Methanogenic Population Dynamics

during Start-Up of Anaerobic Digesters

Treating Municipal Solid Waste
andBiosolids
Matt E. Griffin,
1
KatherineD. McMahon,
1
RoderickI. Mackie,
2
LutgardeRaskin
1
1
Environmental Engineering and Science, Department of Civil Engineering,
3221 Newmark Civil Engineering Laboratory, University of Illinois at
UrbanaChampaign, Urbana, Illinois 61801; telephone: 217-333-6964; fax:
217-333-6968 or -9464; e-mail: lraskin@uiuc.edu.
2
Department 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 used
to initiate codigestion in two anaerobic, continuously
mixed bench-top reactors at mesophilic (37C) and ther-
mophilic (55C) conditions. The digesters were inocu-
lated with mesophilic anaerobic sewage sludge and
cattle 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 loading
rate was 3.1 kg volatile solids/m
3
/day and the retention
time was 20 days. Ribosomal RNA-targeted oligonucleo-
tide probes were used to determine the methanogenic
community structure in the inocula and the digesters.
Chemical analyses were performed to evaluate digester
performance. The aggressive start-up strategy was suc-
cessful for the thermophilic reactor, despite the use of a
mesophilic inoculum. After a short start-up period (20
days), stable performance was observed with high gas
production rates (1.52 m
3
/m
3
/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. After
several weeks of operation, the mesophilic digester be-
came more stable, but propionate levels remained very
high. Methanogenic population dynamics correlated well
with performance measures. Large fluctuations were ob-
served in methanogenic population levels during the
start-up period as volatile fatty acids accumulated and
were subsequently consumed. Methanosaeta species
were the most abundant methanogens in the inoculum,
but their levels decreased rapidly as acetate built up. The
increasein acetatelevels was paralleled by an increasein
Methanosarcina species abundance (up to 11.6and 4.8%
of total ribosomal RNA consisted of Methanosarcinaspe-
cies ribosomal RNA in mesophilic and thermophilic di-
gesters, respectively). Methanobacteriaceae were the
most abundant hydrogenotrophic methanogens in both
digesters, but their levels werehigher in thethermophilic
digester. 1998J ohn Wiley &Sons, Inc. Biotechnol Bioeng 57:
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 anaerobic
digesters 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
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 and
OFMSW 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 of
organisms 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
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)/m
3
/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 and Operation of Anaerobic Digesters
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 N
2
-sparged, distilled deionized water was
added to reach the working volume of 3 L. NaHCO
3
(3 g)
and 6N NaOH (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, NaHCO
3
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 31,000g for 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 discards
a
(%) of total OFMSW % of simulated OFMSW
b
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 rates
c
OFMSW Primary sludge WAS
635 g/capita/day 102 g dry solids/capita/day 57 g dry solids/capita/day
a
Data from the EPA (1992).
b
OFMSW used in this study.
c
OFMSW 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
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 2000g for 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 (T
w
) (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
32
P using bac-
teriophage T
4
polynucleotide kinase and [-
32
P] 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 simulated
waste 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-UpStrategy
No 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
GRIFFIN ET AL.: METHANOGENIC POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 345
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-
nosaetaceae were present at high levels and Methanomicro-
biales constituted the second most important group. The
high levels of Methanosaeta spp. are consistent with the low
acetate concentrations in the anaerobic digester from which
the inoculum was taken (15 mg/L). Methanosaeta spp. 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.
Probe
a,b
T
w
Target group RNA standard Characteristic substrates
c
Universal probe
S-*-Univ-1390-a-A-18
44 Virtually all organisms
Probes for domains
S-D-Arch-0915-a-A-20 58 Virtually all Archaea Methanosarcina acetivorans
or Methanosaeta concilii
GP6
S-D-Bact-0338-a-A-18 55 Virtually all Bacteria Escherichia coli
S-D-Euca-0502-a-A-16 58 Virtually all Eucarya Saccharomyces cerevisiae
Probes for methanogens
S-F-Mbac-0310-a-A-22
d
57 Methanobacteriaceae Methanobacterium bryantii Most use H
2
CO
2
, some use
H
2
CO
2
and formate
S-F-Mcoc-1109-a-A-20
e
55 Methanococcaceae Methanococcus voltae Most use H
2
CO
2
and formate
S-O-Mmic-1200-a-A-21 53 Methanomicrobiales Methanogenium cariaci Most use H
2
CO
2
and formate
S-G-Msar-0821-a-A-21 60 Methanosarcina spp. Methanosarcina acetivorans Use acetate and other substrates
(H
2
CO
2
, methanol, and
methylamines); generally have
high minimum threshold, K, and

max
values for acetate
S-F-Msae-0825-a-A-23
f
59 Methanosaetaceae
g
Methanosaeta concilii GP6 Use only acetate; generally have
low minimum threshold, K, and

max
values
Probes for SRB
S-F-Dsv-0687-a-A-16 47 Desulfovibrionaceae Desulfovibrio desulfuricans H
2
, lactate, formate
S-*-Dsb-0804-a-A-18 47 Desulfobacter group 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
a
Probe names have been standardized as described in Alm et al. (1996).
b
Original 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).
c
Characteristic substrates for methanogens as listed in Raskin et al. (1994) and characteristic substrates of SRB as listed in Devereux et al. (1992).
d
Most representatives of the order Methanobacteriales belong to the family Methanobacteriaceae and are targeted by probe S-F-Mbac-0310-a-A-22. This
probe does not target the thermophilic members of this order (Methanothermus fervidus and M. sociabilis).
e
Probe 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 jannaschii and Methanoignis igneus) have one mismatch with probe S-F-Mcoc-1109-a-A-20.
f
Probe 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).
g
Methanosaeta is the only genus that has been defined so far within the family of Methanosaetaceae (Boone et al., 1993).
346 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 57, NO. 3, FEBRUARY 5, 1998
approximately half of which consisted of Methano-
bacteriaceae SSU rRNA.
Digester Performance and 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 (Poggi
Varaldo 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 NaHCO
3
to 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 butyrate
increases. 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 NaHCO
3
were
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 NaHCO
3
, the pH
dropped to 6.2 and the value increased to 13 on day 5,
prompting further chemical additions. Either NaHCO
3
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 SD
a
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 VFAs
b
587 91 mg/L as acetic acid
Bicarb. alkalinity 158 26 mg/L as CaCO
3
pH 7.1 0.2 SU
Sulfate 53 14 mg/L as sulfate
Nitrogen 1.4 0.3 % of TS
a
Mean values and standard deviations (SD) were obtained by performing
analyses for seven batches of feedstock.
b
Total VFAs equals the sum of the concentrations of acetate, propionate,
butyrate, valerate, isovalerate, and isobutyrate as measured by gas chro-
matography.
GRIFFIN ET AL.: METHANOGENIC POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 347
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 (% CH
4
) in biogas in (E) mesophilic and (F) thermophilic digesters.
348 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 57, NO. 3, FEBRUARY 5, 1998
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/m
3
/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 in
the 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 Performance and Microbial
PopulationDynamics
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 of Bacteria,
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
Specific gas production m
3
/kg/VS
fed
0.29 0.02 10 0.43 0.05 48
Gas production rate m
3
/m
3
/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 18
Isovalerate mg/L as acetic acid 64 6 4 49 22 18
Total VFAs
a
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.
a
Total VFAs equals the sum of the concentrations of acetate, propionate, butyrate, valerate, iso-
valerate, and isobutyrate as measured by gas chromatography.
GRIFFIN ET AL.: METHANOGENIC POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 349
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
Methanosarcina SSU 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 of Methanosaeta spp. and Methanomi-
crobiales SSU 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 that Methanosarcina spp. were
the dominant methanogens during this period (Fig. 2C).
Methanosarcina spp. 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, Archaea and Methanosarcina levels
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 levels
in (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%.
350 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 57, NO. 3, FEBRUARY 5, 1998
decreased as well. Methanosaeta remained present after day
17, but at low levels (mean 0.13 0.02%).
The levels of Methanomicrobiales, 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 of Methanococcaceae
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 on
day 1 (Fig. 1F). As in the mesophilic digester, the levels of
Methanosaeta spp. and Methanomicrobiales SSU 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 Methanobacteriaceae levels.
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) as
acetate 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 Methanosarcina levels. 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.
Methanomicrobiales were present initially in the thermo-
philic digester, but their SSU rRNA levels decreased and
remained low, with the exception of day 13 (Fig. 2F).
Methanococcaceae were 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-
ily Desulfovibrionaceae, the Desulfobacter group, 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 undefined
mixed 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.
GRIFFIN ET AL.: METHANOGENIC POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 351
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, which
allowed 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 sludge
from 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
352 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 57, NO. 3, FEBRUARY 5, 1998
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). Desulfobulbus spp. 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 results
support 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 of Methanosaeta 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 (K
M
)
that is even lower than that of Methanosaeta, perhaps re-
sulting in a competitive advantage at very low acetate lev-
els. Based on our results (high levels of Methanobacteri-
aceae, insignificant levels of Methanosaeta 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-
nobacteriaceae were the dominant hydrogenotrophic meth-
anogens in the thermophilic digester, it is likely that species
of this family, which includes Methanobacterium spp., 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-
GRIFFIN ET AL.: METHANOGENIC POPULATION DYNAMICS IN ANAEROBIC DIGESTERS 353
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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