inhibition of anaerobic digestion by organic priority pollutants
TRANSCRIPT
Inhibition of Anaerobic Digestion by Organic Priority PollutantsAuthor(s): Lyle D. Johnson and James C. YoungSource: Journal (Water Pollution Control Federation), Vol. 55, No. 12 (Dec., 1983), pp. 1441-1449Published by: Water Environment FederationStable URL: http://www.jstor.org/stable/25042127 .
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Inhibition of anaerobic
digestion by organic
priority pollutants Lyle D. Johnson, James C. Young
Instability is a common problem with anaerobic diges tion processes. Organic chemical inhibitors are frequently cited as contributors to this problem. However, anaerobic
process reactions involve the sequential breakdown of
complex organic materials by several interacting groups of microorganisms. Thus, although an organic compound
may be inhibitory to the methanogenic bacteria within the treatment system, there may be other microorganisms present that are capable of degrading the compound and
eliminating the inhibitory effects. Various chemical,
physical, and biological factors also may affect the nature
and extent of inhibition caused by organic compounds. Several researchers1"3 have reported the occurrence
of reversible inhibition during toxicity testing of organic chemicals, but the actual conditions that relate to the
recovery of methane production are not well defined. The term "acclimation" has been applied loosely to this
condition, because microorganisms frequently have the
ability to adapt to some extent to inhibitory concentra
tions of most materials.
Because of the limited knowledge of factors contrib
uting to toxicity and because of the increasing potential for toxic organic compounds to enter wastewater streams,
laboratory tests were initiated to study the inhibition of
anaerobic cultures by toxic organic chemicals and to
examine the conditions affecting recovery from this in
hibition.
EXPERIMENTAL PROGRAM
The experimental program consisted of three phases.
Screening tests were used in Phase I to isolate organic
compounds that had severe inhibitory effects on anaer
obic biological reactions. Subsequent tests were con
ducted in Phase II to investigate the nature of the re
covery of anaerobic cultures from inhibitory effects.
Phase III was designed to help identify factors contrib
uting to recovery of cultures from inhibition. A detailed
description of the test program and results is given by Johnson.4
Selection of test organic compounds. The semivolatile
organic priority pollutants, as defined by the U. S. En
vironmental Protection Agency (EPA), were chosen for use as a test base because these chemicals reflect an over
all environmental concern and occur commonly in in
dustrial waste streams.5 Twenty-four compounds con
sidered to be representative of this group were selected
for testing (Table 1).
Adsorption plays an important role in
reducing the concentration of soluble organic compounds.
A batch bioassay technique using serum bottles3'6 was
used to determine inhibitory effects. This procedure in
volved addition of 60 mL of a nutrient-buffer solution
(5.7 g/L NaHC03 and 0.37 g/L FeCl2) to 250-mL serum
bottles. The bottles were purged with a mixture of 70%
nitrogen/30% carbon dioxide, and 40 mL of anaerobic
seed was added. Absolute ethanol (0.100 mL) was added
to each serum bottle to serve as a substrate for the seed
organisms. Various concentrations of organic test chem
icals were added with the ethanol to selected serum bottles.
Prepared serum bottles were connected to manome
ters for pressure monitoring (Figure 1). Gas production was measured by withdrawing the excess gas in each
bottle into a calibrated syringe until the fluid displace ment in the manometer was the same as for a thermal
barometric control. A reduction in gas production as
compared with controls that received no test chemicals
served as a measure of inhibition. Replicate serum bot
tles were set up to provide separate samples for analysis of individual volatile acids and to determine the soluble
concentration of the organic inhibitor.
Anaerobic seed was obtained from a 25-L laboratory
digester maintained at 37?C. The synthetic substrate for
the digester was a mixture of materials resembling mu
nicipal solid waste (Table 2). The laboratory digester was
December 1983 1441
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Johnson & Young
Table 1?Organic priority pollutants tested for inhibition to anaerobic metabolism.
Phenols
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dichloropheno!
2,4-Dimethylphenol
2-Nitrophenol
4-Nitrophenol Phenol
2,4,6-Trichlorophenol
Phthalate esters
Di-n-butylphathalate
Diethylphathalate
Dimethylphthalate
Nitrosamines
N-Nitrosodimethylamine
Nitroaromatics
Nitrobenzene
PCBs Arochlor 1242 Arochlor 1254
Haloethers
Bis(2-chloroethyl)ether
4-Bromodiphenylether
Chlorinated hydrocarbons
Hexachlorocyclopentadiene
Hexachloro-1,3-butadiene
Hexachloroethane
1,2-Dichlorobenzene
1,3-Dichlorobenzene
Polynuclear aromatic
hydrocarbons
Naphthalene
Pyrene
maintained at a 30-day solids retention time (SRT) for
approximately 2 years prior to use in the inhibition
study.
Analytical procedures. Methane and carbon dioxide in the product gas were measured using a gas Chro
matograph with a thermal conductivity detector. Two
glass columns (3.5 m X 4.0 mm ID) packed with 80/ 100-mesh Poropak Q were used. Column temperature
was 95?C and the detector temperature was 100?C.
Helium (30 mL/min) was used as a carrier gas. Quan titative analysis was based on an area-normalization
A A
CONTROL THERMAL BAROMETRIC CONTROL
? <y ?
Figure 1?Schematic diagram of anaerobic toxicity assay ap
paratus.
Table 2?Components of artificial substrate used in this study.
Component Amount
Kraft paper 26.8 g Newsprint 23.2 g
Dog food 10.2 g Hay 8.3 g Dipotassium hydrogen phosphate 0.2 g Ammonium chloride 0.4 g Sodium bicarbonate 3.5 g
Municipal primary sludge 80.0 ml_
Ethyl alcohol (after first 6 mo) 10.0 mi
Tap water 740.0 mL
Total volume 833.0 mL
procedure7 using standard gas mixtures to determine
response factors.
Samples analyzed for volatile acids were centrifuged (G
= 39 100) for 30 minutes and the centrate was acidified to pH < 2 using concentrated H2S04. The C2 to C6 volatile
acids, including iso-C4 and iso-C5, were analyzed by gas
chromatography8 using column (1.8 m X 2.0 mm ID) packed with 10% SP-1200/1% H3P04 on 80/100-mesh Chromosorb WAW. The column was operated isother
mally at 115?C. Phosphoric acid-treated glass wool was
used to retain the packing in the column and to prevent peak tailing. The injection port was operated at 225 ?C and contained a glass liner that was changed frequently to prevent build-up of organic char. The flame ionization detector was heated to 280?C. Nitrogen (35 mL/min) was
the carrier gas. Quantitative analysis was based on absolute
response factors.7
Samples analyzed for residual priority pollutants were
centrifuged to remove solids. The centrate was acidified to pH <2 for analysis of phenolic compounds. Hexa
chloroethane, hexachloro-l,3-butadiene, hexachlorocy clopentadiene, and nitrobenzene were analyzed imme
diately after collection of centrate without acidification. Direct aqueous injection was used for analysis of all com
pounds, and quantitative analyses were based on absolute
response factors.
The phenolic compounds were analyzed using glass column ( 1.8 m X 2.0 mm ID) packed with 80/100-mesh Tenax GC. The column temperatures were 200?C for
2-nitrophenol, 230?C for 4-nitrophenol, and 215?C for
2,4-dichlorophenol. Nitrogen (15 mL/min) was the car
rier gas for all three compounds. Flame ionization de
tection at 250?C was used for quantitative analysis. Nitrobenzene was analyzed on a glass column (1.8 m
X 2.0 mm ID) packed with 1.5% SP-2250/1.95% SP
2401 on 100/120 Supelcoport. The column was oper ated isothermally at 100?C. Nitrogen carrier gas was
maintained at 30 mL/min, and a flame ionization de
tector was used at 250?C.
Hexachloroethane, hexachlorocyclopentadiene, and
hexachloro-1. ^-butadiene were analyzed on glass columns
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_Process Research
Table 3?Relative activities8 of samples dosed with the test chemicals.
Chemical
Run
time ?
(hr) 1
Dosage (mg/L)
10 50 100
4-Nitrophenol
2-Nitrophenol
2,4-Dichlorophenol
2,4-Dimethylphenol
Hexachloroethane
N-Nitrosodimethylamine
Dimethylphthalate
Diethylphthalate
4-Bromodiphenylether
Di-n-butylphthalate
1,3-Dichlorobenzene
1,2-Dichlorobenzene
Phenol
2,4,6-Trichlorophenol
4-Chloro-3-methylphenol
Naphthalene
Hexachlorocyclopentadiene
Hexachloro-1,3-butadiene
Bis(2-chloroethyl)ether
Nitrobenzene
2-Chlorophenol
Pyrene
Arochlor 1242
Arochlor 1254
5
72
5
100
5 96
5 96
5 193
5 193
5 193
5 193
5 193
5 193
5 193
5 193
5 193
5 193
5 193
5 285
5 285
5 285
5 285
5 285
5 285
5 285
5 167
5 167
88 93
116 102
90 96
92 104
130 154 116 108
99 87 103 100
97 97
107 97
95 93
89 92
89 91
92 93
85 91
84 90
89 91
87 91
79 90
97 99
92 99
99 98
90 101
88 102
92 96
98 96
92 93
90 93
89 91
100 95
90 94
85 91
92 92
85 91
74 87
99 95
92 77 105 104
88 102
97 97
83 100
88 94
100 100 101 100
104 102 102 101
89 99
54
137 107
95 100
73 96
98 95
97 95
93 94
89 91
90 92
90 91
77 88
87 91
87 89
77 87
97 95
65 101
88 100
95 98
81
37 91
35 103
80 99
98 98
14 91
103 96
93 92
93 94
85 90
92 93
85 91
77 87
92 92
80 86
74 85
95 98
24 90
72 68
95 98
54 103 101
88 102
95 104
95 105
83 99
100 ?
100 ?
103 99 103 100
97 99 105 105
0 72
5 103
0 76
95 92
14 90
102 93
92 91
98 95
87 91
92 92
79 86
70 83
85 90
77 79
69 84
18 91
59 41
90 97
20 97
86 93
89 90
95 102
a Percent of control.
(1.8 m X 2.0 mm ID) packed with 3% OV-1 on 100/120
Supelcoport and held at 100?C. The carrier gas was ni
trogen (30 mL/min). An additional 30 mL/min of ni
trogen was used as a makeup gas for the electron capture detector. The detector was operated at 300?C, and the
injection port temperature was 225?C.
The analysis of chlorides in the centrate from samples dosed with chlorinated compounds was performed by the potentiometric titration method described in "Stan
dard Methods."9
TESTING AND RESULTS
Phase I screening tests. Phase I screening tests in volved measuring relative activity?the total gas pro duction in the bioassay samples expressed as a percent age of the gas production in the respective controls?for the compounds listed in Table 3. Chemical dosages of
1,5, 10, 50, and 100 mg/L were used for all test chem
icals except naphthalene and pyrene. Test periods
ranged from 72 to 85 hours.
Relative activities after 5 hours of incubation and at
the termination of the test period are listed in Table 3. None of the compounds was highly inhibitory at the
lower concentrations. Only six compounds?4-nitro
phenol, 2-nitrophenol, 2,4-dichlorophenol, hexachlo
roethane, nitrobenzene, and hexachlorocyclopenta diene?produced significant inhibition (>50% reduction in gas production) after 5 hours at the 100-mg/L test
concentrations (Figure 2). Hexachloro-l,3-butadiene
produced significant inhibition but only in the later
TIME, hours
Figure 2?Relative activity curves for seven organic priority
pollutants causing significant inhibition of methane production at 100 mg/L.
December 1983 1443
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Johnson & Young
stages of the test run. Anaerobic cultures exposed to 2,4
dichlorophenol responded in such a manner as to sug
gest stimulation at low concentrations. Samples dosed
with nitrobenzene, 2,4-dichlorophenol, 4-nitrophenol,
2-nitrophenol, hexachloroethane, and hexachlorocyclo pentadiene exhibited a tendency toward recovery over
the test period. Only hexachloro-1,3-butadiene pro duced an increase in the degree of inhibition during the
testing period. The common element among these com
pounds is the presence of a nitro or chloro functional
group. Inhibition of anaerobic cultures by chlorinated
organic compounds has been well established.10"14 Some
compounds containing a nitro group, such as nitroben
zene, have been identified as being inhibitory;11 but the
nitro group in general has not been linked to inhibition
problems. The screening tests indicated that the relative activity
varied with time, especially for those compounds that
produced significant inhibition (Figure 2). This implies that toxicity data on organic compounds as measured
from batch bioassays must be interpreted carefully. Al
though a short test may identify the compounds as in
hibitory, this indication does not accurately reflect the
long-term response of the culture or the fate of the in
hibitory compound. Phase II recovery tests. Inhibition caused by nitro
benzene, 4-nitrophenol, 2-nitrophenol, 2,4-dichloro
phenol, hexachloroethane, hexachlorocyclopentadiene, and hexachloro-1,3-butadiene was explored further in
Phase II to examine patterns of recovery of the anaerobic culture from the inhibitory effects of the toxic organic compounds. Serum bottles were used as culture vessels, and the tests consisted of monitoring gas production over incubation periods ranging from 10 to 20 days. Concentrations of soluble organic compounds remain
ing in solution were measured at various times through out the test period, and the methane content of the gas was analyzed periodically.
Gas production and soluble concentration data for
cultures receiving nitrobenzene dosages of 100 and 500
mg/L are presented in Figure 3. Active gas production began immediately in the control unit, but only after the concentration of nitrobenzene was reduced signifi cantly in the dosed unit. The disappearance of nitro
benzene coincided with the appearance of aniline, the concentration of which is also shown in Figure 3.
The activity of cultures dosed with nitrobenzene re
flects the detoxification of this compound by the reduc tion of the nitro group to an amine group. Cartwright and Cain15 observed a similar reduction of para-nitro benzoic acid by facultative soil organisms. The recovery observed indicates that aniline is not as toxic as nitro benzene to anaerobic cultures. Chow et aln reported similar results.
Recovery of gas production in samples dosed with 4
nitrophenol and 2-nitrophenol was similar to that for
1000- O CONTROL GAS PRODUCTION A >->?-^i200 O CONTROL 100 mg/L NITROBENZENE ^^------^^^^-^^ '
? SOLUBLE NITROBENZENE ̂-J^-~~~~~ _, ? SOLUBLE ANILINE ^^-^^^S^_
-1
i /V^^^ -* -,25 i ?
SQf^/lf - too ̂
? I / / ?
ocL^_I_I_I_I_I_L-. I I o
500Y- ? C0NTR0L n -j,200 O CONTROL + 500 mg/L NITROBENZENE B ^_______--? ? SOLUBLE NITROBENZENE ^^^-^^ ?-?-* 17c ^ ? SOLUBLE ANILINE
_-^-^^?^^^--?O"""- _.
| 200-4)7 / _ ^
o 100 -/ H / |
0Jo-?^U-gl-1-1-1-1-k?-1-1-lO 0 24 48 72 96 120 144 168 192 216 240
TIME, hours
Figure 3?Effect of 100 mg/L (A) and 500 mg/L (B) of ni trobenzene on gas production and production of soluble aniline.
nitrobenzene. Active gas production coincided with re
ductions in concentrations of both nitrophenols. Com
plete removal of both of these compounds occurred within 2 days. Because the nitro group of nitrobenzene was reduced to an amine group, the same reaction was
expected with the nitrophenols. However, GC analysis of standard solutions of 2-aminophenol and 4-amino
phenol was unsuccessful, and therefore the existence of these compounds could not be confirmed.
Gas production and inhibitor concentration data for
samples dosed with 2,4-dichlorophenol (2,4-DCP) at 100 mg/L are shown in Figure 4A. The concentration
of 2,4-DCP was reduced rapidly from 100 to 60 mg/L, with little change after the initial few hours. This con stant residual soluble concentration of 2,4-DCP shows that it was not biodegraded. The initial gas production rate was very close to that of the control, but the dif ference in total gas production increased with time.
Volatile acids data indicated that metabolism of pro
pionic acid was inhibited, but acetic and butyric acid were present in concentrations similar to those in the control (Figure 5).
A similar response pattern was observed when the cultures were dosed with 500 mg/L of 2,4-DCP (Figure 4B). However, gas production was almost totally inhib
ited, and build-up of all volatile acids occurred at this test concentration (Figure 5).
Samples dosed with 50 and 100 mg/L of hexachlo
rocyclopentadiene were inhibited initially, as indicated
by a reduced rate of gas production, but recovered later
1444 Journal WPCF, Volume 55, Number 12
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Process Research
O 24 48 72 96 120 144 168 192 216 240
TIME, hours
Figure 4?Effect of 100 mg/L (A) and 500 mg/L (B) of 2,4 dichlorophenol on gas production.
in the test run (Figure 6). Although full recovery of total
gas production took about 10 days, the methane content
of the gas remained relatively constant after 3 to 4 days of incubation.
0 2 4 b 8 10
TIME, days
Figure 5?Concentration of organic acids produced during test
ing of 100 and 500 mg/L of 2,4-dichlorophenol.
O CONTROL -O CONTROL & 50 mg/L ? CONTROL & 100 mg/L O CONTROL & 500 rag/L
TIME, days
Figure 6?Total gas production and methane content in the
presence of 50,100, and 500 mg/L hexachlorocyclopentadiene.
Recovery of gas production was not complete at 500
mg/L of hexachloroclopentadiene, but analysis of the centrate indicated a significant increase in the concen
tration of chloride as compared to the controls (Table
4). The increased chloride concentration indicated that
this compound was dechlorinated by the biological re
action. The observed increase of 258 mg/L represents the loss of approximately four chlorine atoms from each
molecule of hexachlorocyclopentadiene. Anaerobic cultures dosed with 50 and 100 mg/L of
hexachloroethane showed almost complete recovery in
18 days, and a tendency toward recovery was apparent in the samples dosed with 164 mg/L of this compound
(Figure 7). Analysis of soluble hexachloroethane showed
that the concentration was reduced to less than 1 mg/ L within 8 hours for all dosages. After that time, the
soluble concentration remained fairly constant (Figure 8). Therefore, recovery occurred in the presence of low
concentrations of hexachloroethane. Because there was
Table 4?Chloride analysis of chlorinated hydrocarbon samples.
Chlorinated
hydrocarbon
Test
concentration
(mg/L)
Chloride (mg/L)
Control Sample
Hexachloro-1,3-butadiene 988 286 288
Hexachlorocyclopentadiene 500 300 558
Hexachloroethane 164 296 291
December 1983 1445
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Johnson & Young
10 12 14 16 TIME, days
Figure 7?Total gas production and methane content in the
presence of 50, 100, and 164 mg/L hexachloroethane.
no detectable build-up of chloride in the sample, this
recovery seemed to be a result of acclimation rather than
biod?gradation (Table 4).
sot
i 0.4
0.3
0.0
100<
V
O HEXACHLOROETHANE 9 50 mg/L
T-?- r?-o-+ J_L
? HEXACHLOROETHANE 0 100 mg/L
-4fvn I J_
O HEXACHLOROETHANE 0 164 mg/L
-4-0-0 I I I I 10 12- 14 16
TIME, days
=___
Figure 8?Soluble concentrations in samples receiving 50,100, and 164 mg/L hexachloroethane.
Inhibition by hexachloro-l,3-butadiene was exam
ined at dosages of 50, 100, 500, and 988 mg/L (Figure 9). Some degree of inhibition was noted at all dosages, but the methane content of the gas remained relatively constant throughout the test run. Soluble hexachloro
1,3-butadiene applied at 100 mg/L was reduced to a
concentration of 1 mg/L in 1 hour and remained at
approximately this concentration throughout the test
period. This rapid removal of hexachloroethane and
hexachloro-l,3-butadiene from solution was similar to the behavior of chlorinated hydrocarbon pesticides in
anaerobic environments.16
Phase II results suggested that the organic test com
pounds were removed from solution by two mecha
nisms?biological decomposition or adsorption?and the response of the anaerobic culture was related wome
what to the mechanism of removal.
Removal by decomposition was illustrated in Figure 3 for nitrobenzene. Here, the rate and magnitude of nitrobenzene removal was proportional to the rate and
magnitude of aniline production. Complete recovery occurred only when the toxic chemical was removed
completely. Similar results were observed with the ni
trophenols.4 The only other toxic compound removed
significantly by biological decomposition was hexa
chlorocyclopentadiene, which seemed to be dechlori nated by biological action (Figure 6).
Phase III adsorption tests. Because adsorption seemed to be a predominant mechanism by which some organic
100
o CONTROL D CONTROL & 50 mg/L ? CONTROL & 100 mg/L "O CONTROL & 500 mg/L ? CONTROL & 988 mg/L
J_I_I_I_I_I_I_I_I_I_L 5 6 7
TIME, days 10 11 12 13
Figure 9?Total gas production and methane content in sam
ples receiving 50, 100, 500, and 988 mg/L hexachloro-1,3 butadiene.
1446 Journal WPCF, Volume 55, Number 12
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_Process Research
compounds were removed from solution, the effect was
explored in greater depth in Phase III. Baseline adsorp tion data were collected by adding 100 mg/L of the test
compounds to sterilized and unsterilized anaerobic cul
tures and by analyzing the supernatant for soluble re
siduals after 48 hours of contact. Removals by adsorp tion were related somewhat to the solubility of these
compounds, but removal of the chlorinated hydrocar bons from the aqueous phase also could have occurred
because of phase separation and removal of the com
pound by centrifugation (Table 5). The effect of adsorption on response of the cultures
illustrated in Figures 3, 4, and 8. Addition of 2,4-DCP was accompanied by a rapid initial decrease in soluble
concentration, certainly caused by adsorption. An equi librium was reached in which the soluble residual was
essentially proportional to the amount added (Figure 4). Inhibition of gas formation increased as the soluble re
sidual increased, and there was no evidence of biological
degradation because the residual soluble concentration
remained essentially constant throughout the test run.
Similar results were observed with hexachloroethane, but much greater amounts were adsorbed initially and
the soluble residual remained toxic to the microbial cul
tures (Figures 7 and 8). If adsorption is a significant mechanism for removing
toxic organic compounds from solution and thereby re
ducing their toxicity, the response of anaerobic cultures to these compounds should be related to the concentra
tion of biological solids present. To test this hypothesis,
biological solids from the anaerobic seed culture reactors were concentrated by centrifugation, dried at 103?C, and pulverized. These inactive solids were added in vary
ing amounts to pairs of serum bottles, each containing nutrients, buffer, and anaerobic seed at a mixed-liquor concentration of about 30 g/L. Ethanol was used as an
organic substrate as in Phase II. Test chemicals were
added to one of the serum bottles in each pair; the other
served as a control. Each serum bottle contained the same amount of active seed microorganisms.
Table 5?Test chemical solubilities and removals during Phase II testing.
Removal (%)a
Solubility Active Sterilized Chemical (mg/L) cultures cultures
Nitrobenzene 1 900.0 100 52
4-Nitrophenol 16 000.0 100 19
2-Nitrophenol 2 100.0 100 51
2,4-Dichlorophenol 4 600.0 40 31
Hexachlorocyclopentadiene 0.085 100 99
Hexachloroethane 50.0 99 99
Hexachloro-1,3-butadiene 2.0 99 96
a Removal based on soluble concentration after 48 hours of contact.
O SEED D SEED & 1 GRAM OF SOLIDS * SEED & 5 GRAMS OF SOLIDS
L-v
O SEED SEED & 1 GRAM OF SOLIDS ? SEED & 5 GRAMS OF SOLIDS
96 120 144 168 216 240 TIME, hours
Figure 10?Relative activity in samples with various solids levels and receiving 100 mg/L nitrobenzene and 2-nitrophenol.
Figure 10 illustrates the relative response of these cul tures to 100 mg/L of nitrobenzene and 2-nitrophenol.
Although the methane production rate decreased ini
tially, recovery was essentially complete in all samples within 72 hours. However, the lag time for recovery
definitely decreased as the level of inactive solids was
increased.
The effect of the increased level of solids was much more pronounced in cultures dosed with hexachloro
ethane than with nitrobenzene and the nitrophenols
(Figure 11). Samples dosed with hexachlorocyclopen tadiene showed slower recovery, but again the recovery
was accelerated as the level of solids increased. A third response pattern was produced by 2,4-DCP
(Figure 12). Although the higher level of solids did reduce
the relative inhibition, complete recovery of gas produc tion did not occur. There was a similar lack of recovery
with hexachloro-l,3-butadiene, but there was no notice
able effect of solids concentration. This response indicated
that adsorption of these compounds was not sufficient to
reduce their residual soluble concentration to below toxic
levels. Because the solubility of 2,4-DCP compound was
so low, the equilibrium soluble concentration was essen
tially the same at all solids levels (Table 5). Phase III results essentially demonstrated that solids
level and consequent adsorption play an important role in reducing the inhibitory effect of toxic organic com
pounds on anaerobic reactions. However, the relative
effect of adsorption varies with biochemical and physical characteristics of the organic compounds. For example,
December 1983 1447
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Johnson & Young
Figure 11?Relative activity in samples with various solids levels and receiving 50 mg/L hexachloroethane and 100 mg/
L hexachlorocyclopentadiene.
the effect of solids level on the reduction of inhibition
from readily biodegradable compounds, such as the ni
trophenols, was slight because these compounds were
removed readily from solution by biological means.
However, the greater adsorption in the samples con
taining the highest levels of solids did shorten the re
covery time (Figure 10). The beneficial role of adsorption was more pro
nounced with the low-solubility compounds that de
graded slowly or not at all, such as hexachlorocyclopen tadiene and hexachloroethane (Figure 11).
Little benefit of adsorption was observed with com
pounds such as 2,4-DCP and hexachloro-l,3-butadiene, which were not biologically degraded.
SUMMARY AND DISCUSSION
Batch bioassays were conducted to determine the in
hibitory effects of various organic priority pollutants on
complex anaerobic cultures at 37?C. Twenty-four com
pounds were tested at levels ranging from 1 to 100 mg/ L. Only seven chemicals produced at least 50% reduc tion in gas production at concentrations of 100 mg/L.
The inhibition caused by these seven chemicals was ex
amined in more detail in a second phase of testing. The results of Phase II indicated that the inhibitory effort of toxic organic compounds was related to their solubility and their biodegradability, and to adsorption. Recovery from inhibitory effects occurred when the toxic chemical
was removed from solution. Phase III testing further substantiated the beneficial role of adsorption in reduc
ing toxicity and enhancing recovery for some of the toxic
compounds.
The recovery patterns shown in Figures 10 through 12 are not expected to represent the effect of solids level
on all toxic organic compounds. In fact, the role of ad
sorption may vary with the amount of toxic compound per unit of biological solids. In Figure 12, for example, it is likely that not all the 100 mg/L hexachloro-1,3
butadiene was either adsorbed or in solution. Thus, the
soluble concentration in contact with the active micro
organisms would be essentially the same at all solids
levels. The recovery pattern would be expected to change toward that shown by 2,4-DCP as the total dosage de
creased?possibly to full recovery, as shown in Figure 10, if biod?gradation occurred at the lower dosages.
Although these tests only scratched the surface of fac
tors affecting inhibition of anaerobic digestion by prior
ity pollutants, the results do permit some practical in
terpretations. First, the inhibitory effect of toxic organic
compounds can be expected to vary with the type of
waste being treated. For example, the response of a di
gester to 100 mg/L of a highly toxic compound such as
hexachloroethane would be much different for treating a wastewater composed of biodegradable soluble organ ics than for treating a municipal sludge of the same
organic strength and at the same loading. In the second
case, adsorption would be expected to play a greater role
than in the first.
Secondly, the mode of operation can be expected to
affect the response of anaerobic digesters to toxic organic
O SEED SEED & 1 GRAM OF SOLIDS
A SEED & 5 GRAMS OF SOLIDS 2,4-DICHLOROPHENOL
O SEED O SEED & 1 GRAM OF SOLIDS ? SEED & 5 GRAMS OF SOLIDS
120 144 168 192 TIME, hours
Figure 12?Relative activity of samples with various solids
levels and receiving 100 mg/L hexachloro-l,3-butadiene and
150 mg/L 2,4-dichlorophenol.
1448 Journal WPCF, Volume 55, Number 12
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_Process Research
compounds. For example, inhibitory effects should be
reduced as solids retention times (SRT) increase because
the equilibrium level of total solids increases as SRT
increases?again, assuming that a given waste load is
imposed at all SRTs.
Finally, although not substantiated by the test work
described in this paper, the relationship between hy draulic and solids retention time is expected to be an
important factor in establishing the inhibitory effect of a toxic organic compound. That is, as the hydraulic re
tention time (HRT) decreases relative to the SRT of a
system, inhibitory effects of organic compounds, at least
with slug doses, should be lessened because the contact
time between biological solids and the organic com
pound would be shortened. Consequently, anaerobic
systems having long SRTs and short HRTs?such as
fixed-film anaerobic filters and fluidized-bed processes? would not be affected to the same degree by slug loads
of toxic organic compounds as mixed digesters in which
the HRT and SRT are substantially the same.
CONCLUSIONS
The results of tests described in this paper form a basis
for the following conclusions:
Concentrations of 100 mg/L of hexachloroethane,
hexachlorocyclopentadiene, hexachloro-1,3-butadiene,
4-nitrophenol, 2-nitrophenol, 2,4-dichlorophenol, and
nitrobenzene inhibited anaerobic cultures at 37?C; Inhibition of anaerobic cultures by 4-nitrophenol,
2-nitrophenol and nitrobenzene was reversible under the
conditions of this study as a result of biological reduction
of the nitro group to the less toxic amine group;
Recovery of gas production in anaerobic cultures
exposed to inhibitory concentrations of hexachloroethane
seemed to be caused by biological acclimation;
Adsorption plays an important role in reducing the
soluble concentration of organic compounds; The rate of recovery for some compounds is related
to the concentration of organic compounds; and
Short-term batch bioassays are adequate for screen
ing of organic compounds for inhibitory effects, but lon
ger bioassays are required to evaluate properly the re
covery of anaerobic cultures from the inhibitory effects
of toxic organics.
ACKNOWLEDGMENTS
Credits. The work reported in this paper was sup
ported in part by the Engineering Research Institute of
Iowa State University and in part by the Ames Labo
ratory of Iowa State University, Ames, as part of contract
W-7405-ENG-82 from the U. S. Department of Energy. Authors. At the time of this study, L. D. Johnson was
a graduate research fellow and James C. Young was a
professor in the Department of Civil Engineering, Iowa
State University, Ames. Johnson presently is superinten dent of water reclamation, Sioux Falls, S. Dak. Young is professor and head of the Department of Civil Engi
neering at the University of Arkansas at Fayetteville. Cor
respondence should be addressed to James C. Young,
College of Engineering, University of Arkansas, 340 En
gineering Bldg., Fayetteville, AR 72701.
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