microbial population dynamics during start-up and overload conditions of anaerobic digesters...

Microbial Population Dynamics DuringStart-Up and Overload Conditions ofAnaerobic Digesters Treating MunicipalSolid Waste and Sewage Sludge

Katherine D. McMahon,1* Dandan Zheng,1** Alfons J.M. Stams,3

Roderick I. Mackie,2 Lutgarde Raskin1

1Department of Civil and Environmental Engineering, University of Illinoisat Urbana-Champaign, 3221 Newmark Civil Engineering Laboratory, 205 NorthMathews Avenue, Urbana, Illinois 61801; telephone: 217-333-6964;fax: 217-333-6968; e-mail: [email protected] of Animal Sciences, 132 Animal Sciences Laboratory,University of Illinois at Urbana-Champaign, Urbana, Illinois3Department of Microbiology, Wageningen Agricultural University,Wageningen, The Netherlands

Received 9 September 2003; accepted 8 April 2004

Published online 18 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20192

Abstract: Microbial population dynamics were investigatedduring start-up and during periods of overload conditions inanaerobic co-digesters treating municipal solid waste andsewage sludge. Changes in community structure weremonitored using ribosomal RNA-based oligonucleotideprobe hybridization to measure the abundance of syntro-phic propionate-oxidizing bacteria (SPOB), saturated fattyacid-beta-oxidizing syntrophs (SFAS), and methanogens.These changes were linked to traditional performanceparameters such as biogas production and volatile fattyacid (VFA) concentrations. Digesters with high levels ofArchaea started up successfully. Methanosaeta conciliiwas the dominant aceticlastic methanogen in these sys-tems. In contrast, digesters that experienced a difficultstart-up period had lower levels of Archaea with propor-tionally more abundant Methanosarcina spp. Syntrophicpropionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in alldigesters, and SPOB appeared to play a role in stabilizingpropionate levels during start-up of one digester. Digesterswith a history of poor performance tolerated a severe or-ganic overload event better than digesters that had pre-viously performed well. It is hypothesized that higher levelsof SPOB and SFAS and their methanogenic partners in pre-viously unstable digesters are responsible for this behavior.B 2004 Wiley Periodicals, Inc.

Keywords: anaerobic digestion; microbial population dy-namics; sewage sludge; solid waste; methanogens; syn-trophic bacteria; ribosomal RNA

INTRODUCTION

Efforts to optimize anaerobic digestion processes for the

treatment of various organic waste streams have focused on

a range of variables, including temperature, organic loading

rate, retention time, solids level, and feed composition.

The evaluation of the effect of a change in one of these oper-

ating parameters so far has mostly been limited to the assess-

ment of operational performance measures, and the impact

on microbial community structure has rarely been investi-

gated. Thus, direct links between operating conditions, per-

formance, and microbial community structure usually are

not available for anaerobic digestion processes. Advances

in molecular microbial ecology have made a more com-

plete characterization of anaerobic digestion systems pos-

sible (Griffin et al., 1998; McMahon et al., 2001; Raskin

et al., 1995).

Stable anaerobic digestion systems employ consortia of

microorganisms to completely degrade organic material to

methane, carbon dioxide, and water. Process imbalances

can result in the accumulation of reduced intermediates

such as volatile fatty acids (VFA) and alcohols (Gujer and

Zehnder, 1983), which must be effectively metabolized to

maintain stable fermentation and process performance. It

was demonstrated in a number of recent studies that sta-

ble performance of anaerobic digestion systems depends

greatly on the establishment of a suitable microbial com-

munity during start-up. For example, Griffin et al. (1998)

correlated operating conditions and the abundance of

specific methanogenic populations during start-up of an-

aerobic digesters treating a mixture of the organic fraction

of municipal solid waste (OFMSW) and sewage sludge. It

was also observed that butyrate, propionate, and acetate ac-

cumulated to high levels in these and similar digesters when

they were subjected to aggressive start-ups (Griffin et al.,

B 2004 Wiley Periodicals, Inc.

Correspondence to: Lutgarde Raskin*Current address: Department of Civil and Environmental Engineering,

University of Wisconsin–Madison, 3204 Engineering Hall, 1415 Engi-

neering Drive, Madison, Wisconsin 53706-1691.**Current address: Grifols Biologicals Inc., 2450 Lillyvale Avenue, Los

Angeles, California 90032.

Contract grant sponsors: Office of Solid Waste Research; University of

Illinois; U.S. National Science Foundation Graduate Fellowship (Katherine

D. McMahon)

Contract grant number: OSWR-12-013

1998; Stroot et al., 2001). While acetate and butyrate were

gradually consumed, propionate often persisted throughout

system operation, leading to suboptimal performance. How-

ever, when mixing levels were reduced, propionate did not

accumulate or the accumulated propionate was consumed,

even under higher loading rates (Stroot et al., 2001), in-

dicating that it would be important to study propionate and

other VFA-consuming microorganisms to fully characterize

these observations.

In anaerobic environments lacking external electron ac-

ceptors, propionate and butyrate are oxidized by syntrophic

propionate-oxidizing bacteria (SPOB) and saturated fatty

acid-beta-oxidizing syntrophs (SFAS), respectively (Schink,

1992; Schink and Stams, 2002; Stams, 1994). Generally,

SPOB and SFAS couple substrate oxidation to interspecies

hydrogen (or formate) transfer (Boone and Bryant, 1980;

Boone et al., 1989; McInerney, 1992; Stams and Dong,

1995). Historically, SPOB were thought to be much more

limited in their ability to use different substrates compared to

SFAS, which have been shown to use a variety of organic

acids for growth (McCarty and Mosey, 1991). However,

some SPOB also can grow on propionate while reducing

sulfate (Van Kuijk and Stams, 1995; Wallrabenstein et al.

1994, 1995), fumarate, or L-malate (Stams et al., 1993), and

several SPOB can ferment fumarate or pyruvate (Stams et al.,

1993; Wallrabenstein et al., 1994). Still, the syntrophic

oxidation of propionate is considered to be the metabolic

pathway of choice for most SPOB in mixed community and

natural systems (Stams, 1994).

The phylogenetic characterization of several SPOB and

SFAS facilitated the development of small subunit (SSU)

rRNA targeted oligonucleotide hybridization probes for

these organisms and the application of these probes to study

anaerobic systems (Hansen et al., 1999; Harmsen et al.,

1995; Harmsen et al., 1996a, 1996b). We recently used

SSU rRNA targeted probes to link population dynamics of

SPOB, SFAS, and methanogens to the performance of co-

digesters treating the OFMSW and sewage sludge operated

under various mixing conditions (McMahon et al., 2001;

Stroot et al., 2001). In the current study, we evaluated the

role of SPOB, SFAS, and their methanogenic partners

during start-up and overload conditions of co-digestion

systems, linking microbial population dynamics to digest-

er performance, and studied the impact of organic loading

rate (OLR) on microbial community structure.

MATERIALS AND METHODS

Digester Operation and Chemical Analyses

Laboratory-scale digesters consisted of 2-L Pyrex bottles

with a 1-L working volume. Two consecutive, long-term

(>90 days) experiments were conducted, as described

below. The feed was a mixture of synthetic OFMSW,

primary sludge, and waste activated sludge (WAS), mixed

in a ratio reflecting actual U.S. production rates for the

respective waste streams (Griffin et al., 1998). Individual

aliquots of premixed feed prepared at the beginning of

each experiment were stored at �20jC. All digesters were

operated at 37jC in a semicontinuous mode with daily

feeding and wasting to achieve a retention time of 20 days,

unless otherwise noted. If necessary, the pH was controlled

by chemical addition or by reducing the daily feed rate. A

detailed description of digester operation and feed compo-

sition is presented elsewhere (Stroot et al., 2001).

For experiment 1, one digester was started without an

exogenous inoculum (Digester 1) and one digester was

inoculated with anaerobic sludge from a sewage sludge

digester (Digester 4). The names selected for the digest-

ers in this article (Digesters 1 and 4) correspond to the

names used for the same digesters described by Stroot

et al. (2001), who discussed different aspects of digester

performance evaluation. The digesters were operated at an

OLR of 3.7 kg volatile solids (VS) m�3 active volume

day�1. They were continuously mixed on a shaker table

for 2 weeks, and were then switched to minimally mixed

conditions (thoroughly shaken by hand for 2 min each

day) (Stroot et al., 2001).

For experiment 2, six digesters were operated to com-

pare digester performance under continuously mixed

(Digesters 5, 6, and 7) and minimally mixed (Digesters 8,

9, and 10) conditions at three different OLRs (3.7, 7.6,

and 9.4 kg VS m�3 active volume day�1). Three differ-

ent feed mixtures were created to obtain the three OLRs,

representing three possible primary sludge and WAS

thickening procedures commonly found at wastewater

treatment plants (Metcalf and Eddy, 1991; Stroot et al.,

2001). Anaerobic digester sludge was used as the inoculum

for all six digesters.

Total VFA concentrations, alkalinity, pH, and biogas

production were measured daily. The parameter ‘‘alpha,’’

defined as the ratio of VFA concentration to bicarbonate

alkalinity, was used as a measure of digester stability

(Poggi-Varaldo and Oleszkiewicz, 1992). An increase in

alpha value above 1.0 can predict an imbalance before pH

or VFA concentrations indicate instability. Biogas com-

position, individual VFA concentrations, and solids lev-

els were measured 2–3 times per week. Analytical

methods were described previously (Griffin et al., 1998;

Stroot et al., 2001).

Quantification of Microbial Populations

The microbial community structure was analyzed during

start-up of Digesters 1, 4, 7, 8, 9, and 10, during steady-state

operation of Digesters 8, 9, and 10, and during overload

conditions for Digesters 1 and 4. Nucleic acids were ex-

tracted from biomass samples using a low-pH hot-phenol

protocol (Griffin et al., 1998; Stahl et al., 1988) and

quantitative membrane hybridizations were conducted as

described previously (McMahon et al., 2001) using the oli-

gonucleotide probes presented in Table I. Pure culture

rRNA was available as rRNA standards for all methanogens

824 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 7, SEPTEMBER 30, 2004

(Table I), Syntrophobacter fumaroxidans MPOB, which

was grown as a pure culture on fumarate (Harmsen, 1996;

Stams et al., 1993), and Syntrophomonas wolfei LYB, which

was grown as a pure culture on crotonate (Boone et al.,

1989; Zhao et al., 1990) and was obtained from the Oregon

Collection of Methanogens (OCM) (http://methanogens.

pdx.edu). At the time of this study, pure cultures were not

available for the SPOB Syntrophobacter wolinii DB,

Syntrophobacter pfennigii KoProp1, and Smithella propion-

ica LYP. Therefore, co-cultures of these organisms with

methanogens were used to synthesize rRNA in vitro for use

as rRNA standards (McMahon et al., 1998; McMahon et al.,

2001). S. wolinii DB and S. pfennigii KoProp1, each in co-

culture with a Methanospirillum species, were grown on

fumarate and on propionate with sulfate, respectively

(Stams et al., 1993; Wallrabenstein et al., 1995). Smithella

propionica LYP was obtained from the OCM and grown in

co-culture with Methanospirillum hungateii with 20 mM

propionate and 20 mM sulfate at 37jC (Boone et al., 1989).

It is necessary to note that the relative concentrations of

SSU rRNA of the organisms determined using in vitro

transcribed rRNA as a standard (Table I) may be under-

estimated, due to a bias created by the use of transcripts as

quantitative standards (McMahon et al., 1998). Therefore,

the data generated with transcripts should only be used to

monitor shifts in population abundance and to compare

levels of these organisms in different systems.

Design of Oligonucleotide Probefor Smithella propionica

A probe, named S-S-S.pro-0450-a-A-23 (Table I), was de-

signed to target S. propionica, a recently identified, meso-

philic, propionate-degrading bacterium phylogenetically

affiliated with the y subclass of the Proteobacteria (Liu

et al., 1999). The probe sequence was 5V-GAAATGCA-

TAGTGGCTAATATCC-3V. The specificity of this probe

was verified using PROBE_MATCH software provided

by the Ribosomal Database Project II (Maidak et al.,

2001) and the BLASTN software available through the

National Center for Biotechnology Information (NCBI)

(Altschul et al., 1990). The dissociation temperature (Td)

was determined using an elution method (de los Reyes

et al., 1997). To eliminate any discrepancies introduced by

the differences in the membrane washing protocols used

during Td studies (elution method) and routine quantitative

membrane hybridizations, the optimal wash temperatures

(Tw) for the probe designed to target S. propionica and for

a number of previously characterized probes (Table I)

were determined using a replicate slot method (with

washes at 52, 54, and 56jC) (Raskin et al., 1994; Zheng

et al., 1996).

Probe specificity was verified experimentally by mem-

brane hybridization (de los Reyes et al., 1997; Raskin et al.,

1994) using nucleic acids from 37 organisms representing

a wide phylogenetic diversity (Rattus norvegicus, Saccha-

romyces cerevisiae, Zea mays, Methanogenium organo-

philum, Methanosarcina acetivorans, Methanobacterium

formicicum, Methanococcus voltae, Methanosaeta concilii,

Cellulophaga lytica, Prevotella ruminicola, Nitrobacter wi-

nogradskyi (ATCC 25391), Maricaulis sp. MCS10, Chro-

mobacterium violaceum (ATCC 12472), Nitrosomonas

europaea, Iodobacter fluviatilis, Pseudomonas putida GS,

Aeromonas hydrophila, Escherichia coli K-12, Thermode-

sulfobacterium thermophilum, Desulfovibrio africanus

(ATCC 27774), Desulfuromonas acetoxidans, Desulfobul-

bus propionicus, Listeria monocytogenes, Staphylococcus

aureus, Bacillus subtilis, Clostridium innocuum, Brevibac-

terium linens (ATCC 9172), Corynebacterium renale

(ATCC 19412), Pseudonocardia autotrophica (ATCC

19727), Mycobacterium vaccae (ATCC 15483), Tsuka-

murella paurometabola (ATCC 8368), Gordonia amarae

SE102 (AT CC 27809), Syntrophobacter pfennigii Ko-

Prop1, Syntrophobacter wolinii DB, Syntrophobacter fu-

maroxidans MPOB, Syntrophomonas wolfei LYB (OCM

65), and Smithella propionica LYP). Stock solutions of

rRNA were denatured and diluted, 30 ng SSU rRNA was

blotted per slot, and hybridizations and washes (using the

Tw given in Table I) were performed as described previously

(Raskin et al., 1994).

Table I. Oligonucleotide probes used in hybridizations.

Probe Tw Target Organism(s) RNA standard Original reference

S-*-Univ-1390-a-A-18 44 Most Organisms Zheng et al., 1996

S-D-Arch-0915-a-A-20 58 Most Archaea Methanosarcina acetivorans Stahl and Amann, 1991

S-D-Bact-0338-a-A-18 55 Most Bacteria S. fumaroxidans MPOB Amann et al., 1990

S-G-Msar-0821-a-A-21 60 Methanosarcina spp. Methanosarcina acetivorans Raskin et al., 1994

S-S-Mst.co-0381-a-A-22 54 Methanosaeta concilii Methanosaeta concilii GP6 Zheng and Raskin, 2000

S-F-Mbac-0310-a-A-22 57 Methanobacteriaceae Methanobacterium wolfei Raskin et al., 1994

S-S-S.fum-0464-a-A-19 52a Syntrophobacter fumaroxidans MPOB S. fumaroxidans MPOB Harmsen et al., 1995

S-S-S.pfn-0460-a-A-21 53a Syntrophobacter pfennigii KoProp1 S. pfennigii transcribed rRNA Harmsen et al., 1995

S-S-S.wol-0223-a-A-19 57a Syntrophobacter wolinii DB S. wolinii transcribed rRNA Harmsen et al., 1995

S-G-Dsbb-0660-a-A-20 57 Desulfobulbus propionicus D. propionicus transcribed rRNA Devereux et al., 1992

S-S-S.pro-0450-a-A-23 54a Smithella propionica LYP S. propionica transcribed rRNA This study

S-F-Synm-0700-a-A-23 54 Syntrophomonadaceae Syntrophomonas wolfei LYB Hansen et al., 1999

aWash temperatures were determined experimentally in this study.

MCMAHON ET AL.: MICROBIAL POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 825

RESULTS

Probe Characterization

Probe S-S-S.pro-0450-a-A-23 was designed to specifically

target S. propionica, based on SSU rRNA sequences

available through public databases. The Td and Tw for

probe S-S-S.pro-0450-a-A-23 were determined to be 56jC

and 54jC, respectively. The organisms used in the

specificity study included five Archaea, three Eucarya,

and 21 members of various phylogenetic groups of Bac-

teria (Bacteroidetes, Proteobacteria, Firmicutes, and Acti-

nobacteria). Seven of the remaining organisms were SPOB

or close relatives of S. propionica. The SSU rRNA of

Iodobacter fluviatilis, a distantly related (80% SSU rRNA

sequence identity) member of the h subclass of the

Proteobacteria, has one mismatch and one deletion in the

probe target region. Therefore, I. fluviatilis was included

to further check for nontarget hybridization. The target,

S. propionica, was also included. A strong hybridization

signal was observed for S. propionica rRNA, while none of

the other rRNA samples resulted in significant hybridization

signals, except for a slight signal observed for I. fluviatilis

rRNA. These results indicate that probe S-S-S.pro-0450-a-

A-23 exhibited the desired specificity.

It should be noted that an additional probe (named 177)

was previously designed to target a very closely related

strain, that appears to also be a SPOB, identified only as

‘‘SYN7’’(Harmsen, 1996; Harmsen et al., 1996a; Liu et al.,

1999). This organism was observed to be present in

anaerobic granular sludge and is not available in pure or

enrichment culture. At the time probe S-S-S.pro-0450-a-A-

23 was designed, the SSU rRNA sequence of SYN7 was not

available in GenBank and information on probe 177 was not

available to us. Recent analyses indicate that probe S-S-

S.pro-0450-a-A-23 is a perfect match to the SSU rRNAs of

SYN7 as well as an uncultivated bacterium present in anae-

robic granular sludge (clone R4b16, Genbank accession

number AF482441; Hofman-Bang and Ahring, unpublished

data). In addition, several ‘‘Syntrophus-like’’ SSU rRNA se-

quences retrieved from contaminated aquifers recently have

become available (Genbank accession numbers AJ009471,

AJ1333796, AF014287, AF050534). These sequences con-

tain a single mismatch near the middle of probe S-S-S.

pro-0450-a-A-23 and it is therefore possible that probe S-S-

S.pro-0450-a-A-23 will hybridize to these sequences under

low stringency conditions. The use of the experimentally

determined Tw should preclude a substantial hybridization

signal for these sequences.

Microbial Community Structure in Inocula

The results of quantitative oligonucleotide probe hybrid-

izations performed on the anaerobic digester sludges

used to inoculate the digesters in experiments 1 and 2

are shown in Table II. These data originally were re-

ported in a previous study (McMahon et al., 2001), but

are presented here to facilitate the discussion of micro-

bial population dynamics during start-up (below). A de-

tailed discussion of these results is provided by McMahon

et al. (2001).

Microbial Population Dynamics DuringStart-up—Continuously Mixed Conditions

Samples were taken from Digesters 1 and 4 during the

period that they were continuously mixed (first 14 days

of operation) and from Digester 7 (first 6 days of operation)

to link microbial population dynamics to performance

during start-up of continuously mixed systems. VFA

concentrations and biogas production data are shown in

Figure 1A–C. Biogas production data are presented as spe-

cific gas production (SGP) in units of m3 biogas produced

per kg VS fed during the previous day, allowing

comparisons of gas production between digesters with

different loading rates. Additional start-up performance data

are presented elsewhere (Stroot et al., 2001).

Table II. Microbial community structure in anaerobic digester sludge inocula used for start-up (% SSU rRNA F SD). Data were originally reported in a

previous study (McMahon et al., 2001).

Probe Target organisms Experiment 1 Digester 4 Experiment 2 Digesters 7,8,9,10

S-D-Bact-0338-a-A-18 Most Bacteria 73.9 F 9.0 63.7 F 8.7

S-D-Arch-0915-a-A-20 Most Archaea 4.52 F 0.46 8.33 F 0.95

S-G-Msar-0821-a-A-21 Methanosarcina spp. <0.12a 0.24 F 0.13

S-S-Mst.co-0381-a-A-22 Methanosaeta concilii 1.54 F 0.10 3.32 F 0.38

S-F-Mbac-0310-a-A-22 Methanobacteriaceae <0.12a <0.15a

S-S-S.fum-0464-a-A-19 Syntrophobacter fumaroxidans MPOB 0.27 F 0.14 0.37 F 0.12

S-S-S.pfn-0460-a-A-19 Syntrophobacter pfennigii 0.33 F 0.08 0.48 F 0.08

S-S-S.wol-0223-a-A-19 Syntrophobacter wolinii 0.44 F 0.12 0.43 F 0.11

S-G-Dsbb-0660-a-A-20 Desulfobulbus spp. 0.53 F 0.08 0.43 F 0.06

S-S-S.pro-0450-a-A-23 Smithella propionica < 0.20a 0.28 F 0.29

S-F-Synm-0700-a-A-23 Syntrophomonadaceae 0.42 F 0.16 1.76 F 0.25

aThe relative hybridization signals from some samples were below the detection limit. Detection limits vary with probe labeling efficiency, nucleic acid

loading, and length of hybridization signal exposure and are therefore different from each sample and probe (McMahon et al., 2001).


Digester 1 performed poorly during start-up: Acetate

built up to high levels, propionate accumulated steadily,

and the SGP was relatively low until day 13 (Fig. 1A).

In contrast, Digester 4 performed quite well: Acetate did

not accumulate, propionate was effectively degraded after

a slight build up during the first 7 days, and the SGP was

high and remained relatively stable throughout the 2 weeks

analyzed (Fig. 1B). Digester 7 exhibited very poor start-

up performance, as indicated by an immediate accumu-

lation of acetate to very high levels, a rapid increase in

propionate concentration, and a very low SGP (Fig. 1C).

This poor performance appeared to be linked to the detri-

mental effects of continuous mixing at high OLR (Stroot

et al., 2001).

A selection of oligonucleotide probes (Table I) was used

to characterize shifts in microbial community structure

during the start-up of the three continuously mixed

digesters (Fig. 1D–O). Methanogen (archaeal) rRNA was

present at substantially higher relative levels on day 0 in

Digester 4 (Fig. 1E) than in Digester 1 (Fig. 1D), because

Figure 1. Volatile fatty acids (VFAs), specific gas production (SGP) in m3 biogas produced per kg VS fed during the previous day, and microbial

population dynamics in Digesters 1 (A, D, G, J, M), 4 (B, E, H, K, N), and 7 (C, F, I, L, O) during start-up. The organic loading rate (OLR) is given in kg

VS per m3 active volume per day. Some standard deviations are not reported in panels J– O to improve the clarity of presentation. Generally, coefficients of

variation (COVs) among replicates were between 20% and 30%, except for the probe targeting Syntrophobacter fumaroxidans in Digester 7 (panel L),

which had very high COVs. The detection limit for these samples was approximately 0.2%, indicating that the hybridization response for these samples was

below the detection limit.


archaeal rRNA was present in the sludge used to inoculate

Digester 4 (Table II), while Digester 1 was started without

an exogenous inoculum. Levels of archaeal rRNA were

also high in Digester 7 on day 0 (Fig. 1F), but their relative

abundance decreased immediately, implying that metha-

nogens were severely inhibited by the high VFA concen-

trations produced during start-up in this digester (Fig. 1C).

This is consistent with the very low SGP observed in

Digester 7 (Fig. 1C).

Methanosaeta concilii rRNA was the most abundant type

of methanogen rRNA in Digester 4 (Fig. 1E), and its levels

increased steadily through the first 2 weeks of operation.

Methanosaeta concilii is a mesophilic, aceticlastic metha-

nogen of the family Methanosaetaceae and is known to be

competitive in environments with low acetate concentra-

tions (Griffin et al., 1998; Zinder, 1993). Consistent with

this, the relative levels of rRNA from Methanosarcina spp.,

which can also use acetate as their growth substrate, were

very low during the start-up of Digester 4 (Fig. 1H), im-

plying these organisms were outcompeted by M. concilii.

In contrast, the relative abundance of M. concilii rRNA

did not increase in Digester 1 (Fig. 1G), but levels of Meth-

anosarcina spp. rRNA increased substantially (Fig. 1G)

soon after an increase in the acetate concentration was

observed (Fig. 1A). This relatively rapid growth of

Methanosarcina spp. is consistent with its high maximum

specific substrate utilization and growth rates compared to

those of M. concilii, which provide a competitive advantage

for Methanosarcina spp. at high acetate concentrations

(Zinder, 1993). In Digester 7, levels of both M. concilii

rRNA (Fig. 1F) and Methanosarcina spp. rRNA (Fig. 1I)

decreased substantially, suggesting that these organisms

were inhibited by the very high acetate concentrations.

During this period, Methanobacteriaceae rRNA accumu-

lated in Digesters 1 (Fig. 1D) and 7 (Fig. 2), suggesting that

representatives of this group grew rapidly due to the in-

creased supply of H2/formate, which was probably formed

as a result of the unstable fermentation conditions in these

digesters. The very high VFA concentrations in Digester 7

did not appear to affect Methanobacteriaceae, in contrast

to the inhibition experienced by Methanosarcina spp. and

M. concilii. The relative abundance of Methanobacteria-

ceae rRNA also increased in Digester 4 (Fig. 1H), but not as

much as in Digester 1.

Syntrophobacter fumaroxidans and S. wolinii rRNAs were

present at higher levels in Digesters 1 and 4, as compared to

Digester 7, while S. pfennigii rRNA was present at compa-

rable and constant levels in all three continuously mixed

digesters (Fig. 1J–L). Syntrophobacter fumaroxidans rRNA

relative abundance increased dramatically in Digester 4

(Fig. 1K) as propionate was turned over between days 6 and

10 (Fig. 1B), suggesting that these SPOB were (partly)

responsible for propionate degradation. In contrast,

S. propionica rRNA was not present at detectable levels in

any of the digesters during start-up. Syntrophomonadaceae

rRNA was present at comparable levels in Digesters 1 and 4,

though their levels were not as high as in Digester 7 on

day 0, which had received a Syntrophomonadaceae-rich

inculum (Table II). Notably, their rRNA abundance in

Digester 7 dropped precipitously during start-up, suggesting

that the continuously mixed conditions and high VFA

concentrations were inhibiting the growth and/or activity of

these SFAS (see below). Desulfobulbus spp. rRNA was

present at low levels (Fig. 1J–L), and its abundance did not

appear to change much during start-up. It is unlikely that

these organisms were responsible for appreciable propio-

nate degradation, since sulfate was shown to be limiting

in similar systems (Griffin et al., 1998).

Microbial Population Dynamics DuringStart-Up—Minimally Mixed Conditions

Changes in VFA concentrations, SGP, and microbial

community structure during start-up of Digesters 8, 9, and

10 are shown in Figure 2. Digester 8, which was operated

with the same OLR as the continuously mixed Digesters 1

and 4, did not experience any appreciable VFA accumu-

lation during start-up (Fig. 2A). The other two digesters,

which were operated at higher OLRs, also performed

well during the first 14 days of start-up. Acetate and pro-

pionate increased transiently in these digesters, but the

VFAs were effectively metabolized before their levels

reached approximately 500 mg/L as acetic acid. (Fig. 2B

and C; Stroot et al., 2001). All three digesters exhibited high

and stable SGP.

Significant differences in microbial community struc-

ture and population dynamics were not observed among

the three minimally mixed digesters during start-up

(Fig. 2D–L). The levels of methanogen (archaeal) rRNA

started out relatively high in all three digesters due to their

apparent high activity in the inoculum used (Table II;

Fig. 2D–F). Their levels gradually increased during start-

up similar to observations made for Digester 4, which

had been started up successfully under continuously mixed

conditions (Fig. 1E; Stroot et al., 2001). Methanosaeta

concilii was very active in all three minimally mixed

digesters, while Methanosarcina spp. rRNA levels were

below the detection limit (around 0.12 % SSU rRNA),

consistent with the low acetate concentrations. Methano-

bacteriaceae rRNA was also below the detection limit

(around 0.10% SSU rRNA).

SPOB and SFAS rRNAs were also found at compa-

rable levels in all three digesters. The relative abundance

of S. fumaroxidans rRNA was lower in these systems than

in Digesters 1 and 4, and was similar to the levels observed

in the more highly loaded Digester 7. No major changes

were observed for S. fumaroxidans, S. pfennigii, and

S. wolinii populations, though their rRNA levels decreased

slightly in Digesters 9 and 10 between days 0 and 12

(Fig. 2H and I).

Syntrophomonadaceae rRNA abundance initially was

quite high in all three digesters (Fig. 2J–L) because their

levels were high in the inoculum (Table II). Their levels

gradually decreased, possibly because saturated fatty acids


were not produced in appreciable amounts in these stable

systems. However, Syntrophomonadaceae rRNA abun-

dance did not drop as much as in Digester 7, which had

received the same inoculum and was operated at the same

OLR as Digester 10, but was continuously mixed.

Desulfobulbus spp. and S. propionica rRNAs were present

at low levels (Fig. 2J–L), and their abundance did not

change substantially.

Steady-State Microbial Community Structure

After approximately 40 days of operation, the minimally

mixed systems (Digesters 8, 9, and 10) achieved steady state

with respect to performance (Stroot et al., 2001). The

microbial community structures in these digesters were

analyzed to evaluate the effect of different OLRs. The

results of oligonucleotide probe (Table I) hybridizations

with samples collected on days 51, 55, and 56 are presented

as averages in Table III. Archaeal rRNA was present at

high levels (ranging from 12.7–17.3%) in all three

digesters. Methanosarcina spp. were essentially absent

from Digesters 8 and 9 because acetate levels were low

(Stroot et al., 2001). These aceticlastic methanogens were

slightly more prevalent in Digester 10, which is consistent

with the higher acetate levels in this digester (Stroot et al.,

2001). Methanosaeta concilii rRNA abundance was high in

all three digesters, indicating that under stable conditions

with relatively low acetate levels, M. concilii were out-

competing Methanosarcina spp. for acetate. Members of the

family Methanobacteriaceae were present in all three

digesters, but their rRNA levels were lower than observed

for other less stable continuously mixed digesters (1.1% in

Digester 7-CM described by McMahon et al., 2001 and

0.8% in a digester operated by Griffin et al., 1998).

Figure 2. Volatile fatty acids (VFAs), specific gas production rate (SGP) in m3 of biogas produced per kg of volatile solids fed during the previous day,

and microbial population dynamics in Digesters 8 (A, D, G, J), 9 (B, E, H, K), and 10 (C, F, I, L) during start-up. The organic loading rate (OLR) is given in

kg volatile solids per m3 active volume per day. Some standard deviations are not reported in panels G– L to improve the clarity of presentation. Generally,

coefficients of variation (COVs) among replicates were between 20% and 30%, except for the probe targeting Smithella propionica (panels J– L), which

had very high COVs. The detection limit for these samples was approximately 0.2%, indicating that the hybridization response for these samples was below

the detection limit.


SPOB and Desulfobulbus spp. were present during stable

operation of Digesters 8, 9, and 10 with rRNA levels that

were comparable to those observed during their start-up

period (compare Table III to Fig. 2G–L). This could be

attributed to the relatively low levels of propionate

produced in these minimally mixed digesters during stable

operation. Syntrophomonadaceae rRNA was present at

slightly lower levels than observed in these digesters during

their start-up period (compare Table III to Fig. 2J–L),

though at levels still higher than observed in Digester 7

(compare Table III to Fig. 1[O]).

Microbial Community Structure DuringOverload Conditions

On Day 75, the OLR of Digesters 1 and 4 was raised to

5.6 kg VS (m3 active volume)�1day�1 by decreasing the

retention time from approximately 20 days to 13 days

(Stroot et al., 2001). The digesters appeared to tolerate this

increase well and maintained a high SGP and low VFA con-

centrations (Stroot et al., 2001). On Day 87, the digesters

were exposed to extreme overload conditions by a further

reduction in retention time to 4 days and increase in OLR

to 18.8 kg VS (m3 active volume)�1day�1. The reactors

were fed until Day 91 and monitored for two additional

days (i.e., the digesters were not fed on Days 92 and 93).

Performance and microbial community structure data

for this period are shown in Figure 3. The parameter

‘‘alpha’’ can be used to predict stability, since it is defined as

the ratio of VFA concentration to bicarbonate alkalinity

(Poggi-Varaldo and Oleszkiewicz, 1992). An alpha value

of 1.0 corresponds to the threshold of stability above which

the system is considered unstable because VFAs are

accumulating. Prior to overload, the alpha value in both

digesters was well below 1.0 (Fig. 3A and B). Immediately

following the OLR increase, alpha values increased in

both digesters. VFA concentrations also rose during this

period. During these overload conditions, Digester 4 per-

formed worse than Digester 1, as reflected in a much higher

acetate concentration and alpha value on day 92 for Digester

4 (Fig. 3A and B).

Microbial population dynamics are presented in Fig-

ure 3C–H. Archaea were very active in both digesters prior

to overload, especially in Digester 4 (Figure 3C–D). Their

relative rRNA levels decreased sharply in Digester 1 and

slightly in Digester 4, following the OLR increase, implying

that the rapid increase in VFA concentrations inhibited

methanogen growth and/or activity. Methanosarcina spp.

were well represented in Digester 1, though their rRNA

levels fell during overload conditions in parallel with the

levels of archaeal rRNA (Fig. 3C). In contrast, these

organisms’ rRNA was below the detection limit (around

0.15% SSU rRNA) in Digester 4 throughout this period.

Methanospirillum concilii rRNA was also present in

Digester 1 at low and steady levels (around 0.5% SSU

rRNA, data not shown), though they were markedly more

active in Digester 4 (Fig. 3D). Although Methanobacte-

riaceae rRNA abundance was fairly high earlier during

Digester 1 operation (4.30 F 0.51% SSU rRNA on day 39),

it had dropped to below 0.26% SSU rRNA by day 86. These

hydrogenotrophic methanogens were undetectable in Di-

gester 4 during this same period (below around 0.16%

SSU rRNA).

Significant differences in SPOB and SFAS rRNA levels

between Digesters 1 and 4 were observed. SPOB pop-

ulations were fairly steady in Digester 1 (Fig. 3E), though

levels of S. wolinii rRNA were markedly lower than those

seen on day 39 in this digester (1.55 F 0.22% SSU rRNA;

McMahon et al., 2001). In Digester 4, the relative

abundance of this SPOB’s rRNA doubled between days

88 and 91 (Fig. 3F), implying this population responded to

the increased supply of propionate. Syntrophobacter

pfennigii rRNA was present at higher levels in Digester 1

than in Digester 4 (Fig. 3E and F). Smithella propionica

was more active in Digester 1 (Fig. 3G) than observed in

any other co-digesters at any time analyzed (McMahon

et al., 2001) and was present at levels nearly twofold

higher than in Digester 4. However, this population’s

Table III. Steady-state microbial community structure in minimally mixed digesters (% SSU rRNA F SD). Averages calculated using hybridization re-

sults from three samples (days 51, 55, 62) each blotted in triplicate. The SD represents a composite estimate of the overall error associated with the con-

struction of a standard curve for quantification and the averaging of the three samples. OLR is organic loading rate [kg VS (m3 working volume)�1 day�1].

Probe Target group Digester 8 OLR = 3.5 Digester 9 OLR = 7.6 Digester 10 OLR = 9.4

S-D-Bact-0338-a-A-18 Most Bacteria 60.4 F 3.2 59.4 F 3.0 56.6 F 3.4

S-D-Arch-0915-a-A-20 Most Archaea 12.7 F 0.7 15.2 F 0.7 17.3 F 0.5

S-G-Msar-0821-a-A-21 Methanosarcina spp. 0.06 F 0.11 0.06 F 0.03 0.26 F 0.07

S-S-Mst.co-0381-a-A-22 Methanosaeta concilii 6.6 F 0.5 6.9 F 0.5 5.7 F 0.6

S-F-Mbac-0310-a-A-22 Methanobacteriaceae 0.21 F 0.03 0.09 F 0.01 0.12 F 0.02

S-S-S.fum-0464-a-A-19 Syntrophobacter fumaroxidans 0.24 F 0.03 0.19 F 0.03 0.25 F 0.05

S-S-S.pfn-0460-a-A-19 Syntrophobacter pfennigii 0.65 F 0.04 0.57 F 0.05 0.67 F 0.07

S-S-S.wol-0223-a-A-19 Syntrophobacter wolinii 0.40 F 0.05 0.39 F 0.06 0.47 F 0.06

S-G-Dsbb-0660-a-A-20 Desulfobulbus propionicus 0.49 F 0.04 0.41 F 0.05 0.46 F 0.06

S-S-S.pro-0450-a-A-23 Smithella propionica 0.20 F 0.04 0.17 F 0.04 0.23 F 0.04

S-F-Synm-0700-a-A-23 Syntrophomonadaceae 0.77 F 0.02 0.70 F 0.02 0.65 F 0.03


rRNA levels were also high in Digester 4, as com-

pared to all other digesters during start-up (above and

Fig. 3J–L), and to the minimally mixed digesters at

steady state (Table III). Desulfobulbus spp. rRNA was

present at comparable levels in Digesters 1 and 4 and

remained constant (Fig. 3G and H). The relative abun-

dance of Syntrophomonadaceae rRNA decreased slightly

in both digesters during this period, and rRNA from these

Figure 3. Volatile fatty acids (VFAs), alpha (see text for definition), and microbial population dynamics in Digesters 1 (A, C, E, G) and 4 (B, D, F, H)

during organic overload conditions. Methanosaeta concilii and Methanobacteriaceae rRNAs were detected at low and steady levels (around 0.5% and 0.2 %

SSU rRNA, respectively) in Digester 1, but these results are omitted from panel C in the interest of clarity. Methanosarcina spp. and Methanobacteriaceae

rRNAs were not detectable in Digester 4 during this period. Some standard deviations are not reported in panels E – H to improve the clarity of presentation.

Generally, coefficients of variation were between 20% and 30%.


SFAS was almost twice as abundant in Digester 1 than in

Digester 4 (Fig. 3G and H).

DISCUSSION

The motivation for this study came from our previous work

with anaerobic co-digestion systems. It was observed that

performance during start-up under continuously mixed

conditions varied with the inoculum source (Stroot et al.,

2001). Also, minimally mixed conditions promoted more

rapid start-ups, particularly for high OLRs. Digesters with a

history of VFA accumulation appeared to perform better

under severe organic overload conditions. These observa-

tions prompted us to further analyze samples collected and

preserved during the study by Stroot et al. (2001), to

investigate the microbial community structure and popula-

tion dynamics in those digesters. The results presented here

support hypotheses developed during analysis of traditional

physical and chemical performance data (Stroot et al.,

2001), and allow us to further link community structure to

digester behavior.

Our data suggest that adequate levels of methanogens are

essential for the rapid start-up under continuously mixed

conditions. The anaerobic digester sludge used to inoculate

Digesters 4 and 7 provided sufficient levels of methanogens

to facilitate a rapid start-up for Digester 4, but not under the

higher OLR administered to Digester 7. It is possible that

the observed difference could be attributed to the fact that

the two digesters received slightly different inocula (col-

lected from the same municipal anaerobic sludge digester,

but on different dates) (Stroot et al., 2001). In general,

however, these results imply that a rapid start-up under

continuously mixed conditions can be achieved through the

use of an appropriate inoculum only if the OLR is not too

high. For higher OLRs, minimally mixed conditions result

in a more rapid start-up, possibly because this mode of

operation promotes the development of microenvironments

allowing spatial juxtapositioning of syntrophic bacteria and

methanogens. Notably, Syntrophomonadaceae were not

maintained in Digester 7, even though these SFAS were

present at significant levels in the inoculum. In contrast,

levels of both Syntrophomonadaceae and SPOB did not

vary much during start-up under minimally mixed con-

ditions (Digesters 8–10). This appeared to be the case in-

dependent of the OLR, at least in the range we investigated

(3.7, 7.6, and 9.4 kg VS m�3 active volume day�1).

The lack of any apparent correlation between the OLR

and the relative abundance of individual microbial pop-

ulations present in Digesters 8–10 during start-up (Fig. 2)

and steady state (Table III) speaks to the overall stability of

the minimally mixed systems. Stroot et al. (2001) proposed

that minimally mixed conditions promote lower rates of

hydrolysis and fermentation, which enable the use of

higher OLRs while maintaining stable performance. Fur-

thermore, if the higher OLRs corresponded to a larger (but

still balanced) flux of carbon and reducing equivalents

through the microbial community, this would translate to a

higher absolute abundance of participating populations.

Indeed, differences in overall activity, reflected in

correlating biogas production rates, were reported previ-

ously (Stroot et al., 2001). However, since the SSU rRNA-

targeted hybridization data generated in this study assess

relative abundances, differences in absolute abundance at

the entire community level could not be determined.

The fact that Methanobacteriaceae SSU rRNA levels

were low during start-up (and steady-state conditions) in the

minimally mixed digesters suggests that these methanogens

were not the dominant H2-consuming organisms. It is pos-

sible that other hydrogenotrophs (such as Methanomicro-

biales) were consuming most of the H2 in these digesters,

which were presumably experiencing low H2/formate

concentrations as a result of their stable operation. Since

members of the order Methanomicrobiales were found to be

the dominant hydrogenotrophs in stable anaerobic sewage

sludge digesters in which H2/formate concentrations were

low (Griffin et al., 1998; Raskin et al., 1995), these methano-

gens also may have been the main consumers of H2/formate

in the stable systems operated in this study. This hypothe-

sis is supported by the hybridization data: Probe nesting

results indicate that the high levels of archaeal rRNA

cannot be accounted for by totaling the levels of Meth-

anosarcina, M. concilii, and Methanobacteriaceae rRNAs,

suggesting that levels of Methanomicrobiales rRNA (for

which hybridization data are not available in this study)

account for the difference. The approach of probe nesting

worked well for previous studies in which probes for all

major groups of methanogens were included (McMahon

et al., 2001; Zheng and Raskin, 2000) and provide

confidence that the discrepancy in the current study is attrib-

utable to Methanomicrobiales.

It was previously hypothesized that digesters experienc-

ing a period of elevated VFAs caused by unstable operation

(e.g., during start-up) are more resistant and resilient to

subsequent short-term organic overload conditions (Stroot

et al., 2001). VFA accumulation encourages the growth of

SPOB and SFAS populations, which when still present at

substantial levels during organic overload conditions, could

limit VFA buildup. The data reported here support this

hypothesis: Digester 1 experienced a difficult start-up pe-

riod with substantial VFA accumulation compared to

Digester 4, which was attributed to a difference in inoc-

ulum (none for Digester 1 vs. anaerobic digester sludge for

Digester 4). Thus, although inoculation with anaerobic

digester sludge appeared to enhance start-up in Digester 4,

the slow start-up of Digester 1 presumably caused by low

levels of methanogens may have eventually lead to the

observed higher levels of SPOB and SFAS. Indeed,

Syntrophobacter wolinii, S. pfennigii, S. propionica, and

Syntrophomonadaceae may have contributed to the better

performance of Digester 1 during overload conditions

by consuming some of the VFAs that were produced during

the first few days of overload. The higher levels of Metha-

nosarcina spp. rRNA in Digester 1 suggest that Methano-

sarcina spp. played a role in controlling the acetate


accumulation in this digester. In contrast, M. concilii were

not able to consume acetate sufficiently fast in Digester 4

to prevent acid accumulation and subsequent digester

failure. Thus, a history of high acetate concentrations

appears to select for a population of methanogens capable of

more rapid acetate turnover. This effect was observed

previously in similar systems (Griffin et al., 1998). There-

fore, we propose that a digester’s history is an important

predictor of performance during organic overload condi-

tions, and that this can be linked to the maintenance, at

sufficient levels, of certain groups of methanogens, SPOB,

and SFAS. Although initial microbial community compo-

sition is determined by which organisms are present in

the inoculum, subsequent exposure to periodic upsets will

select for populations more capable of responding to, and

recovering from, sudden overload conditions.

CONCLUSIONS

We demonstrated links between digester operating con-

ditions, physical and chemical performance parameters,

and microbial population dynamics. Digesters that started

up successfully contained high levels of archaeal rRNA,

with significantly more rRNA from M. concilii than from

Methanosarcina spp. Methanobacteriaceae rRNA levels

were low in these digesters, suggesting that the low levels of

H2/formate produced in these systems were used by other

hydrogenotrophic methanogens. SPOB rRNA was present

at comparable levels in digesters that failed to successfully

start up and that operated well. However, S. wolinii

appeared to play a major role in propionate consumption

in the only continuously mixed digester that started up

successfully. Syntrophomonadaceae rRNA was present at

modest levels in digesters exhibiting good performance

during start-up.

In contrast, poorly performing digesters with high VFA

concentrations and low SGP contained low levels of

archaeal rRNA, but relatively high levels of Methano-

sarcina spp. and Methanobacteriaceae rRNA. Furthermore,

digesters with a history of poor performance tolerated

subsequent organic overload conditions better than digest-

ers that had previously performed very well. Specifically,

while Digester 1 exhibited a poor start-up, it was less sus-

ceptible to short-term overload conditions than Digester 4,

which had performed very well throughout start-up and the

rest of the operational period. The SPOB, SFAS, and

methanogen populations that had developed in Digester 4

presumably were not capable of handling the overload. The

results presented here confirm those presented elsewhere

(McMahon et al., 2001) and support hypotheses presented

by other researchers who have speculated that digesters with

a history of good performance may be more susceptible to

upset because of low levels of key populations (McCarty

and Mosey, 1991).

The observations reported here provide further evidence

for the importance of mixing levels during start-up and

operation of anaerobic co-digesters, particularly for sys-

tems operated at high OLRs. As discussed previously

(Stroot et al., 2001), digesters operated with high OLRs

started up successfully only under minimally mixed

conditions. In these minimally mixed systems, no correla-

tion was observed between OLR and the abundance of the

individual microbial populations analyzed, even though the

biogas production rate (Stroot et al., 2001) and total archaeal

rRNA abundance increased proportionally with OLR. This

provides further evidence for the hypothesis that minimally

mixed conditions promote balanced degradation for a

broader range of OLRs.

In summary, these results indicate that if high OLRs

must be used during digester start-up, minimally mixed

conditions will lead to faster start-up and better long-term

performance. However, we also observed that a history of

poor performance promotes the establishment of microbial

communities better equipped to deal with extreme organic

overload conditions. Taken together, these findings sug-

gest that the relationship between mixing levels and overall

performance should be investigated further in existing

digesters and for the design of new digesters.

We are thankful to David Boone for providing synthrops; to Kaare

Hansen for cloning SSU rDNA from S. wolfei LYB; to Peter Stroot,

Jim Danalewich, Jose Barrios-Perez, and David Schumacher for

help with digester maintenance and analyses; to Bryan White for

access to laboratories; and to Donna Hilton for VFA analyses.

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