
-160
-160
-150
+20
-190
-270
-270
-280
-280
2100
2100
2000
2500
2300
1900
1800
1700
1700
64
77
115
76
105
41
55
86
58
68
104
67
95
35
44
79
49.5
42.2
32.7
10.7
7.24
4.01
2.68
3.41
2.08
2.01
5.87
2.68
14.7
87.8
206
318
0.95
0.48
N.D.
0.64
0.54
0.25
0.25
0.21
0.24
N.D.
N.D.
2390 882 1870 522
3300 1400 2430 687
4240 1780 3260 1030
2490 846 2410 590
2490 846 2100 490
2490 846 1980 466
2490 846 1950 480
-------
TABLE 7. Cont.
CO
Feed Cone.
No.
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
DNOC
(mg/1)
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
0
Sucrose
(9/1)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
1.0
1.0
0.5
0.5
0.5
0.25
0.25
0.25
0.25
0.25
Influent
Q HRT*
(I/day) (days)
13
13
17
17
17
21
21
21
28
28
28
43
43
72
72
26
26
26
26
26
26
26
26
26
26
26
26
0.93
0.93
0.72
0.72
0.72
0.58
0.58
0.58
0.43
0.43
0.43
0.28
0.28
0.17
0.17
0.47
0.47
0.47
0.47
0.47
0.47
0.47
0.47
0.47
0.47
0.47
0.47
pH"
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
Effluent
ORP Alkalinity SS VSS DNOC
(MV)(mg/l as CaC03) (mg/1) (mg/1) (mg/1)
-270
-260
-270
-270
-270
-270
-280
-230
-140
-120
-79
-67
+7
1700
1700
1700
1700
1700
1700
1700
1000
580
420
1600
79
85
87
54
56
42
25
47
70
76
61
45
45
35
21
42
N.D.
N.D.
0.01
0.60
0.12
0.08
0.05
0.14
N.D.
0.12
0.09*
0.02
0.03
0.06
N.D.
N.D.
N.D.
1.03
1.38
14.0
19.9
21.0
61.8
112
126
133
--
Sucrose
(9/D
0.24
0.24
0.24
0.24
0.22
0.22
0.24
0.24
0.22
0.20
Influent
COD TOC
(mg/1) (mg/1)
2490
2490
2490
2490
2490
2490
2490
1290
850
850
621
2200
846
846
846
846
846
846
846
421
291
291
135
654
Effluent
CODs TOC
(mg/1) (mg/1)
1780
1870
1800
1880
1980
1840
1910
1100
733
707
557
1920
450
518
450
425
500
548
562
288
215
229
167
522
Average gas
production
(I/day)
3.8
8.2
9.2
0.2
*HRT Is based on Influent flow rate, Q.
-------
TABLE 8. THE PERFORMANCE OF THE ACTIVATED SLUDGE REACTOR
to
Feed to the system
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
DNOC
(mg/1 )
10.0
20.0
30.0
50.0
75.0
100
125
150
200
250
325
400
500
600
500
500
500
500
500
500
500
500
500
500
500
500
500
500
Sucrose
(9/1)
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
2.0
2.0
1.5
1.5
1.0
1.0
1.0
0.75
0.75
0.75
0.75
1.5
Flow
Rate .
(I/day)
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
Reactor
pH MLSS
(mg/1)
8.5 1800
8.2 2000
7.9 2400
8.6 2800
8.8 2000
1500
1400
1100
1100
1100
Influent
MLVSS
(mg/1)
1500
1600
2000
2100
1200
850
780
570
580
520
DNOC
(mg/1)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.45
0.55
2.17
3.11
2.15
46.7
55.5
101
104
62.8
CODS
(mg/1)
2000
2350
2500
2800
2960
2850
2170
1670
1260
1170
1240
TOC
(mg/1)
--
--
_-
1300
1220
705
605
585
444
494
Effluent
CODs
(mg/1)
80
70
65
80
386
460
440
420
380
484
522
TOC
(mg/1 )
__
--
__
-_
_»
130
175
180
146
158
-------
TABLE 8. Cont.
CO
00
No.
29
30
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
Feed to
DNOC
(mg/1 )
500
500
500
500
500
500
500
500
500
500
600
750
750
750
750
750
250
250
250
250
250
250
250
250
250
250
250
the system
Sucrose
(g/D
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
2.0
2.0
2.0
2.0
3.0
3.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Flow
Rate
(I/day)
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.8
4.8
6.0
6.0
7.5
7.5
10
10
Reactor
pH MLSS MLVSS
(mg/1) (mg/1)
8.5 250 220
8.9 450 390
1180 970
8.6 1100 920
1000 860
1400 1160
8.5 1770 1440
DNOC
(mg/1)
49.5
42.2
32.7
10.7
7.24
4.01
2.68
3.41
2.08
1 2.01
5.87
2.68
14.7
87.8
206
318
0.95
0.48
N.D.
0.64
0.54
0.25
0.25
0.21
0.54
N.D.
N.D.
Influent
CODs TOC
(mg/1) (mg/1)
1870 522
2430 687
3260 1030
2410 590
2100 490
1980 466
1950 480
Effluent
CODs TOC
(mg/1) (mg/1]
510 215
475 240
700 185
425 111
310 68
230 36
220 30
-------
TABLE 8. Cont.
CO
to
Feed to the system
No.
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
DNOC
(mg/1)
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
0
Sucrose
(9/1)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
1.0
1.0
0.5
0.5
0.5
0.25
0.25
0.25
0.25
0.25
Flow Reactor
Rate pH MLSS MLVSS
(I/day) (mg/1) (mg/1)
13
13
17
17
17 2300 1700
21
21
21
28
28
28
43
43 8.5 3700 2500
72
72
26
26 2200 1860
26
26
26
26
26 8.2 1750 1100
26
26
26
26
26
Influent
DNOC
(mg/1)
N.D.
N.D.
0.01
0.60
0.12
0.08
0.05
0.14
N.D.
0.12
0.09
0.02
0.03
0.06
N.D.
N.D.
N.D.
1.03
1.38
14.0
19.9
21.0
61.8
112
126
133
CODs
(mg/1)
1780
1870
1800
1880
1980
1840
1910
1100
733
707
557
1920
TOC
(mg/1)
450
518
450
425
500
548
562
288
215
229
167
522
Effluent
CODs
(mg/1)
__
230
--
220
755
1720
385
180
«._
245
293
94
TOC
(mg/1)
__
43
_.
33
245
512
200
26
__
56
70
42
-------
over influent concentration range of 10 mg/1 to 750 mg/1. As shown in
Table 9, the DNOC was not detected in the anaerobic column effluent with
DNOC concentrations as high as 600 mg/1, when the sucrose concentration
in the influent was maintained at 3.0 g/1 and the hydraulic retention time
(HRT) at 3 days.
The Effect of Hydraulic Retention Time on the Removal of DNOC Under
Anaerobic Conditions
The effect of the HRT on the performance of the anaerobic column
was determined under the following conditions:
sucrose concentration = 2.0 g/1
DNOC concentration = 250 mg/1
HRT = 0.17 to 3.0 days
As seen in Table 10, the removal of DNOC by the anaerobic column was > 99.7%
even for HRT as short as 0.17 days. Hence, under these given operational
conditions, no effect of HRT on the removal of DNOC was observed. However,
the anaerobic gas production was increased (Table 7 runs No. 66-70) when
the HRT decreased at constant influent concentration.
The By-products Resulting From the Removal of DNOC Under Aanaerobic
Conditions
Acid extracted samples (Appendix C) from anaerobic shaker bottle test,
anaerobic continuously-stirred batch test, anaerobic column effluent and
activated sludge reactor effluent were analyzed by GC/MS to identify the
anaerobic biodegradation by-products of DNOC. From these results (Table 11),
it is postulated that under anaerobic conditions the DNOC underwent an
initial breakdown that resulted in molecular structural changes, primarily
in the loss of its nitro groups, with one of them substituted by a methyl
group. The GC/MS results also showed that the breakdown of sucrose under
anaerobic conditions resulted, as was expected, in the formation of organic
40
-------
TABLE 9. REMOVAL OF DNOC IN THE ANAEROBIC COLUMN*
DNOC inf
(rag/1)
10
20
30
50
75
100
125
150
200
250
325
400
500
600
DNOC eff Removal of DNOC
(mg/l) (%)
Not Detected >99.9
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
*Sucrose influent concentration = 3 g/1,
hydraulic retention time = 3 days.
41
-------
Table 10. THE EFFECT OF HYDRAULIC RETENTION TIME (HRT)
ON THE ANAEROBIC COLUMN PERFORMANCE*
HRT (days) DNOC-jnf ("'S/1)
3.0 250
"
2.5
"
2.0
"
1.6
"
1.2
"
0.93
"
0.72
"
11
0.58
"
"
0.43
n
"
0.28
"
0.17
"
DNOCoff (mg/1)
c T T
0.48
Not Detected
0.64
0.54
0.25
0.25
0.21
0.54
N.D.
N.D.
N.D.
N.D.
0.01
0.60
0.12
0.08
0.05
0.14
N.D.
0.12
0.09
0.02
0.03
0.06
N.D.
DNOC Removal (%)
99.8
>99.9
99.7
99.8
99.9
99.9
>99.9
99.8
>99.9
>99.9
>99.9
>99.9
>99.9
99.8
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
*Influent sucrose concentration = 2.0 g/1.
42
-------
CO
TABLE 11. BY-PRODUCTS OF THE DEGRADATION OF DNOC AND SUCROSE AS IDENTIFIED
BY GC/M$1»2
Sample (add extracted)
Compound Anaerobic shaker
bottle batch test
DNOC X
2-Methyl-5-(l-methylethane)- X
phenol
Pentanolc add X
Butanolc acid
Acetic add denvatlves
Some alkanes
3,4-D1methy1 phenol
l,l-B1cyclohexyl
Anaerobic contlnu- Anaerobic column
ously-stlrred batch effluent3
test I
X
X X
X
X
X
X
X
II
X
X
X
X
X
Activated sludge
effluent
X
1. GC/MS analysis performed by D. (Campbell at the U.S. E.P.A. RSKERL, Ada, OK.
2. Identification criteria: matching base peaks, matching mass spectra and fit of >900.
3. The two anaerobic column effluent samples were taken from 2 different runs.
-------
acids: pentanoic and butanoic acids and an array of acetic acids deriva-
tives. The subsequent aerobic biodegradation in the activated sludge
process resulted in the elimination of most of the anaerobic degradation
by-products that were present in the anaerobic column effluent. The GC
analysis performed on grab samples of gas produced in the anaerobic column
provided further information on the by-products of the anaerobic breakdown
process. Gas analyses were performed when gas production exceeded 3
liters/day. Based on these limited data, the gas composition was 82 to 85%
C02, 17 to 15% N2 and less than 1% CH4. These data indicate that acid-
producing bacteria, rather than the more sensitive methane-producing
bacteria, dominated the microorganisms population in the anaerobic column.
The production of nitrogen gas also indicate that a denitrification process
also occurred in the anaerobic column. Thus the denitrifying microorganisms
are responsible for the reduction of the nitro groups that originated from
the DNOC molecule, to nitrogen gas.
B) THE CO-METABOLISM OF DNOC IN ANAEROBIC CONDITIONS
Sucrose as a Co-substrate
The continuously-stirred batch test results indicated that the
degradation of DNOC occurring under anaerobic conditions was a result
of the co-metabolism of the nitroaromatic compound and depended upon the
presence of a co-substrate. In the presence of the co-substrate sucrose,
DNOC was anaerobically co-metabolized. As can be seen from Tables 6, 12,
13, and 14 there is a direct correlation between the concentration of
sucrose and the removal of DNOC in the anaerobic batch tests. The results
presented in Figure 5 and Table 6 demonstrated the phenomenon which is the
essence of any co-metabolic process; namely, the inability of the micro-
organisms to utilize the DNOC as its sole source of carbon and energy. As
observed from Figure 5, when there is no sucrose present, the concentration
44
-------
TABLE 12. THE EFFECT OF SUCROSE CONCENTRATIONS ON THE CO-METABOLISM
OF DNOC IN CONTINUOUSLY-STIRRED BATCH TEST, RUN 1*
Sample Time
(days)
M 0
1
2
3
4
5
6
7
N 0
1
2
3
4
5
6
7
0 0
1
2
3
4
5
6
7
pH
8.0
7.0
7.0
7.0
6.9
6.9
6.9
6.9
7.9
6.8
6.6
6.6
6.6
6.6
6.6
6.6
8.1
7.1
7.0
7.0
6.9
6.9
6.9
6.9
SS
(mg/1)
35
122
144
152
197
160
168
223
35
107
70
72
110
85
96
110
41
96
68
62
115
71
67
124
VSS
(mg/1)
13
102
110
126
173
140
140
171
8
88
60
64
98
74
78
87
10
78
52
50
97
58
48
84
CODs
(mg/1)
5400
4940
5040
4470
4530
4500
4600
4680
2130
1910
1890
1800
1850
1820
1840
1870
6590
6560
6310
6120
5910
5840
5850
6080
TOC
(mg/1)
2880
2440
2010
2260
2120
1850
2290
2220
1010
950
810
810
890
800
880
890
4090
3700
3310
3290
3250
3330
3440
3500
DNOC
(mg/1)
250
229
168
130
57.8
11.7
0.4
N.D.
250
230
215
206
201
193
186
183
250
223
171
135
83.8
9.2
0.3
N.D.
Sucrose
(g/D
4.50
2.68
2.04
1.00
0
0
0
0
1.50
0.52
0.20
0
0
0
0
0
6.00
4.10
3.60
3.16
0.20
0.16
0.02
0.02
* In.itial concentrations:
Sample
M
N
0
DNOC
(mg/1)
250
250
250
Sucrose
(g/D
4.5
1.5
6.0
NaHOh
(9/1)
11.25
3.75
13.75
(NH4)2HP04
(g/D
1.875
0.625
1.875
45
-------
TABLE 13. THE EFFECT OF SUCROSE CONCENTRATIONS ON THE CO-METABOLISM
OF DNOC IN CONTINUOUSLY-STIRRED BATCH TEST, RUN 2*
Sample Time pH
(days)
P 0
1
2
3
4
5
6
7
Q 0
1
2
3
4
5
6
7
R 0
1
2
3
4
5
6
7
8.1
7.8
7.3
6.6
6.4
6.4
6.4
6.4
8.2
8.0
6.9
6.7
6.5
6.4
6.4
6.4
8.1
7.9
7.0
6.8
6.8
6.7
6.7
6.7
ss
(mg/1)
110
113
109
128
154
161
152
150
108
141
101
62
80
72
66
64
120
154
117
68
99
107
106
99
VSS
(mg/1 )
52
56
62
88
118
116
115
120
58
72
70
40
58
45
42
40
65
84
78
46
63
74
71
68
CODs
(mq/1 )
5620
5610
5600
5280
5150
5200
5020
5100
7260
7210
7050
6790
6560
6600
6400
6400
8780
8730
8580
8230
3470
8240
7940
7850
TOC
(mq/1 )
2670
2670
2610
2650
2390
2190
2260
2190
3130
3000
3010
2950
2810
2790
2680
2870
3900
3600
3610
3500
3570
3440
3570
3450
DNOC
(mq/1 )
500
500
500
490
413
327
275
256
500
500
474
394
308
181
69.6
14.9
500
500
455
387
359
272
136
42.0
Sucrose
(g/1)
4.50
4.47
3.96
2.84
1.71
1.52
1.45
1.45
6.00
6.00
4.62
3.84
2.07
1.75
1.46
1.41
7.50
7.50
5.93
5.14
4.61
3.51
3.00
2.57
* Initial concentrations:
Sample
P
Q
R
DNOC Sucrose
(mg/D (9/1)
500
500
500
4.5
6.0
7.5
NaHCOa
(g/i)
5.0
6.5
8.75
(NH4)2HP04
(971)
1.25
1.75
2.25
46
-------
TABLE 14. THE EFFECT OF SUCROSE CONCENTRATIONS ON THE CO-METABOLISM
OF DNOC IN CONTINUOUSLY-STIRRED BATCH TEST, RUN 3*
Sample Time pH
(days)
V 0
1
2
3
4
5
6
7
W 0
1
2
3
4
5
6
7
X 0
1
2
3
4
5
6
7
7.7
6.1
6.1
6.1
6.1
6.1
6.1
6.1
7.8
6.6
6.6
6.6
6.6
6.6
6.6
6.6
7.9
6.9
6.9
6.9
6.9
6.9
6.9
6.9
ss
(mg/1 )
68
170
156
161
162
159
166
158
66
113
109
121
120
120
116
128
70
115
97
98
97
94
103
97
VSS
(mg/1)
30
138
116
134
137
133
133
134
30
81
78
88
89
91
88
97
20
70
56
61
63
55
69
64
CODs
(mg/1)
2480
2140
2030
2080
2070
2070
2100
2080
1360
1170
1100
1090
1070
1050
1050
1020
847
732
676
652
636
610
597
594
TOC
(rag/1 )
1160
900
950
930
890
923
917
927
695
608
663
616
633
618
644
688
512
522
517
530
541
497
591
575
DNOC
(mg/1)
100
99.0
80.4
74.6
64.3
58.0
56.5
52.9
100
100
91.6
87.9
79.3
66.2
62.6
60.6
100
100
100
100
96.5
93.3
89.9
88.1
Sucrose
(g/1)
2.00
0.20
0.16
0.16
0.16
0.16
0
0
1.00
0.23
0.14
0
0
0
0
0
0.50
0.28
0.16
0
0
0
0
0
* Initial concentrations:
Sample
V
W
X
DNOC
(mg/1)
100
100
100
Sucrose
(g/i)
2.0
1.0
0.5
NaHCO-j
(9/1)
3.5
3.5
3.5
(NH4)2HP04
(9/1)
0.5
0.5
0.5
47
-------
200r
Initial Conctntrationt
Symbols
DNOC
_
A
vss
o
A
Q
ONOC
(ma/I)
0
100
100
Sue rote
(o/l)
S.O
3,0
0
e
O
to
o
o
Figure 5. The effect of sucrose concentration on the removal of
DNOC in continuously-stirred batch test.
48
-------
of DNOC remained unchanged. This also resulted in a drastic inhibition
of biomass growth; i.e. no change in VSS concentration was observed over
time, showing that the DNOC itself is toxic to the anaerobic microor-
ganisms when a readily biodegradable co-substrate is not available.
The Effect of Sucrose and DNOC Concentrations on the Performance of the
Anaerobic Column in the Co-metabolism of DNOC
Early results from anaerobic continuously-stirred batch tests
(Tables 15 and 16) showed that the anaerobic process failed to completely
remove DNOC over a period of 7 days when the DNOC initial concentrations
exceeded 200 mg/1 and the sucrose concentration was maintained at 3.0 g/1.
These data indicated that the ratio of sucrose to DNOC concentrations might
be an important parameter controlling the efficiency of the anaerobic co-
metabolism of DNOC. The removal of DNOC in the anaerobic column was there-
fore studied under the following conditions:
I. Influent DNOC concentration = 250 mg/1
Influent sucrose concentration = gradually reduced from
2.0 g/1 to 0.25 g/1, HRT = 0.47 days.
The results of these studies are shown in Figure 6.
II. Influent DNOC concentration = 500 mg/1
Influent sucrose concentration = gradually reduced from
3.0 g/1 to 0.75 g/1, HRT = 3.0 days.
The results of these studies are shown in Figure 7.
The performance of the anaerobic column in the removal of DNOC was
highly dependent on the influent concentration of the co-substrate, sucrose.
A ratio of influent sucrose to DNOC of 2:1 or higher resulted in 95-100%
removal (or conversion) of DNOC in the anaerobic process. However, when
influent sucrose to DNOC ratio was less than 2:1 the anaerobic microor-
ganisms failed to co-metabolize DNOC.
49
-------
TABLE 15. THE EFFECT OF DNOC CONCENTRATIONS ON THE CO-METABOLISM
OF DNOC IN CONTINUOUSLY-STIRRED BATCH TEST*
en
O
Sample
F
G
H
*In1tial
Sample
F
G
H
Time
(days)
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
PH
7.8
7.0
7.0
6.9
6.9
6.9
6.9
6.9
7.9
7.0
6.9
6.9
6.9
6.9
6.9
6.9
7.8
7.1
7.0
6.9
6.9
6.9
6.9
6.9
SS
(mg/1)
48
151
122
116
116
96
106
146
34
144
103
90
110
106
110
111
21
128
123
113
122
114
137
151
VSS
(mg/1)
22
130
93
92
100
82
86
122
15
119
78
71
90
89
93
91
6.5
98
94
92
105
97
124
127
TOA
(mg/1)
2630
2450
--
--
--
.
2710
2530
--
_
2010
2500
CODs
(mg/1)
3500
3140
3240
3030
3040
3080
3140
3150
3630
3290
3350
3140
3170
3210
3260
3290
3680
3390
3390
3110
3160
3160
3240
3360
TOC
(mg/1)
1620
1380
1360
1390
1300
1500
1580
1600
1720
1390
1420
1380
1490
1490
1680
1690
1660
1520
1540
1340
1410
1480
1590
1750
DNOC
(mg/1)
50.0
19.3
1.4
N.D.
--
--
150
51.4
15.0
1.0
0.3
0.1
N.D.
N.D.
200
84.0
26.4
0.6
0.3
0.2
N.D.
N.D.
Sucrose
(9/1)
3.0
0
0
0
0
0
0
0
3.0
0
0
0
0
0
0
0
3.0
0
0
0
0
0
0
0
Concentrations:
DNOC
(mg/1)
50
150
200
Sucrose
(g/D
3.0
3.0
3.0
NaHCOo
(9/17
10.7
10.7
10.7
(NH4)2HP04
(g/D
1.25
1.25
1.25
-------
TABLE 16. THE EFFECT OF HIGH DNOC CONCENTRATIONS ON THE CO-METABOLISM
OF DNOC IN CONTINUOUSLY-STIRRED BATCH TEST*
en
Sample
J
K
L
Time
(days)
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
PH
8.0
6.8
6.8
6.8
6.8
6.8
6.8
6.8
7.9
7.5
7.3
6.9
6.9
6.9
6.9
6.9
8.0
7.9
7.8
7.7
7.7
7.5
7.5
7.4
SS
(mg/1)
35
132
102
73
65
57
51
59
37
64
62
43
32
34
41
62
32
58
45
48
54
54
48
56
vss
(mg/1)
21
102
86
57
52
50
47
49
20
32
35
32
26
30
34
51
15
25
19
20
34
33
25
34
CODs
(mg/1)
3730
3450
3400
3330
3410
3450
3470
3370
4040
4000
3940
3660
3720
3740
3790
3690
4290
4350
4300
4180
4300
4260
4300
4140
TOC
(mg/1)
1850
1400
1320
1350
1400
1620
1700
1530
1520
1680
1600
1530
1360
1660
1660
1590
1560
1600
1690
1660
1600
2020
1940
1920
DNOC
(mg/1)
250
201
152
115
108
100
94.5
83.0
500
500
493
470
390
311
298
253
750
750
750
750
750
739
726
712
Sucrose
(9/1)
3.00
0.88
0.285
0
0
0
0
0
3.00
2.57
2.40
0.67
0.65
0.59
0.56
0.49
3.00
3.00
3.00
2.96
2.93
2.90
2.88
2.79
*Initial
Sample
J
K
L
concentrations:
DNOC
(mg/1)
250
500
750
Sucrose
(9/1)
3.0
3.0
3.0
NaHCO.
(9/1 r
10.0
10.0
10.0
(NH4)2HPO,
(g/T) <
1.25
1.25
1.25
\
-------
150
129
100
o
o
z
o
. 75
u
I 50
"o
u
u
Z
e
5 25
Znflu«n( DNOC Cone* *250 mg/l
HRT sQ.47 Days
01234
Anoirobic Column Influent
Sucre** Conei/
/ONOC Cone.
Figure 6. The effect of sucrose/DNOC concentration ratio on the
removal of DNOC in the anaerobic column, run 1.
52
-------
~ "25
E
e
100
o
o
8 75
2 50
e
E
3
e
o
o 25
Influent DNOC Cone. * 500 mg / I
HRT *3,0 Ooys
I
Anatrobic Column Influtnt
Suero«« Cone
/
DNOC Cone, l
Figure 7. The effect of sucrose/DNOC concentration ratio on the
removal of DNOC in the anaerobic column, run 2.
53
-------
C) THE REMOVAL OF COD IN THE ANAEROBIC-AEROBIC SYSTEM
It has been established that DNOC will be degraded or converted
under anaerobic conditions in the presence of sucrose as a co-substrate.
The degradation of the DNOC molecule in the anaerobic process, monitored
by GC, was not associated with an appreciable decrease in soluble COD.
Early batch tests indicated in one case (Table 4) that over a period of
7 days a DNOC removal of 85% was associated with 16% decrease in soluble
COD, which was coupled with a 500% increase in TOA, or in another case
(Table 6) that over the same period of time a removal of 99.8% of DNOC
was associated with only an 11% decrease in soluble COD, while the TOA
level increased by 700%. These early data demonstrated that in the pre-
sence of sucrose, the anaerobic microorganisms co-metabolized the DNOC
molecule and, thereby, caused some initial structural change(s) in or
around its benzene nucleus. However, since no appreciable COD reduction
occurred in the anaerobic process and a very large increase in TOA
occurred, the two main processes which took place in the anaerobic system
were: a) the co-substrate, sucrose, was degraded by the anaerobic bacteria
to organic acids resulting in a large increase in TOA concentration and
an insignificant decrease in soluble COD and b) the structural conversion
of the DNOC molecule via co-metabolism by the anaerobic bacteria which was
dependent on the presence of the co-substrate sucrose.
The reduction in COD levels across the entire anaerobic-aerobic
pilot plant was studied under varied conditions of hydraulic retention
time in the anaerobic column and varied influent sucrose concentrations.
The Effect of Hydraulic Retention Time on the Removal of Soluble COD
The effect of the hydraulic retention time (HRT) in the anaerobic
column on the removal of soluble COD is shown in Table 17. Influent
sucrose and DNOC concentrations were maintained at 2.0 g/1 and 250 mg/1,
54
-------
TABLE 17. THE EFFECT OF ANAEROBIC COLUMN HYDRAULIC RETENTION TIME (HRT)
ON SOLUBLE COD REMOVAL BY THE ANAEROBIC-AEROBIC SYSTEM
HRT
(days)
0.16
0.28
0.43
0.72
1.62
2.0
2.5
3.0
nwnr
removed
(X)
»99.9
99.9
99.9
99.9
99.8
99.9
99.8
99.8
AC
(X)
26.1
20.5
24.6
24.9
21.7
20.5
15.7
3.2
COD Removal in
AS
(X)
24.0
49.2
66.6
65.9
69.6
68.6
76.8
86.7
Total
(X)
50.1
69.7
91.2
90.8
91.3
89.1
92.5
89.9
Influent sucrose concentration = 2.0 g/1
Influent DNOC concentration = 250 mg/1
55
-------
respectively. The HRT in the anaerobic column was varied from 0.16 days
to 3.0 days and the soluble COD removal in the anaerobic column varied from
26% to 3%, respectively. The soluble COD removal achieved in the subsequent
activated sludge reactor varied from 24% to 87% resulting in an overall
soluble COD removal of 50% to 90%, respectively. The results demonstrated
that within the operational conditions of the system, a short HRT in the
anaerobrc column such as 0.16 days and 0.28 days had an adverse effect
on the overall removal of COD, 50% and 70% removal respectively, while a
HRT of 0.43 days and longer resulted in overall COD removals of about 90%.
The Effect of Influent Sucrose Concentration on the Removal of Soluble COD
The effect of influent sucrose concentration on the removal of
soluble COD through the entire anaerobic-aerobic system was studied under
two sets of conditions:
I. For the first set of conditions, the effect of influent sucrose con-
centration was monitored at moderate influent DNOC concentration and low HRT
in the anaerobic column. The influent DNOC concentration was maintained at
250 mg/1, the HRT in the anaerobic column was maintained at 0.47 days and
influent sucrose concentration varied from 0.25 g/1 to 2.0 g/1.
II. For the second set of conditions, the effect of influent sucrose
concentration was monitored at relatively high influent DNOC concentration
and high HRT in the anaerobic column. The influent DNOC concentration
was maintained at 500 mg/1, the HRT in the anaerobic column was main-
tained at 3.0 days and influent sucrose concentration varied from 0.75
g/1 to 3.0 g/1. The results for both sets of conditions appear in Table 18.
When the ratio of influent sucrose concentration to influent DNOC
concentration was maintained at 2:1 or higher, the degradation (or
conversion) of DNOC in the anaerobic column was between 92% and 100%.
However, even when provided with adequate supply of co-substrate, sucrose
56
-------
TABLE 18. THE EFFECT OF INFLUENT SUCROSE CONCENTRATION ON SOLUBLE
COD REMOVAL BY THE ANAEROBIC-AEROBIC SYSTEM
CODs removal in
ACinf
Set sucrose cone
no. (g/1)
1*
2***
2.00
1.00
0.50
0.25
3.0
3.0
2.0
1.5
1.5
1.0
0.75
0.75
DNOC
removed
(*)
»99.9
99.4
91.6
50.4
»99.9
»99.9
»99.9
99.6
99.9
99.6
88.9
79.8
AC
(%)
23.3
14.7
16.8
10.3
27.3
30.0
26.4
21.8
30.1
30.8
24.0
19.5
AS
(%)
61.2
71.3
54.3
42.5**
63.2
58.7
58.7
56.9
52.3
48.4
44.6**
46.6**
Total
(X)
84.5
86.0
71.1
52.8**
90.5
88.7
85.1
78.7
82.4
79.2
68.6**
66.1**
* Influent DNOC concentration = 250 mg/1
HRT =0.47 days
** The activated sludge removal efficiency was significantly affected
by the high concentrations of DNOC in the anaerobic column effluent.
*** Influent DNOC concentration - 500 mg/1
HRT = 3.0 days
57
-------
to achieve more than 90% conversion of DNOC, the anaerobic microorganisms
were unable to decrease the soluble COD by more than 23% in the first
set of conditions and 30% in the second set of conditions. The importance
of the subsequent aerobic stage in the removal of the remaining high COD
is clearly demonstrated. The soluble COD removal that was achieved in
the activated sludge process (following an anaerobic DNOC conversion of
90% or higher) ranged from 54 to 71% under the first set of conditions
and from 48 to 63% under the second set of conditions.
It is also important to note that the performance of the activated
sludge process was adversely affected when the ratio of sucrose:DNOC in
the anaerobic column influent was less than 2:1 as shown in Figures 8
and 9. This resulted in failure of the anaerobic microorganisms to
satisfactorily co-metabolize DNOC, and, subsequently, high concentrations
of DNOC were present in the anaerobic column effluent and entered into
the activated sludge reactor.
D) CHANGES IN MICROTOX TOXICITY THAT WERE ASSOCIATED WITH THE REMOVAL
OF DNOC IN THE ANAEROBIC-AEROBIC SYSTEM.
The effect of influent sucrose concentration on Microtox toxicity
through the anaerobic-aerobic system was studied under the following set
of conditions: the influent DNOC concentration was maintained at 250 mg/1,
the HRT in the anaerobic column was maintained at 0.47 days and the
influent sucrose concentration was varied from 2.0 g/1 to 0.25 g/1.
As can be seen from Table 19, when comparing the Microtox toxicities
of the anaerobic column influent, the anaerobic column effluent and the
activated sludge effluent, the anaerobic column effluent was more toxic to
the Microtox microorganisms than the anaerobic column influent; however,
the activated sludge effluent was less toxic to the Microtox bacteria
than the anaerobic column effluent was suggesting that the aerobic biode-
58
-------
100
E
K
O
O
u
75
50
25
I
o
I
I
I
% of COD Removal (AC +AS)
% of COO Rtmoval (AC)
O AC Efflutnt DNOC Concentration
Operational Condition:
Influent ONOC * 250 mg/l
HRT «0,47dayi
ISO
125 1
J
100
u
o
o
u
o
75
E
50 »
o
u
u
.A
O
25 S
0,25 0.5 I 1.5 2.0
Influent Sucrott Concentration (g/l)
Figure 8. The effect of influent sucrose concentration on the
removal of COD by anaerobic column and activated
sludge, run 1.
59
-------
100
~ 75
e
E
§ 50
29
Operational Condition:
Influent DNOC * 500 mo,/l
HRT > 3.0 days
% of COD Removal (AC * AS)
A % of COD Removal (AC)
O AC Effluent DNOC Concentration
125
100 =
o
75
u
e
e
O
X
o
50 £
w
e
E
£
'o
o
25
0.75 1.0 1.5 2,0 3.0
Influent Sucrose Concentration(g/l)
Figure 9. The effect of influent sucrose concentration on the
removal of COD by anaerobic column and activated
sludge, run 2.
60
-------
gradation that occurred in the activated sludge process was principally re-
sponsible for the observed reduction in Microtox toxicity. However, from
the results presented in Tables 19 and 20 it is also clear that the Microtox
toxicities, that were demonstrated by the anaerobic-column effluent and the
activated sludge effluent were mainly a result of the anaerobic degradation
of the sucrose and the build up of organic acids in the anaerobic column.
These organic acids were subsequently further degraded to C02 and HgO in the
activated sludge process, when the hydraulic retention time in the system
v/as long enough and the initial sucrose:DNOC ratio was 2:1 or higher.
Since DNOC did not undergo biodegradation in the absence of the co-substrate
sucrose, it was impossible to monitor the Microtox toxicity changes that
were associated with the biodegradation of DNOC only, without interference
of the toxicity effects resulting from the degradation of sucrose to organic
acids in the anaerobic column. Accordingly, no conclusions were drawn
about the changes in DNOC toxicity from the Microtox data.
61
-------
TABLE 19. THE EFFECT OF INFLUENT SUCROSE CONCENTRATION ON MICROTOX
TOXICITY THROUGH THE ANAEROBIC-AEROBIC SYSTEM
Influent
DNOC Sucrose
(rag/1) (g/1)
250
250
250
250
0
2.0
1.0
0.5
0.25
2.0
HRT
in AC
(days)
0.47
0.47
0.47
0.47
0.47
DNOC removal
1n AC
<*)
»99
99
91
49
-
.9
.4
.6
.6
AC Influent
EC50*(5) EC5Q(15)
19.
7.
10.
10.
63.
36
34
77
92
16
13.84
6.18
9.74
8.90
51.57
AC effluent
EC50(5) EC50(15)
0.673
0.865
5.2
5.95
4.3xlO'5
0.44
0.62
5.3
5.22
7,9xlO~2
AS effluent
EC5Q(5) EC50(15)
9.97
10.25
5.73
3.63
2.78
8.48
8.27
5.55
3.52
2.28
01
ro
1s expressed in units of % of Initial sample concentration.
-------
TABLE 20. MICROTOX TOXICITY DATA FOR THE ANAEROBIC-AEROBIC SYSTEM.
en
CO
Influent
DNOC
(mg/1)
750
500
500
250
250
250
50
Sucrose
(9/1)
2.0
1.5
1.5
2.0
2.0
2.0
2.0
HRT
in AC
(days)
3.0
3.0
3.0
0.43
0.28
0.7
0.28
AC influent
EC5ot5)
5.69
6.44
9.60
6.83
19.4
19.4
19.36
14.7
EC50(15)
5.37
4.72
6.52
4.62
13.8
13.8
13.84
13.1
AC effluent
EC50(5)
2.26
2.45
0.67
0.26
0.707
0.69
EC50(15)
2.21
2.60
0.43
<0.1
0.40
0.52
AS effluent
EC50(5)
4.75
2.12
2.12
13.1
7.53
16.5
EC50(15)
4.49
2.03
2.03
9.18
10.6
14.0
is expressed in units of % of initial sample concentration.
-------
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69
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APPENDIX A
EXPERIMENTAL APPARATUSES AND PROCEDURES
ANAEROBIC SHAKER BOTTLE BATCH TEST APPARATUS AND PROCEDURES
Apparatus
a) Glass bottles with screw caps, each with 240 ml capacity.
b) Shaker water bath with temperature control.
Procedures
a) Each batch test lasted seven days with a single DNOC concentration
in each batch test run. A total of eight bottles were needed for each run
to supply eight incubation time samples, each one taken once every 24 hours,
for incubation duration periods of 0 to 7 days.
b) Purge the bottles with oxygen-free nitrogen gas.
c) Add in 170 ml of DNOC-feed solution. For composition of DNOC-feed
solution see Table A-l.
DNOC is added into the feed solution from an aqueous alkaline stock
solution of 10,000 mg/1 or 20,000 mg/1 DNOC, to make the desired final
DNOC concentration with a total volume of 200 ml.
d) Mix the solution in the bottles with nitrogen gas.
e) Collect a sufficient amount of anaerobic upflow column, mix with
oxygen-free nitrogen gas to ensure homogeneous distribution of its content.
f) Carefully transfer 30 ml of anaerobic effluent into each bottle to
serve as seed. Each bottle contains now a total volume of 200 ml.
g) After inoculation quickly purge the bottle top space with nitrogen
gas and immediately seal the top of the bottle with a sheet of parafilmR,
then screw the cap tight.
h) Use one of the eight bottles as time 0 sample, subject it to
70
-------
TABLE A-l. COMPOSITION OF DNOC-FEED SOLUTION
Compound Concentration (mg/1)
DNOC varies
sucrose varies
NaHCOa varies*
(NH4)£HP04 varies**
minerals:
FeCl3-6H20 10
CaCl2-6H20 5
MgCl2-6H20 5
NiCl2-6H20 5
ZnClo 5
(NH476M07024.4H20 1.5
CuCl2 0.5
MnCl2-4H20 0.5
Na2B407-H20 0.5
dilution water dechlorinated tap water
Concentration of NaHCO^ added is adjusted to maintain desired pH.
**Concentration of (NH4)2HP04 added is adjusted to maintain COD:N=100:5.
71
-------
analyses.
i) Put the remaining seven bottles in the 36.5°C water bath. Turn on
the shaker and adjust the shaking speed to 100 strokes per minute.
j) Stop the incubation of one bottle every 24 hours by removing it
from the water bath and subject it to analyses, continue incubation of re-
maining bottles with the last bottle removed at the end of the seventh day.
k) Right after the daily sample is taken, it is analyzed for pH,
oxidation-reduction potential (ORP), suspended solids (SS), volatile sus-
pended solids (VSS), and toxicity. Samples for soluble chemical oxygen
demand (COD), soluble total organic carbon (TOC), and total organic acids
(TOA) are also prepared. A sample is extracted and then analyzed by gas
chromatography (GC) for determination of DNOC concentration. When necessary,
an additional sample is hydrolysed and analyzed for residual sucrose concen-
tration. Details of analytical methods are in Appendix C.
1) Note: during the first few days of the incubation period there is
considerable gas production. It is, therefore, important to relieve the
gas and pressure produced by slightly opening the bottles and closing them
tight again every day.
ANAEROBIC CONTINUOUSLY-STIRRED BATCH TEST APPARATUS AND PROCEDURES
Apparatus
a) Three amber glass bottles with 4.2 liters capacity, each fitted with
a three-hole rubber stopper, serve as batch-test bottles.
b) Heat tapes wrapped around the amber glass bottles.
c) Powerstat autotransformers to manually adjust the voltage applied to
the heat tapes and maintain the temperature in the bottles at 36.5°C 1°C.
d) Heavy duty magnetic stirrer and a large stir bar for each batch test
bottle.
e) Three additional bottles containing gas displacement fluid (aqueous
72
-------
solution prepared following Method 511A, Standard Methods (53))
each filled with a two-hole rubber stopper.
f) Container to collect displaced fluid from e.
g) Three thermometers, each one fitted into the rubber stopper of each
of the three batch test bottles.
h) Tubing and clamps.
i) Apparatus set-up is shown in Figure A-l.
Procedures
a) To each batch test bottle add 3.75 liter of dilution water (tap
water), minus the volume for DNOC stock solution.
b) Turn on the autotransformers and the magnetic stirrers, equilibrate
to 36.5°C+1°C. Adjust the autotransformers if necessary.
c) Purge the batch test bottles as well as the bottles containing the
gas displacement fluid with oxygen-free nitrogen gas.
d) For each batch test bottle add in the desired amounts of sucrose,
NaHC03, (NH^HPCty and trace minerals to give the final concentrations as
listed in Table A-l.
e) Add in DNOC stock solution (10,000 mg/1 or 20,000 mg/1, in aqueous
alkaline solution) to make the desired DNOC concentration with total volume
of 4 liters.
f) Collect enough volume of anaerobic effluent from anaerobic upflow
column, mix with oxygen-free nitrogen gas during collection time.
g) Carefully transfer 250 ml of anaerobic effluent into each batch test
bottle for inoculation.
h) After inoculation, quickly purge the top space of the batch-test
bottles with nitrogen gas and stop the bottles with the rubber stoppers.
i) Use parafilm^ sheet to build up the lip of the bottles and add water
to ensure a water seal.
73
-------
thermometer
displaced,-
liquid
siphoning tubing
«*
F \vpld space
rt
\ (a + b +
power
supply
displacing
liquid
Figure A-l. Schematic diagram of continuously-stirred batch test
apparatus.
-------
j) Withdraw 250 ml immediately after inoculation, to serve as time 0 sample,
by opening the clamp and siphoning with a section of tubing filled with water.
1) Withdraw 250 ml sample once every 24 hours for the following seven days.
m) Right after the daily sample is taken, it is analyzed for pH,
oxidation-reduction potential (ORP), suspended solids (SS), volatile suspended
solids (VSS), and toxicity. Samples for soluble chemical oxygen demand (COD),
soluble total organic carbon (TOO, and total organic acids (TOA) are also
prepared. A sample is extracted and then analyzed by gas chromatography (GO for
determination of DNOC concentration. When necessary, an additional sample is
hydrolysed and analyzed for residual sucrose concentration. Details of analytical
methods are in Appendix E.
n) Note: the withdrawal of the samples from the batch test bottles will
cause the gas displacement fluid to flow from container (f), back to the gas
collecting bottles (e). To prevent air from getting into the system at the time
when a sample is withdrawn, the end of the tubing in container (f) should be well
submerged at all times.
75
-------
PILOT PLANT PROCEDURES
a) The anaerobic upflow sand filter column was started up by several
inoculations with seed from a sludge digester of a local municipal sewage
treatment plant. Operating parameters such as organic loadings, hydraulic
retention times, loading concentrations of DNOC and of sucrose were carefully
monitored and were changed when changes in operating parameters were called
for by the protocol of a particular experiment, which was usually no less
than three hydraulic retention time periods under previous operating con-
ditions. These changes were introduced only when the column was operating
under steady-state conditions.
b) The performance of the anaerobic column in the pretreatment process
of DNOC was monitored by sampling its effluent and analyzing it for pH,
alkalinity, oxidation-reduction potential (ORP), suspended solids (SS),
volatile suspended solids (VSS), and toxicity. Samples for soluble chemical
oxygen demand (COD) and soluble total organic carbon (TOC) were also prepared.
A sample was extracted and then analyzed by gas chromatography (GC) for deter-
mination of DNOC concentration. When necessary, an additional sample was
hydrolyzed and analyzed for residual sucrose concentration. When gas pro-
duction in the column was significant, the volume of the off-gas produced was
analyzed and its volume was measured. Details of analytical methods used are
in Appendix C.
c) The performance of the activated sludge process in the treatment of
the anaerobic-degradation-products was monitored by sampling its effluent and
analyzing it for pH, COD, TOC, and toxicity.
76
-------
APPENDIX B
ANAEROBIC CONTINUOUSLY-STIRRED BATCH TEST RESULTS
tfio
^^^-- ^ .-"'**'4 H te"-»"~ 4- .^".' t
J I"
_- "_:i-^ » ___; * i _: ;;:_: ; JL - ..^. .. . :_:,:_ : . ;
1 :..j T - .... f i. \ »- i -- r. .- i. j* ~ *- -*
-100
-i-'. -*
^:z:r~~i:^;:
6 i i
Figure B-l.
pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations. ___ _
"Reproduced irom
; best available_copy.
77
-------
Figure B-2. COD and TOA changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
78
-------
mj--.p.ii
-I----1F-- ---i
Figure B-3. DNOC and VSS changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
Reproduced from
best available
79
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^^^ '"-'""";:rr:r:i."r7T±7±::::!-.ri±:i:-.-im-:i::i ::--...:.:r:"::
Figure B-4. pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
Reproduced from
best available copy.
80
-------
4«0
MOOT
Figure B-5. COD and TOC changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
81
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='::Ir.j:-
TJE
==3-:=~;c=iV::
I I | li'
--i__^l '---Ij ^ ----Ji -_;^ - "-!"'_" "".'*' ' '^_'"'' ' ^^C~ 'j"~"' ' "~ ' ' "" ' t'- ' '
390*
Figure B-6. DNOC changes vs. time in continuously-stirred
batch test under the given initial concentrations.
Reproduced from
best available copy.
82
-------
p: L^^rrTtT^J^ry--:).--^=:i::::::... :
-------
-«50
-3oo
izriviit
Figure B-8. pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
84
Reproduced from
^available copy
-------
Soco
Figure B-9. COD and TOC changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
85
-------
:_^-T7i- 4--- -; : ~."| TJjn"^
OX "'
Figure B-10. Sucrose changes vs. time in continuously-stirred
batch test under the given initial concentrations.
86
-------
KD»
i~j|:-^--
-"--:=?F-"~7i;:~:£:-i--" ::"~:£r=rt::::. -- r--*
.1 : :I:T_.J-:_:J: : J~:4-"-"-- -' J.nuL.
=3=
Figure B-ll. DNOC changes vs. time in continuously-stirred batch
test under the given initial concentrations.
87
-------
300
VSS g^lfS'ijj
..iil^f::: i^j-:::; /.. /.liT. I^UII^V~!>
- ~ "' """" ' '"
^"I'-iTl*:.'. ~i~:i:~.~.r,:~:~ ;r _:r:ii":":"::
Figure B-12. VSS changes vs. time in continuously-stirred batch
test under the given initial concentrations.
-------
^j^^g
.O
=^'j::::i:;^^-==f=^-"jr-^^^:r---^^
j. ' p::ia-..-::...-::::. \ ::»"..::-;
l-i:T ..il
-*»,-
3
TI'
Figure B-13. pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
89
-------
"""" -'-^- " -
rrhrrTi-T:
ini: :~;.:
TOC
iHCfl,
3135**
"_.. T]. ii, --~1'.' ' ..j.-'.^....'. ...'... I.....':
E~=TTr-"
:i._:::zi.:
E^:
_ _i
' ' I ..,f t _ !
__.: r.:ir^. : ^^ t^= - iJ= ;". :~:p=-.- , ..-^
I I I .'_ : _t~ I" --*--; l
^_-_^ r-~~ '^-^:-|
9ooo
Figure B-14. COD and TOC changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
90
-------
-I.--.._...4 .._.--) -i- irr* l J
"- , ~ ,"" - '*". ;' -"
1 ' ' a -.~ rH ^^.'--^H^HEE^;;^';: :""r~.;;:::;;::.- .-j
v ; ' '~ *
>-^^'\TJ^=:Fr3=~=i=---^r^^ira .-V~3 ,-I|....J=:;;.= fc";:u.rj.;--::-J-.:.-: ;.:..'t:'..-3
Figure B-15. Sucrose changes vs. time in continuously-stirred
batch test under the given initial concentrations
91
-------
.. , , | , , , , ,_
-t--r=TJ.- :1-.TTA azsij::.;;:.^!-.^^^^ r~" I"
I I.-.TT-.'-:.)-... :..=?
£^
^___^.::r...^-..."r:.^.. .^..:^_:.jl-r.-y
'
4.016.
See*
DMOC.
"~'J"JH.Ti'.T -=^ : i r ; " '-N i""'7yr : ^
1 ..._._i-.-t; %...1 a^L
see
i r- ^ * -^ y ( y
~ T
UUy)
Figure B-16. DNOC changes vs. time in continuously-stirred
batch test under the given initial concentrations,
92
-------
i .; ;... j.--. ;..... ..: .- :-.. .. -.._. i ..-.*_..... ( - - --.. .... .;
m
*»***&»»<* ~-~-i
__:n.Tr:i::".:.._~T::T..nti .L._^':';i-....,. .liuTT'::::
^=-4 ;^_.^=^^-::z^=:3-=^^=
L.^r.-'r-.-.-....-^-^^-^.rrr^ii...... -Ui:-::::;
lj.. 1..
£*»*»(»
^>}nf-
^SJLTTolt;
Si^M^il
HWca,
50^.
feft^
fk"afc
500^
:*5:?t:
f>5lfc
=e=
100
r^i_~L.TT^.ii: '^rmij-i m: **t-" r f. irt ^; _; 3 :"
1 j p^ ~/'" I ~
V5S
VK=
. ..._.,.....
==jfto»^
50'
i^i^HH-i
;_ni; :-:;;V:!-:^:T^
")"::=:'
Figure B-17. VSS changes vs. time in continuously-stirred batch
test under the given initial concentrations.
93
-------
-loo.
~^---- '**-± r-* "t*" :n^"l.^"iiu ,-i-
.-::~\^-r^^:?.:V:tti^.-.l-~ : T!".-.::.-^"..-- ^t~:
r~T"Tii^_.T:-7ir~rrr:r._3'. ::nn^;L".::t~i- ".
3 *
TIM t^Uf.)
i ^_.rj
Figure B-18. pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
94
-------
BttO
' ' r " --
r:.^=7l::^.-:-:ir=::r:L^;::ri-^;:Ir:: i=^i3_^;;^r_^::^4_^
s*»-
woe
,-..,!. ^ -..!! . -»-'j'~ I | ^ T ? . . - , . ^ I \
^^^ii^v - ~*"~~~""*^^^sJ:.^Lr"j !* i »^ _!_" *' iffl" ~*' '* ' ' '*'
Figure B-19. COD and TOC changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
-------
Figure B-20. Sucrose changes vs. time in continuously-stirred
batch test under the given initial concentrations,
96
-------
(rrr
:! --i~-r-::- -~:--i :.-:-: -.:::
=^.Jg.-?fe '
t~ '
1^, ^.^,
=^:-~ln~;-^fe::-=n=-^L-=;:^=H.-=- -^:--;
~ """:."r:'._:-r^L-."^i.'i'.'.'..~ "~i
Figure B-21. DNOC changes vs. time in continuously-stirred batch
test under the given initial concentrations.
97
-------
.i:-.":.:::i.:::::.-.r:
TOHfi"
.15.'
~'T~" ' ..~t 1 ^^
... .^ : m x~ : ^. - ^^ .y -.- _^--. J= w ^=>
:r:-.-71i: \. i-- ::; :.-:-.-|-:r:- .-i'NJ:. .7..":::. j'V.-:.. yT: -;^ j;J:-.:.;:. .!._.;. '.'.J.: .':....-
vss L --.;-//
i==
r.:--^
Hn==]
.-.:--::::
- ;
r_-.-
::::..
. . -"-
-: TTTT
T^=:
£^--~^ r,
: -: --I = V--~=-
- ' '- p -
Tm^^ i7=rr-:r -.:.-: -"i
L:_I_,,_.."' ~TTTT."". ".rr. ",j~ .'[". ;i*rini
1 ''JT'.' -.3
.....J..::: i:. :;:J :;:^
-r"^^==?^j:^^^:^:S::j::^;:;ini^:J^^rU:;ifi
Figure B-22. VSS changes vs. time in continuously-stirred batch
test under the given initial concentrations.
98
-------
T~I1:'n';r:":"':':"'4"~::^i
,_ ;._{ ;.. .;.. j ; j.j.^jrj
Figure B-23. pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
99
-------
Figure B-24. COD and TOC changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
100
-------
-^ rrrrf-TT^rri: ''--Jl^^r^" ^'~" T-~;:l-i---r^^r"~
Sr~:k:
., ..... ..._.-,- I
Figure B-25. Sucrose changes vs. time in continuously-stirred
batch test under the given initial concentrations,
101
-------
:.l:3t:'-:::.i::-Sj.::i:"
IT._n^T.;~'. .........
=§=
.-itrrir;::
-- ^--
^
rziTzt-T~r_l inr i^
3.5 »ic
UDO.
^:&>
as %
-2~=i ^------f-'-^P^=^-^^--|--=---E...::...y.^^r-a
Figure B-26. DNOC changes vs. time in continuously-stirred batch
test under the given initial concentrations.
102
-------
;U-:-3
r.Ttr=rf--7-=fc -.-^±.-^=lr.----l:r.-z=l-~:
---j :4| j^-^Vji-^--^^----
too-^
telO-^
'*6W
r3.5%
-=*
r- a
-:---[--:^-T|-. -rrl ^t-i-.^^N^;:!:;:;:^:;
Figure B-27. VSS changes vs. time in continuously-stirred batch
test under the given intial concentrations.
103
-------
'&+*
-to;
-.^-, ...-. ..... ^., , .__ r.. ~^..,. .. r-.--^j------ -T - :;jj:_.__- -;^ L^_^i
(vT1^3^HniH2^;^^fH^f^^:n^"^^
s +
T«W o<«j«>
Figure B-28. pH and ORP changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
104
-------
-T7~E~^:^:-T7=:::i;. ' ' -:-J---"--g:
Figure B-29. COD and TOC changes vs. time in continuously-
stirred batch test under the given initial
concentrations.
105
-------
Figure B-30. Sucrose changes vs. time in continuously-stirred
batch test under the given initial concentrations.
106
-------
APPENDIX C
METHODS USED IN ANALYTICAL PROCEDURES
Experimental procedures employed in this study conformed to established
laboratory techniques, and were in accordance with those described in
"Standard Methods" (53), "Methods for Chemical Analysis of Water and Waters"
(55), or "Test Methods: methods for organic chemical analysis of municipal
and industrial wastewater" (56).
CHEMICAL OXYGEN DEMAND (COD) DETERMINATION
Chemical oxygen demand (COD) was measured by the modified semi-micro tube
method (54,55). This method uses the same reagents as listed in Standard
Methods (53), but uses smaller volumes. It has been approved by the EPA for
NPDES compliance monitoring.
DNOC DETERMINATION BY GAS CHROMATOGRAPH
Equipment
Varian Aerograph 1440 gas chromatograph with flame ionization detector.
Column: 3 ft x 2 mm ID Glass. Packing: 1% SP-1240 DA on 100/120
supelcoport. Supplier: Supelco, Inc.
Carrier gas: Prepurified N2 at 20 ml/min and 24 psig.
Flame ionization detector fuel: prepurified H2 (29 ml/min, 15 psig) and
air (300 ml/min, 30 psig).
Temperatures: 180°C injector and 180°C detector.
Column temperature program: 2 min initial hold at 150°C, 15°C/min to
170°C and hold.
Procedure
The procedures for sample preparation, extraction, quality control and
107
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data interpretation followed Method 604-Test Method for Phenols (56).
OFF-GAS DETERMINATION
Volumetric measurements of off-gas produced in the anaerobic column
were done following volumetric method-511A of Standard Methods (53)»
Off -gas composition was determined following gas chromatographic method
511B of Standard Method (53), using 1 1 sealed gas sampling bags for sample
collection.
Equipment
Baseline Industries, Inc. Model 1030A gas chromatograph equipped with a
thermal conductivity detector. Helium was used as carrier gas, analytical
grade C02, Cfy and N2 were used as calibration gases.
OXIDATION-REDUCTION POTENTIAL (ORP) DETERMINATION
The determination of oxidation-reduction potential (ORP) was done by
the use of Sensorex reference combination ORP electrode (Sensorex Inst. Co.,
Westminister, California) along with a Fisher Accumet^ 325 pH/Ion Meter
(Fisher Scientific Co., Pittsburg, Pennsylvania).
pH DETERMINATION
pH was measured with a Fisher Accumet 325 pH/Ion Meter (Fisher
Scientific Co., Pittsburg, Pennsylvania).
SUCROSE DETERMINATION
The determination of sucrose concentration was done by using the indirect
method in which sucrose is inverted by hydrolysis to give equal parts of
glucose and fructose. Samples and sucrose standard curve samples were hydro-
lyzed with ^$04 and the concentration of glucose formed after the inversion
was determined by Beckman Glucose Analyzer II.
108
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SUSPENDED SOLIDS (SS) AND VOLATILE SUSPENDED SOLIDS (VSS) DETERMINATIONS
Suspended solids (SS) were determined using Method 209D of Standard Method
(53) for total nonfiltrable residue dried at 103-105°C, and volatile suspended
solids (VSS) were determined using Method 209G for volatile and fixed matter in
nonfiltrable residue and in solid and semi sol id samples.
TOTAL ORGANIC ACIDS (TOA) DETERMINATION
Total organic acids (TOA) were measured by the chromatographic separation
method-504A of Standard Methods (53). However, when a sample contains a high
concentration of DNOC or its by-products, the identification of the final
titration endpoint is not clear due to interference by the yellowish or
reddish color of DNOC or its anaerobic degradation products.
TOTAL ORGANIC CARBON (TOC) DETERMINATION
Total organic carbon (TOC) analyses were made in a Beckman Carbonaceous
Analyzer Model 915B equipped with total and inorganic carbon channels.
TOXICITY DETERMINATION
Toxicity measurements were done by using the Beckman Microtox toxicity
analyzer. The bioassay method used is based on the light output of lumines-
cent bacteria, as measured by a temperature controlled photometric device.
The bioassay microorganisms are handled like a chemical reagent.
Toxicity analysis procedures as described in Microtox system operating
manual (51) were followed.
The ECso, which is the effective concentration that reduces the light
output by 50%, was determined by plotting gamma (the ratio of the amount of
light lost to the amount of light remaining) as a function of sample concen-
tration on log-log graph paper. The ECso value was determined by the inter-
section of the best fit line with gamma = 1.0.
109
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