inhibition of anaerobic digestion by organic priority pollutants

10
Inhibition of Anaerobic Digestion by Organic Priority Pollutants Author(s): Lyle D. Johnson and James C. Young Source: Journal (Water Pollution Control Federation), Vol. 55, No. 12 (Dec., 1983), pp. 1441- 1449 Published by: Water Environment Federation Stable URL: http://www.jstor.org/stable/25042127 . Accessed: 10/07/2014 09:37 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Water Environment Federation is collaborating with JSTOR to digitize, preserve and extend access to Journal (Water Pollution Control Federation). http://www.jstor.org This content downloaded from 46.109.220.238 on Thu, 10 Jul 2014 09:37:13 AM All use subject to JSTOR Terms and Conditions

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Page 1: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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 .

Accessed: 10/07/2014 09:37

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Water Environment Federation is collaborating with JSTOR to digitize, preserve and extend access to Journal(Water Pollution Control Federation).

http://www.jstor.org

This content downloaded from 46.109.220.238 on Thu, 10 Jul 2014 09:37:13 AMAll use subject to JSTOR Terms and Conditions

Page 2: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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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Page 3: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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

1442 Journal WPCF, Volume 55, Number 12

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Page 4: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

_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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Page 5: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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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Page 6: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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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Page 7: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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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Page 8: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

_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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Page 9: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

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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Page 10: Inhibition of Anaerobic Digestion by Organic Priority Pollutants

_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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