FINAL REPORT
on
DEVELOPMENT OF .TEST FOR DETERMINING AN AEROBIC
BIODEGRADATION POTENTIAL
TECHNICAL DIRECTIVE 16
by
D. R. Shelton and J. M. Tiedje
Dept. Crop and Soil Sciences
Michigan State University
East Lansing, MI 48824
Contract No. 68-01-5043
R. G. Wilhelm, Project Officer
R. Brink, Task Officer
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
December 30, 1981
-------
Table of Contents
Summary [[[ i i i
Introduction [[[ *
Background [[[ 1
Survey of organic compounds for anaerobic biodegradation ........... 2
Comparison of parent compound disappearance in whole sludge
vs mineralization in 10% sludge ................................... 4
Comparison of degradation results among sludges .................... 8
Effect of length of storage on sludge viability .................... 10
Effect of mineral salts media on gas production from
si udge [[[ 12
Effects of Og on gas production in 10* sludge .................... 17
Effect of chemical concentration on gas production in
si udge .............................................. . ............. 19
Reproducibility of results ......................................... 20
Discussion of significant test parameters .......................... 22
Appendix I. Calculation of percent theoretical degradation ......... 28
f
-------
Summary
The goal of this research was the development and validation of
a screening level test for determining anaerobic biodegradation
potential. The requirements of such a screening level test are that
the method be easily and rapidly performed with a minimum of
equipment, that the results be reliable and easily interpreted, and
that the method be able to accomodate a wide variety of chemical
substances. This necessarily eliminates the use of more
sophisticated, analytical techniques which, although more definitive,
are too costly and too specific. Measurement of ultimate
biodegradation or mineralization is appropriate for such a screening
method because the products of mineralization (CH4 and C02 in
anaerobic habitats) can be easily monitored and are independent of
parent chemical structure. This is also desirable in that only
compounds which are mineralized will give positive results; it is
unlikely that compounds which readily undergo mineralization will
pose a significant environmental threat.
We have conducted experiments which we feel adequately define
and validate the significant test parameters of this method. Based
on the results of these experiments and our experience with the
method, we feel that the proposed method will yield consistent and
reliable results. The salient features of the recommended test
method are given below.
1) A 10% sludge inoculum consisting of primary anaerobic sludge
with 15-30 day retention time and total organic solids of
approximately 1.0-2.0% is recommended. Sludges can be stored for up
to 4 weeks at 4°C in tightly capped containers; however, fresh sludge
should be used whenever possible.
2) Any standard anaerobic mineral salts medium with a 2 mM
phosphate buffer and 1.2 g NaHCOs/liter with a 103 C02/90% N2
headspace is recommended. Sulphide can be added to ensure reducing
conditions in concentrations not to exceed 1 mM. Since active sludge
has a strong 02 consuming capacity we have not found this necessary.
-------
IV
3) Chemicals should be added to achieve a final
concentration of 50 ppm carbon. Chemicals should be introduced into
test bottles, prior to addition of 10« sludge, in one of four ways
depending on phase and solubility: (1) liquids should be dispensed
into bottles with a microsyringe, (2) water soluble solids should be
dissolved in water, then dispensed into bottles, (3) water insoluble
solids should be dissolved in di-ethylether (or comparable solvent),
the ether solution dispensed into bottles and then allowed to
evaporate for at least 2 hours, and (4) insoluble polymers should be
weighed out and added to bottles.
4) One hundred milliliters of 10% sludge solution in 160 ml
serum bottles with new butyl rubber stoppers should be used.
5) Serum bottles should be incubated at 35°C for at least 8
weeks or until biodegradation is complete. If gas production is
still in progress at the eighth week, then incubations should be
continued until 'gas production is complete.
6) Gas production should be measured using a pressure
transducer with multimeter which is connected to the test bottle with
a 3-way valve and 20-gauge needle. Measurements should be made on a
weekly basis; excess gas pressure should be vented.
7) Reference chemical(s) should be included with each series of
incubations in order to characterize sludge activity. p-Cresol
and/or phthalic acid are suggested as a more rigorous test of
biodegradtion capacity. Ethanol, which is easily degraded, can be
used as a positive control.
-------
Introduction
The Toxic Substance Control Act was passed with the intent of
preventing new chemical products from posing an unreasonable risk to health
or to the environment. In order to assess such risks, a knowledge of the
fate of chemicals in the environment, including whether or not degradation
occurs, is necessary. Hydrolytic, photochemical, and aerobic microbial
degradation are processes which have been fairly well studied and for which
reasonable test methods exist. This is not the case for anaerobic
biodegradation; no proven methods for a generalized test exist in the
scientific literature. The purpose of this project was the development and
validation of a general screening method for determining the anaerobic
biodegradation potential of organic compounds.
Background
Few studies have been conducted in the past on the anaerobic
biodegradation of organic compounds of industrial or environmental
significance. Earlier work focused primarily on the metabolism of model
substrates such as benzoic acid, using sludge as an inoculum
(2,5,6,10,15,18), or the transformation of pesticides in flooded soils
(1,13,20,23,24). None of these studies provided the necessary experimental
framework for describing a general anaerobic biodegradation test method.
Recently, Healy and Young have described the anaerobic biodegradation of
several hydroxy and/or methoxy substituted benzoic acids and substituted
phenols (7,8). They used a 10% sludge inoculum with mineral salts medium
and assessed ultimate biodegradation (mineralization) by measuring gas
production. Based on the work of Healy and Young, a committee of the Amer.
Soc. for Testing Materials (ASTM) has proposed a test method. The salient
features of the ASTM method are: (1) a 4 week incubation period, (2) a
test compound concentration of 50 ppm carbon, (3) slightly modified Healy
and Young medium and headspace of 30»-70* C02/N2. (4) introduction of
water-insoluble materials into sludge bottles by disolving them in dioxane,
and (5) assessment of biodegradation by measuring gas production using a
pressure transducer (with multimeter). We have used the proposed ASTM
method as a starting point for our own studies in modifying, refining,
and/or validating the significant test parameters.
-------
Survey of organic compounds for anaerobic biodegradation (compound survey)
As previously stated, there is little information in the literature
concerning the anaerobic biodegradation of synthetic organic chemicals.
(xenobiotics). One of our first priorities was to conduct a biodegradation
survey of a variety of organic chemicals with the following goals in mind:
(1) attempt to establish a structure vs. biodegradability scheme, (2) use
the results to select compounds for future methods testing, (3) gain
experience handling xenobiotics with different physical properties and
working with anaerobic sewage sludge, and (4) assess the appropriateness of
a 10% sludge inoculum.
Secondary anaerobic sewage sludge from Adrian and Jackson waste
treatment plants was collected in 1 liter jars, tightly capped, and stored
at 4°C until use (fresh sludge was collected once a month). A 10% sludge
inoculum was prepared by adding sludge, filtered through one layer of
cheesecloth, to 1-2 liters of mineral salts medium consisting of (per
liter) 0.30 g KH2P04, 0.35 g K2HP04, 0.5 g NH4C1, 0.1 g Mgd2 • 6H20, 70 mg
CaCl2 -2H20, 20 mg FeCl2 • 4H20, 1 ml of a trace metals solution (25), 1.2
ag MaHCOs, and 120 mg Na2S • 9H20, which had been autoclaved (to drive off
02), then cooled to approximately 35°C while sparging with a 10% C02-90% N2
gas mixture. One hundred milliliter aliquots of 10% sludge were dispensed
into 160 ml serum bottles (in the Wheaton catalogue these are Ifsted as 125
ml bottles) while sparging with a 10% C02-90% N2 gas mixture. Test bottles
had previously been amended with 50 ppm test compound as follows: liquids
were dispensed via syringe, water-soluble solids were dissolved in water,
then dispensed, while water-insoluble solids were dissolved in
diethylether, dispensed, and the ether allowed to evaporate (1-2 hours).
Bottles were incubated at 35°c from 4 to 14 weeks. All compounds were
tested in duplicate. Methane production was monitored weeky; if methane
production was observed in test bottles over controls (no chemical added),
those bottles were again amended with substrate as before, except that
water-insoluble solids were dissolved in dioxane to facilitate additions.
Methane was quantified using a FID equipped gas chromatograph with a 2 m
Tenax-GC column; the carrier gas flow rate was 30-40 ml/min N2.
Degradation is expressed as percent theoretical methane production based on
the stoichiometry of mineralization (see Appendix I for calculations). Lag
-------
time represents the minimum time period (in weeks) before significant
methane production over controls was observed.
A total of 98 compounds were tested. Thirty-nine were degraded to
some extent (in at least one sludge) (Figure 1), 49 were neither degraded
nor inhibitory (Figure 2), and 10 were inhibitory (Figure 3). Based on
these data, it is difficult to make generalizations regarding structure vs.
biodegradability. Of the substituted phenols, m- and £-methylphenol were
degraded, chloro- and aminophenols were not, while of the substituted
benzoates none of the methylbenzoates were degraded but several chloro- and
aminobenzoates were; of the chlorobenzoates, only m-chl.orobenzoate was
degraded, while of the aminobenzoates, jn-aminobenzoate was the only one not
degraded. Also, 2-octanoJ was degraded while 2-hexanol was not; benzyl
alcohol and 4-hydroxybenzyl alcohol were degraded while 2-hydroxybenzyl
alcohol was not. It does appear that the sole presence of hydroxy and/or
carboxy substituents (regardless of number or position) usually confers
biodegradability on the aromatic nucleus. Although we feel confident of
the data indicating biodegradability, we cannot be certain that those
compounds for which we found no evidence of biodegradation are, in fact, '
persistent. This is based, in part, on the observation that degradation
results for a few compounds which were particularly difficult to degrade
(long lag times) were not consistent. For example, 4-hydroxybenzyl alcohol
was not degraded initially, however, when retested, biodegradation was
apparent by the 8th week (Ja'ckson sludge only); jD-aminobenzoate and
jn-chlorobenzoate acid were degraded initially in both Adrian and Jackson
sludges, however, when retested, jn-chlorobenzoate was not degraded and
£-aminobenzoate was degraded in Jackson sludge only. After gaining
experience with sludges we now believe that secondary anaerobic sludge,
such as used in the survey experiment, does not have the metabolic
diversity and activity of primary anaerobic sludge (see Appendix II),
particularly when used as a source of inoculum up to 30 days after
collection. Early length of storage experiments with secondary anaerobic
sludge indicated that a 30 day storage period was not detrimental to the
degradation of glucose, ethanol, propionic acid, or butyric acid. However,
we now question whether degradation results for easily degraded substrates
such as glucose, etc. can be extrapolated to compounds that are more
difficult to degrade. Considering that Adrian and Jackson secondary
anaerobic sludges are unmixed and unheated (these experiments were
-------
conducted during the summer months) with retention times of 60-90 days, it
seems almost certain that some loss of viability would have occurred,
particularly after a 30 day storage period. Since serum bottles were
generally discarded after 8 weeks of incubation in the absence of net
methane production, any extension of the lag time beyond 8 weeks would have
been regarded as lack^of degradation.
Comparison of parent compound disappearance in whole sludge vs.
mineralization in 10% sludge
Since the purpose of the test method is to accurately assess anaerobic
biodegradation potential of xenobiotics, it is important that
biodegradation results obtained with a 10% sludge inoculum be consistent
with or representative of the fate of xenobiotics in actual anaerobic
habitats*. In particular, we felt it important to establish the degree of
correlation between degradation in 10% sludge vs. whole sludge for selected
compounds. Compounds for study were chosen so as to meet one or more of
the following criteria: (1) compounds of some environmental concern, (2)
compounds of similar structure such that extraction and analyses of several
compounds could be accomplished simultaneously, and (3) compounds which
would most likely (in our opinion) be completely mineralized if parent
compound disappearance occurred and not simply transformed into some
persistent product (we made no attempt to follow mineralization in 100%
sludge since methane production from test compounds was an insignificant
fraction of total methane production). The 18 compounds chosen were
divided into two groups based on structure:
Group I Group II
di-n-methy!phthal ate £-methylphenol (o_-cresol)
di-n-ethylphthalate m-methyl phenol On-cresol)
di-n-butylphthalate £-methylphenol (£-cresol)
butyl benzylphthalate o-chlorophenol
di-(2-ethylhexyl)phthalate m-chlorophenol
di-n-octylphthalate £-chlorophenol
tri-m-cresyl phosphate m-methylbenzoate
jT-methylbenzoate
o-chlorobenzoate
m-chlorobenzoate
£-chlorobenzoate
*Dr. Ami Horowitz, working in our laboratory, has investigated the degradation of
aromatic compounds in anaerobic hypereutrophic lake sediments. Based on a
comparison of 28 compounds incubated in both anaerobic lake sediment and 10%
secondary sludge, there was approximately 65% agreement between the two habitats
with regard to degradation or persistence.
-------
For group I chemicals, 6 liters of anaerobic sludge were collected in
8-liter glass digestor bottles equipped with glass spigots for sampling,
and stoppers with 1 psi check values for venting excess gas pressure.
Phthalate esters plus tri-m-cresylphosphate were dissolved in acetone and
added to digestor bottles in an amount to give 20 ppm of each compound.
Digestor bottles were prepared in duplicate and incubated at 35°C for 8-10
weeks. One hundred milliliter samples were withdrawn routinely from the
digesters, mixed with 10 ml toluene (to inhibit further biological
activity) and dried for 17-23 hours at 50°C. Dried sludge was extracted in
a Vir-Tis Omni mixer with two 150 ml aliquots of dichloromethane. The
dichloromethane solution was centrifuged at 23,000 x _£ for 15 min to remove
particulates and then filtered through a 0.2 urn pore size Teflon filter.
The filtered solution was concentrated by roto-evaporation to approximately
2-3 ml and applied to a florisil column (approximately 1 cm X 15 cm). The
column was washed with 20 ml of hexane to elute extremely non-polar
components, then with 20 ml of acetone. The phthalate esters plus
tri-m-cresylphosphate were eluted in the acetone fraction. The acetone was
evaporated to approximately 2 ml, internal standard added, and injected
onto the GC column. A significant amount of colloidal material remained in
the sample, but it was deposited onto the glass sleeve of the GC injector
and did not interfere with the analyses; glass sleeves were changed
periodically to avoid occlusion. Phthalate esters plus
tri-m-cresylphosphate were quantified using a FID equipped GC with a 2 m 7*
Dexsil glass column; the carrier gas flow rate was 35 ml/min ^2- Injector
and manifold temperatures were 350° and 400°C respectively; the oven was
temperature programmed from 150°-330°C at 12°/min. Phthalate esters plus
tri-m-cresylphosphate concentrations were determined using known response
factors to phenanthrene (internal standard). The limit of detectability
was approximately 0.5 ppm.
Extraction efficiencies varied among compounds and among sludges
(Figure 4). Recovery of di-n-methylphthalate and di-ethylphthalate was
relatively poor. It appears that loss of these compounds was due both to
volatilization during drying and loss during sample cleanup. Sludges also
contained background peaks which co-chromatographed with certain phthalate
esters. We suspect that some of these may be phthalate esters indegenous
-------
in the sludge, however, we cannot be certain.
A total of three sludges were tested (Figures 5-7). Degradation is
expressed as percent parent compound remaining with time; percentages -have
been corrected for extraction efficiencies and background peaks.
Di-n-methyl-, di-n-ethyl-, di-n-butyl-, and butyl benzyl phthal ate were
degraded in all sludges although the relative rates varied among compounds
and among sludges. We cannot be certain whether or not
di-(2-ethylhexyl )phthalate, di-n-octylphthalate, or tri-m-cresylphosphate
were degraded (considering the accuracy and precision of extraction),
however, concentrations in most sludges tended to decrease with time. Data
for di-(2-ethylhexyl )phthalate were confounded due to the fact that a
certain amount of leaching of this plasticizer from tygon tubing (attached
to the spigot for sampling) undoubtedly occurred. 2 in general, relative
rates of phtha.late ester degradation, from fastest to slowest, were
di -n-methyl phthal ate _>_ di-n-ethyl phthal ate > di-n-butyl phthal ate _>_
butyl benzyl phthal ate.
A comparison of phthal ate ester degradation, assessed by monitoring
CH4 production in 10* secondary sludge vs. parent compound disappearance in
whole sludge revealed that degradation results were generally consistent,
although not always comparable (Figure 8). Only di-n-methyl phthal ate in
Adrian secondary sludge and di-n-butyl phthal ate in Jackson secondary sludge
yielded comparable results in both test systems. Low methane recoveries
(di-n-ethyl- and butyl benzyl phthal ate in Jackson sludge) or lack of methane
production (di-n-ethyl- and butyl benzyl phthal ate in Adrian sludge) may, in
large part be, attributable to lack of, or variability in, the viability of
secondary sludge; separate phthalate esters were tested at different times
using different batches of sludge.
In order to reassess the biodegradabil ity of phthalate esters in 10%
anaerobic sludge, separate experiments were initiated monitoring
production and substrate disappearance in 10% Jackson primary sludge.
Serum bottles of 10% sludge were prepared and CH4 production monitored as
previously stated for the compound survey. Five milliliter samples of 10%
sludge were periodically withdrawn from serum bottles and frozen until
extracted. Individual replicate samples from bottles containing
di-n-methyl-, di-n-ethyl-, and di-n-butyl phthal ate or butyl benzyl-,
-GC analysis of hexane extracts of tygon tubing indicated the presence of
significant amounts of di-(2-ethylhexyl) phthalate.
-------
di-(2-ethylhexyl)-, and di-n-octylphthalate were combined and extracted
with 30 ml of hexane. After standing overnight to allow for phase
separation, 25 ml of hexane (out of 30 ml) was withdrawn, evaporated to 2
ml and injected into a FID equipped GC with a 50 m SE-54 fused silica
capillary column. Injection and manifold tempreatures were 300°C; initial
oven temperature was 100°C for 5 min with a temperature program of 20°/min
to 290°C; the carrier gas flow rate was 1 ml/min helium. Concentrations of
phthalate esters were determined from standard curves. All incubations
were run in triplicate.
Results for phthalate ester disappearance in 10% sludge were
consistent with those using whole sludge (Figures 8 and 9), although the
relative rates varied somewhat (Figure 9). Di-n-methylphthalate was
degraded rapidly while di-n-ethyl-, di-n-butyl-, and butylbenzyl phthalate
were all degraded at a slower rate. There was no evidence for the
degradation of di-(2-ethylhexyl)- or di-n-octylphthalate. Methane
production mirrored substrate disappearance (Figure 10) with percent
theoretical Cfy production ranging from 76%-lQ3% (Figure 8). Based on CH^.
production and substrate disappearance data there does not appear to be a
substantial lag time for phthalate ester degradation. With the exception
of di-n-methyl- and di-n-ethylphthalate (40* and 30% loss respectively)
there was no loss of extractable parent compound in autoclaved controls
over the course of the incubation.
For group II chemicals, sludges were set up as previously stated,
except that digester bottles were fed 30 ppm of each compound. One hundred
milliliter samples were routinely withdrawn and frozen until extracted.
Fifty milliliter aliquots of sludge were adjusted to pH 2.0 and phenols and
benzoates extracted with three 100-ml aliquots of dichloromethane. The
dichloromethane solution was concentrated by roto-evaporation down to 3 ml
and injected into GC for analysis of phenols. The dichloromethane solution
was then taken to dryness, taken up in 5 ml of methanol and injected into
HPLC for analysis of benzoates. Phenols were quantified using a FID
equipped GC with 2 m Carbopak C-0.1% SP1000 glass column. Injector and
manifold temperatures were 250°C, the oven was run isothermally at 205°C;
the carrier gas flow rate was 20ml/min N£. Benzoates were quantified using
a HPLC with UY detector (set at 230 nm) and Partisil PXS 10/25 PAC column.
-------
Samples were run at ambient temperature using a 95% methanol/5% 0.05 M Na
acetate (pH = 4.5) buffer solvent system; the flow rate was 3.0 ml/min.
Concentrations of compounds were determined from standard curves.
Extraction efficiencies for substituted phenols were determined from 0 time
samples (Figure 4).
A .total of four sludges were tested. Data for Holt and Jackson
sludges are shown in Figures 11 and 12; data for Mason and Ionia sludges
were comparable. We were surprised by the degradation of £-chlorophenol in
all four sludges since we had no prior evidence of its degradation. It is
possible that _p_-chlorophenol may be transformed into some other persistent
product, however it seems most likely to us that a dechlorination occurs
leaving a readily degradable phenol moiety. We were disappointed in that
compounds which should have been degraded — ni-cresol and £-cresol —
generally were not. Only in Mason sludge did £-cresol degradation occur,
and then only after a 3 week lag. We are convinced that the sludges were
viable, so presumably certain of the compounds which we added were
inhibitory to degradation of m-cresol and £-cresol. We are suspicious of
jn-chlorophenol and £-chlorophenol. Both compounds were inhibitory to
methane production in 10* secondary sludge at concentrations of 89 ppm
(Figure 3). We have also observed that ni-chlorophenol and £-chlorophenol
were inhibitory to phenol degradation at 64 ppm in a phenol enrichment
which we have maintained for 1 1/2 years. Data for methyl and chloro
substituted benzoates are not shown because no degradation was observed for
any of the compounds in any of the four sludges. Since we had prior
evidence for degradation of m-chlorobenzoate in Holt sludge, degradation of
this compound may also have been inhibited. However, at this time, we can
make no general conclusions regarding the biodegradability of
methylbenzoates or chlorobenzoates in anaerobic sewage sludge.
Comparison of degradation results among sludges (sludge survey)
Since potential users of this test method will presumably be obtaining
anaerobic sludges from waste treatment plants in their own immediate area,
it is important to have some understanding of how sludges vary with respect
to their ability to degrade organic chemicals. In particular, we were
interested in determining what types of compounds were consistently
-------
degraded (such information would be extremely useful in selecting
reference chemicals) and also attempting to evaluate the differences,if
any, between "industrial" and "residential" sludges.
Bottles were set up as previously stated for the compound survey with
the following exceptions: primary anaerobic sludge was used instead of
secondary sludge and test bottles were run in triplicate. Twelve sludges
were obtained from the mid-Michigan area. The sewage treatment plants from
which these sludges were obtained vary considerably in size (inflow and
number of people served) and percent industrial input (personal
communication, John Phillips, former graduate, Pesticide Research Center,
MSU) (Figure 13). Twelve compounds, all previously tested, were chosen to
satisfy the following conditions: (1) diversity of compounds with respect
to rate and extent of biodegradation, and (2) diversity of chemical
structure and bond type. The compounds were:
polyethylene glycol-20,000 phthalic acid
2-octanol di-n-butylphthalate
ethanol di-n-octylphthalate
£-cresol m-chlorobenzoic acid
jn-cresol . propionanil ide
£-cresol chloroform
Degradation is expressed as percent theoretical methane production based on
the stoichiometry of mineralization (see Appendix I). Lag time is the
period of time (in weeks) required for approximately 20% of theoretical Cfy
production.
It is apparent that sludges varied in their ability to degrade
different compounds in an 8-week incubation (Figure 14). All sludges were
able to degrade ethanol, polyethylene glycol-20,000, _p_-cresol, and phthalic
acid (with the exception of Charlotte), while jn-cresol was degraded in at
least six sludges. 2-Octanol, di-n-butylphthalate, and £-chlorobenzoate
-------
10
proved more difficult to degrade; each compound was degraded by only 3-5
sludges. No sludge degraded all three compounds, although Jackson, Ann
Arbor, and Adrian degraded two of the three. Propionanilide probably was
partially degraded (in most sludges there was greater methane production in
propionanilide bottles vs. control bottles); however, increased methane
production was generally less than 20% theoretical, hence not significant
enough to conclude degradation (we suspect that the propionic moiety is
cleaved and degraded leaving a persistent aniline molecule). Percent
degradation was generally greater than 70% for jn-cresol, _p-cresol, phthalic
acid, and _m-chlorobenzoate. Lower percent degradation for polyethylene
glycol-20,000, 2-octanol, and di-n-butylphthalate may be, in part, due to
their slower rates of degradation such that an 8 week incubation was not
sufficient to allow for complete methane recovery. We are not certain why
CH4 recoveries from ethanol were low. It does appear that certain sludges
were less active than usual, i.e. di-n-butylphthalate is normally
completely degraded in 10% Jackson sludge with no apparent lag, yet in this
study only 49% of theoretical CH4 was recovered with a lag time of 5 weeks.
It is not obvious why sludges vary in their ability to degrade
different compounds, other than the obvious yet unverifiable reason that
different sludges have become acclimated to different chemicals due to
prior exposure. With the possible exception of di-n-butylphthalate, there
does not appear to be any apparent correlation between compound degradation
and percent industrial input. With the exception of Pontiac, sludges with
high input flows (_> 5- 106 gal/day) degraded more compounds (seven),
although Holt (flow = 1.1 • 10*> gal/day) also degraded seven compounds.
Based on these results, there does not appear to be any single criterion
predictive of sludge versatility with respect to biodegradation.
Effect of length of storage on sludge viability
Ideally, fresh sludge inoculum should be obtained for use in all test
incubations; however, depending on the distance between potential user and
a suitable source of anaerobic sludge, this may be impractical. It is
important then to know how long anaerobic sludge can be stored and under
what conditions to maintain its viability.
Length of storage experiments were initiated using £-cresol, phthalic
-------
11
acid (mono potassium phthalate), di-n-butylphthalate, and ethanol as test
substrates with Jackson, Holt, Adrian, and Chelsea primary anaerobic
sludges. Individual sludge samples were stored in tightly capped 200 ml
bottles at 4°C until use, such that all incubations were begun with sludge
from.previously unopened bottles. Test bottles were set up as described in
the sludge survey, except that NaS • 9H2 was not added to the medium. Gas
production was monitored using a pressure transducer with multimeter and
3-way valve. The 3-way valve (with 25-gauge needle) was attached to the
pressure transducer using stainless steel 1/16" tubing. At the beginning
of all incubations, bottles were allowed to equilibrate at 35°C, for 2-4
hours, then vented to atmospheric pressure to equalize initial pressures.
Gas pressure in bottles was measured and vented weekly. Experiments were
begun weekly up to the 4th week of storage. Degradation is expressed as
percent theoretical gas production based on the. stoichiometry of CH4 and
C02 production and taking into account gas solubilities (see Appendix I).
Based on percent theoretical degradation (after an 8 week incubation),
it is not apparent that a 4 week storage period had any detrimental effects
on sludge viability (Figure 16). However, based on the lag time before
significant gas production occurred, sludge viability was affected. This
effect was particularly pronounced in Holt sludge (Figures 16-19). Lag
times for degradation of £-cresol, phthalic acid and di-n-butylphthalate in
10* Holt sludge were significantly lengthened by storage of sludge beyond
the second week, although rates of degradation after the lag were generally
unaffected. It was also common for sludge stored for 3-4 weeks to show an
initial net negative gas production before the onset of degradation. We
are not certain as to the causes of this effect; we have no evidence for.
toxicity with any of these compounds. Ethanol degradation was unaffected
by sludge storage with the exception of the fourth week where rate of
degradation was somewhat slower. Jackson sludge was less affected by the 4
week storage (Figures 20-23). Lag times for degradation of £-cresol and
phthalic acid were only slightly affected with rates of degration remaining
approximately equal. Degradation of di-n-butylphthalate and ethanol were
little affected, if at all. Again, in the case of phthalic acid, increased
sludge storage resulted in initial net negative gas production. The
effects of length of storage on lag times for all four sludges (weeks
-------
12
required for 5Q% of net gas production) are summarized in Figures 24-26:
Lag times for p-cresol degradation increased from 4 to 7 weeks for Holt
sludge, 2-3 weeks for Jackson sludge, and 4-4.5 weeks for Adrian and
Chelsea sludge. Lag times for phthalic acid degradation increased from 3
to 6 weeks for Holt sludge, 3 to 4 for Jackson sludge and 4 to 4.5 for
Adrian and Chelsea sludges. Lag times for ethanol degradation were
relatively constant. Based on these data, a 4 week storage period does not
appear to be excessive for most sludges. We suspect that the viability of
Holt sludge was adversely affected, primarily as a result of the long
retention times (38-39 days) and low total solids (approximately 1.7%) of
the primary sludge; in essence, the organisms were starved for carbon. We
feel that the use of fresh sludge is always preferrable, if it can be .
conveniently obtained.
Effect of mineral salts media on gas production from 10% sludge
Since the purpose of the test method is to assess anaerobic
biodegradation potential it is desirable that the rate and extent of
xenobiotic degradation be maximized. In order to maximize degradation it
is necessary that the nutritional requirements for each component of the
methanogenic consortium be supplied. Presumably, in whole sludge this is
not a problem, however, in IQ% sludge it is possible that some critical
mineral nutrient may be diluted below its optimum concentration. We chose
to attack this question from three directions: (1) review the literature
on. methanogensis in search of studies demonstrating nutritional
requirements, (2) survey the anaerobic literature for content and
concentration of mineral salts and metals commonly found in anaerobic media
and compare these with minerals and metals found in 10* sludge, and (3)
based on 1 and 2, compare experimentally those media most likely to be
proposed for the test method.
We are aware of few studies where the mineral nutritional needs of
methanogens or methanogenic consortia have been extensively investigated
(most studies have concentrated on organic nutritional requirements).
Taylor and Pirt (21) reported that growth of Methanobacterium
thermoautotrophicum (a lithotrophic methanogen) was enhanced by 11 mM NH^
and 0.05 mM Fe^+ as compared to concentrations five times lower. Patel, et
-------
13
al. (17) reported that growth of Methanobacterium strain M.O.H.,
Methanospiril Turn hungatii GPI, and an unidentified strain similar to
Methanobacterium formicicum (all lithotrophic methanogens) was optimal at
iron concentrations in the range of 0.3-0.9 mM. Khan, et al. (12) reported
that maximum rates of methane production from cellulose by a methanogenic
consortium were achieved at HC03" and Fe^+ concentrations of 16-24 mM and
0.4-0.6 mM respectively, while nitrogen concentrations ranging from
3.5-30.0 mM and phosphate from 1.5-12.0 mM had little or no effect on
cellulose degradation. Hoban and Berg (9) reported that iron additions of
5-20 mM resulted in stimulation of methane production from acetate up to
100» in cultures enriched for heterotrophic methanogens. Such large
additions were necessary in order to increase the concentration of soluble
iron; iron was precipitated in the form of FeS, FeHP04 and FeC03. We were
particularly interested in this observation since carbon flow through
acetate is of key importance in methanogenic consortia. We repeated the
work of Hoban and Berg using IQ% sludge and our enrichment medium to
determine if increased iron additions would result in an increased rate of
methanogenses.3
Test bottles were set up as previously described for the sludge
survey, except that a 3 mM phosphate buffer was used. Adrian (no substrate
added) and Jackson (50 ppm carbon diethylene glycol added) 10% sludge
inocula were prepared, with addition of 0, 5, 10 or 20 mM FeClg ' 4^0.
Concentrations of iron, phosphorous, and other minerals in the 10* sludge
solution were measured at the start of the incubation. Two milliliters of
sludge solution were removed from each bottle, filtered through a 0.45 urn
pore size Millipore filter, acidified with HC1, and stored at room
temperature until analyzed for metal content on a plasma emission
spectrometer. Methane production was monitored weekly using a FID equipped
GC. At the termination of the Adrian experiment, the pH of bottles was
recorded.
Methane production in both Adrian and Jackson sludge was inhibited by
addition of iron to the medium (Figures 27-28). The level of inhibition
appeared to be directly correlated with the amount of iron added. With
higher iron additions, levels of soluble iron also increased but the
majority of iron was precipitated out in the form of FeP04 (soluble P04 was
^These experiments were performed primarily by Susan Frasier,- under the
direction of Dr. Tiedje for the undergraduate course MPH 490: Special Problems,
-------
14
almost completely absent at 20 mM Fe), and probably as FeC03 as well
(Figure 29). The pH of medium receiving 20 mM iron was depressed (6.1-6.2)
well below the optimum for methanogenesis which probably caused the
inhibition of methane production. Apparently, our buffering capacity (3 mM
P04 and IQ% C02) was insufficient to offset the effect of the iron
addition. This can be visualized from the following scheme (assuming
FeHP04 and FeC03 are the primary precipitates)
-> FeHP04 + H*
—^ » FeC03 + H+
This is probably oversimplified, however, it illustrates how the buffer
capacity could have been exceeded. • Hoban and Berg used a 40% COg/SO^ N£
gas mixture such that their total bicarbonate buffer was approximately 56
mM, sufficient to buffer the pH. Based on results with our present medium,
we feel that the addition of high levels of iron is not advisable.
A survey of the anaerobic literature reveals that the concentrations
of minerals in media routinely used for culturing methanogens and
methanogenic consortia varies from one to two orders of magnitude (Figure
30) (4,12,14,17,18,21,22,25,26). This is probably indicative of the fact
that the concentration of most minerals and metals is, within reason, not
particularly critical. Sommers (19) conducted a survey of sewage sludges
from New Hampshire, New Jersey, Indiana, Illinois, Michigan, Wisconsin,
Minnesota and Ohio in which he reported the range, median, and mean
concentration for several elements (Figure 30). A comparison of anaerobic
media with 10% sludge indicates that, with the exception of perhaps K, NH4+
and Co, all elements are present in 103 sludge in ample supply.
Experiments were initiated with Jackson and Holt sludges to directly
compare the ASTM medium, our enrichment medium, and phosphate buffer alone.
Bottles were set up as previously described for length of storage except
that 0.5 mM sulphide was included in our enrichment medium; phosphate
-------
15
buffer medium contained no added sulphide (Figure 30). Gas production was
monitored using a pressure transducer with multimeter and 3-way valve (a
20-guage needle was used in place of a 25-guage needle). Unfortunately, it
was necessary to terminate this experiment after 6 weeks of incubtion due
to extreme variability and anamolous data from all three media. New
experiments were initiated with Jackson, Holt, Mason and Ionia sludges to
compare the ASTM medium, our enrichment medium (O.E.M.) and a supplemental
medium (S.M.) consisting of 3 mM P04= buffer, 9 mM K, 10 mM NH4+, and 10 uM
Co (those elements most likely to be in low concentrations in 10* sludge).
Mineral and metal composition of 10* Jackson, Holt, Ionia and Mason sludges
are listed in Figure 31. One gram samples of dried sludge were digested
sequentially with liberal amounts (20-30 ml) of HN03, HF and HC103 in 50 ml
Teflon beakers at 110°-130°C. The remaining metals and minerals were
dissolved in 6N HN03 with 1000 ppm LiCl and analyzed on a Plasma Emission
Spectrometer. Substrates tested were £-cresol (introduced directly into
serum bottles with a 10 ul microsyringe), phthalic acid (introduced into
serum bottles in 0.5 ml water as monopotassium phthalate),
di-n-butylphthalate (introduced directly into serum bottles with a 10- ul
syringe) and m-chlorobenzoic acid (introduced into serum bottles in 0.5 ml
di-ethylether and the ether allowed to evaporate for ^ 2 hours). Medium
performance was judged.based on three criteria: (1) maximum background gas
production (we assume that any inhibition of gas production is potentially
deleterious to biodegradation), (2) maximum gas production from test
compounds, and (3) minimum lag times.
In the aborted medium experiment (6 week incubation) we observed that
gas production was markedly inhibited in control bottles containing the
ASTM medium relative to our enrichment medium, and in both media relative
to phosphate buffer .alone (Figure 32). This was not unexpected for the
ASTM medium, because the C02 gas phase and NaHCOs called for in the medium
are not in equilibrium (see Appendix I). Although we stir our 10% sludge
solution before dispensing bottles, this agitation is neither sufficient
nor prolonged enough to allow for C02(g) to establish equilibrium with
C02(aq) and HC03"; the rate of phase transfer is too slow. When bottles
containing the ASTM medium were measured prior to venting, after
equilibration at 35°C, they consistently pulled a vacuum; bottles
-------
16
containing our enrichment medium or phosphate buffer consistently had
overpressures of 1-3 ml. In the completed medium experiment, we again
observed an inhibition of gas production in control bottles containing the
ASTM medium relative to our medium, even though a sufficient amount of
NaHCOs had been added to bring aqueous and gas phases into equilibrium (3.6
g NaHC03/liter). We also observed a slight inhibition of gas production in
control bottles containing our enrichment medium relative to the
supplemental medium. In an attempt to understand this phenomenon, 100 ml
aliquots of 10* sludge were dispensed sequentially from the same batch of
10% sludge (10% C02/90% N2 headspace) containing phosphate buffer alone,
phosphate buffer + our mineral salts, and phosphate buffer + our mineral
salts + 0.5 mM sulphide, or (separate batch of 10% sludge) no addition,
ASTM mineral salts, ASTM mineral salts + 2 mM sulphide. We found that
there was no effect of mineral salts additions on gas production after a 2
week incubation (Figure 32); however, addition of both 0.5 mM and 2 mM
sulphide resulted in inhibition of gas production with the level of
inhibit-ion inversely proportional to sulphide concentration. We are not
certain if this is primarily an effect on the C02(g) bicarbonate
equilibrium (sodium sulphide solutions are extermely alkaline) or on the
biology. Sulphide is known to inhibit methanogenesis at higher
concentrations, however 2 mM is generally considered to be a safe
concentration.
Percent theoretical gas production for substrates incubated in serum
bottles containing the ASTM medium was uniformly higher in all four sludges
than with either our enrichment medium (O.E.M.) or the supplemental medium
(S.M.) (Figure 33). This effect was particulrly pronounced for phthalic
acid and m-chlorobenzoic acid. Since the same stock solutions of phthalic
acid and ni-chlorobenzoic acid were used for amending bottles receiving the
three different media, it is highly unlikely that ASTM medium bottles were
consistently overfed. It is more likely that the over production of gas
was attributable to abiological rather than biological causes. This is
particularly true in the case of apparent gas production from
ni-chlorobenzoic acid in sludges for which we had no prior evidence of
degradation. Both phthalic acid and ni-chlorobenzoic acid were amended into
serum bottles as mono-acids (the second carboxyl group of monopotassium
phthalate has a pKa of 4.75 while m-chlorobenzoic acid has a pKa of 4.5)
-------
17
such that at a pH of 7.0, both compounds would be expected to be
deprotonated. Since the ASTM medium's sole buffer system consists of 30X
€02(9) - HC03", any introduction of protons would result in a flux of-C02
gas into the headspace (HCOs~ + H+i=?H2C03=*=?H20 + C02). Our enrichment
medium consists of a 2 mM P04= system in conjunction with 10% C02(g)-HC03~,
while the supplemental medium consists of 3 mM P04= in conjunction with 10%
C02(g) - HC03~. The phosphate ions would form an alternate (or competing)
buffer system, such that fluxes of C02 in the headspace would be
diminished. That this may be the case is supported by the fact that much
or all of the difference in gas production between the ASTM medium and our
enrichment medium in serum bottles containing phthalic acid or
jn-chlorobenzoic acid with 10% Holt or 10% Ionia sludge can be accounted for
in the first 1-2 weeks of incubation (Figures 35,37,39,and ;41). Based on-
past experience, this is considerably shorter than the expected lag period.
With the exceptions of £-creso1 and di-n-butylphthalate in 10% Holt sludge
(Figures 34 and 36), there does not appear to be any significant effects of
different mineral salts media on lag times or rates of degradation (Figures
35 and 37-41). Results for Jackson and Mason 10% sludges are consistent
with those for Ionia 10% sludge.
We feel that the most important components of the medium are the
buffer system and the reductant. Based on these experiments, we feel that
a 2 or 3 mM phosphate buffer system is advisable in conjunction with a 10%
C02(g) - HC03" system. We do not feel that the addition of sulphide is
necessary, however, if added, we recommend that the concentration not
exceed 1 mM. Iron concentrations greater than 2 mM are also not
recommended. We have obtained satisfactory results with our enrichment
medium; however, any standard anaerobic mineral salts medium would probably
be acceptable.
Effects of 0? on gas production in 10% sludge
Since the purpose of this test method is to assess anaerobic
biodegradation, it is important that 02 be excluded from test bottles. In
the case of accidental 02 intrusion, however, it is important to have some
means of detecting the extent of 02 contamination in bottles so that they
can be ignored or discarded. Almost all anaerobic media contain resazurin
-------
18
as a redox indicator (resazurin is colorless at Eh < -0.100 mv and pink
when oxidized). Although resazurin is a very sensitive indicator of 02
intrusion in anaerobic media, we were not aware of any information
indicating the sensitivity of resazurin to 02 intrusion in 10% sludge.
Based on experience with injecting 02 into bottles with 10% sludge, we have
found that a minimum of 5% (generally 5%-10%) 02 in the headspace is
required for a pinkish color to appear in the water column, with or without
sulphide. A 5% 02 atmosphere corresponds to a headspace composed of 25%
air. Using even poor anaerobic technique, it is unlikely that 02
contaminated bottles would be detected by this indicator.
We were interested in the capacity of 105 sludge to consume 02 and
whether slight 02 additions might effect gas production. Bottles were set
up as previously stated in the compound survey, except that 70 ml bottles
were filled with 40 ml of 10% Adrian sludge and 0-5 ml of 02 injected into
the headspace. Oxygen concentrations were quantified daily using a Carle
GC equipped with 2 m Porapak-Q column and microthermister detector; the
carrier gas flow rate was 40 ml/min He. All bottles were maintained
static. Oxygen was consumed at a rate of approximately 1 ml per day
(Figure 42). Bottles containing 3, 4 or 5 ml of 02 had a pink gradient at
the.top of the water column at zero time. After 24 hours a pinkish color
was visible in bottles having received 4 and 5 ml of 02 and after 48 hours
the water column in all bottles was colorless.
In a separate experiment bottles with 10% Jackson sludge were set up
as previously stated for length of storage experiments. After equilibrium
at 35°C and venting of bottles, 0, 1, 2, 3 or 4 ml of gas was removed from
the headspace and replaced with an equal amount of 02- Half of the bottles
were incubated with benzoate as substrate. After 16 hours, bottles
receiving 4 ml of 02 (6.7% 02 in the headspace) were slightly pink at the
water-gas interface, however, if the bottles were even slightly agitated
the pink disappeared. No pink was obvious after 40 hours. Oxygen addition
affected gas production data significantly (Figure 43). Gas production
after one week decreased proportionally to increasing 02 additions. This
was expected since the 02 would have been consumed leaving a partial
vacuum. A decrease in gas production was still evident after two weeks and
was particularly pronounced in bottles receiving benzoate as substrate.
-------
19
Presumably, this was due to inhibition of methanogenesis and loss of
benzoate carbon to aerobic metabolism. Although it is clear that
significant variability can be introduced as a result of 03 intrusion in
the absence of its detection, we do not feel that this presents a serious
problem if careful anaerobic technique is observed.
Effect of chemical concentration on gas production in 10% sludge
An objective of this research was to optimize the conditions under which
anaerobic biodegradation can occur, yet retain a relatively simple assay for
biodegradation which can be performed quickly and inexpensively. The only
sure way to maximize biodegradation potential is to work with fresh whole
sludge, while the most simple and inexpensive procedure for assessing
biodegradation is to monitor gas production. Unfortunately, the two are
incompatible. Total gas production from whole sludge is much too great to
allow for quantisation of small amounts of gas from xenobiotic mineralization.
Conversely, in order to decrease background gas production, it is necessary to
age or dilute whole sludge; both result in a decrease or dilution of the
biological activity. The goal, then is to find some suitable compromise
between maximizing viability and diversity, hence, maximizing chances for
biodegradation to occur, and minimizing background gas production such that
gas production from xenobiotics can be accurately quantified. The crucial
question in determining this compromise is, what concentration of xenobiotic
is most appropriate for such a screening method. If low concentrations of
xenobiotics are to be tested then steps must be taken to decrease background
gas production, while if higher concentrations are acceptable less dilution is
necessary and desirable to minimize potential toxic effects. The ASTM method
proposes that compounds be tested at a concentration of 50 ppm carbon,
however, we were not aware of any experimental justification for this
recommendation.
We initiated experiments with 10% Jackson sludge to investigate the
possibility of using lower concentrations of substrate, and to investigate the
potential toxic effects of phenol, £-cresol, benzoate, and phthalate at higher
concentrations (benzoate-and phthalate were added in the deprotonated form).
Bottles were set up as previously described for the length of storage
experiment. Substrates were tested at 25 ppm, 50 ppm, 100 ppm, and 200 ppm
carbon.
-------
20
Percent theoretical gas production was not appreciably affected by
initial substrate concentration; apparently toxicity was not a problem
(Figure 44). Standard deviations were greatest for low substrate
concentrations and tended to decrease with increasing substrate
concentrations. Based on these results, it is possible that substrates
could be tested at 25 ppm carbon; however, this concentration is near the
limits of resolution for the test. If lower concentrations were
recommended it would be necessary to pre-age the sludge before use to
reduce background gas production. This could be accomplished by incubating
whole sludge at 35°C for approximately 5 days prior to use. Within this
period of time, the majority of gas production should occur (personal
communication, John Eastman, Assistant Professor, Department of Civil and
Sanitary Engineering, MSU). If a concentration of 50 ppm carbon is
acceptable, however, we do not feel that a pretreatment is necessary. Test
compound concentrations of greater than 50 ppm carbon may also be
acceptable, however, they do not appear necessary.
Reproducibility of Results
One of the most important aspects of any test method is the ability to
obtain reproducible results. Observations which cannot be repeated are of
little interpretive value and represent a waste of time and resources. We
have evaluated the variability associated with the analytical aspects of
the method, the variability among replicate determinations using the same
batch of sludge, and the variability in the final interpretation among
different batches of sludge.
We have found the pressure transducer to be a reliable instrument.
Gas pressure measurements can be made with extreme accuracy and precision
(Figure 45). The standard curve shown in Figure 45 is the average of 13
different determinations over an 8 week period. As illustrated by Figure
45, there is no significant variability associated with the analytical
method. We have had, from time to time, difficulty with variability among
replicates, signified by high standard deviations (Figure 15). During the
length of storage experiments we observed that water vapor was condensing
in the 25-gauge needle and 3-way valve such that needle and valve had to be
flushed with air periodically to avoid occlusion. For the effect of
-------
21
mineral salts medium experiments, a 20-gauge needle was used. Either as a
result of this switch or closer attention to detail, standard deviations
for the effect of mineral salt medium experiments were somewhat improved,
although there was still occasional instances of high variability among
replicates. The source of this variability is not clear. It does not
appear to be associated with substrate degradation; standard deviations
(average for all four sludges in ml of gas) for separate substrates were
control (0.56), £-cresol (0.64), phthalic acid (0.72), di-n-butylphthalate
(0.52) and m-chlorobenzoic acid (0.46). Neither does the variability
appear to be associated exclusively with gas pressure measurements as
opposed to methane measurements; the standard deviation for methane
production in control bottles (average of 12 sludges in sludge survey) was
0.38 ml of CH4. We suspect that the greater part of this variability is
due to lack of complete homogeneity in the 10% sludge. Although we
vigorously stir the 10% sludge suspension while dispensing 100 ml aliquots
into serum bottles, small discrepencies in the distribution of degradable
organic matter would be sufficient to cause variability in gas production.
Reproducibility of results using the same batch of sludge does not
appear to be a problem. With the exception of a few anomalous results in
the length of storge experiments, no sludge failed to degrade or not to
degrade test compounds when incubated using the same batch of sludge. Even
more importantly, there was reproducibility between batches of the same
sludge, collected over a 1 1/2 year span. When degradation results for
several substrates used as test compounds in as many as five separate
experiments are compared (compound survey, sludge survey, effect of length
of storage, effect of mineral salts media, and effect of substrate
concentration), there are only four discrepancies out of a possible 66
comparisons (Figure 46). This does not include the data from the aborted
effect of mineral salts media experiment.
We feel that with careful technique and a minimal knowledge of
anaerobic sewage sludge, this method should yield relatively consistent and
reproducible results. We do not believe there should be any difficulty
with making reliable conclusions using this method.
-------
22
Discussion of Significant Test Parameters
and the Rationale for Our Recommendations
What should be the ratio of inoculum to test compound concentrations?
We have routinely used a 10% sludge inoculum with 50 ppm carbon test
compound in our studies, and see no reason to recommend other conditions.
Based on our experience, a 10% sludge inoculum will, in general, yield
10-40 ml of gas, depending on the retention time and total solids of the
sludge. A 50 ppm carbon addition will yield between 5.5 to 8.5 total ml of
gas, depending on the stoichiometry of mineralization. Even though the
background gas production may be significantly higher than the gas
production from the test compound, we feel that the accuracy and precision
of the test are sufficient to allow for assessment of biodegradation. As
stated previously, we feel that 50 ppm carbon substrate is the lowest
reasonable concentration which yields sufficient gas production over
controls to be quantifiable. Concentrations greater than 50 ppm carbon are
more easily quantifiable, however, they are not necessary and may lead to
toxicity. It is quite possible that some compounds which are relatively
slowly degraded in whole sludge may not be appreciably degraded in 10%
sludge; however, certain compromises are necessary in order to decrease
background gas production.
What should be the source of inoculum?
We have had only limited experience with anaerobic lake sediments but
have found them more difficult to work with in this test system. Also,
based on work by Dr. Horowitz in our laboratory, lag times in sediment are
generally longer than in sludge (10). Primary anaerobic sludge with a
retention time of 15-30 days is most desirable. Shorter retention times
may result in high background gas production, while longer retention times
may result in attenuation of sludge viability, particularly during storage.
Although total solids and volatile solids measurements are not always
indicative of background gas production, in general, we have found that
sludges with a percent organic matter (total solids X volatile solids) of
approximately 1.0%-2.0% provide satisfactory results. We strongly
recommend that potential users find a waste treatment plant where anaerobic
-------
23
digesters have been operated successfully in the past and are likely to be
so in the future. Although this is no guarantee that different batches of
the same sludge will always perform identically, results are more likely to
be consistent over time. At present we have insufficient data to allow us
to predict which types of anaerobic sludges are most likely to degrade the
widest range of xenobiotics.
We have not experimented with the mixing of different sludges together
(or sludge with sediment and/or soil) in an attempt to increase the
xenobiotic degrading versatility of the anaerobic inoculum. However, we
feel that this approach is promising. Since sludges do vary with respect
to their xenobiotic degrading capacity, a combination of the most active
sludges should, ideally, result in a maximizing of the biodegradation
potential.
How should the inoculum be stored or pretreated?
Based on our results discussed previously, we feel that most sludges
can be stored for up to 4 weeks at 4°C (in tightly capped containers) if
necessary; however we recommend that fresh sludge be obtained whenever
possible. It is clear, based on lag times for substrate degradation, that
storage can have a detrimental effect on sludge activity, particularly for
those sludges with long retention times and low organic matter contents.
If, however, sludges with retention times of.less than 30 days and percent
organic matter of greater than 1.0% are used, attenuation in activity is
not likely to be a serious problem. Based on our experience working with a
10* sludge inoculum, we do not feel that a pretreatment of the sludge is
warranted.
What should be the composition of the test medium?
Based on a survey of the literature and our own experience, we do not
feel that there is a strong basis for preferring any particular anaerobic
medium. In most cases, 10% sludge alone is likely to contain all mineral
and metal nutrients in ample supply, with the possible exceptions of K,
NH4+, and Co. However, inclusion of a complete mineral salts medium is
probably advisable as insurance. We recommend the use of a 2 or 3 mM
phosphate buffer system in conjunction with 10% C02(g) - HC03'. We do not
-------
24
feel that the addition of sulphide is necessary; however, if used,
concentrations should not exceed 1 mM.
What should be the length of incubation?
We feel that an 8 week incubation is advisable. Based on our own work
and that of Healy and Young, a 4 week incubation is simply not sufficient
to allow for biodegradation of more persistent chemicals. Longer
incubations are probably not justifiable. Ten percent sludge, like whole
sludge, has a finite shelf life beyond which components of the methanogenic
consortium cannot be expected to remain viable. Although we have not
performed experiments designed specifically to answer this question, we
have observed that background gas production in all control bottles has
ceased by the 8th week. We recommend that compounds be incubated for 8
weeks or until biodegradation is complete, defined as two consecutive weeks
without net gas production. If gas production is still in progress at the
8th week, incubations should be continued until gas production has ceased.
We recommend that incubations be conducted at 35°C, since most primary
anaerobic digesters are operated in this temperature range.
How should test substrates be introduced into sludge bottles?
The ASTM method proposes that bottles be incubated overnight before
testing begins to assure anaerobiosis, and that water insoluble materials
first be dissolved into dioxane,. then introduced into 10% sludge bottles.
Based on our Og intrusion experiments, we do not feel that it is necessary
to hold bottles overnight to check for 02 contamination because the redox
indicator (resazurin) is not sensitive to low levels of 03 contamination.
With proper anaerobic technique, D£ instrusion should not pose a serious
problem.
We have not extensively investigated the effect of potential -organic
diluents on methane and/or gas production in 10% sludge. Based on a
limited survey of common organic solvents incubated in 10* sludge (amended
with 1 mM glucose) at a concentration of 0.1%, we found four solvents
(acetonitrile, aniline, dioxane and pyridine) which were neither inhibitory
to methanogenesis nor degraded (Figure 47). Aside from the fact that some
of these compounds have undesirable characteristics i.e., dioxane is an
-------
25
animal carcinogen, it is not possible to definitely assert that the
introduction of these solvents into 10% sludge at concentrations probably
approaching 0.1% would have no effect on the fermentative populations of
the methanogenic consortium. We prefer to first dissolve water insoluble
solids into di-ethylether, dispense the ether solution into serum bottles,
and allow the ether to evaporate for >_ 2 hours prior to filling with 10%
sludge. We have found no residual effects of the ether after a'2 hour
evaporation period. Liquids can be dispensed directly with a microsyringe
while water soluble solids should be dissolved in water, then dispensed
into bottles. Unfortunately, insoluble polymers must be weighed out
individually.
How should the volume of gas be measured?
We have had considerable experience in monitoring both CH4 production
(via gas chromatography) and gas production (via pressure transducer) as a
means of assessing biodegradation, and feel that there are advantages and
disadvantages to both methods. Pressure transducer measurements are
extremely accurate and precise, easily and rapidly obtained, and the
required instrumentation (pressure transducer, multi-meter, 3-way valve,
and 1/16" tubing) relatively inexpensive. Unfortunately, accurate
measurement of biological C02 and Cfy production is complicated by the fact
that C02 is highly soluble in and reacts with water, such that small
variations in conditions affecting C02 solubilities can result in errors in
accurately assessing the extent of biodegradaticn. Gas chromatography
allows for accurate and sensitive quantisation of methane gas. Also, since
methane is produced solely as a result of biological activity and is
relatively insoluble in and does not react with water, measurements of
methane production are less sensitive to abiological fluctuations.
Monitoring of methane production also appears to be a more sensitive
indicator of inhibition due to toxicity. Based on our limited experience,
compounds which cause complete inhibition of methane production
(chloroform) show moderate levels of gas production, although significantly
less than controls. This could be due to C02 flux into the headspace as a
result of acid production by fermenters or simply a result of volatility of
the compound itself. However, measuring methane via gas chromatography is
-------
26
relatively slow, and requires moderately sophisticated instrumentation (a
gas chromatograph and accessories). Considering the speed, accuracy,
precision and cost effectiveness with which gas pressure measurements can
be made, we feel that the use of pressure transducer and multimeter is the
method of choice, particularly for a general screening method such as this.
We have routinely corrected for gas solubilities when calculating percent
theoretical degradation though this is not necessary in making a decision
as to whether compound degradation has occurred (See Appendix I).
Gas pressure measurements were made by inserting a 20-guage needle,
attached to a Hamilton Miniature Inert 3-way valve, into serum bottles.
The 3-way valve was attached to a Unimeasure pressure transducer (Grants
Pass, Oregon) equipped with a P-3 adapter (8 psi maximum response) via 10
cm of 1/16 in stainless steel tubing (fitted to pressure transducer) and a
16-guage luer needle (fitted to 3-way valve) connected by a 1/16" in
.Swagelok union. The signal from the transducer was quantified using a
Fluke multimeter (Mountlahe Terrace, Washington). Serum bottles were
shaken vigorously before pressure measurements were taken, and excess gas
vented after measurements were taken to avoid cumulative gas pressures
beyond the response range of the P-8 adapter.
What should the reference chemical(s) be?
The purpose of incubating sludges with reference chemicals is to
ascertain that the sludges are biologically active and to characterize the
sludges with respect to their overall versatility. Any readily degradable
substrate such as ethanol can be used to quickly determine whether or not a
sludge is active. However, a more rigorous test of activity and viability
may be desirable. We have used £-cresol and phthalic acid throughout most
of our testing. We feel that one or both of these compounds may be useful
as reference chemicals because (1) both compounds appear to be uniformly
degraded in anaerobic sewage sludge (sludge survey), (2) degradation
results for both compounds have, been consistently reproducible
(reducibility of results), and (3) lag times before degradation have varied
from 2 to 6 weeks depending on the age and source of sludge. We do not
feel that we have sufficient knowledge or experience to recommend a group
of chemicals which could be used to characterize sludges with respect to
-------
27
their biodegradational versatility. Although it is clear that sludges vary
with respect to their ability to degrade specific compounds, it is not
apparent that this is predictive of their ability to degrade general
classes of compounds of like structure.
-------
28
Appendix I
Calculation of percent theoretical degradation
' In order to ascertain CH4 and C02 production from a particular
substrate, the stoichiometry of mineralization must be determined. This
can be done as proposed in the ASTM method. Using benzoic acid as an
example:
12 H20-^7C02 + 15H?
7C02 + 15H2— >3.75 CH4 + 3.25 C02 + 7.5 H20
C7H6U2 + 4.5 H20— *3./5 CH4 + 3.Zb C02
At a concentration of 50 ppm carbon in a 100 ml aqueous phase and 350C, the
volume of gas produced is:
5 mg carbon benzoic acid x 5/0.6885 = 7.27 mg benzoic acid
(0.6885 = fraction of molecular weight due to carbon)
7.27 mg benzoic acid = 0.0595 m moles benzoic acid/100 ml
0.2232 m moles CH4 + 0.1934 m moles C02 = 0.4167 m moles total gas
0.4167 m moles gas at 35°C = 10.5 ml of gas
Since all substrates are provided at 50 ppm carbon, the theoretical
gas yield for all substrates, regardless of structure, is 10.5 ml. If
methane production is monitored (as opposed to total gas) the stoichiometry
of substrate conversion to CH4 must be determined. Examples of
determinations involving different structural groups are given below.
o-, m-, p- aminobenzoic acid
<• 12H20—»7C02 + 14H2 + NK3
7C02 + 14H2—»3.5 CH4 + 3.5 C02 + 7H20
«• 5H20—*3.5 CH4 + 3.5 C02 + NIH3
-------
29
o-, m-, p-chlorobenzoic acid
C7H502C1 + 12H2— >7C02 +'l4Hz + HC1
7C02 + 14H2~^>3.5 CH4 + 3.5 C02 + 7H20
C7H502C1 + 5H20 — »3.5 CH4 + 3.5 C02 + HC1
tn'cresyl phosphate
C21H21°4P + 42 H2°~ *21C02 + 51 H2 + H3P04
21 C02 + 51 H2— s-12.75 CH4 + 8.25 C02 + 25.5 H20
C21H21°4P + 16-5 H2°~~ >12.75 CH4 + 8.25 C02 + H3P04
parathion
ClOH14°5NPS + 15H20— >10 C02 + 18 H2 + NH3 + H3P04 +
10 C02 + 18 H2— *4.50 CH4 + 5.50 C02 + 9.0
C10H14°5NPS + 6H20— »4.5 CH4 + 5.5 C02 + NH3 + H3P04 +
-------
30
As calculated above, 50 ppm carbon of any chemical yields a total of 10.5
ml gas (CH4 + C02). However, since both CH4 and C02 are soluble in water, one
does not actually measure 10.5 ml of gas (assuming 100% mineralization) in the
headspace. The quantity of methane dissolved in the aqueous phase is a
function of temperature and can be calculated according to the equation:
Vt
vg =
1 + L-A
TT
where Vg = volume of gas in headspace
Vt = total volume of gas in bottle
A = volume of aqueous headspace
G = volume of headspace
L = Ostwald coefficient^- (defined as mis gas dissolved in
1 ml of water at 1 atm partial pressure of gas)
Assuming a 100 ml aqueous phase, 60 ml gas phase, and 35°C (L = 0.0296), if Y^
is arbitrarily set to 100 Vg = 95.3; or 95.3% of total CH4 in the bottle will
be in the headspace.
Unfortunately, the loss of C02 to the aqueous phase is considerably
greater. Not only is COg highly soluble in water (L = 0.666 at 35°C), it also
reacts chemically with water to form carbonates according to the following
scheme:
C02(g) gas phase
II +H + H
C02(aq) * H20*^H2C03<'!=^HC03-*=!1?C03= (dissolved) liquid phase
lr
C03= (precipitate) solid phase
The quantity of C02 dissolved in the aqueous phase is a function of both
pH and temperature. At a given pH, one can calculate the fraction of
dissolved carbonate present as C02(aq) according to the equation (from Aquatic
Chemistry, Stumm and Morgan, Wiley-Interscience, 1970, p 120):
Although the Bunsen coefficient (°f) is generally more familiar and widely
used than the Ostwald coefficint (L), we chose to use the Oswald coefficient
because it gives the volume of dissolved gas at temperature (35°C), whereas
the Bunsen coefficient gives the volume of gas assuming 0°C, regardless of the
actual temperature. Since theoretical gas production is calculated at 35°C,
the Ostwald coefficient is more accurate. The two can be equated through the
expression
L = T -or
273.15
-------
31
Ki-K
where pKi = 6.3, p'<2 = 10.25
Then, using the equation:
Vg =
vt
the percent of total inorganic carbon (excluding precipitates) present as C02
in the headspace at a given pH and temperature, can be calculated. The effect
of pH on C02 solubilities is illustrated by the following calculations:
Percent of C02
£H_ Qfo in headspace at 35QQ
7.2 0.117 9.1
7.1 0.1367 11.0
7.0 0.1663 (0.1900a) 13.0 (14.63)
6.9 0.2007 15.3
6.8 0.2402 17.8
a = adjusted for approximate ionic strength
Assuming a pH of 7, and 10% C0£ headspace in equilibrium with the aqueous
bicarbonate pool, one can calculate that there should be a total of 41.1 mis
of gas in the bottle. We add 1.2 g NaHCOs/liter medium which would give a
total of 42.1 mis of gas in the bottle. We have found that after dispensing
medium into bottles and allowing for equilibration at 35°C, there are 1-3 ml
of gas above atmospheric pressure in the headspace.
We have determined empirically that approximately 37% of C02(g) added to
the headspace of serum bottles containing 10% sludge (to simulate biological
C02 production) remains in the headspace; the rest is dissolved in the aqueous
phase. Since this is considerably greater than predicted for pure water, it
would appear that precipitated carbonates are involved in establishing
equilibrium conditions for dissolved C02 and bicarbonates in 10* sludge. Gas
production from substrates with acidic groups is a function of whether or not
these groups are protonated, as illustrated below.
-------
32
CH3COOH - >CH4 + C02
CH3COO" +
In some experiments, the pH of stock solutions of benzoic acid or phthalic
acid was increased to maximize their solubility in water for dispensing into
serum bottles.
All theoretical gas production data have been corrected for CH4 and C02
solubilities and where applicable, deprotonation effects. Theoretical gas
production from compounds whose biodegradation we have studied is tabulated
below:
Substrate Theoretical gas production (ml)
p-Cresol 6.4 ml CH4 + 4.1 ml C02
x.95 .37
FTT TT5" 7.6
Monobasic phthalate 4.9 ml CH4 + 4.3 ml C02
x.95 .37
375" T7o 6.2
Dibasic phthalate 4.9 ml Cfy + 3.0 ml C02 5.7
Di-n-butylphthalate 5.9 ml Cfy + 4.6 ml C02 7.6
Ethanol 7.9 ml CH4 + 2.6 ml C02 8.5
Phenol 6.1 ml CH4 + 4.4 ml C02 7.4
Benzoate 5.6 ml CH4 + 3.4 ml C02 6.5
m-Chlorobenzoic acid 5.25 ml Cfy + 5.25 ml CC>2 6.9
-------
33
Appendix II
Characteristics of Sewage Treatment Plants and Anaerobic Sewa,ge
Sludge
Anaerobic digestion is only one of several methods for treating
raw sewage. As such not all waste treatment plants have anaerobic
sludge. Anaerobic digestion is used to stabilize raw solid waste,
i.e., render it safe for handling and disposal. The flow of sewage
through anaerobic digesters is illustrated in the following
simplified diagram:
raw sewage.
primary settling
tank
solids from
activiated sludge
(optional)
thickening
(optional)
primary digestor-^ secondary .(storage^
digestor
land application
drying bed &
eventual landfill
The primary anaerobic digestor is where principal degradation and
stabilization of organic matter occurs. Primary digestor(s) are
heated (between 85-100°F) and mixed to provide a consistent
environment for digestion to occur. From the primary digestor,
sludge is pumped to secondary digestbr(s) for storage. Secondary
digesters are not heated (not necessary since stabilization has
already occurred) or mixed. Solids are allowed to settle; then,
periodically, supernatant is pumped back through the plant while
solids are disposed of either by land application or landfill.
The flow of carbon in methanogenic fermentations can be
diagrammed as follows:
CO.
Fermenters Fermenters
omplex organic ) Simple organic > Volatile fatty acids
carbcn ' carbon
"€
\ f \ C0
structural!^ ponomers, structurally!
Acetogenic
bacteria
polymers
complex colecules
^
\fimple moledules j
* CH,
Utnotropnic methanogens
-------
34
In considering the overall viability of sludge, one must be concerned
with at least three groups of organisms: a group of organisms loosely
referred to as fermenters which is responsible for breaking down more
complex organic molecules into volatile fatty acids, H2 and C02; the
fatty acid degraders, which are responsible for degrading short chain
volatile fatty acids (propionate, butyrate, etc.) into acetate, H2, and
C02; and the methanogens (heterotrophic and lithotrophic) which produce
CH4. The loss of viability of any one component of the consortium will
result in a cessation of methanogensis.
Primary sludge, because of heating and mixing, is generally
consistent over time and is suitable for use in this method. Secondary
anaerobic sludge is not suitable because of greater heterogeneity and
because digestor temperatures can drop to 40-50°F during winter months.
Also, due to long retention times, bacterial viability is likely to be a
problem.
The parameters most important in characterizing sludge are
retention time, pH, total solids (drying at 100°C), and volatile solids
(ashing at 550°C). The minimum retention time required for complete
digestion of sewage (greatest gas production) at 35°C is approximately
15 days (16). Retention times for sludges used in our survey varied from
17 to 33 days. Total solids of sludges vary depending on whether or not
solids from the primary settling tank are prethickened before pumping
into the digesters, and whether or not flocculant from activated sludge
is pumped into the digestor. In our sludges, total solids varied from
1.7% to 5.5% (excluding Leslie). Volatile solids is a measure of
organic matter present in the sludge and is useful in estimating the
degree of digestion which has occurred, however, depending on the input
of nonbiodegradable organic matter (eg., lignin) volatile solids can
vary. Volatile solids, as percent of total solids in our sludges,
varied from 49% to 57% (excluding Leslie). The pH of the digestor
indicates whether or not fermentation is coupled with methanogenesis.
If the temperature drops or digesters are overloaded they can go "sour"
ie., the rate of fermentation to fatty acids exceeds the rate of
methanogenesis and the pH drops. This pH drop will destroy methanogenic
activity. The optimum pH for digestion is approximately 6.8-7.2.
-------
35
Sewage treatment operators routinely determine each of these
parameters for their digesters such that they should be readily
available to any potential user.
Based on our experience with sludge we recommend a retention
time of 15-30 days, pH of 6.8-7.2 and a percent organic matter
content of approximately 1.0-2.0 (total solids X volatile solids).
This should result in good quality sludge and based on correlation
between CH4 production and percent organic matter (see below) a
background CH4 production in control bottles of 10-25 ml.
Background Percent
Sludge3 CH4 production0 organic matter (total solids X volatile solids)
1.53
1.99
1.73
1.82
1.47 r = 0.85
1.19
0.98
0.89
aData for Pontiac, Leslie, Ann Arbor, and Charlotte sludges are not
reported due to lack of data or known irregularities in digestor
operation.
"Data from sludge survey.
clf Adrian is dropped r = 0.97
A more thorough treatment of theory and practice of anaerobic
digestion can be found in EPA publication 625/1-79-011: Process
Design Manual, Sludge Treatment and Disposal. Municipal
Environmental Research Laboratory. Cincinnati, Ohio 45268.
Adrian0
Jackson
Mason
Ionia
St. Johns
Portland
Chelsea
Holt
23.3
21.7
19.7
18.0
14.2
12.9
11.7
8.4
-------
36
Literature Cited
1. Ambiosi, D., P. C. Kearney, and J. A. Macchis. 1977. Persistence and
metabolism of phosalone in soil. J. Agr. Food Chem. 25:342-347.
2. Evans, W. C. 1977. Biochemistry of the bacterial catabolism of
aromatic compounds in anaerobic environments. Nature. 270:17-22.
3. Ferry, J. G. and R. S. Wolfe. 1976. Anaerobic degradation of benzoate
to methane by a microbial consortium. Arch. Microbiol. 107:33-40.
4. Ferry, J. G. and R. S. Wolfe. 1977. Nutritional and biochemical
characterization of Methanospirill urn hungatii. Appl. Environ.
Microbiol. 34:371-375";
5. Fina, L. R. and A. M. Fishin. 1960. The anaerobic decomposition of
benzoic acid during methane fermentation. II. Fate of carbons one and
seven. Arch. Biochem. Biophys. 91:163-165.
6. Fina, L. R., R. L. Bridges, T. H. Coblentz, and F. F. Roberts. 1978.
The anaerobic decomposition of benzoic acid during methane fermentation.
III. The fate of carbon four and the identification of propanoic acid.
Arch. Microbiol. 118:169-172.
7. Healy, J. B. and L. Y. Young. 1978. Catechol and phenol degradation by
a mechanogenic population of bacteria. Appl. Environ. Microbiol.
35:216-218.
8. Healy, J. B. and L. Y. Young. 1979. Anaerobic biodegradation of eleven
aromatic compounds to methane. Appl. Environ. Microbiol. 38:84-89.
9. Hoban, D. J. and L. Berg. 1979. Effect of iron on conversion of acetic
acid to methane during methanogenic fermentations. J. Appl. Bacteriol.
47:153-159.
10. Horowitz, A., D. R. Shelton, C. P. Cornell, and J. M. Tiedje. 1981.
Anaerobic degradation of aromatic compounds in sediments and digested
sludge. Proc. Soc. for Indust. Microbiol. (in press).
11. Keith, C. L., R. L. Bridges, L. R. Fina, K. L. Clierson, and J. A.
Cloran. 1978. The anerobic decomposition of benzoic acid during
methane fermentation. IV. Dearomatization of .the ring and volatile
fatty acids formed on ring rupture. Arch. Microbiol. 118:173-176.
12. Khan, A. W., T. M. Trotlier, G. B. Patel, and S. M. Martin. 1979.
Nutrient requirement for the degradation of cellulose to methane by a
mixed population of anaerobes. J. Gen. Microbiol. 112:365-372.
13. MacRae, I. C., K. Raghu, and T. F. Castro. 1976. Persistence and
biodegradation of four common isomers of benzene hexachloride in
submerged soils. J. Agr. Food Chem. 15:911-194.
-------
37
14. Mclnerney, M. J., M. P. Bryant, and N. Pfennig. 1979. Anaerobic
bacterium that derades fatty acids in syntrophic association with
methanogens. Arch. Microbiol. 122:129-135.
15. Nottingham, P. M. and R. E. Hungate. 1969. Methanogenic fermentation of
benzoate. J. Bacteriol. 98:1170-1172.
16. O'Rourke, J. T. 1968. Kinetics of anaerobic treatment at reduced
temperatures. Unpublished doctoral dissertation, Stanford University.
Palo Alto, California. 94305.
17. Patel, G. B., A. W. Khan, and L. A. Roth. 1978. Optimum levels of
sulphate and iron for the cultivation of pure cultures of methanogens in
synthetic media. J. Appl. Bacteriol. 45:347-356.
18. Shlomi, E. R., A. Lankhorst, and R. A. Prins. 1978. Methanogenic
fermentation of benzoate in an enrichment culture. Microbiol. Ecol.
4:249-261. .
19. Sommers, L. E. 1977. Chemical composition of sewage sludges and
analysis of their potential use as fertilizers. J. Environ. Qua!.
6:225-232.
20. Tahase, Iwao, Takeyoshi Nakahara, and Kozo Ishizuka. 1978. Degradation
of 3-(3-chloro-4-chlorodifluoromethylthiophenyl)-l,l-dimethylurea
(Clearcide) in paddy soils. J. Pesticide Sci. 3:9-19.
21. Taylor, G. T. and S. J. Pirt. 1977. Nutrition and factors limiting the
growth of methanogenic bacterium (Methanobacterium thermoautotrophtcum).
Arch. Microbiol. 13:17-22.
22. Toerien, D. F. 1970. Population description of the non-methanogenic
phase of anaerobic digestion - 1. Isolation, characterization, and
identification of numerically important bacteria. Water Research.
4:129-148.
23. Van A!fen, N. K. and T. Kosuge. 1976. Metabolism of the fungicide
2,6-dichloro-4-nitroaniline in soil. J. Agr. Food Chem. 24:584-588.
24. Walter-Echols, G. and E. P. Lichtenstein. 1978. Movement and metabolism
of C^C] phorate in a flooded soil system. J. Agr. Food Chem.
26:599-604.
25. Zehnder, A. J. B. and K. Wuhrmann. 1977. Physiology of Methanobacterium
strain AZ. Arch. Microbiol. 111:199-205.
26. Zeikus, J. G., P. J. Weimer, D. R. Nelson, and L. Daniels. 1975.
Bacterial methanogenesis: acetate as a methane precursor in pure
culture. Arch. Microbiol. 104:129-134.
-------
38'
Figure 1. Survey of organic compounds for biodegradation in 10* sludge.
Adrian Jackson
Substrate Lag time
(weeks)
Benzoic acid
o-Hydroxybenzoic acid
p-Hydroxybenzoic acid
3,4-Dihydroxy
benzoic acid
p-Methoxybenzoic acid
3-Methoxy, 4-hydroxy
benzoic acid
o-Aminobenzoic acid
p-Aminobenzoic acid
ra-Chlorobenzoic acid
Benzyl alcohol
4-Hydroxybenzyl alcohol
- Propionanilide
Phthalic acid
0 i -methyl phthal ate
Oi-ethylphthalate
Oi-n-butyl phthal ate
Butyl benzyl phthal ate
Phenol
m-Methyl phenol
p-Methyl phenol
1,2-Dihydroxyber.zene
1 ,3-Oihydroxybenzene
1 .2,3-Dihydroxybenzene
1 , 3 , 5-0 i hy droxybenzene
Oiethylene glycol
Polyethylene
glycol-1000
Polyethylene
glycol -20,000
Geraniol
Amy! alcohol
1-Octanol
2-Octanol
2-Hexanone
Acrylic acid
Ethyl acetate
3-Hydroxy-2-butamone
2,3-3utanedio1
Acetyl salicylic acid
4-Hydroxyacetanilide
4-Chl oroacetanil ide
1
4
2 '
3
1
1
1
6
5
2
2
5
1
2
2
4
4
3
3
1 "
1
1
1
2
4
1
1
2
1
1
1
1
2
2
Percent incubation
degradation3 (weeks)
102°
99
115
89
106
103
104
61
68
103
0
31
99
88
0
32
0
91
92
51
98
98
102
126
158
155
112
73
95
110
0
0 .
92
91
90
111
93
27
18
4
8
4
8
8
4
8
10
10
8
10
8
8
4
4
6
4
4
4
4' •
' 4
4
4
4
8 .
7
7
8
8
7
7
8
9'
4
Lag time
(weeks)
1
2
2
3
1
1
1
8
8
1
3
3
4
1
4
3
4
2
5
3
3
1
1
1
1
2
4
1
1
7
7
2
1
1
1
2
2
2
Percent
degradation
96
86
133
80
91
106
127
60
47
100
• 61
29
100
58
32
85
24
99
90
121
91
0
97
170
162
180
50
72
100
88
65
- 87
83
88
82
75
108
39
25
incubation
(weeks)
4
8
4
8
8
4
8
. 10
10
8
8
10
8
8
8
4
8
4_
6
4
4
4
4
4
4
4
8
7
7
14
12
8
8
7
7
8
9
4
^Percent theoretical methane production.
^Values are approximate. Average of two determinations.
-------
39
Figure 2. Compounds for which we have no evidence of degradation in 10% sludge.
Chloronaphthol (4)a
DDT (4)
PBB (4)
Phenoxyacetic acid (4)
Methyl-IDA (4)
IDA (4)
N-methylacetanilide (4)
5-Ch1oro-2-hydroxyanil ine (4)
2,6-Diethylaniline (4)
3,5-Dichloroaniline (4)
p-Chloroaniline (4)
NTA (8)
Di-ethylhexylphthalate (8)
Di-octlylphthalate (8)
4-Aminoacetophenone (8)
4-Chlororesorcinol (8)
o-Chlorophenol (8)
Acrolein (8)
Acrylamide (8)
Dimethylformamide (8)
Naphthol (8)
2-Hexene (8)
3-Hexanol (8)
2-Hexanol (8)
3-Henanone (8)
2-Hydroxybenzyl alcohol (8)
Tricresylphosphate (8)
Cresyldiphenylphosphate (8)
o-Aminophenol (8)
p-Aminophenol (8)
3-Methylsalicylic acid (8)
Orcinol (8)
4-Methylcatechol (8)
2-Methylresorcinol (8)
4-Chlorocatechol (8)
m-Aminobenzoic acid (8)
o-Chlorobenzoic acid (8)
p-Chlorobenzoic acid (8)
3,4-Dichlorobenzoic acid (8)
Benzene (8)
Toluene (8)
o-Methylbenzoic acid (8)
m-Methylbenzoic acid (8)
p-Methylbenzoic acid (8)
2-Amino-5-chlorobenzoic acid (8)
3-Chloro-4-hydroxybenzoic acid (8)
Benzyl acetone (8)
o-Cresol (8)
Anisole (8)
aNumber in parenthesis indicates weeks of incubation.
-------
40
Figure 3. Compounds which were inhibitory to methanogenesis in 10% sludge,
Percent inhibition3
Substrate Adrian Jackson
m-Chlorophenol
p-Chlorophenol
5-Chlorosalicyl ic acid
p-Nitrophenol
Chloroform
Pentachlorophenol
Hexachlorocyclopentadiene
Trictiloroacetic acid
4-Phenoxybutyric acid
Atrazine
14
7
78
97
98
94
88
96
64
76
58
41
85
97
96
84
95
98
90
80
aAfter a 4 week incubation
-------
41
Figure 4. Extraction efficiencies for phthalate esters, tri-m-cresylphosphate,
and monochlorophenols in whole sludge.
Percent recovery
Compound
Di-n-methyl phthal ate
Di-n-ethyl phthal ate
Di-n-butyl phthal ate
Butyl benzyl phthal ate
Di-(2-ethylhexyl )phthalate
Di-n-octyl phthal ate
Tri-m-cresyl phosphate
o-Cresol
m-Cresol
p-Cresol
o-Chlorophenol
m-Chlorophenol
_p_-Chlorophenol
Jackson
40
57
97
101
116
103
98
69
72
65
74
84
73
Holt
40
41
69
91
109
127
88
61
67
51
69
80
76
Adrian
49
64
102
94
100
106
102
-------
FIGURE 5. DEGRADATION OE PHIHALATE ESTERS IN JACKSON SLUDGE
CM
S3
ex.
QI-(2-ETHYLHEXYL)PHTHALA"
I-N-OCTYLPHTH/lLAiE
DI-N-BUTYLPHTHALATE
D i -N-ETHYLPHTHALATE:
DI-N-METHYLPHTHALATE
•TRI-M-CRE3YLPH09PHATE
BUTLYBENZYLPHTHALATE
-------
FIGURE 6. DEGRADATION OF PHTHALATE ESTERS IN ADRIAN SLUDGE
cn
c5
5
01- (8-ETHYLHEXYUPHTHALATE
TRI-M-CRESYLPHOSPHA
DI-N-OCTYLPHTHALATE
BUTYLBENZYLPHTHALATE
DI-N-BUTYLPHTHALATE
DI-N-ETHYLPHTHALATE
DI-N-METHYLPHTHALATE
WEEKS
-------
FIGURE 7 DEGRADATION OF PHTHALATE ESTERS IN HOLT SLUDGE
CZJ
s
13
0_
TRI-M-CRESLYPH09PHATE
DI-ca-ETHYLHCXYL)PHTHALATE
DI-N-OCTYLPHTHALATE
BUTYLBENZYLPHTHALATE
DI-N-BUTYLPHTHALATE
DI -N-ETHYLPHTHAUTE
DI-N-METHLYPHTHALATE
VELXS
-------
Figure 8. Comparison of degradation measured by methane production vs substrate disappearance in Adrian and
Jackson sludge.
Adrian i „.!,,„..
Phthlate esters
Di-n-methyl
Di-n-ethyl
Di-n-butyl
Butyl benzyl
Di-(2-ethylhexyl)
Di-n-octyl
CH4 production
in 10% secondary
sludge
88a
0
32
0
0
0
Substrate
disappearance
in whole sludge
100b
100
90
93
28
44
CH4 production
1n 10% secondary
sludge
58a
32
85
24
0
0
CH4 production
in 10% primary
sludge
82a
76
80
103
9
13
Substrate
disappearance
in whole sludge
100b
100
100
98
0
27
Substrate
disappearance
in 10% sludge
100b
100
100
100
_
-
aPercent of theoretical methane production
DPercent disappearance
in
-------
FIGURE 9 . PHTHALATE ESTER DEGRADATION IN WZ JACKSON SLUDGE
-BUTYLBENZYLPHTHALATE
DI-N-OCTYLPHTHALATE
DI-C2-ETHYLHEXYL)PHTHA
6
DI-N-ETHYLPHTHALATE
I-N-BUTYLPHTHALATE
DI-N-METHYLPHTHALATE
e &T e 1—e
DAYS
-------
FIGURE 10. PHTHAIATE ESTER DEGRADATION IN IflZJACKSON SLUDGE
O
<-BUTYLBENZYLPHTHALATE
DI-N-BUTYLPHTHALATE
DI-N-METHYLPHTHALATE
-N-ETHYLPHTHALATE
DI- (a-ETHYLHEXYL)PHTHALATE
DI-N-OCTYLPHTHALATE
DAYS
-------
FIGURE I) . DEGRADATION OF CRESOLS AND MONOCHLOROPHENOLS IN JACKSON SLUDGE
CO
M-CHLOROPHENOL
0-CHLOROPHENOL
-------
FIGURE 12 . DEGRADATION OF CRE50LS AND MONOCHLOROPHENOLS IN HOLT SLUJff
-CHLOROPHENOL
-CHLOROPHENOL
en
0-CHLOROPHENOL
-------
50
Figure 13. Characterization of sludges.
Percent
C industrial input
-
an
tckson
n Arbor
tntiac
. Johns'
tnia
It
Kson
slie
K el sea
arlotte
flprtland
17.5
17.1
2.0
39.7
28.3
34.9
0.0
19.4
0.0
18.0
4.2
13.6
Number of
people served
20,410
60,000
95,260
92,170
5,200
5,200
17,000
1,170
2,040
3,950
8,200
3,500
Flow Total
(lO^gal/day) solids(%]
5.0
17.0
15.0
20.0
1.0
1.2
1.1
0.8
0.2
0.4
1.2
0.6
2.8
4.0
4.0
n.d.
- 3.0
3.5
1.7 .
3.8
10.2
1.8
4.3
2.3 .
Volatile
L solidsU)
55
55
55
n.d.
49
52
57
50
40
56
55
50
Average
retention
time (days)
20
20
20
17
38-39
21
30
19
I
Calculated by dividing total known industrial input by total flow
-------
Figure 14. Comparison of degradation results for b*lve sludges.
Substrate
Ethariol
Polyetiylene
glycol-20,000
jvCresol
Phthalic acid
nvCresol
Di-n- butyl
phthalate
2-Octanol
nvChlorobenzoic
~acid
Propionanilide
o-Cresol
Di-n-octyl
phthalate
Chloroform
Adrian
Lag3 X
time deg.
(1) 62
(2) 61
(4) 91
(4) 88
(6) 82
(8) 24
0
(4) 85
0
0
0
Jackson
Lag %
time deg.
(1)
(2)
(4)
(5)
(4)
(5)
(8)
78
43
88
80
103
49
22
0
0
0
0
<0C O
Ann Arbor
Lag %
time deg.
(1) 86
i.d.b
(4) 101
(4) 132
(4) 85
(4) 91
(8) '58
0
(4) 36
0
0
O
Pontiac
Lag I
time deg.
(1) 62
(2) 55
(3) 72
(3) 74
(4) 55
0
0
0
0
0
0
-------
52
Figure 15. Effect of length of sludge storage on gas production in 10% sludge.
Mean percent of theoretical gas production + S.D.a
Length of
storage p-Cresol
Phthalic acid
Background
gas
Di-n-butylphthalate Ethanol production (ml)
Jackson
Week 0
Week 1
Week 2
Week 3
Week 4
Avg.
Week 0
Week 1
Week 2
Week 3
Week 4
Avg.
Week 0
Week 1
Week 2
Week 3
Week 4
Avg.
103 +_
76 +_
133 +_
93 +_
105 +
106
112 +_
109 +_
120 +_
—
118 +
115
83 +_
112 +_
89 +_
100 +_
132 +
103
7.83
3.8
3.2
12.1
11.0
7.6"
2.2
15.1
6.0
6.3
7.4
9.9
5.2
16.5
18.1
2.7
10.5
111
122
64
96
77
94
98
101
116
98
113
105
69
93
99
111
123
99
+_ 27.4
i 12.6
+_ 25.3
+_ 14.7
+ 7.7
17.5
+_ 2.6
+_ 13.7
± 15-8
+_ 13.6
+ 46.4
18.6
± 16<1
± l'2
+_ 10.4
i 12.3
+ 4.8
9.0
76
97
109
78
93
91
Hoi
30
36
57
48
52
44
Che!
51
39
22
46
38
+ 5.
+_ 22.
+ 14.
+ 2.
+ 12.
11.
_t
+ 9.
+_ 10.
+ 8.
+ 7.
+ 10.
9.
sea
+_ 20.
+ 4.
1 1'
+ 7.
32C
8.
6
0
8
5
4
5
4
0
2
0
4
0
1
1
0
7
2
99
88
104
78
83
90
96
88
113
98
107
100
88
123
91
127
108
+_ 13.5
+_ 3.4
+_ 2.4
+_ 4.8
+ 6.8
6.2
+_ 16.6
+_ 11.4
+_ 12.3
+_ 17.0
+ 2.0
11.9
+_ 18.2
+_ 13.2
+_ 7.3
i 8.9
llic
11.9
44
44
40
44
43
17
17
15
14
14
6
12
13
12
8
.1
.5
.2
.1
.8
.2
.2
.2
.0
.0
.5
.5
.6
.4
.4
Adrian
Week 0
Week 1
Week 2
Week 3
Week 4
Avg.
105 +_
128 +_
108 +_
—
138 +
120
14.7
10.5
7.1
9.6
10.5
97
63
83
102
86
+_ 24.1
i 14.5
+_ 57.8
+_ 25.6 '
87C
30.5
37
86
92
98
71
+ 7.
+_ 37.
± 8-
+ 11.
41C
16.
1
7
2
6
1
off scaleb
60
62
5y
66
58
.7
.3
.5
.6
.2
aGas production within 8 weeks of incubation after the indicated length of storage prior to
incubation.
^Background gas production plus gas from ethanol exceeded upper limit of pressure transducer
cGas production in two out of three bottles at or below controls (probably due to leaks).
-------
FIGURE 16. EFFECT OF LENGTH OF SLUDGE STORAGE ON DEGRADATION OF P-CRESOL
IN m HOLT SLUDGE
oo
m
CZJ
CD
CX.
CO
UJ
18
8
6
4
2
0
-2
VEF1S
-------
FIGURE IT. EFFEC! OF LENGTH OF SLUDGE STORAGE ON DEGRADATION
PHTHALIC ACID IN 101 HOLT SLUDGE
8
6
en
4
ex.
CO
-=c
0
-1
2 WEE*
VEEKS
-------
FIGURE 18. EFFECT OF LENGTH OF SLUDGE STORAGE ON DEGRADATION OF
Dl-N-BUHLPHTHALATE IN 10JHOLT SLUDGE
LT>
U")
CD
CX-
CO
LJP
-------
VO
ID
FIGURE 19. EFFECT OF LENGTH OF SLUDGE STORAGE ON THE DEGRADATION OF ETHANOL
IN 107 HOLT SLUDGE
-------
in
FIGURE a EFFECT OF LENGTH OF SLUDGE STORAGE ON DEGRADATION OF KRESOL
IN 107 JACKSON SLIM
FRESH
I WEEK
2 WEEK-
-------
FIGURE 21. EFFECT OF LENGTH OF SLUDGE STORAGE ON DEGRADATION
PHTHALIC ACID IN 107 JACKSON SLUDGE
00
in
-------
FIGURE 22. EFFECT OF LENGTH OF SLUDGE STORAGE ON DEGRADATION OF
DI-N-BUTYLPHTHALATE IN 197 JACKSON SLUDGE
-------
FIGURE 23. EFFECT OF LENGTH OF SLUDGE STORAGE ON THE DEGRADATION OF ETHANOL
IN 18?JACKSON SLUDGE
err
CO
-a:
LJP
4 WEEK
3 WEEK
I WEEK
-------
FIGURE 21 EFFECT OF SLUDGE STORAGE ON LAG TIMES FOR DEGRADATION OF P-CRESOL
0
1 2 3
LENGTH OF STORAGE WEEKS)
H
f •
CD
I —
C_J
-— -->
6-
. — >
cn
CD
C£
f~l
1 1 1
ss 5
c_u
1 —
b_
Lu_
4
CD
LO Q
CD
U_
CD
UJ
Q±
f --. 1
ZZ3
y •!
2-
k , 7-»
Lj_J
cc:
CO [ .
u_
2»
J = JACKSON
H = HOLT
A = ADRIAN u
C = CHELSEA
J
HAC
J
•—
H
AC
J
* 1
AC
J
C
J.
AC
4
-------
FIGURE 25. EFFECT OF SLUDGE STORAGE ON LAG TIES FOR KGRADATION OF PHTHALIC ACID
IN 187 SLUDGE
H
CM
o-
cu
t J p
Q 5
CZD
o±:
CO
3j 4.
UJ
u_
o^ 0 '
CO
cc:
I''
U_J
" 1-
UJ
LtJ
J = JACKSON
H = HOLT
A = ADRIAN
C
= C
JH
HELSEA
AC
J H
AC
J
H
A .C
J
H
AC
J
AC
1 2 3
LENGTH OF STORAGE WEEKS)
-------
FIGURE 26. EFFECT OF SLUDGE STORAGE ON LAG TIMES FOR DEGRADATION OF ETHANOL
IN 107 SLUDGE
CD
C_J
Q_
cn
n
<£>
LO
ce:
CD
CO
JwiT
LU
LZJ
J = JACKSON
H = HOLT
C = CHELSEA
J H
0
JH C
JH C
1 2 3
LENGTH OF STORAGE WEEKS)
4
-------
FIGURE ' EFFECT OF IRON ON ffl» PRODUCTION IN 187 ADRIAN SLUDGE
18
15
CD
C3
CD
5
9
6
3
0
2
4
VEEKS
6
8
-------
FIGURE EFFECT OF IRON ON OL PRODUCTION IN VK JACKSON SLUDGE
CD
ex.
J.
5
15
121
9
6
3
0
4
VEEKS
6
8
-------
66
Figure 29. Effect of iron additions on soluble minerals and
pH in 10% Adrian sludge.
Iron addition (uM)
20
5
20a
0
Fe (uM)
250
1.9
290
0.39
P (mM)
0.07
3.9
0.08
>4.0
pH
6.1-6.2
6.8-6.9
6.1-6.2
7.0-7.1
aAccidently doubled 10 mM Fe addition
-------
67
Figure 30. Comparison of mineral salts and metals in anaerobic media
vs. 10% sludge.
Our
enrichment ASTM Survey of Survey of sludges
medium (O.E.M.) medium anaerobic media Range Median Mean
Mineral
K
NH4+
PO^
Ca
Mg
Fe
S2'
Metal s
Mn
Zn
Cu
Co
N1
B
Mo
Se
NaHC03
10%
s (mM)
6.0
10.0
> 4.0
0.5
0.5
0.1
0.5
(uM)
2.53
0.37
0.22
2.10
0.21
0.81
0.04
0.29
1.2 g/1
C02/90% HZ
Resazurin 1 mg/1
3.5
4.3
0.3
0.3
1.8
1.85
2.0
101.0
14.7
17.2
126.0
—
97.1
41.7
—
2.64 g/1
30% C02/70%
1 mg/1
2.0-36.2
3.2-42.4
0.3-23.6
0.05-1.0
0.05-1.8
0.007-1.85
—
0.15-101.0.
0.35-15.4
0.06-20.1
0.42-126
0.08-0.21
0.81-97.1
0.04-41.7
0.29-119.4
N2
0.02-2.8a
0.03-15.4
0.7-18.9
1.9-20.5
0.05-2.3
0.07-11.2
—
4.0-530
7.0-1,740
5.0-650
0.2-1.2
0.1-240
5.0-280
1.0-1.3
.
0.3
0.4
0.9
5.0
0.8
0.9
—
21
120
60
0.5
6.0
14.0
1.3
—
0.5
2.1
4.4
5.9
1.0
1.2
—
30
210
90
0.6
30
37
1.2
--
aCalculations based on a median -total solids of 4.1%.
bTotal phosphorous
-------
68
Figure 31. Mineral and metal concentrations in 10* sludge.
Source of Sludge
Minerals (mM)
K
Total P
Ca
Mg
Fe
Metals (uM)
Mn
In
Cu
Ni
B
Mo
Cd
Cr
Pb
Holt
0.25
3.13
1.67
0.17
0.65
43.4
47.5
22.5
1.8
4.7
1.1
0.47
5.9
3.1
Ionia
0.16
6.03
1.83
0.72
0.56
.14.7
75.3
25.5
2.2
6.7
1.3
0.80
16.7
5.1
Jackson
- 0.45
9.32
3.68
1.97
4.39
66.1
738.7
57.1
39.3
29.0
5.2
8.3
908.6
12.5
Mason
0.22
3.60
3.37
1.01
1.66
18.0
28.1
28.2
2.2
11.9
1.9
0.70
10.6
3.4
Total Solids (%)
2.2
3.0
5.5
2.3
-------
69
Figure 32. Effect of mineral salts media on gas production in absence of
added substrate by 10% sludge.
Medium
Source of sludge
Jackson Holt Mason
Ionia
ASTM medium9 13.5C
Our enrichment medium 18.8
Supplemental medium^3 17.9
ASTM medium 13.8
Our enrichment medium 21.8
4 mM P04 = 29.3
No addition 12.1
ASTM medium-sulphide3 11.5
ASTM medium + 2 mM sulphide 6.7
4 mM P04= 11.9
Our enrichment medium-sulphide 12.8
Our enrichment medium + 0.5 mM sulphi-de 10.1
8 week incubation
9.6 9.9
18.1 18.8
19.0 21.2
6 week incubation
-2.7
9.4
13.1
2 week incubation^
16.2
20.1
21.6
aAdded 3.6 g NaHC03/liter instead of 2.64 g
b6 mM P04, 9 mM K+, 10 mM NH4+, 10 uM Co++, 10% C0£/90%
cGas production (ml)
^Average of 5 replicates
-------
70
Figure 33. Effect of mineral salts media on gas production in 10% sludge.
Mean percent of theoretical gas production + S.D.
Medium
ASTM3 medium
Our enrichment medium
Supplemental medium^
ASTM medium
Our enrichment medium
Supplemental medium
ASTM medium
Our enrichment medium
Supplemental medium
ASTM medium
Our Enrichment medium
Supplemental medium
p-Cresol
Phthalic acid Di-n-butylphthalate
m-Chlorobenzoic ac
Jackson
102
108
79
106
104
89
+
+
+
+
+
+
3.
3.
7.
10.
4.
4.
2
9
9
3
5
6
156
130
85
145
112
96
± °
± 5-
± 7-
Hoi
1 19-
+_ 13.
± 2-
0
7
_t
0
7
9
128
101
89
59
46
19
± 3
± 9
± 14
± °
± 3
± 5
.9
.7
.8
.2
.4
30 +_ 8.6
0
0
153 +_ 6.1
101 +_ 3.5
65 +_ 5.2
Ionia
127
115
99
+
+_
+
7.
9.
6.
1
5
7
183
118
104
± 9-
± U'
+_ 24.
5
4
8
117
72
77
± 4
± 3
± 16
.7
.6
.7
50 +_ 9.4
15 +_ 10.4
0
Mason
114
96
104
+
+_
+
36.
5.
0.
0
0
9
139
98
108
+_ 13.
± 7-
+ 24.
7
2
7
111
70
93
± 14
± 3
+ 2
.2
.2
.4
37 +_ 8.5
22 +_ 8.9
0
aAdded 3.6 g NaHCOs/liter instead of 2.64 NaHCOs/liter
b6 mM P04=, 9 mM K, 10 mM NH4+, 10 uM Co, 10% C02/90S N2 headspace
-------
FIGURE 34. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF P-CRESOL IN
W HOLT SLUDGE
VEEKS
-------
CM
FIGURE 35. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF PHTHALIC ACID
IN 10 HOLT SLUDGE
c_o
-------
FIGURE 36. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF OI-N-BUTTLPHTHALATE
IN 18Z HOLT SLUDGE
6
4
CH
CD
ce:
2
U_J
0
A3TM
6
8
-------
FIGURE 37. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF M-CHLOROBENZOIC ACID
IN 101 HOLT SLUDGE
-------
FIGURE 38. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF P-CRESOL
IN 182 IONIA SLUDGE
10
8
era
6
CD
Q_
CO ^
2-
0
8
4
VEEKS
6
8
-------
FIGURE 39. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF PHTHALIC ACID
IN W7 IONIA SLUDGE
era
CZ5
ex.
CO
-a:
10
8
6
4
4
6
8
-------
FIGURE 40. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION OF Dl-N-BUTTLPHTHALATE
IN iar IONIA SLUDGE
err
Cl_
CO
-------
oo
FIGURE 41. EFFECT OF MINERAL SALTS MEDIA ON DEGRADATION M-CHLOROBENZOIC ACID
IN 18? IONIA SLUDGE
8
6
a_
CO
-------
FIGURE f;,. 0, CONSUMPTION BY W ADRIAN SLUDGE
-------
o
oo
3
E5
CO
fei
NO BENZOATE
FIGURE :' EFFECT OF 0, ON GAS PRODUCTION IN 107HOLT SLUDGE
20
50 PPM-C BENZOATE
3
0
2
3
-------
81
Figure 44. Effect of substrate concentration on gas production in 10%
Jackson sludge
Mean percent of theoretical degradation + S.D.
Substrate concentration
(ppm carbon)
25
50
100
200
Background gas
production (ml )
Theoretical gas production
from 50 ppm carbon (ml )
Phenol
100 +_ 9.9
104 +_ 8.0
106 +_ 6.3
113 +_ 3.1
31.4
7.4
p-Cresol
98
86 +_ 13.5
98 +^ 5.5
95 +_ 4.5
31.6
7.6
Benzoate
92 +_ 18.0
92 +_ 6.0
96 +_ 4.7
98 +_ 3.6
24.5
6.5
Phthalic acid
105 +_ 4.8
104 ^ 18.7
109 +_ 3.7
100 +_ 1.5
28.4
5.7
-------
FIGURE 45. STANDARD CURVE EOR MEASURING GAS PRODUCTION IN SERUM BOTTLES
USING PRESSURE TRANSDUCER
si 1 150
SLOPE -- 8.52
T-1NTERCEPT = .025
5
10 15 20
VOLUE OF GAS (ML)
25
30
35
-------
84
Figure 47. Effect of solvents on CH4 and gas production from 1 mM glucose
in 10% Jackson sludge.
Solvent9 _ CHfl _ Gas pressure
(% of unamended control )
Acetonitrile
An i 1 i ne
Chloroform
Cyclohexane
Dioxane
DMSO
Ether
Hexane
Methyl ene chloride
Pentane
Pyridine
Toluene
Xylene
96 b
109
<0.01
0.03
102
98
0.03
60
0.01
74
106
0.02
0.01
98
111
33
45
93
157
65
99
23
143
95
34
33
aTested at concentration of 0.1% v/v
^Average of two replicates.
-------
|