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Methanogenic PopulationDynamicsduring Start-Up of Anaerobic DigestersTreating Municipal SolidWasteandBiosolids

MattE.Griffin,1KatherineD.McMahon,1Roderick I.Mackie,2 LutgardeRaskin1

1Environmental Engineering and Science, Department of Civil Engineering,3221 Newmark Civil Engineering Laboratory, University of Illinois atUrbanaChampaign, Urbana, Illinois 61801; telephone: 217-333-6964; fax:217-333-6968 or -9464; e-mail: [email protected] of Animal Sciences, 132 Animal Sciences Laboratory,University of Illinois at UrbanaChampaign, Urbana, Illinois 61801

Received 14 February 1997; accepted 12 J uly 1997

Abstract: An aggressive start-up strategy was usedto initiate codigestion in two anaerobic, continuouslymixed bench-top reactors at mesophilic (37C) and ther-mophilic (55C) conditions. The digesters were inocu-

lated with mesophilic anaerobic sewage sludge andcattle manure and were fed a mixture of simulated mu-nicipal solid waste and biosolids in proportions that re-flect U.S. production rates. The design organic loadingrate was 3.1 kg volatile solids/m3/day and the retentiontime was 20 days. Ribosomal RNA-targeted oligonucleo-tide probes were used to determine the methanogeniccommunity structure in the inocula and the digesters.Chemical analyses were performed to evaluate digesterperformance. The aggressive start-up strategy was suc-cessful for the thermophilic reactor, despite the use of amesophilic inoculum. After a short start-up period (20days), stable performance was observed with high gasproduction rates (1.52 m3/m3/day), high levels of meth-ane in the biogas (59%), and substantial volatile solids

(54%) and cellulose (58%) removals. In contrast, the me-sophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids in-creased dramatically and pH control was difficult. Afterseveral weeks of operation, the mesophilic digester be-came more stable, but propionate levels remained veryhigh. Methanogenicpopulation dynamics correlated wellwith performance measures. Large fluctuations were ob-served in methanogenic population levels during thestart-up period as volatile fatty acids accumulated andwere subsequently consumed. Methanosaeta specieswere the most abundant methanogens in the inoculum,but their levels decreased rapidly as acetate built up. Theincrease in acetate levels was paralleled by an increase inMethanosarcina species abundance (up to 11.6 and 4.8%of total ribosomal RNA consisted ofMethanosarcina spe-cies ribosomal RNA in mesophilic and thermophilic di-gesters, respectively). Methanobacteriaceae were themost abundant hydrogenotrophic methanogens in bothdigesters, buttheir levels were higher in the thermophilicdigester. 1998 J ohn Wiley & Sons, Inc. Biotechnol Bioeng57:342355, 1998.

Keywords: methanogenic population dynamics; anaero-bic digesters; solid waste; biosolids

INTRODUCTION

Production of municipal solid waste (MSW) in the United

States is expected to increase from 209 million tons in 1994

to 262 million tons in 2010 (EPA, 1996). Even though the

amount of MSW requiring disposal continues to grow, land-

fill and incinerator capacities are not increasing (Williams,

1994). Consequently, the interest in alternative large-scale

waste processing technologies will continue to grow. Re-

covery through recycling and composting has grown sig-

nificantly since the late 1980s and early 1990s and is ex-

pected to divert 3040% of MSW streams in 2010 (EPA,

1996). Anaerobic biological treatment, either in anaerobicdigesters or in landfill bioreactors, may serve to further

reduce the percentage of MSW disposed of in traditional

landfills or treated by incinerators. Anaerobic digestion can

be an attractive MSW treatment strategy because it reduces

the volume of MSW, stabilizes MSW, produces a residual

that can be used for soil conditioning, and recovers energy

from MSW in the form of methane (Tchobanoglous et al.,

1993).

Historically, the potential for energy recovery, rather than

environmental benefits, has driven much of the interest in

anaerobic digestion technology. As a result, interest has

been cyclic, reflecting the price of petroleum and the status

of fuel reserves (Hobson and Wheatley, 1993; Pfeffer,

1987). Some of the technical challenges associated with

anaerobic digestion of MSW can make the process less

attractive economically, especially when fuel prices and

landfill tipping fees are low. In the past, technical difficul-

ties have been associated mostly with the quality of the

MSW feedstock and materials handling (e.g., mechanical

separation of various waste components for RefCoM, Pom-

pano Beach, FL) (Pfeffer, 1987). These challenges have

Correspondence to: L. Raskin

Contract grant sponsor: Office of Solid Waste Research, Univ. of IL

Contract grant number: OSWR-12-013

1998 J ohn Wiley & Sons, Inc. CCC 0006-3592/98/030342-14


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limited the applications of MSW anaerobic digestion at the

full-scale level in the United States. As MSW streams

change through implementation of recycling and source

separation programs, the potential for successful applica-

tions of anaerobic digestion of the remaining organic frac-

tion of MSW (OFMSW) increases (Chynoweth and Pul-

lammanappallil, 1996). Moreover, the technical feasibility

of nutrient-deficient MSW anaerobic digestion can be im-

proved through the addition of biosolids to the waste stream

(PoggiVaraldo and Oleszkiewicz, 1992; Rivard et al.,

1990). Biosolids supply the nutrients and moisture lacking

in MSW. Codigestion of MSW and biosolids by a central-

ized facility may be an attractive alternative for the man-

agement of two separate waste streams that are produced in

every community. The feasibility of this technology has

been demonstrated in Europe where mixtures of biosolids

and solid wastes from households, agriculture, and industry

are processed at centralized digestion plants (Cecchi et al.,

1988; Rintala and Ahring, 1994).

This study evaluates anaerobic digestion of a feedstock

consisting of a mixture of biosolids and OFMSW, with

characteristics typical for the United States, combined in

proportions reflecting production rates of biosolids andOFMSW typical for most U.S. communities (EPA, 1992;

Metcalf and Eddy, 1991). Although several other research-

ers have studied MSW digestion (Cecchi et al., 1991; Kay-

hanian and Hardy, 1994; Rivard et al., 1993; Six and de

Baere, 1992) and codigestion systems (PoggiVaraldo and

Oleszkiewicz, 1992; Rivard et al., 1990; Stenstrom et al.,

1983), none have characterized changes in microbial com-

munity structure during start-up of these systems. Here, we

emphasize the importance of evaluating the microbial com-

munity structure in combination with chemical and physical

parameters to assess digester performance during the start-

up period.

Anaerobic digestion involves numerous interactions be-tween the four major metabolic groups that are generally

accepted as present in anaerobic digesters (Chynoweth and

Pullammanappallil, 1996; Zinder et al., 1984b). These

groups consist of hydrolytic-fermentative bacteria, proton-

reducing acetogenic bacteria, aceticlastic methanogens, and

hydrogenotrophic methanogens. These microorganisms cat-

alyze the mineralization of waste components to carbon

dioxide, methane, and water through a cascade of biochemi-

cal reactions. It is often assumed that the depolymerization

reactions at the top of the mineralization cascade are the

rate-limiting steps of the anaerobic digestion process and

that the methane yield is determined by the efficiency of

depolymerization (Chynoweth and Pullammanappallil,

1996; Eastman and Ferguson, 1981; Noike et al., 1985).

This is likely correct during stable operation of an anaerobic

digestion system when acetate, formate, hydrogen, and car-

bon dioxide are the main products of balanced carbohydrate

fermentation (Chynoweth and Pullammanappallil, 1996)

and the electron flux through reduced intermediates is small.

At higher substrate loadings, such as during start-up and

periods of overload, more reduced metabolites (e.g., pro-

pionate, butyrate, lactate, ethanol) accumulate because hy-

drogenotrophs fail to consume all of the hydrogen produced

during fermentation and acetogenesis. The presence of lip-

ids in some feedstocks (e.g., food waste) also results in the

production of fatty acids through hydrolysis of triglycerides.

Volatile fatty acid (VFA) accumulation can lead to a drop in

pH and inhibit methanogenesis, causing an even greater

imbalance. Methanogens, sulfate-reducing bacteria (SRB),

and acetogens are believed to be responsible for the removal

of hydrogen in most anaerobic systems (Schlegel and Jan-

nasch, 1992; Zehnder and Stumm, 1988). The presence of

methanogens is particularly desirable, because they gener-

ate methane, which can be used to produce energy. Thus,

during start-up and periods of overload, the microbial com-

munity should contain sufficient levels of methanogens to

prevent digester failure and maximize energy recovery.

Herein, we evaluate methanogen population dynamics

during start-up of a mesophilic and a thermophilic system.

In the past, such studies have been difficult because of the

long recognized limitations of traditional culture based

methods (Amann et al., 1995; Ward et al., 1992). Applica-

tions of molecular based methods to studies of microbial

community structure have eliminated some of these prob-lems because these techniques allow the direct identification

and enumeration of microbial populations in complex en-

vironments (Ahring, 1995; Amann et al., 1995; Macario and

Conway de Macario, 1988; Raskin et al., 1996; Ward et al.,

1992). Immunological methods have been used to track

methanogen populations in various anaerobic systems (Ah-

ring, 1995; Ney et al., 1990; Visser et al., 1991). However,

these methods usually still rely on the availability of pure

cultures for the production of antibodies. In addition, be-

cause antibodies bind to surface markers on target cells

(active and inactive), the direct measurement of metabolic

activity is impossible. Ribosomal RNA (rRNA) based meth-

ods can be used to detect phylogenetically defined groups oforganisms and quantify metabolic activity, because activity

is directly related to ribosome production (Poulsen et al.,

1993). Previously, we used several oligonucleotide probes

targeting the small-subunit (SSU) rRNA of phylogenetic

groups of methanogens (Raskin et al., 1994) to determine

their abundance in grab samples obtained from biosolids

digesters and gastrointestinal environments (Lin et al.,

1997; Raskin et al., 1995). We also demonstrated their util-

ity for studies of population dynamics in anaerobic biofilm

reactors fed a simple glucose-nutrient solution (Raskin et

al., 1996). In this study, we used these probes to relate

methanogenic population dynamics to traditional perfor-

mance parameters in order to better evaluate the start-up of

anaerobic codigestion systems.

MATERIALS ANDMETHODS

Apparatus

Two continuously mixed (400600 rpm), 5-L Microferm

bench-top fermentors (New Brunswick Scientific Co., New

GRIFFIN ET AL.: METHANOGENIC POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 343


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Brunswick, NJ) with a 3-L working volume were operated

in semicontinuous mode at a retention time of 20 days. One

digester was maintained at thermophilic conditions (55C)

and the other one at mesophilic conditions (35C). The de-

sign organic loading rate for both digesters was 3.1 kg vola-

tile solids (VS)/m3/day. Gas production was measured by

wet tip meters (Cambro, Huntington Beach, CA).

Feedstock

The feed was a mixture of simulated OFMSW, primary

sludge, and waste activated sludge (WAS). The simulated

OFMSW was prepared by collecting different paper frac-

tions (Community Recycling Center, Champaign, IL) and

food waste from several local restaurants and grocery stores

and combining these components in proportions that reflect

their presence in actual OFMSW (Table I). Yard waste was

not included in the simulated OFMSW because most com-

munities have established separate composting facilities for

this waste stream (Steuteville, 1995). The paper waste and

packaging were shredded using an industrial paper shredder

(model AZ-15, ShredPax Corp., Chicago, IL), and the food

waste was blended in a commercial blender (model 91-215,

Waring, New Hartford, CT). Single batches of WAS, thick-

ened by dissolved air flotation, and primary sludge were

obtained from the Urbana & Champaign Sanitary District

Sewage Treatment Plant (Urbana, IL). The simulated

OFMSW, primary sludge, and WAS were combined ac-

cording to typical production rates (Table I) and blended in

the commercial blender. The ratio of simulated OFMSW to

sewage sludge was 3.3:1 on a dry solids basis, with primary

sludge solids making up 64% of the sludge solids and WAS

solids making up the balance. The WAS was diluted with

tap water to obtain a solids level similar to that of unthick-

ened WAS. Aliquots (150 mL) of the blended feedstock

mixture were measured into screw-cap plastic bottles to

facilitate the daily feeding and stored at 20C.

Start-Up andOperationof AnaerobicDigesters

The inoculum for each digester consisted of 500 mL of a

mixture of anaerobic sludge (75% w/w) (Urbana & Cham-paign Sanitary District Sewage Treatment Plant) and cattle

manure (25% w/w) (feces and urine without bedding) (De-

partment of Agriculture, University of Illinois at Urbana

Champaign). One daily feed loading (150 mL) was added to

this inoculum and N2-sparged, distilled deionized water was

added to reach the working volume of 3 L. NaHCO3 (3 g)

and 6NNaOH (2 mL) were added to raise the initial pH to

7.2.

Wasting and feeding of the anaerobic digesters began 24

h after inoculation. Each day, 150 mL of digester contents

were removed from the digesters and 150 mL of thawed

(overnight at 4C) feed was added. If pH control measures

were necessary, NaHCO3 or NaOH was added to the feed.In the event of a more severe pH imbalance, the organic

loading rate was decreased.

Chemical Analyses

Supernatant for VFA, sulfate, and alkalinity analyses was

obtained by centrifuging a portion of the 150-mL digester

sample at 31,000gfor 15 min. The total VFA concentration

and alkalinity were measured daily for the first 2 weeks and

Table I. Composition of U.S. MSW, U.S. OFMSW, simulated OFMSW, and typical U.S. production rates of OFMSW, primary sludge, and WAS.

Component % of total discardsa (%) of total OFMSW % of simulated OFMSWb

Paper waste 19.4 49.1 50.0

Newspaper 4.6 11.6 18.2

Magazines 1.5 3.8 12.9

Office paper 2.9 7.3 13.9

Tissue paper and paper towels 2.0 5.1 5.0

Miscellaneous paper 8.4 21.3

Packaging 12.0 30.4 29.9

Corrugated boxes 7.7 19.5 29.9

Bags and sacks 1.4 3.5

Miscellaneous 2.9 7.3

Food waste 8.1 20.5 20.1

Total 39.5 100 100

Production ratesc

OFMSW Primary sludge WAS

635 g/capita/day 102 g dry solids/capita/day 57 g dry solids/capita/day

aData from the EPA (1992).bOFMSW used in this study.cOFMSW production rates were calculated using MSW discard rates (after materials recovery and composting), the U.S. population in 1990 (EPA, 1992),

and the fraction of discarded MSW that is organic (39.5%) as calculated above. Sludge production rates were calculated using typical values for the solids

content in primary sludge and WAS (Metcalf and Eddy, 1991) and per capita wastewater flow rates (McGhee, 1991).

344 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 57, NO. 3, FEBRUARY 5, 1998


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34 times per week thereafter. Methane content in the bio-

gas, individual VFA concentrations, and sulfate concentra-

tions were measured daily for the first week and 23 times

per week thereafter. Solids and fiber content were measured

23 times per week. Biogas production and pH were mea-

sured daily.

The total VFA concentration was measured by a titration

technique that accounts for approximately 7080% of the

total VFA concentration (DiLallo and Albertson, 1961). Bi-

carbonate and total alkalinity were determined by titrating

to pH 5.8 and 4.3, respectively (Greenberg et al., 1992). The

biogas composition was analyzed using a gas chromato-

graph (Series 580, Gow-Mac Instrument Co., Bridgewater,

NJ) equipped with a thermal conductivity detector. Indi-

vidual VFA concentrations (acetate, propionate, butyrate,

isobutyrate, valerate, and isovalerate) were measured using

a gas chromatograph equipped with a flame ionization de-

tector (model 5830A, HewlettPackard, Palo Alto, CA).

Sulfate concentrations were determined using a high per-

formance liquid chromatograph (AI-450 model II, Dionex,

Sunnyvale, CA) equipped with an ion conductivity detector

(Pfaff et al., 1989). Cellulose, hemicellulose, and lignin

were determined as described by Goering and Van Soest(1970) and total solids (TS) and VS were measured accord-

ing to Greenberg et al. (1992).

Microbial Analyses

Samples were collected at least twice a week in preweighed

2.2-mL screw-cap centrifuge tubes (Fisher, St. Louis, MO)

filled with approximately 0.5 g baked 0.1-mm zirconium

silica beads (Biospec Products, Bartlesville, OK). The tubes

were centrifuged at 2000gfor 5 min at 4C, the supernatant

was removed, and the samples were immediately placed in

a liquid nitrogen freezer. Samples were stored for up to 1

year in liquid nitrogen or at 80C.Nucleic acid was extracted in duplicate from 14 meso-

philic and 16 thermophilic digester samples and from the

two inoculum sources (anaerobic digester sludge and cattle

manure) using a low pH, hot phenol procedure described

elsewhere (Raskin et al., 1995; Stahl et al., 1988) with the

following modifications. The samples were bead beaten

in intervals of 2 min instead of 3 min. Nucleic acid was

precipitated using 0.5 vol of 7.5M ammonium acetate and

23 vol of absolute ethanol. Samples obtained from dupli-

cate extractions were pooled, nucleic acid quality was in-

spected, and concentrations were estimated using polyacryl-

amide gel electrophoresis (Alm and Stahl, submitted).

Membrane hybridizations were conducted using Magna

Charge membranes (Micron Separation Inc., Westboro,

MA) as previously described (Raskin et al., 1997) with mi-

nor changes. Samples were applied in triplicate with a di-

lution series of pure culture target rRNA on each membrane

(Table II). Membranes were prehybridized for 26 h at

40C, hybridized at 40C for 1619 h, washed twice at 40C

for 1 h, and washed for 30 min at a previously determined

wash temperature (Tw

) (Table II). Oligonucleotide probes

(Table II) were synthesized and purified with OPC columns

(PerkinElmer, Foster City, CA) at the University of Illinois

Biotechnology Center Genetic Engineering Facility (Ur-

bana, IL). Probes were 5 end labeled with 32P using bac-

teriophage T4 polynucleotide kinase and [-32P] ATP

(Raskin et al., 1994). The amount of probe hybridized was

quantified using PhophorImaging (Molecular Dynamics,

Sunnyvale, CA). The abundance of each phylogenetic group

was expressed as a fraction of total SSU rRNA, determined

using a universal probe, S-*-Univ-1390-a-A-18, while tak-

ing into account the variability of the hybridization response

from the pure culture rRNA used to construct a standard

curve (Raskin et al., 1997; Zheng et al., 1996).

RESULTS

Feedstock

A mixture of simulated OFMSW, primary sludge, and WAS

was used as the feed for the laboratory scale digesters

(Table I). The chemical characteristics of this feedstock are

presented in Table III. The feed had an average TS concen-

tration of approximately 70 g/kg (7% TS), which is signifi-

cantly higher than the %TS of sewage sludge typically di-

gested in an anaerobic digester [solids content of the pri-

mary sludge, unthickened WAS, and thickened WAS are

approximately 5, 0.8, and 5%, respectively (Metcalf and

Eddy, 1991)], but lower than the values reported in some

recent publications on high solids digestion of OFMSW

[30% TS (Rivard, 1993), 2030% TS (Kayhanian and

Hardy, 1994), and 1623% TS (Cecchi et al., 1991)]. A

large percentage of the TS was organic (88% VS). This

organic fraction was composed mainly of paper waste and

thus contained high levels of cellulose, hemicellulose, and

lignin. The levels of these compounds in our simulatedwaste were consistent with other studies (Palmisano and

Barlaz, 1996).

The pH of the feedstock was 7.1 (Table III), but the

bicarbonate alkalinity was relatively low, indicating that the

buffering capacity of the feed was minimal and that the

daily additions of feed did not aid much in maintaining a

stable pH in the digesters. The levels of VFAs (in particular,

acetate, propionate, and butyrate) were relatively high

(Table III) and may have contributed to start-up problems

(discussed below).

Inocula and Start-UpStrategyNo inoculum that had been acclimated to the feedstock was

available to accommodate the start-up of the digesters.

Therefore, a combination of inocula obtained from two dif-

ferent anaerobic environments was used to seed the digest-

ers. Anaerobic sludge from a stable sewage sludge digester

was used as the main inoculum to provide a balanced mi-

crobial community consisting of fermenters, acetogens, and

methanogens. Because the feedstock contained a high level



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of cellulose, cattle manure was chosen as a secondary in-

oculum to provide sufficient levels of cellulolytic microor-

ganisms. Because there was no thermophilic inoculum

readily available, we used these two mesophilic inocula to

seed the mesophilic as well as the thermophilic reactor.

Because methanogenesis is critically important during

start-up, we determined methanogen levels in the inocula

using oligonucleotide hybridization probes (Table II) that

target most currently known methanogens. An Archaeal

specific probe (S-D-Arch-0915-a-A-20) was used to deter-

mine total methanogen levels (Raskin et al., 1995), and

specific probes were used to quantify various phylogenetic

groups of methanogens within four taxonomically defined

orders:Methanobacteriales, Methanococcales, Methanomi-

crobiales,and Methanosarcinales (Boone et al., 1993). No

probes were used to target the hyperthermophile Methano-

pyrus kandleri, the only currently known representative of

the fifth order (Methanopyrales).

Table IV presents the hybridization results for the two

inocula. The anaerobic sludge contained a large fraction of

methanogens (approximately 12% of total SSU rRNA con-

sisted of methanogen SSU rRNA). Members of the Metha-

nosaetaceaewere present at high levels andMethanomicro-

biales constituted the second most important group. The

high levels ofMethanosaetaspp. are consistent with the low

acetate concentrations in the anaerobic digester from which

the inoculum was taken (15 mg/L). Methanosaetaspp. have

a low threshold for acetate and therefore have a competitive

advantage over Methanosarcina spp. at low acetate con-

centrations (at high levels of acetate, Methanosarcina spp.

generally dominate) (Zinder, 1993). The cattle manure

contained low levels of methanogen SSU rRNA (2.4%),

Table II. Oligonucleotide probes used in hybridizations.

Probea,b Tw

Target group RNA standard Characteristic substratesc

Universal probe

S-*-Univ-1390-a-A-18

44 Virtually all organisms

Probes for domains

S-D-Arch-0915-a-A-20 58 Vi rt ually all Archaea Methanosarcina acetiv orans

or Methanosaeta concilii

GP6

S-D-Bact-0338-a-A-18 55 Vi rt ually all Bacteria Escherichia coli

S-D-Euca-0502-a-A-16 58 Vi rt ually all Eucarya Saccharomyces ce re visiae

Probes for methanogens

S-F-Mbac-0310-a-A-22d 57 Methanobacteriaceae Methanobacterium bryantii Most use H2CO2, some use

H2CO2 and formate

S-F-Mcoc-1109-a-A-20e 55 Methanococcaceae Methanococcus voltae Most use H2CO2 and formate

S-O-Mmic-1200-a-A-21 53 Methanomicrobiales Methanogeni um cariaci Most use H2CO2 and formate

S-G-Msar-0821-a-A-21 60 Methanosarcina spp. Methanosarcina acetivorans Use acetate and other substrates

(H2CO2, methanol, and

methylamines); generally have

high minimum threshold, K, and

max values for acetate

S-F-Msae-0825-a-A-23f 59 Methanosaetaceaeg Methanosaeta concilii GP6 Use only acetate; generally have

low minimum threshold, K, and

max values

Probes for SRBS-F-Dsv-0687-a-A-16 47 Desulfovibrionaceae Desulfovibrio desulfuricans H2, lactate, formate

S-*-Dsb-0804-a-A-18 47 Desulfobactergroup Desulfobacterium

vacuolatum

Various

S-G-Dsbm-0221-a-A-20 57 Desulfobacterium spp. Desulfobacterium

vacuolatum

Various

S-*-Dcoc-0814-a-A-17 47 Desulfosarcina variabilis,

Desulfococcus multivorans,

Desulfobotulus sapovorans

Desulfosarcina variabilis Various

aProbe names have been standardized as described in Alm et al. (1996).bOriginal citations are as follows: universal probe (Zheng et al., 1996); archaeal probe (Stahl and Amann, 1991); bacterial and eucaryal probes (Amann

et al., 1990); methanogen probes (Raskin et al., 1994); and SRB probes (Devereux et al., 1992).cCharacteristic substrates for methanogens as listed in Raskin et al. (1994) and characteristic substrates of SRB as listed in Devereux et al. (1992).dMost representatives of the orderMethanobacterialesbelong to the familyMethanobacteriaceaeand are targeted by probe S-F-Mbac-0310-a-A-22. This

probe does not target the thermophilic members of this order (Methanothermus fervidusand M. sociabilis).eProbe S-F-Mcoc-1109-a-A-20 targets the family Methanococcaceae, which groups most members of the order Methanococcales. Two thermophilic

representatives of this order (Methanocaldococcus jannaschiiand Methanoignis igneus) have one mismatch with probe S-F-Mcoc-1109-a-A-20.fProbe S-F-Msae-0825-a-A-23 may not target some Methanosaeta spp. due to a possible deletion in the 825 region (Raskin et al., 1994).gMethanosaetais the only genus that has been defined so far within the family ofMethanosaetaceae (Boone et al., 1993).



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approximately half of which consisted of Methano-

bacteriaceaeSSU rRNA.

Digester Performanceand Chemical Parameters

The chemical parameters used to provide an indication of

digester stability and methanogen activity include VFA con-

centrations, pH, alkalinity, gas production rate, methane

content of biogas, specific gas production, TS and VS de-

struction, and fiber (cellulose, hemicellulose, lignin) re-

moval. A derived parameter, , was also used to predict

changes in the relationship between buffering capacity and

VFA concentration; is the ratio of the VFA concentration

(estimated by the difference in the bicarbonate alkalinity

and the total alkalinity) to the bicarbonate alkalinity (PoggiVaraldo and Oleszkiewicz, 1992). For anaerobic codiges-

tion of OFMSW and sewage sludge, PoggiVaraldo and

Oleszkiewicz (1992) determined that an value of 1.0 cor-

responds to the threshold of stability. An increase in the

value precedes a pH decrease, making it possible to predict

an imbalance before the VFA concentrations or pH reveal

the instability and, consequently, to prevent digester upset.

In the event of instability, McCarty (1964) recommends

adding NaHCO3to maintain the pH near neutrality, decreas-

ing the rate of feeding, or both. On the other hand, Hobson

and Wheatley (1993) discourage the practice of adding

chemicals to restore the pH and suggest simply reducing the

feed rate. We followed the method of pH control proposed

by McCarty. The amount of Na+ added was monitored to

prevent Na+ toxicity that starts at approximately 3500 mg/L

(Parkin and Owen, 1986).

Figure 1 presents the important performance parameters

for the mesophilic and thermophilic digesters. In the meso-

philic digester, the gas production rate was low, while the

percentage of methane in the biogas was high (58%) on day

1 (Fig. 1E). The methane content of the biogas then gradu-

ally decreased and remained low until day 14 (1728%).

The gas production rate also remained low. In the thermo-

philic digester, the methane content of the biogas was very

low on day 1 (2%), but increased dramatically during the

first 7 days of operation to 57% methane on day 7 (Fig. 1F).

The concentrations of VFA increased in both digesters

during the first 3 days of operation (Fig. 1A,B), indicating

that hydrolytic and fermentative bacteria were active. Ac-

etate and propionate levels increased significantly in the

mesophilic digester, while the bulk of the VFA increase in

the thermophilic digester was due to acetate and butyrateincreases. As a result of the initial VFA accumulation, the

pH dropped and the value became greater than the thresh-

old value of 1.0 (Fig. 1C,D). On day 3, 6 g of NaHCO3were

added to both digesters to restore the pH and provide buff-

ering capacity. This strategy worked well for the thermo-

philic digester: the pH remained above 7.0 for the rest of the

experiment, the value dropped below 1.0 on day 11, the

concentration of VFA decreased, the methane content of the

biogas continued to rise, and the gas production rate in-

creased. The maximum measured acetate concentration was

1620 mg/L on day 4, after which its concentration gradually

decreased. Butyrate concentrations remained relatively high

until day 9 but then decreased. Propionate gradually accu-mulated, while valerate, isovalerate, and isobutyrate re-

mained at low concentrations for the entire period. Follow-

ing the initial start-up period, the thermophilic digester

achieved a relatively stable performance after approxi-

mately day 19.

In contrast, the mesophilic digester exhibited poor per-

formance during start-up. Despite recovery of the pH and

values on day 4 after the addition of NaHCO3, the pH

dropped to 6.2 and the value increased to 13 on day 5,

prompting further chemical additions. Either NaHCO3 or

NaOH were added on days 5, 7, 9, 10, 11, 13, and 15 to

restore the pH and to provide buffering capacity. Feeding

was suspended from days 5 to 8 and again from days 11 to

18 to prevent further buildup of VFA and to allow conver-

sion of the accumulated VFA. The mesophilic digester fi-

nally showed signs of improvement between days 14 and

20: the pH and values approached satisfactory levels, the

methane content in the biogas increased significantly while

the gas production rate increased slowly, acetate decreased

from approximately 5000 to 2000 mg/L, and total VFA

levels decreased from approximately 5600 to 3600 mg/L.

Table IV. Methanogenic population levels in inocula, expressed as per-

centage of specific SSU rRNA of total SSU rRNA (% SSU rRNA SD).

Probe

Cattle

manure

Anaerobic

digester sludge

S-F-Mbac-0310-a-A-22 1.0 0.1 0.17 0.16

S-F-Mcoc-1109-a-A-20 0.19 0.05 0.27 0.11

S-O-Mmic-1200-a-A-21 0.14 0.01 3.3 0.4

S-G-Msar-0821-a-A-21 0.19 0.04 0.19 0.09

S-F-Msae-0825-a-A-23 0.02 0.03 9.4 0.9

S-D-Arch-0915-a-A-20 2.4 1.5 12.1 1.4

Table III. Chemical characteristics of feedstock.

Parameter Mean SDa Units

TS 7.0 0.6 %

VS 88.0 0.9 % of TS

Cellulose 55.5 4.4 % of TS

Hemicellulose 10.7 1.1 % of TS

Lignin 8.9 0.4 % of TS

Acetate 372 66 mg/L as acetic acid

Propionate 136 36 mg/L as acetic acid

Butyrate 47 29 mg/L as acetic acid

Total VFAsb 587 91 mg/L as acetic acidBicarb. alkalinity 158 26 mg/L as CaCO3pH 7.1 0.2 SU

Sulfate 53 14 mg/L as sulfate

Nitrogen 1.4 0.3 % of TS

aMean values and standard deviations (SD) were obtained by performing

analyses for seven batches of feedstock.bTotal VFAs equals the sum of the concentrations of acetate, propionate,

butyrate, valerate, isovalerate, and isobutyrate as measured by gas chro-

matography.



7/14

Feeding was resumed on day 19 at an organic loading rate

that was approximately one-third of the design rate. By day

29 the concentrations of acetate (184 mg/L) and total VFAs

(2631 mg/L) had decreased further, and the percentage of

methane in the biogas exceeded 50%. Therefore, the organic

loading rate was increased to its original value. Acetate

levels remained low but propionate concentrations gradu-

ally increased after this perturbation. After a slow start-up

that required several chemical additions and feed suspen-

sion, the mesophilic digester demonstrated relatively stable

operation for a short period of time and the design retention

time of 20 days was maintained between days 31 and 48.

After this period, the mesophilic digester entered another

period of instability (days 5080). Chemical additions again

were required as well as temporary reduction and suspen-

sion of daily feeding.

Although temperature was the only design variable and

both digesters received similar treatment, the performance

of the mesophilic digester was distinctly different from that

of the thermophilic digester. To better compare digester

performance, representative chemical characteristics were

calculated by averaging the values obtained from days 41

50 and 1975 for the mesophilic and thermophilic digesters,

respectively (relatively stable periods for both digesters)

(Table V). The average pH and values (and their relatively

low standard deviations) indicate that the thermophilic di-

gester was stable. The mesophilic digester appeared to be on

the verge of instability, even during the relatively stable

period between days 41 and 50. The average value for this

period was greater than 1.0 and the pH was slightly less than

7.0. Values for the methane content of the biogas, the spe-

cific gas production, and gas production rate also demon-

strate that the thermophilic digester performed better than

the mesophilic digester. The methane content, the specific

gas production, and the gas production rate were 9, 48, and

58% greater in the thermophilic digester compared to the

mesophilic digester.

Table V also provides information on the ability of the

digesters to remove TS, VS, and fiber components (hemi-

cellulose, cellulose, and lignin). Because the mesophilic di-

Figure 1. Chemical performance characteristics for mesophilic and thermophilic digesters: VFA concentrations and organic loading rate (OLR) in (A)

mesophilic and (B) thermophilic digesters; pH and parameter in (C) mesophilic and (D) thermophilic digesters; gas production rate (GPR) and percent

methane (% CH4) in biogas in (E) mesophilic and (F) thermophilic digesters.



8/14

gester was fed irregularly, it was difficult to perform a mass

balance on the feed and effluent streams to determine the

removal of fibers. During the period when the mesophilic

digester was being fed at the design organic loading rate of

3.1 kg VS/m3/day, the cellulose concentration climbed to a

maximum of 12,000 mg/L on day 42 before the concentra-

tion decreased to 8700 mg/L on day 50, which corresponds

to 63% cellulose removal over this 8-day period. Although

the thermophilic digester was able to utilize the cellulose inthe feed and accomplished 58% removal, the steady-

state cellulose concentration in the thermophilic digester

(17,000 mg/L) was higher than the maximum cellulose level

found in the mesophilic digester. A possible explanation for

the apparent higher level of cellulose degradation in the

mesophilic digester is that cellulolytic bacteria were able to

increase to higher concentrations during periods when feed-

ing was suspended and no wash-out occurred. Rivard et al.

(1990) demonstrated that cellulose conversion is influenced

by the solids retention time (SRT): they observed a higher

conversion efficiency of cellulose at a 30-day SRT (80%)

than at 20-day (75%) and 14-day (70%) SRTs. Although the

design SRT was the same for both digesters (20 days), the

feeding of the mesophilic digester was suspended or greatly

reduced at times during the first 30 days of operation, while

the design feeding schedule was maintained for the thermo-

philic digester. The resulting SRT for this period was ap-

proximately 40 days for the mesophilic digester. This longer

SRT may have facilitated growth and development of a

consortium of microbes that were able to effectively de-

grade the cellulose. On the other hand, it is possible that the

mesophilic inocula contained cellulolytic bacteria, which

were able to perform better at mesophilic conditions. The

apparent lignin removal rate was relatively low in both di-

gesters, which is consistent with previous findings that lig-

nin is essentially refractory to microbes under anaerobic

conditions (Jung and Deetz, 1993; Palmisano and Barlaz,

1996).

Digester Performanceand MicrobialPopulationDynamics

A selection of SSU rRNA-targeted oligonucleotide probes

(Table II) was used to determine the relative concentrations

of the three domains (Bacteria, Archaea,and Eucarya) and

different phylogenetic groups of methanogens in samples

collected from each digester during the course of the ex-

periment. Figure 2 presents the hybridization results for the

two reactors. The sum of the relative amounts ofBacteria,

Archaea, and Eucarya (presented as a percentage of the

total SSU rRNA) should equal 100% because all known

organisms are contained within these three domains (Woese

et al., 1990). Figure 2A,B shows that this nesting re-

quirement was relatively well met throughout the experi-

ment.Bacteria constituted the majority of the microorgan-

isms in the reactors, Archaea were present in smaller

amounts (below 10% in most cases), and Eucarya were

present at very low levels (the means for both digesters were

below 0.8%). The low amounts of Eucarya indicates that

anaerobic protozoa likely were not abundant in our digest-

ers, even though they are thought to play a role in a variety

of anaerobic environments (Fenchel and Finlay, 1995).

Table V. Performance of mesophilic and thermophilic digesters.

Parameter Units

Mesophilic

(Days 4150) N

Thermophilic

(Days 1975) N

Methane % 54 4 4 59 6 20

Spe cific gas production m3/kg/VSfed 0.29 0.02 10 0.43 0.05 48

Gas production rate m3/m3/day 0.96 0.07 10 1.52 0.12 48

Acetate mg/L as acetic acid 143 12 4 90 28 18

Propionate mg/L as acetic acid 2043 177 4 492 149 18

Butyrate mg/L as acetic acid 60 9 4 12 5 18

Isobutyrate mg/L as acetic acid 79 4 4 46 21 18

Valerate mg/L as acetic acid 184 10 4 11 4 18Isovalerate mg/L as acetic acid 64 6 4 49 22 18

Total VFAsa mg/L as acetic acid 2572 162 4 700 192 18

pH SU 6.7 0.1 10 7.1 0.2 55

1.7 0.2 7 0.7 0.1 33

Sulfate mg/L as sulfate 3.4 2.2 4 2.7 1.1 16

TS removal % 48 0.5 3 53 8.9 20

VS removal % 53 0.2 3 54 8.9 20

Cellulose removal % ND 58 5.4 15

Hemicellulose removal % ND 40 15 15

Lignin removal % ND 19 15 15

Max. cellulose concentration mg/L 12,023

(day 42)

1 19,244

(day 37)

1

Min. cellulose concentration mg/L 8693

(day 50)

1 14,161

(day 67)

1

N,number of analyses performed to calculate means and standard deviations. ND, not determined.aTotal VFAs equals the sum of the concentrations of acetate, propionate, butyrate, valerate, iso-

valerate, and isobutyrate as measured by gas chromatography.



9/14

The archaeal domain probe was used as an approximate

measure to evaluate total methanogen SSU rRNA levels in

the digesters. Initially, significant levels of methanogen

SSU rRNA were present in both reactors (Fig. 2C,D), which

can be attributed to the anaerobic sludge and animal manure

inocula (Table IV). The ratio between Methanosaeta and

MethanosarcinaSSU rRNA levels on day 0 roughly corre-

sponded to those in the anaerobic sludge, and Methanosaeta

SSU rRNA levels were higher than those of all other meth-

anogens in both systems (Fig. 2C,D). Methanobacteriaceae

and Methanomicrobiales were present in both systems on

day 0 (Fig. 2E,F) due to their presence in animal manure

and anaerobic sludge, respectively. Methanococcaceae lev-

els were very low on day 0 (Fig. 2E,F), which is consistent

with their low concentration in both inocula.

The presence of methanogens in the mesophilic digester

resulted in an immediate production of methane (Fig. 1E).

By day 5 the levels ofMethanosaeta spp. and Methanomi-

crobialesSSU rRNA had decreased significantly. Thus, the

rate at which these methanogens were removed through

wash-out was greater than their growth rates. A peak in

archaeal abundance occurred near day 17 (Fig. 2C), corre-

sponding to the acetate turnover between days 14 and 20

(Fig. 1A). A corresponding decrease in relative bacterial

abundance was observed on day 17. The archaeal peak was

likely the result of the high methanogenic activity respon-

sible for the turnover of large quantities of acetate, because

specific probe data indicated thatMethanosarcinaspp. were

the dominant methanogens during this period (Fig. 2C).

Methanosarcinaspp. have higher growth rates than Metha-

nosaeta spp. (Zinder, 1993) and thus are generally more

competitive at high acetate concentrations. This explains

why, as the acetate concentration increased, Methanosaeta

levels decreased to 0.2% on day 17 and Methanosarcina

levels increased to 11.6%. When the acetate concentration

decreased after day 20, Archaeaand Methanosarcinalevels

Figure 2. Microbial community structure in mesophilic and thermophilic digesters: Archaeal, bacterial, and eucaryal levels in (A) mesophilic and (B)

thermophilic digesters; archaeal and aceticlastic methanogen levels in (C) mesophilic and (D) thermophilic digesters; hydrogenotrophic methanogen levelsin (E) mesophilic and (F) thermophilic digesters. The error bars in (AD) indicate standard deviations. Standard deviations are not reported in (E) and (F)

to improve the clarity of presentation; coefficients of variation were generally between 5 and 30%.



10/14

decreased as well.Methanosaetaremained present after day

17, but at low levels (mean 0.13 0.02%).

The levels ofMethanomicrobiales, which were present

initially due to their abundance in the anaerobic sludge in-

oculum, decreased shortly after start-up and remained low

for most of the operating period (Fig. 2E). Methanobacte-

riaceae were the most abundant hydrogenotrophic meth-

anogens after day 0, and the levels ofMethanococcaceae

remained low (below 0.4%) for the duration of the experi-

ment.

The methanogenic population dynamics in the mesophilic

digester can be correlated to biogas and VFA data (Fig.

1A,E). As discussed above, the percentage of methane in the

biogas was high on day 1, but decreased rapidly (Fig. 1C).

This corresponds well with the reduction in methanogen

abundance. The percentage of methane in the biogas stead-

ily increased after day 10 and peaked near day 17 when

aceticlastic methanogens were most active and large

amounts of acetate were consumed.

Even though significant levels of methanogens were

present in the thermophilic digester, they were apparently

not able to adjust within 1 day to thermophilic conditions, as

demonstrated by the low methane levels in the biogas onday 1 (Fig. 1F). As in the mesophilic digester, the levels of

Methanosaetaspp. andMethanomicrobialesSSU rRNA de-

creased during the first few days, indicating that the removal

rate through wash-out was greater than their growth rates.

The total methanogen concentrations remained relatively

constant during the first few days of operation, because the

loss in Methanosaeta and Methanomicrobiales was com-

pensated by an increase in the Methanobacteriaceaelevels.

Methanobacteriaceae apparently served as the main hydro-

gen scavengers during this period of rapidly increasing ac-

tivity, reflected by rapid increases in the gas production rate

and the level of methane in the biogas. Around day 7 an

increase in archaeal abundance was observed (Fig. 2D) asacetate accumulated and then turned over (Fig. 1B), but the

maximum archaeal SSU rRNA concentration was much

smaller than the level observed in the mesophilic digester

(Fig. 2C). Specific probe analyses indicated that this peak

also corresponded to elevated Methanosarcinalevels. After

the high levels of acetate were consumed, Methanosarcina

spp. remained present throughout the experiment and

Methanosaeta levels did not increase appreciably (mean

0.07 0.01%), despite the low acetate concentrations.

Methanomicrobialeswere present initially in the thermo-

philic digester, but their SSU rRNA levels decreased and

remained low, with the exception of day 13 (Fig. 2F).

Methanococcaceaewere also present throughout the experi-

ment, but at low levels (0.171.4%). Methanobacteriaceae

were generally the most abundant hydrogenotrophic meth-

anogens in the thermophilic digester. Their relative SSU

rRNA levels varied considerably throughout the experiment

with peaks on days 1, 10, 23, and 44. It is not clear what

caused these fluctuations and whether they can be linked to

digester performance.

The sulfate concentration decreased from 12.5 mg/L in

the mesophilic digester and from 17 mg/L in the thermo-

philic digester to levels close to the detection limit (1 mg/L)

from day 0 to 1 and remained low throughout the rest of the

experiment (data not reported). Given that the sulfate con-

centration in the feed averaged 53 14 mg/L, it is apparent

that at least some SRB were active in both digesters. How-

ever, the low sulfate concentrations and relatively high level

of methane in the biogas suggest that sulfate concentrations

were limiting and that SRB were not able to compete ef-

fectively with methanogens. This is confirmed by the con-

sistently low levels of SRB in both digesters (Table VI)

determined by hybridizations using a selection of oligo-

nucleotide probes for phylogenetic groups of SRB (De-

vereux et al., 1992). Table II lists the various probes, the

target groups for each probe, and some characteristic sub-

strates for each target group. Target groups include the fam-

ilyDesulfovibrionaceae,theDesulfobactergroup, the genus

Desulfobacterium,and an assemblage of several other gen-

era. The most abundant SRB present in the digesters were

members of the family Desulfovibrionaceae (Table VI),

while the levels of other target groups were always below

1.0% and usually below 0.2%. No apparent trends were

observed in SRB SSU rRNA levels in either digester.

DISCUSSION

The start-up is generally considered the most critical step in

the operation of anaerobic digesters. Once an anaerobic di-

gester has been started up successfully, it is expected to run

without much attention as long as operating conditions are

not significantly altered (Hobson and Wheatley, 1993). The

source of microorganisms, the size of the inoculum, and the

initial mode of operation are important factors during start-

up (Cecchi et al., 1992; Hobson and Wheatley, 1993). Usu-

ally, the inoculum volume is at least 10% of the new di-

gester volume and the inoculum consists of an undefinedmixed culture from an equivalent system that is actively

digesting a similar feedstock (Hobson and Wheatley, 1993).

Ahring (1994) discusses that the start-up of thermophilic

digesters can be problematic when no thermophilic inocula

are readily available, as was the case for our study. In ad-

dition, she illustrates that even if a thermophilic inoculum

from a similar system is available, the start-up method is

critical for success (Ahring, 1994). She compared two start-

Table VI. Mean levels of SRB in mesophilic and thermophilic digesters,

expressed as percentage of specific SSU rRNA of total SSU rRNA (% SSU

rRNA SD).

Probe

Mesophilic

digester N

Thermophilic

digester N

S-F-Dsv-0687-a-A-16 1.8 0.2 3 1.7 0.2 8

S-*-Dsb-0804-a-A-18 0.18 0.03 7 0.05 0.03 8

S-G-Dsbm-0221-a-A-20 0.03 0.05 7 0.02 0.03 8

S-*-Dcoc-0814-a-A-17 0.90 0.34 13 0.61 0.08 15

N, the number of days used to compute the means and standard devia-

tions.



11/14

up strategies for thermophilic manure digesters. The first

method involved mixing a thermophilic inoculum in equal

amounts with manure. Approximately 50 days were needed

for VFA concentrations to decrease to levels that allowed

the initiation of feeding. The second strategy consisted of

the addition of a thermophilic inoculum in an amount equal

to 10% of the reactor volume. After 1 day of operation

without feeding, the reactor was fed 36% manure per day,

expressed as a percentage of the biomass volume in the

reactor. No increase in VFA levels was observed, and full

capacity was reached after 23 days.

The results obtained in our study indicate that a much

more aggressive start-up strategy can be used for codiges-

tion of OFMSW and sewage sludge at thermophilic condi-

tions, even when a thermophilic inoculum is not available.

We added a mixture of two mesophilic inocula (anaerobic

sludge and cattle manure) at a combined level of 17% of the

final digester volume and started the daily feeding schedule

immediately. This strategy resulted in satisfactory perfor-

mance for the thermophilic digester (Table V) after a short

period (approximately 20 days) with high VFA levels. Key

to the success of this aggressive start-up method was the

daily monitoring of important control parameters, whichallowed us to practice pH control as necessary and pre-

vented digester failure.

Our results also indicate that the mesophilic digester did

not respond favorably to an aggressive start-up method; a

more gradual start-up [e.g., similar to the one suggested by

Ahring (1994)] would have improved the digestion process

at mesophilic conditions. A more gradual approach during

the first 3 days of operation may have also further reduced

the start-up time for the thermophilic digester. The prob-

lems during start-up were likely the result of an imbalance

in the activities of hydrolyticfermentative bacteria, proton-

reducing acetogenic bacteria, and methanogens, which are

typical for anaerobic systems with high substrate inputs(Schink, 1988). Fermenters can acclimate more quickly to

new conditions because of their relatively high growth rates,

while proton-reducing acetogens and methanogens grow

much slower. Thus, shortly after start-up (day 1), when

hydrogen partial pressures were likely low (hydrogen was

not measured), hydrogen, carbon dioxide, and acetate (Fig.

1A,B) were the main products of the fermentative pathways

of hydrolyticfermentative bacteria. Because of the rela-

tively low levels of methanogen rRNA present in the start-

up mixture (Fig. 2), the metabolic capacity of the methano-

gens was initially not sufficient to balance the increasing

activity of the fermenters. As a result, acetate and hydrogen

were not consumed at the same rate at which they were

produced. Under these conditions, the electron flux through

reduced intermediates (propionate and butyrate) increased.

For example, over a time period of just 1 day, propionate

levels increased significantly in the mesophilic digester

(Fig. 1A), whereas butyrate concentrations rose in the ther-

mophilic reactor (Fig. 1B). Subsequently, butyrate and pro-

pionate levels also increased slightly in the mesophilic and

thermophilic digesters, respectively. The increase in VFA

levels caused a decrease in pH (Fig. 1C,D), which further

inhibited methanogenesis. The oxidations of propionate and

butyrate by proton-reducing acetogens are exergonic only at

low partial pressures of hydrogen. These bacteria are thus

obligately syntrophic and dependent on the activity of hy-

drogenotrophic microorganisms, such as methanogens. The

fermenters shifted their metabolism to the production of

more reduced compounds because the hydrogenotrophic

methanogens were not able to keep the hydrogen concen-

tration low; these reduced intermediates subsequently built

up, because the proton-reducing acetogens also required a

low hydrogen partial pressure. Gradually, the levels of

methanogens increased (Fig. 2C,D) and excess acetate (Fig.

1A,B) and hydrogen were consumed. As a result, butyrate

levels also began to decrease after about 10 and 20 days of

operation in the thermophilic and mesophilic digesters, re-

spectively. However, propionate levels persisted at a rela-

tively high level in both digesters (approximately 500 and

2000 mg/L in the thermophilic and mesophilic reactors,

respectively).

Thus, during the first day the most critical step in both

digesters was shown to be methanogenesis. After the initial

part of the start-up period, the removal of reduced interme-diates (especially propionate) appeared to be the critical

process as the levels of acetate were effectively reduced.

Approximately 20 days after start-up, propionate levels

remained high in both digesters, especially in the meso-

philic digester. Our observations are consistent with condi-

tions of digester overload, which have been shown to result

in the initial presence of high levels of VFA and sometimes

in the persistence of propionate even after other VFAs

have been consumed (McCarty and Mosey, 1991). Propio-

nate-degrading syntrophs (e.g., Syntrophobacter wolinii)

were likely not present in high numbers in our inoculum,

because these syntrophs only can use a very limited range of

substrates (Schink, 1992) and because anaerobic sludgefrom a stable digester was used, in which propionate con-

centrations were below the detection limit of 1 mg/L. There-

fore, an extensive amount of time would have been neces-

sary to reduce propionate concentrations because propio-

nate-degrading syntrophs were initially present at very low

levels and because they have very low specific growth rates.

Butyrate-degrading syntrophs (e.g., Syntrophomonas wol-

fei) compete better because they have a higher specific

growth rate and a much wider substrate spectrum (McIner-

ney, 1992; Schink, 1992). Thus, while butyrate was con-

sumed relatively rapidly in both digesters, the accumulated

propionate was removed very slowly by wash-out and/or

conversion to acetate by propionate-degrading syntrophs.

Based on our results, we speculate that use of an inocu-

lum from a well-balanced, stable anaerobic digester may not

promote the rapid start-up of anaerobic systems. For a

gradual start-up, as suggested by Ahring (1994), the pres-

ence of significant levels of methanogens is most critical to

prevent the formation of reduced intermediates. On the

other hand, an aggressive start-up will benefit more from the

presence of significant levels of proton-reducing acetogens



12/14

because the formation of reduced intermediates cannot be

completely avoided. An inoculum from a digester that has

been exposed to periods of overload contains higher levels

of propionate-degrading syntrophs and may be more suit-

able for rapid digester start-up. To further investigate this

hypothesis, our studies of methanogenic population dynam-

ics in digester systems need to be complemented with stud-

ies of population dynamics of propionate-degrading syn-

trophs and other syntrophic fatty acid oxidizing bacteria.

The levels of SRB, for which oligonucleotide probe hy-

bridizations were performed, were consistently low in both

digesters. In terms of digester performance, these results are

positive because they indicate that the feed sulfate levels

were low enough to discourage the proliferation of SRB.

Because SRB can compete with methanogens for hydrogen

and acetate, a high level of SRB might decrease the overall

methane yield. It is somewhat surprising that SRB levels

were so low, because previous studies indicate that SRB are

often present in sulfate-limited environments (Barlaz et al.,

1989; Raskin et al., 1996; Yoda et al., 1987) due to their

ability to grow syntrophically with methanogens in the ab-

sence of sulfate (Widdel, 1988). However, it is possible that

other SRB, not detected by the probes used in this study,were present at significant levels in the reactors (e.g., De-

sulfobulbus).Desulfobulbusspp. degrade propionate to pro-

duce acetate in the presence of sulfate. However, they can

also produce propionate by fermenting lactate in the ab-

sence of sulfate (Widdel, 1988). Thus, future studies on the

population dynamics of propionate-degrading syntrophs

should be complemented with probes for Desulfobulbus

spp.

In general, the thermophilic digester performed better,

had a shorter start-up period, and was more stable than the

mesophilic digester. The gas production rate and specific

gas production for the thermophilic digester were more than

1.5 times those of the mesophilic digester. These resultssupport earlier findings that thermophilic digestion is more

efficient compared to mesophilic conditions (Cecchi et al.,

1991; Pfeffer, 1974). Previous studies on similar systems

report comparable gas production rates and specific gas pro-

ductions (Pfeffer, 1974; Rivard et al., 1993; Stenstrom et al.,

1983). Profiles of VFA accumulation and consumption

similar to those observed in this study have also been re-

ported previously (Cecchi et al., 1991; Zinder et al., 1984b).

Based on a comparison of changes in VFA concentrations

in the mesophilic and thermophilic digesters (discussed

above), it appears that enhancement of the growth of pro-

pionate-degrading syntrophs at thermophilic conditions may

be a critical factor in these observations. Although propio-

nate also persisted in the thermophilic digester, it did not

continue to accumulate after start-up as it did in the meso-

philic digester. In general, the thermophilic digester main-

tained a much more balanced fermentation system, capable

of consistently withstanding a higher organic loading rate

without a significant accumulation of VFAs or a decrease

in pH.

The thermophilic digester contained higher levels of hy-

drogenotrophic methanogens than the mesophilic digester.

This allowed the fermenters to channel more electrons to

hydrogen and fewer to organic compounds. The relatively

high levels of hydrogenotrophic methanogens and low lev-

els ofMethanosaeta spp. in the thermophilic digester are

consistent with previous studies on acetate metabolism in

thermophilic systems with low acetate concentrations (Lee

and Zinder, 1988a; Petersen and Ahring, 1991; Zinder et al.,

1984a). These studies suggest that a syntrophic relationship

between an acetate-oxidizing organism and a hydrogenotro-

phic methanogen is the major route of acetate degradation

when acetate concentrations are low. Zinder and Koch

(1984) originally obtained such syntrophic partners from a

thermophilic solid waste digester. The hydrogenotrophic

methanogen was identified as Methanobacterium strain

THF and the acetate oxidizer was later isolated (Lee and

Zinder, 1988b). Ahring has several thermophilic acetate-

degrading enrichment cultures containing acetate-oxidizing

cocultures available in her laboratory, but so far she has not

been successful in isolating these organisms (Ahring, 1995).

Unlike the acetate oxidizer isolated by Lee and Zinder

(1988b), the organisms in Ahrings lab are not homoaceto-

gens. Petersen and Ahring (1991) suggest that the acetate-oxidizing organisms have a half-maximum constant (KM)

that is even lower than that ofMethanosaeta, perhaps re-

sulting in a competitive advantage at very low acetate lev-

els. Based on our results (high levels ofMethanobacteri-

aceae, insignificant levels ofMethanosaeta spp., and low

acetate concentrations), it is likely that a significant fraction

of acetate consumption in the thermophilic reactor also pro-

ceeded through syntrophic interactions between acetate oxi-

dizers and hydrogenotrophic methanogens. Because Metha-

nobacteriaceaewere the dominant hydrogenotrophic meth-

anogens in the thermophilic digester, it is likely that species

of this family, which includesMethanobacteriumspp., were

the syntrophic partners of the acetate oxidizers.As discussed above, the application of oligonucleotide

probes to study methanogenic population dynamics has pro-

vided valuable insights in the start-up of complex digester

systems. Nevertheless, much work remains to optimize

these methods for studies of complex environments, such as

MSW digesters or landfill bioreactors. The accurate char-

acterization of microbial communities using methods of oli-

gonucleotide probe hybridizations depends on the unbiased

recovery of nucleic acids from different populations. Dif-

ferential recoveries could result from sampling problems

(e.g., heterogeneity of environmental matrices) (Palmisano

and Barlaz, 1996) and/or problems related to nucleic acid

extraction procedures (Raskin et al., 1997). Currently, the

nucleic acid extraction step is the limiting factor with re-

spect to the incorporation of replication (to address variabil-

ity) in experimental design (Raskin et al., 1997). To address

this problem, we performed duplicate extractions for each

sampling day. However, given the potential introduction of

bias and variability during the extraction, the collection and

analysis of several more samples would have been preferred

but not feasible using state of the art nucleic acid hybrid-



13/14

ization techniques. We should point out that the limitations

related to sampling are also of concern for other microbio-

logical detection methods, which often introduce significant

other biases (Palmisano and Barlaz, 1996; Ward et al.,

1992).

In conclusion, this study showed that anaerobic codiges-

tion can be a feasible method for the treatment of MSW

with a high content of nutrient-deficient paper combined

with biosolids in proportions reflecting typical U.S. produc-

tion rates. An aggressive start-up strategy using a mixture of

two mesophilic inocula was successful for the rapid start-up

of a thermophilic digester. The steady-state performance

compared favorably to results from previous studies. The

aggressive start-up of an anaerobic codigestion system at

mesophilic conditions proved to be more difficult. A more

gradual start-up strategy would have been beneficial to

achieve a rapid steady state.

Our results suggest that the effects of other operational

parameters on performance should be explored. For ex-

ample, to accomplish a rapid start-up using a strategy simi-

lar to the one used in this study, inocula obtained from

unstable digesters may provide better results. Further

research using SSU rRNA based probes for propionate-degrading syntrophs and SRB and other syntrophic fatty

acid oxidizing bacteria in combination with the use of

probes for methanogenic populations and performance mea-

sures will allow us to further explore the start-up behavior

of codigestion systems.

We are thankful to: Jim Danalewich, Kristin Eder, Bradley

Grens, Jose BarriosPerez, David Schumacher, Peter Stroot, and

Tetsuo Wada for help with digester maintenance and chemical

analyses; Biswarup Mukhopadhyay and Dandan Zheng for ad-

vice on culturing methanogens; Bryan White and the Staff at the

Department of Animal Sciences (University of Illinois) for ac-

cess to laboratories; and the Community Recycling Center of

Champaign for help with collection of paper and food waste.This research was supported by the Office of Solid Waste Re-

search (Project OSWR-12-013), University of Illinois. K. D.

Sauer was supported by a NSF Graduate Fellowship.

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