Vol. 47, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1984, p. 850-8570099-2240/84/040850-08$02.00/0Copyright C) 1984, American Society for Microbiology

General Method for Determining Anaerobic BiodegradationPotentialt

DANIEL R. SHELTON't AND JAMES M. TIEDJE12*Departments of Crop and Soil Sciences' and Microbiology and Public Health,2 Michigan State University, East Lansing,

Michigan 48824Received 3 October 1983/Accepted 10 January 1984

A simple, generalized method was refined and validated to test whether an organic chemical wassusceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to10% and incubated anaerobically in 160-ml serum bottles with 50 pug of C per ml of test chemical.Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over thepressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by apressure transducer. The latter method is recommended because of its speed, accuracy, and low cost.Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. Thesludge could be stored anaerobically at 4C for up to 4 weeks with satisfactory test results. p-Cresol,phthalic acid, and ethanol are suggested as reference chemicals to confirm sludge activity and methodreliability. A revised anaerobic salts medium was developed which minimizes problems of abiological gasproduction (CO2), avoids precipitation, and meets the requirements of the anaerobic microbiota. When>75% of the theoretical gas production was observed, the chemical was judged to be degradable, and when30 to 75% of the expected gas was produced, it was termed partially degradable. This method has beentested on more than 100 chemicals of various physical properties and found to reproducibly determineanaerobic biodegradation potential. Of the chemicals tested, 46 were found to be anaerobically degraded.Sludges from nine different municipal treatment plants were surveyed for their ability to degrade ninechemicals which differed in susceptibility to degradation. As expected, the sludges varied in whichsubstrates they degraded, but this could not be correlated with influent waste properties of the particulartreatment plants.

Most manufactured chemicals will pass through anoxicenvironments, and in some cases they will reside in thesehabitats for long periods of time. These anoxic habitatsinclude sediments of all types, anaerobic waste treatmentsystems, gastrointestinal tracts, poorly drained or floodedsoils, and some landfills and groundwaters. To make anenvironmental risk assessment for a chemical, it may beimportant to determine the chemical's susceptibility to an-aerobic biodegradation. Furthermore, information on anaer-obic degradability is also requested under EnvironmentalProtection Agency guidelines implementing the U.S. ToxicSubstances Control Act and is of interest to the Organizationfor Economic Cooperation and Development. To obtain thisinformation, a simple general screening method for assessinganaerobic biodegradability is needed.Owen et al. (12) provided the first description of such a

test method, drawing on previous gas measurement (11) andincubation bottle (10) methods. They based their method onmeasurement of the excess gas volume (CH4 + C02) pro-duced after addition of a test chemical to an anaerobic seedincubated in sealed bottles. The gas volume was measuredfrom the displacement of the piston in a glass syringe whoseneedle had been inserted into the bottle. Subsequently,Gledhill improved the method with the goal of defining asimple protocol that could be established by the American

* Corresponding author.t Published as Journal Article no. 11032 of the Michigan Agricul-

tural Experiment Station.t Present address: Department of Soil and Environmental Sci-

ences, University of California, Riverside, CA 92521.850

Society for Testing Materials (ASTM) as a standard method(4). He introduced the use of a pressure transducer tomeasure the gas pressure, recommended 50 mg of carbon perliter to be the test chemical concentration, and used 10%anaerobic sludge as described by Healy and Young (5). Hedid not, however, evaluate each aspect of the method todetermine whether it was optimum, evaluate the variabilityand reproducibility, or evaluate the method against othermethods, all of which are necessary to validate a standardmethod. In this paper we report on this evaluation anddescribe the further refinements which we feel are benefi-cial. Furthermore, we report on chemicals which we foundto be degraded anaerobically, on an improved anaerobicmedium, and on the differences among municipal anaerobicsludges in their degradation capacities.

MATERIALS AND METHODS

Source and characteristics of anaerobic sludge. Sludgesamples were collected from primary or secondary anaero-bic digesters in 1- or 2-liter jars, tightly capped, and stored at4C until use. Sludges were from waste treatment plants inthe following mid-Michigan communities: Adrian, Ann Ar-bor, Chelsea, Holt, Ionia, Jackson, Mason, Portland, and St.Johns. Percent organic matter (total solids x volatile solids)varied from 0.89% (Holt) to 1.99% (Jackson) with a medianvalue of 1.53%. Average retention times varied from 17(Ionia) to 39 (Holt) days with a median value of 20 days.Inflow varied from 1.6 x 106 (Chelsea) to 68 x 106 (Jackson)liters/day with a median value of 4.4 x 106 liters/day.

Preparation of test bottles. Serum bottles of 160-ml capaci-ty (described as 125-ml Wheaton serum bottles; American

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ASSESSING ANAEROBIC BIODEGRADABILITY 851

Scientific Products, McGraw Park, Ill.) were amended asfollows with 50 ,ug of carbon per ml of the test compound:liquids were dispersed via microsyringe; water-soluble solidswere dissolved in water and then dispensed; water-insolublesolids were dissolved in diethyl ether and dispensed, and theether was allowed to evaporate (-2 h); insoluble polymerswere weighed out and added to serum bottles as solids. Therevised anaerobic mineral medium (RAMM) developed forthis study consisted of (per liter): phosphate buffer, 0.27 g ofKH2PO4 and 0.35 g of K2HPO4 (adjusted to pH 7.0); mineralsalts, 0.53 g of NH4CI, 75 mg of CaCl2 * 2H2O, 100 mg ofMgCl * 6H20, and 20 mg of FeCl2 * 4H20; and trace metalsmodified from Zehnder and Wuhrmann (20), 0.5 mg ofMnCl2 * 4H,O, 0.05 mg of H3B03, 0.05 mg of ZnCl,, 0.03 mgof CuCl2, 0.01 mg of NaMo4 2H20, 0.5 mg ofCoCl2 * 6H20, 0.05 mg of NiCl2 6H20, and 0.05 mg ofNa-SeO3. The medium was autoclaved for 5 to 10 min todrive off 02 and then cooled to approximately 35C whilesparging with a 10% CO-90% N2 gas mixture passedthrough copper filings at 300C to remove traces of 02 (7).After cooling, 1.2 g of NaHCO3 and 0.5 g of Na2S * 9H2O(optional) per liter were added to the medium. A 10% dilutedsludge was prepared by adding 1 part of sludge filteredthrough one layer of cheesecloth to 9 parts of cooled mineralmedium while stirring. The diluted sludge was dispensed intothe serum bottles while sparging with an 02-free 10% CO-90% N2 gas mixture. Methods for anaerobic gassing ofbottles and for preparation of 02-free gases were essentiallythose of Hungate (7). Serum bottles were sealed with 1-cm-thick butyl rubber stoppers (Bellco Glass, Inc., Vineland,N.J.) and capped with aluminum crimp seals (AmericanScientific Products). All compounds were tested in triplicatewith the exception of the compound survey, for whichcompounds were tested in duplicate. Bottles were incubatedstationary and in the dark at 35C.Measurement of gas production. After the medium had

equilibrated to 35C, the bottles were vented by syringeneedle to atmospheric pressure (generally there was 1- to 3-ml overpressure). Total gas production (CH4 + CO2) wasmeasured by a UniMeasure pressure transducer (GrantsPass, Ore.) equipped with a P-8 adapter (bellows) capable ofmeasuring up to 8 lb/in2 of gas pressure (Fig. 1). The needlewas inserted through the stoppers of the serum bottles andthe signal (in milliohms) from the transducer was quantifiedwith a Fluke multimeter (Mountlake Terrace, Wash.). Serumbottles were shaken vigorously before pressure measure-ments were taken, and excess gas pressure was ventedafterwards through the three-way valve to avoid cumulativegas pressures beyond the response range of the P-8 adapter.The milliohm response was related to milliliters of gasproduced by a standard curve constructed by adding knownquantities of gas to serum bottles by syringe; the r2 was>0.999%. Net gas production was calculated by subtractinggas produced in unamended bottles from that produced intest bottles. Degradation is expressed as percentage oftheoretical gas production based on the stoichiometry ofmineralization to CH4 + CO2 and correcting for gas solubili-ties.

Methane production was quantified by injecting 0.3 ml ofheadspace gas from serum bottles into a gas chromatographequipped with a flame ionization detector. Net methaneproduction was calculated by subtracting background meth-ane production in unamended bottles from that in testbottles. Degradation is expressed as percentage of theoreti-cal methane production based on the stoichiometry of degra-dation.

-D

Qe

FIG. 1. Gas production was measured with a pressure transduc-er (A) equipped with a P-8 bellows (B). The transducer wasconnected via 10 cm of 1/16-in. (ca. 0.16-cm) stainless-steel tubingand a 1/16-in. Swagelok union to a Hamilton three-way valve (C)with a 20-gauge needle attached. The signal from the transducer wasquantified with a multimeter (D).

Experimental. To determine the effect of length of sludgestorage on assay results, sludge from primary digesters inJackson, Holt, and Chelsea sewage treatment plants wasstored, sealed at 4C. Incubations were begun by taking newcontainers of sludge from storage after 0, 1, 2, 3, and 4weeks. Ethanol, p-cresol, phthalic acid, and di-n-butylphtha-late at 50 p.g of C per ml were used as test compounds.The effect of oxygen intrusion on the assay was investigat-

ed by replacing equal volumes of headspace gas with oxygenadded by syringe. Oxygen concentrations were quantifiedwith a gas chromatograph equipped with a thermal conduc-tivity detector.

Calculations. Since all substrates are provided at 50 pg ofC per ml, the theoretical gas yield from 100 ml of medium forall substrates is 10.5 ml. This gas will be divided betweenCO2 and CH4 based on the stoichiometry of the reaction,which can be calculated by the Buswell equation (17):

a bCnHaOb + n ----H.O0

n a b n_ _

- + -CO,+_2 8 4 2

a b+ -- - CH48 4

Knowledge of the mole fraction of each gas is necessary ifonly CH4 is measured or if total gas is measured because ofthe high water solubility of CO2. To correct for solubility,multiply the theoretical milliliters of CH4 x 0.95 and those ofCO2 x 0.35 to obtain the expected gas in the headspace.These constants were determined empirically and are accu-rate only for the temperature (35C), aqueous and gasvolumes, and medium composition of the method recom-mended here. If the substrate has carboxyl groups that wereneutralized when added to the test bottles, 1 CO2 perneutralized carboxyl group should be subtracted from thestoichiometry given by the Buswell equation since this groupdoes not contribute to the gas phase; if the group(s) was inthe acid form, it should remain in the equation.

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852 SHELTON AND TIEDJE

RESULTSPreliminary experiments were initiated to obtain degrada-

tion data for a wide variety of organic compounds to selectcompounds for future testing of assay parameters and todetermine the minimum length of incubation. The assayconditions were as described except that sludges fromsecondary digesters in Adrian and Jackson were used.Compounds were initially incubated for 4 weeks; however,this proved to be inadequate. Subsequently, all compoundswere incubated for 8 weeks, or until net methane productionhad ceased. Of 94 compounds tested, 27 were mineralized(>75% of theoretical methane production) in at least onesludge (Table 1); of these, 8 had lag times of >2 weeks. Tencompounds were partially mineralized (>30 to

ASSESSING ANAEROBIC BIODEGRADABILITY 853

TABLE 2. Effect of substrate concentration on gas production in 10% sludge from the Jackson digesterMean % of theoretical degradation t SD

Substrate concn(~Lg of carbon per ml) Phenol p-Cresol Benzoic Phthalicgocarbnperml)Penol

-Cresoacid acid25 100 9.9 98 92 18.0 105 4.850 104 8.0 86 13.5 92 6.0 104 + 18.7100 106 6.3 98 5.5 96 4.7 109 3.7200 113 3.1 95 4.5 98 3.6 100 1.5

Background gas production 31.4 31.6 24.5 28.4(ml)

Theoretical gas production 7.4 7.6 6.5 5.7from 50 ,ug of carbon perml (ml)"a Corrected for gas solubilities.

times, and extent of degradation in Jackson, Ionia, and Holt virtually all of the excess gas production was observed in theprimary sludges. An inhibition of background gas production first week of incubation, well before the end of the typical lagwas observed in the ASTM medium (Table 4). Further period for this compound (Fig. 3).experimentation indicated that an inverse correlation existed Sludge storage had no significant effect on extent ofbetween added sulfide and background gas production (data degradation, but lag times before degradation began werenot shown). There was no significant effect on lag times with affected for compounds that were degraded more slowly.any of the media; however, the extent of degradation did This is illustrated for p-cresol (Fig. 4), where lag timesvary (Table 4). Percent theoretical gas production was increased from 4 to 7 weeks for Holt sludge, 2 to 3 weeks forconsistently higher in the ASTM medium than in the Jackson sludge, and 4 to 4.5 weeks for Chelsea sludge, lagRAMM, whereas the supplemental medium generally yield- times for ethanol, which is readily degraded, were unaffecteded the lowest percent theoretical gas production. The effect (data not shown).was particularly pronounced with phthalic acid and m- Effect of oxygen on gas production. The rate of oxygenchlorobenzoic acid (both amended into sludges as the mon- consumption by 10% sludge was investigated since oxygenoacid), where percent theoretical gas production in the could inadvertently enter the test bottles and affect testASTM medium exceeded 150%. With m-chlorobenzoic acid results. Adrian sludge consumed approximately 1 ml of 02

TABLE 3. Comparison of mineral salts and metals in anaerobic media versus 10% sludgeConcn (mM, minerals; ,uM, metals) in:

MineraUmetal ASTM Survey of Survey of sludges'RAMM medium' anaerobic

media' Range Median Mean

MineralK 6.0 3.5 2.0-36.2 0.02-2.8 0.3 0.5NH4+ 10.0 4.3 3.2-42.4 0.03-15.4 0.4 2.1P042- 4.0 0.3 0.3-23.6 0.7-18.9' 0.9' 4.4"Ca 0.5 0.3 0.05-1.0 1.9-20.5 5.0 5.9Mg 0.5 1.8 0.05-1.8 0.05-2.3 0.8 1.0Fe 0.1 1.85 0.007-1.85 0.07-11.2 0.9 1.2S2- 0.5 2.0

MetalMn 2.53 101.0 0.15-101.0 4.0-530 21 30Zn 0.37 14.7 0.35-15.4 7.0-1,740 120 210Cu 0.22 17.2 0.06-20.1 5.0-650 60 90Co 2.10 126.0 0.42-126 0.2-1.2 0.5 0.6Ni 0.21 0.08-0.21 0.1-240 6.0 30B 0.81 97.1 0.81-97.1 5.0-280 14.0 37Mo 0.04 41.7 0.04-41.7 1.0-1.3 1.3 1.2Se 0.29 0.29-119.4

NaHCO3 1.2 g/liter 2.64 g/literResazurin 1 mg/liter 1 mg/literCO,-N2 10%:90% 30%:70%a From reference 4.b From references 3, 5, 6, 9, 13, 15, 18, 19, 20, and 21.' Calculations based on a median total solids of 4.1%.d Total phosphorus.

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854 SHELTON AND TIEDJE

TABLE 4. Effect of three mineral salts media on gas production in 10% sludgeMean % of theoretical gas production (+ SD)

Sludge/medium p-Cresol Phthalic acid Di-n-butyl- n-Chlorobenzoic Background gas

Jackson sludgeASTM medium" 102 3.2 156 0 128 3.9 30 + 8.6 13.5RAMM 108 3.9 130 5.0 101 9.7 0 18.8Supplemental medium' 79 7.9 85 7.7 89 14.8 0 17.9

Holt sludgeASTM medium 106 10.3 145 + 19.0 59 0 153 6.1 9.6RAMM 104 4.5 112 13.7 46 3.2 101 3.5 18.1Supplemental medium 89 4.6 96 2.9 19 5.4 65 5.2 19.0

lonia sludgeASTM medium 127 7.1 183 9.5 117 4.7 50 9.4 16.2RAMM 115 9.5 118 11.4 72 + 3.6 15 10.4 20.1Supplemental medium 99 6.7 104 24.8 77 + 16.7 0 21.6" Added 3.6 g of NaHCO3 per liter to be in equilibrium with the 30% CO2 specified instead of the 2.64 g of NaHCO3 per liter indicated in ref-

erence 4.bContained 6 mM P0427 9 mM K+, 10 mM HN4', 10 ,uM Co, 10% CO,-90% N2 headspace.

per day (Fig. 5). The upper portion of the sludge suspensionin serum bottles containing a headspace gas mixture of -8%02 was lightly pink due to the oxidation of resazurin (Fig. 5).The effect of 0, intrusion on gas production was tested in

sludge from the Holt primary digester. In serum bottleswithout substrate, approximately 4 ml less gas was producedin bottles injected with 4 ml of 0, than in bottles with no O2,due to 02 consumption (Fig. 6). In serum bottles containingbenzoic acid plus 4 ml of 0, approximately 6 ml less gas wasproduced. With serum bottles receiving no benzoic acid ascontrols, the percent theoretical degradation of benzoic acidwas 115% in bottles receiving no 02 and 87% in bottlesreceiving 4 ml of 02. After 16 h, bottles receiving 4 ml of 02(7% O, in the headspace) were slightly pink at the gas-waterinterface; however, the pink disappeared if the bottles wereslightly agitated. No pinkish color was evident after 40 h.

Capacity of different anaerobic sludges to degrade selectedcompounds. Sludges from primary digesters at nine sewagetreatment plants were compared for their ability to degradenine test compounds (Table 5). Ethanol, polyethylene glycol

11

9E]ASTM4.. HOLTE0LD 7 /5-RAMM

5-0~

z1/3-D

z 1/ 7 RAMM .- _

20,000, p-cresol, and phthalic acid were degraded in allsludges. m-Cresol and di-n-butylphthalate were degraded inapproximately half of the sludges, whereas 2-octanol, m-chlorobenzoic acid, and propionanilide were degraded inthree sludges or less. Percent degradation was generally>75% for m-cresol, p-cresol, phthalic acid, and m-chloro-benzoic acid. The lower percent degradation for polyethyl-ene glycol 20,000 and 2-octanol may be in part due to theirslower rates of degradation such that an 8-week incubationwas not sufficient to allow for complete methane recovery.We suspect that low methane recoveries for propionanilidewere due to metabolism of only the propionate moiety.

DISCUSSIONThe only feasible approach to a generalized method of

screening organic compounds for anaerobic biodegradationis to measure their common terminal products, CH4 + CO2.

H

6-IoQ H

28 |5 l |0a.

cot;wWLI '

1 2 3 ALENGTH OF STORAGE (weeks)

FIG. 3. Comparison of two mineral salts media (RAMM versusASTM) on gas production from mn-chlorobenzoic acid in Holt andIonia sludges.

FIG. 4. Weeks required for 50% of net gas production from p-cresol as affected by length of storage of sludges from Jakcson (J),Holt (H), and Chelsea (C).

-14 5WEEKS

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ASSESSING ANAEROBIC BIODEGRADABILITY 855

w

< 13.3

10.0 }

COLORLESS0

ui. 6.70

z

w

3.3

w

00 2 4 6 8

DAYSFIG. 5. Rate of consumption of different 0 concentrations by

10% sludge. The conditions where the resazurin was pink is shownby the stippled area.

A number of approaches to measuring these digestion prod-ucts have been used, but only recently have these evolvedtowards a routine test method. We began our work byevaluating the Owen et al. (12) and closely related ASTMprovisional (4) methods since these seemed to be the mostpromising of the existing methods.We found that a 10% sludge inoculum will generally yield

10 to 40 ml of gas, depending on the retention time andpercent organic matter of the sludge. An addition of 50 pg ofcarbon per ml will theoretically yield 10.5 ml of gas, whereasthe actual gas yield (corrected for solubility) may range from5.5 to 8.5 ml depending on the stoichiometry of mineraliza-tion. Concentrations of 50 pg of C per ml are notneeded for reliability and could lead to more cases of toxicityby the test chemical. Although a preincubation of the sludgeto reduce background gas production would allow for use ofa less dilute inoculum or of concentrations of test compoundof

856 SHELTON AND TIEDJE

TABLE 5. Percent theoretical methane production from nine substrates by sludges from nine municipalities"% Theoretical methane production

Sludge Ethanol Polyethylene p-Cresol Phthalic m-Cresol Di-n-butyl- 2-Octanol m-Chlorobenzoic Propionanilideglycol 20,000 acid phthalate acid

Adrian 62 61 91 88 82 24 0 85 0Jackson 78 43 88 80 103 49 22 0 0Ann Arbor 86 IDb 101 132 85 91 58 0 36St.Johns 78 78 79 113 0 37 0 0 33Ionia 71 51 80 73 0 36 0 0 0Holt 33 53 88 86 77 ID 0 85 23Mason 94 82 94 96 91 0 87 0 0Chelsea 54 38 62 60 ID 0 0 0 0Portland 52 98 77 96 0 0 0 0 0

"Fresh sludge (10%) from primary digesters incubated for 8 weeks, after which methane was measured by gas chromatography.b ID, Insufficient data; generally due to leaky bottles or only one bottle showing evidence for degradation.

retention time and 0.89% organic matter. We recommendthat serum bottles be incubated for a minimum of 8 Weeks.Lag times for p-cresol and phthalic acid in fresh sludgevaried from 2 to 4 weeks; thus, shorter incubation times arenot advisable.Anaerobic methods are always subject to error from 02

contamination; however, the high O2-consuming capacity ofmost sludges, the use of new, thick butyl rubber stoppers,and the use of standard anaerobic methods should preventany serious errors. If 02 intrusion does occur it will reducethe gas pressure somewhat (due to respiratory activity). Wedid not find it necessary to add reductant (sulfide) to theanaerobic medium because of the high 02 scavenging capaci-ty of the sludge. Therefore, use of a reductant is optional. Ifused, it should be in concentrations of

ASSESSING ANAEROBIC BIODEGRADABILITY 857

We have found gas production at -75% of theoretical (aftercorrection for gas solubilities) to be indicative of completemineralization.Summary of recommended protocol for anaerobic biodegra-

dation test. (i) Use primary anaerobic sludge with 15- to 30-day retention time and total organic solids of approximately1.0 to 2.0%. Sludges can be stored for up to 4 weeks at 4C intightly capped containers; however, fresh sludge should beused whenever possible. Since sludges vary in their selectedpopulations, it may be useful to use more than one sludgewhen working with more persistent compounds.

(ii) We suggest use ofRAMM since it has been thoroughlyevaluated under the test conditions and minimizes precipita-tion. If another anaerobic mineral medium is used, a 4 mMphosphate buffer and 1.2 g of NaHCO3 per liter with a 10%C02-90% N2 headspace is recommended. Sulfide can beadded to ensure reducing conditions in concentrations not toexceed 1 mM, but it is not necessary.

(iii) Incubate a 10% homogeneous sludge solution with 50,ug of C per ml of test chemical in 160-ml serum bottles withnew butyl rubber stoppers and aluminum crimp seals. Eachchemical should be tested in triplicate. A standard deviationof