The Roles of Acetotrophic and Hydrogenotrophic Methanogens During Anaerobic Conversion of Biomass to...

REVIEW PAPER

The roles of acetotrophic and hydrogenotrophicmethanogens during anaerobic conversion of biomassto methane: a review

Burak Demirel Paul Scherer

Published online: 31 January 2008

Springer Science+Business Media B.V. 2008

Abstract Among different conversion processes for

biomass, biological anaerobic digestion is one of

the most economic ways to produce biogas from

various biomass substrates. In addition to hydrolysis

of polymeric substances, the activity and perfor-

mance of the methanogenic bacteria is of paramount

importance during methanogenesis. The aim of this

paper is primarily to review the recent literature about

the occurrence of both acetotrophic and hydrogeno-

trophic methanogens during anaerobic conversion of

particulate biomass to methane (not wastewater

treatment), while this review does not cover the

activity of the acetate oxidizing bacteria. Both

acetotrophic and hydrogenotrophic methanogens are

essential for the last step of methanogenesis, but

the reports about their roles during this phase of

the process are very limited. Despite, some conclu-

sions can still be drawn. At low concentrations of

acetate, normally filamentous Methanosaeta species

dominate, e.g., often observed in sewage sludge.

Apparently, high concentrations of toxic ionic agents,

like ammonia, hydrogen sulfide (H2S) and volatile

fatty acids (VFA), inhibit preferably Methanosaetaceae

and especially allow the growth of Methanosarcina

species consisting of irregular cell clumps, e.g., in

cattle manure. Thermophilic conditions can favour

rod like or coccoid hydrogenotrophic methanogens.

Thermophilic Methanosarcina species were also

observed, but not thermophilic Methanosaetae. Other

environmental factors could favour hydrogentrophic

bacteria, e.g., short or low retention times in a biomass

reactor. However, no general rules regarding process

parameters could be derivated at the moment, which

favours hydrogenotrophic methanogens. Presumably, it

depends only on the hydrogen concentration, which is

generally not mentioned in the literature.

Keywords Acetotrophic Anaerobic Biogas Biomass Energy Hydrogenotrophic Methane Methanogens Renewable

1 Introduction

The use of renewable energy sources is becoming

increasingly essential, in order to reduce emissions

from fossil fuel sources that have impacts on global

warming. Therefore, biomass seems one of the most

common form of renewable energy source for

feasible utilization (McKendry 2002). Biomass is

widely available, and its utilization for energy

production has a great potential to reduce carbon

dioxide (CO2) emissions and consequently to prevent

B. Demirel (&) P. SchererLifetec Process Engineering, Faculty of Life Sciences,

Hamburg University of Applied Sciences (HAW

Hamburg), Lohbruggerkirchstrasse 65,

21033 Hamburg, Germany

e-mail: [email protected]

123

Rev Environ Sci Biotechnol (2008) 7:173190

DOI 10.1007/s11157-008-9131-1

global warming (Claassen et al. 1999). It is antici-

pated that biomass will have a major role in

substitution of fossil fuels with renewable sources,

and will presumably contribute 83% to the increased

use of renewable sources by the year 2010 (Karpen-

stein-Machan 2001). Biomass can biologically be

converted to biogas, namely methane (CH4) and

hydrogen (H2), using anaerobic digestion process.

Production of methane via anaerobic digestion of

energy crops and organic agro-wastes would benefit

society by providing a clean fuel form renewable

sources, replacing fossil fuel-derived energy and

reducing environmental impacts, such as global

warming and acid rain (Chynoweth et al. 2001).

Besides, the circuit of minerals as fertilizers would

also be enabled in parallel. Hand- and mechanically-

sorted municipal solid waste, fruit and vegetable solid

wastes, leaves, grasses, woods, weeds, marine and

freshwater biomass have previously been reviewed

for their anaerobic conversion potential to methane

(Gunaseelan 1997). Recently, both conventional

single-phase and high-rate two-phase anaerobic

digestion systems have been widely used, for pro-

duction of biogas-methane from various substrates,

such as the organic fraction of the municipal solid

waste (OFMSW), spent tea leaves, grass, food waste,

fodder beet silage, fruit and vegetable waste, kitchen

waste, crop residues, solid slaughterhouse waste,

manure, potato waste, waste activated sludge and

sugar beet silage (Rao et al. 2000; Ghosh et al. 2000;

Goel et al. 2001; Hai-Lou et al. 2002; Lastella et al.

2002; Scherer et al. 2003; Bouallagui et al. 2003;

Neves et al. 2004; Moller et al. 2004; Scherer and

Lehmann 2004; Wilkie et al. 2004; Parawira et al.

2005; Li et al. 2005; Svensson et al. 2005; Bohn

et al. 2005; Carpentier et al. 2005; Angelidaki et al.

2005; Alvarez et al. 2006; Angelidaki et al. 2006;

Linke et al. 2006; Zhang et al. 2007; Svensson et al.

2007; Demirel and Scherer 2008; Parawira et al.

2008).

Furthermore, biomass has also the potential to

become a significant source of renewable hydrogen as

well, via anaerobic acidogenesis. Therefore, much

attention has recently been focused also on biological

production of hydrogen from different substrates,

such as the OFMSW, food waste, sewage sludge,

paper mill wastes, palm oil effluent, brewery waste,

glucose and etc. (Okamoto et al. 2000; Hawkes et al.

2002; Brown 2004; Shin et al. 2004; Oh et al. 2004;

Kim et al. 2004; Kawagoshi et al. 2005; Valdez-

Vazquez et al. 2005; Atif et al. 2005; Gong et al.

2005; Lay et al. 2005; Shizas and Bagley 2005; Fan

et al. 2006; Gavala et al.2006; Cooney et al. 2007).

Earlier findings on methane fermentation, the basic

biochemistry and microbiology of the organisms

involved, and the microbial generation of hydrogen

have already been discussed previously (Boone et al.

1993a, b; Klass 1998; McKendry 2002).

However, in spite of an increasing attention on

anaerobic digestion of biomass for production of

renewable energy in the form of biogas, there still

exists relatively less information, particularly about

the activity and the performance of both acetotrophic

and hydrogenotrophic methanogens, during anaerobic

conversion of biomass to methane. Actually, the

behaviour and activity of both acetotrophic and

hydrogenotrophic methanogens have been exten-

sively investigated, during conventional single-

phase and high-rate two-phase anaerobic treatment

of simple types of soluble substrates, mostly syn-

thetic, and also complex types of industrial

wastewaters. However, studies covering particularly

the fate of both acetotrophic and hydrogenotrophic

methanogens during anaerobic methanogenesis of

particulate solid biomass for biogas production are

relatively scarce in literature. Biogas plants receive

various types of energy crops or organic wastes as

substrates, and they are operated as co-digesters or

mono-digesters. Besides, each biogas plant is obvi-

ously operated under different operational and

environmental conditions, depending on the type of

substrate(s) to be digested.

The differences in operational and environmental

conditions definitely affect the behaviour and the fate

of both groups of methanogens present in a biogas

digester. A recent study showed that the biogas plant

performance data collected during the winter month

(December, 24C) indicated a significant reduction indaily biogas production as compared to the summer

month (April, 36C), due to decrease in ambienttemperature and the associated shift in the microbial

community, since highly diverse and complex meth-

anogenic communities are present in a biogas plant

(Rastogi et al. 2007). Therefore, the current research

activities, focusing on the special roles of both groups

of methanogens, during anaerobic conversion of

biomass to methane are discussed in this paper. The

key purpose is to point out the behaviour and the fate

174 Rev Environ Sci Biotechnol (2008) 7:173190

123

of both groups of methanogens with respect to the

changes in biogas digester operation.

2 Theory of the anaerobic bacterial food chain

The performance of an anaerobic digestion system is

primarily linked to the structure of the microbial

community present in the digester. On the other hand,

the operational and environmental parameters of the

process obviously affect the behaviour, performance

and eventually the fate of the microbial community in

anaerobic digesters. Furthermore, the nature and

influence of the seed sludge used for inoculation

should also be accounted for as well (Guyot et al.

1993). An overall scheme for anaerobic conversion of

organic substrates to methane is indicated in Fig. 1.

Characteristical morphology of acetotrophic and

hydrogenotrophic methanogens is shown in Fig. 2.

Anaerobic microbial communities can be classi-

fied into two domains, Bacteria and Archaea. Stable

anaerobic digestion is accomplished by representa-

tives of four major metabolic groups: hydrolytic-

fermentative bacteria, proton-reducing acetogenic

bacteria, hydrogenotrophic methanogens, and aceti-

clastic methanogens (Zinder et al. 1984). During

anaerobic degradation of particulate organic materi-

als, particulate biopolymers (carbohydrates, proteins

and lipids) are firstly hydrolyzed to organic mono-

mers, which can be utilized either as substrates by

fermentative organisms (amino acids, sugars) or by

anaerobic oxidizers (fatty acids). The carbonic prod-

ucts from these reactions are either acetate and

hydrogen or intermediate compounds, such as propi-

onate and butyrate, which may later be converted to

acetate and hydrogen. Methane is mostly produced

from acetate or hydrogen (H2) and carbon dioxide

(CO2) or formate. Methyl groups are converted to

methane, too (methanol, methylamines). As shown in

Fig. 1, the acetotrophic methanogens can compete

with the acetate oxidizing bacteria. The acetate

oxidizing bacteria can convert acetate to H2 + CO2or they can also use the reverse reaction to produce

acetate from H2 + CO2. Therefore, the term revers-

ible acetogenesis was created, as it was assumed

that it was even the same Reversibacter enabling

both reactions (Zinder 1994). At high concentrations

of H2 (e.g., C500 Pa), acetogenesis is favoured (or

the methanogenesis from H2 + CO2), and at low

concentrations (e.g., B40 Pa), the acetate oxidation

occurs. Further details about the activity of the

acetogenic bacteria can be found elsewhere (Dolfing

1988; Schink 1994).

Hydrogen is used as an electron acceptor to form

methane, by hydrogenotrophic methanogens, while

many H2-using methanogens can also use formate as

an electron donor for the reduction of CO2 to CH4.

Formate can also be an important substrate, even

though its concentrations in methanogenic environ-

ments are low, since it is rapidly produced and

consumed (Boone et al. 1993a). On the other hand,

acetate is cleaved to form methane from the methyl

group by methyltrophic methanogens and carbon

dioxide from the carboxyl group by acetotrophic

methanogens. Classification of methanogenic bacte-

ria is outlined in Table 1 (Whitman et al. 2001;

Garrity et al. 2004). Properties of some methanogens

are briefly summarized in Table 2 (Vogels et al.

1988; Boone et al. 1993a). A more detailed summary

about the diversity of methanogens can also be found

Particulate Organic Matter

Carbohydrates Proteins Lipids

Hydrolysis

Amino Acids, Sugars, Alcohols, Fatty Acids

Acidogenesis

Intermediary Products Acetate, Propionate, Ethanol, Lactate

Acetogenesis

AcetateHydrogen

CO2

ReductiveHomoacetogenesis

Homoacetogenic oxidation

MethanogenesisCH4 + CO2

Fig. 1 Anaerobic conversion of biomass to methane

Rev Environ Sci Biotechnol (2008) 7:173190 175

123

elsewhere (Mah and Smith 1981; Boone et al.

1993a). The typical methanogenic reactions that take

place during anaerobic digestion process are dis-

played in Table 3 (Chynoweth 1996). Additionally, a

summary of the kinetic data for several methane

forming bacteria are given in Table 4. More infor-

mation for kinetic parameters of acetate and

hydrogen utilizing methanogens can also be found

elsewhere (Koster and Koomen 1988; Van Lier

1996).

Several investigations have already been reported,

which have covered the activity and the behaviour of

the microbial communities in anaerobic biogas reac-

tors running on various kind of substrates (Petersen

and Ahring 1991; Hedrick et al. 1992; Angelidaki

and Ahring 1993; Raskin et al. 1995; Ahring 1995;

Chanakya et al. 1997; Clarens et al. 1998; Vavilin

et al. 1998; Oude Elferink et al. 1998; Hansen et al.

1999). In the following sections of this paper, the

roles of acetotrophic and hydrogenotrophic methano-

gens during anaerobic conversion of particulate

biomass to methane will be discussed, respectively,

in view of the works recently published in literature.

3 The role of acetotrophic methanogens

The acetotrophic methanogens are obligate Archaea

anaerobes, which convert acetate to methane (CH4)

and carbon dioxide (CO2) (Ferry 1992). The activity

and the performance of the acetotrophic methanogens

are of paramount importance during anaerobic con-

version of acetate. In this section of the paper, a

summary of recent investigations about the aceto-

trophic methanogens reported in literature will be

presented.

3.1 The influence of operational

and environmental parameters

In an earlier work, the methanogenic population

dynamics in anaerobic digesters treating municipal

solid waste and biosolids were investigated during

start-up (Griffin et al. 2000). Two laboratory-scale

anaerobic continuously mixed reactors were operated

at 37 and 55C, and both digesters were inoculatedwith mesophilic anaerobic sewage sludge and cattle

manure. Methanosaeta species were observed to be

most abundant in the seed sludge, but their numbers

decreased fast as the acetate concentration increased.

The increase in acetate levels was accompanied by an

increase in Methanosarcina species.

During quantification of Methanosaeta species in

anaerobic bioreactors, Methanosaeta spp. was found

to be the dominant aceticlastic methanogen in a

variety of anaerobic reactors at low acetate concen-

trations (Zheng and Raskin 2000). Relatively,

Methanosaeta spp. levels were higher in bioreactors

with granular sludge than in those with flocculent

sludge.

Anaerobic co-digestion of the OFMSW, primary

sludge and waste activated sludge was evaluated in

mesophilic laboratory-scale anaerobic digesters, in

terms of digester performance and microbial popula-

tion dynamics (Stroot et al. 2001; McMahon et al.

2001). Methanosarcina spp. was determined to be the

most abundant aceticlastic methanogens in unstable

codigesters with high acetate concentrations.

A mathematical model was developed, in order to

describe the dynamic behaviour of mesophilic and

thermophilic anaerobic sewage sludge digestion, with

a special emphasis on acetotrophic methanogenesis

(Siegrist et al. 2002). At a hydraulic retention time

Fig. 2 Morphology of acetotrophic and hydrogenotrophicmethanogens as seen by fluorescence microscopy (excitation

at 420 nm, emission at 480 nm); (a) Acetotrophic methano-gens; Methanosaeta DSMZ 6752 (chains 6150 lm) or

Methanosarcina DSMZ 804 (irregular clumps), (b) Hydro-genotrophic methanogens; rods or cocci (26 lm), authenticsample of a biogas plant with renewable biomass (Pictures

provided by the authors)


123

(HRT) range of from 6 to 20 days (mesophilic) and 2

to 8 days (thermophilic), the observed maximum

growth rates for acetotrophic methanogens were

determined to be 0.33 and 1.3 l/day, respectively,

with a 15% of decay rate. It was additionally reported

that the optimum pH range for acetotrophic metha-

nogens was between 6.6 and 7.3. Acetotrophic

methanogens were inhibited strongly below a pH of

Table 1 Classification ofmethanogenic bacteria

(Whitman et al. 2001;

Garrity et al. 2004)

Class I. Methanobacteria (known to grow on H2/CO2 and formate as C source)

Order I. Methanobacteriales

Family I. Methanobacteriaceae

Genus I. Methanobacterium

Genus II. Methanobrevibacter

Genus III. Methanosphaera

Genus IV. Methanothermobacter

Family II. Methanothermaceae

Genus I. Methanothermus

Class II. Methanococci (known to grow on H2/CO2 and formate as C source)

Order I. Methanococcales

Family I. Methanococcaceae

Genus I. Methanococcus

Genus II. Methanothermococcus

Family II. Methanocaldococcaceae

Genus I. Methanocaldococcus

Genus II. Methanotorris

Class III. Methanomicrobia (known to grow on H2/CO2 and formate as C source)

Order I. Methanomicrobiales

Family I. Methanomicrobiaceae

Genus I. Methanomicrobium

Genus II. Methanoculleus

Genus III. Methanofollis

Genus IV. Methanogenium

Genus V. Methanolacinia

Genus VI. Methanoplanus

Family II. Methanocorpusculaceae

Genus I. Methanocorpusculum

Family III. Methanospirillaceae (known to be hydrogenotrophic)

Genus I. Methanospirillum

Order II. Methanosarcinales (known to be acetato- and methylotrophic)

Family I. Methansarcinaceae

Genus I. Methanosarcina

Genus II. Methanococcoides

Genus III. Methanohalobium

Genus IV. Methanohalophilus

Genus V. Methanolobus

Genus VI. Methanomethylovorans

Genus VII. Methanimicrococcus

Genus VIII. Methanosalsum

Family II. Methanosaetaceae

Genus I. Methanosaeta


123

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123

6.2, while free ammonia (FA) concentrations could

also be inhibitory at pH levels above 7.4.

During two-stage anaerobic digestion of grass, it

was observed that the stability of the methanogenic

reactor, which was inoculated with fresh rumen

resulted due to the dominance of Methanosaeta

species (mainly the filamentous type) (Raizada et al.

2003).

Changes in methanogenic population levels were

examined during start-up of a full-scale anaerobic

sequencing batch reactor (ASBR) treating swine

waste (Angenent et al. 2002). At a volatile solids

(VS) loading rate of 1.7 g/l/day and a HRT of

15 days, and with a total ammonia-N level of about

3600 mg/L, the levels of acetate-utilizing methano-

gens (Methanosarcina) decreased from 3.8 to 1.2%

(expressed as a percentage of the total 16S rRNA

levels), while the levels of Methanosaeta concilii

remained below 2.2%.

Microbial population dynamics of anaerobic co-

digesters treating municipal solid waste and sewage

sludge were investigated during start-up and overload

conditions (McMahon et al. 2004). The authors

observed that the digesters, which contained high

levels of Archaea, started up successfully, and

Methanosaeta concilii was found to be the dominant

acetotrophic methanogen in these digesters. On the

other hand, digesters, which experienced trouble

during the start-up period, had lower levels of

Archaea, with an abundant population of Methano-

sarcina spp. Furthermore, digesters with a poor

performance history tolerated quite unusual severe

organic load even better than the digesters with a

satisfactory performance history (McMahon et al.

2004).

The influence of environmental parameters on the

diversity of methanogenic communities in 15 full-

scale biogas plants treating either manure or sludge as

substrates under different conditions were examined

(Karakashev et al. 2005). The findings of this study

indicated that the methanogenic diversity was

broader in plants operating at mesophilic ranges than

the thermophilic plants. The dominance of Methan-

osaetaceae was observed in digesters fed with sludge.

However, Methanosaetaceae was never found to be

the dominant in digesters treating manure. According

to the authors, inoculum and loading rates did not

affect the diversity of methanogens in biogas reac-

tors, but the concentrations of ammonia (NH3) and

volatile fatty acids (VFA). At high levels of NH3 and

VFA, the dominance of Methanosarcinaceae in

manure digesters was observed, while in sewage

sludge digesters with low levels of NH3 and VFA,

Methanosaetaceae dominated. Acetate-utilizing

methanogens offering thin filaments with a great

surface seemed to be more sensitive to ammonia

concentrations than hydrogenotrophic methanogens

growing as rods or Methanosarcinaceae consisting of

thick clumps. Therefore, Methanosaeta is not

observed to be dominant, particularly in swine

manure biogas reactors (Schmidt et al. 2000; Mlade-

novska et al. 2003). Karakashev et al (2006) also

claimed that in the absence of Methanosaetaceae, the

acetate oxidation to H2/CO2 with the subsequent

generation of methane by hydrogenotrophic metha-

nogens should be the dominant pathway. These

results seem in agreement with the other studies,

too (Angelidaki and Ahring 1993; Shigematsu et al.

2004).

An analysis of community structure of a full-scale

digester was carried out using a quantitative real-time

PCR method (Yu et al. 2005). The anaerobic digester

treating waste activated sludge was operated at a

HRT of 28 days, a pH of 7 and 33C, with an OLR of

Table 3 Methanogenic reactions (Chynoweth 1996)

1. Hydrogen

4 H2 + CO2 ! CH4 + 2 H2O2. Acetate

CH3COOH ! CH4 + CO23. Formate

4 HCOOH ! CH4 + 3 CO2 + 2 H2O4. Methanol

4 CH3OH ! 3 CH4 + CO2 + 2 H2O5. Carbon monoxide

4 CO + 2 H2O ! CH4 + 3 H2CO36. Trimethylamine

4 (CH3)3N + 6 H2O ! 9 CH4 + 3 CO2 + 4 NH37. Dimethylamine

2 (CH3)2NH + 2 H2O ! 3 CH4 + CO2 + 2 NH38. Monomethylamine

4 (CH3)NH2 + 2 H2O ! 3 CH4 + CO2 + 4 NH39. Methyl mercaptans

2 (CH3)2S + 3 H2O ! 3 CH4 + CO2 + H2S10. Metals

4 Me0 + 8 HCO2 ! 4Me+ CH4 + 2H2O


123

1.32 g COD/l/day. Methanosaetaceae was found to

be dominant in the full-scale anaerobic digester. A

relatively long HRT of 28 days, accompanied with

low concentrations of acetate in a full-scale digester,

would provide an advantage to the growth of

Methanosaetaceae family. The authors also con-

cluded that the operating conditions, such as HRT,

along with the type of substrate used, were important

parameters for development of a methanogenic

community.

3.2 The effect of reactor configuration

The application of the UASB reactors is widespread

to treat soluble and solid types of wastes and to

recover energy, through high-rate methane fermenta-

tion (Fang 2000).

The microbial investigation of an UASB reactor

treating effluent from an acidogenic reactor fermenter

during two-phase mesophilic anaerobic digestion of

food waste was carried out in a laboratory-scale work

(Shin et al. 2001). The authors reported that the

typical UASB reactor granules were found to be

mainly composed of microcolonies of Methanosaeta.

Microscopic examination of biogranules sampled

from different UASB reactors indicated that the

microbes were densely packed and the microbial

distribution was strongly dependent upon the degra-

dation thermodynamics and kinetics of individual

substrates (Nishio and Nakashimada 2004). Interior

of the biogranule layers was found to be occupied by

acetotrophic methanogens.

During investigation of acetate conversion in

anaerobic continuous stirred tank reactors (CSTRs)

inoculated with digested cattle manure, the only

recognized acetate utilizing methanogen was

observed to be Methanosarcina-related microorgan-

isms, independent of whether the reactors were fed on

cattle manure alone, or on mixtures of manure and

industrial organic waste (Schmidt et al. 2000). Both

laboratory-scale reactors had a working volume of

3 l, and operated at a hydraulic retention time (HRT)

of 15 days, at 55C.Anaerobic digestion of manure and a mixture

of manure with lipids was investigated, using

Table 4 A summary of kinetic data for several methane-forming bacteria

Bacteria lmax (h-1) Ks (mM) Reference

M. concilii 0.032a 1.5 Schmidt and Ahring 1999

M. mazeii 0.06a 3.6 Schmidt and Ahring 1999

Methanosarcina Barkeri 0.019b Yang and Okos 1987; Schonheit et al.1982; Smith and Mah 1978 3

0.023 320 (as mgCOD/l)

Methanosarcina sp. MSTA-1 0.0500.055 Clarens and Moletta 1990 Lundback et al.1990; Schauer and Ferry 1980Methanobacterium formicicum 0.053

0.082

Methanosarcina spp. 0.0440.064 6.524.7 Mladenovska and Ahring 2000

Methanosarcina CALS-1 0.058 Zinder and Koch 1984

Methanosarcina thermophila 0.0.58 288 (as mgCOD/l) Zinder and Mah 1979; Zinder et al. 1985

Methanosarcina CHTI55 0.085 614 (as mgCOD/l) Touzel et al. 1985

Methanosaeta Jetten et al. 1990; Ohtsubo et al. 1992;Oude Elferink et al. 1994M. soehngenii 0.080.29 (day-1) 0.40.7

M. concilii 0.210.69 (day-1) 0.81.2

Methanospirillum hungatei 0.053 Robinson and Tiedje 1984

Methanobacterium bryantii M.o.H. 0.029 Dubach and Bachofen 1985

Methanomicrobium paynteri 0.144 Dubach and Bachofen 1985

a Growth on acetateb At an acetic acid concentration of 3.6 g/l


123

laboratory-scale mesophilic CSTRs (Mladenovska

et al. 2003). Microbial population analysis indicated

an uniform distribution of activity of acetotrophic

methanogens in biomass from the co-digester.

Besides, the vast majority of clones was phylogenet-

ically most closely related to Methanosarcina

siciliae. The influence of temperature variations

between 50 and 60C on anaerobic digestion of cattlemanure was also evaluated in CSTRs, operated at

HRTs of 10 and 20 days (El-Mashad et al. 2004).

The authors reported that the activity of acetate

utilizing bacteria was affected by free ammonia

(FA) concentrations, rather than high ammonium

concentrations.

In a relatively recent investigation, laboratory-

scale CSTRs were operated with manure, at 55C,a HRT of 18 days, and an organic loading rate

(OLR) of 2.5 g VS/l/day (Mladenovska et al. 2005).

Microbial community analysis indicated that

Methanosarcina thermophila were present in diges-

ters, along with high level of acetate. The archaeal

species exhibited a lower degree of diversity than

that of the bacterial species.

In the view of the literature information reported

above, the adjustment of the hydraulic retention time

(HRT) seems to be a significant operational param-

eter, while adjustment and monitoring of both pH and

temperature seem to be the most important environ-

mental parameters that affect the presence and the

activity of the acetotrophic methanogens. Our exper-

imental findings at Hamburg University of Applied

Sciences (HAW Hamburg) also showed that the

control of pH was the most significant factor in a

biogas digester operating with a mono-substrate for a

safe, stable and efficient biogas production, in

addition to the type of substrate digested and the

HRT range used (data not shown). The fluorescence

pictures taken during mesophilic anaerobic digestion

of sugar beet silage in our laboratory at various HRTs

(25 and 15 days), using two different harvests of the

same substrate type are given in Fig. 3.

Furthermore, the source and type of the sludge

used for inoculation, changing VFA concentrations in

the biogas digester, the concentrations of free

ammonia (FA) resulting from high pH and use of

manure, and finally the success of the start-up phase

were reported to be important factors, which affect

the fate of the acetotrophic methanogens in biogas

digesters during the conversion process.

4 The role of hydrogenotrophic methanogens

The hydrogen partial pressure is an important

parameter, which defines process stability or upsets

in an anaerobic digestion process. Therefore, the

activity of the hydrogenotrophic methanogens are

crucial for a stable and efficient process performance.

The performance and activity of hydrogenotrophic

methanogens in anaerobic conversion of simple-

soluble type of substrates (such as acetate, ethanol,

methanol, glucose and propionate) and various types

of wastewaters (such as olive mill) have been covered

and discussed in recent research activities selected

here (Gijzen et al. 2000; Imachi et al. 2000; Gonz-

alez-Gil et al. 2001; Syutsubo et al. 2001; Delbes

et al. 2001; Paulo et al. 2002; Paulo et al. 2003;

McHugh et al. 2004; Bertin et al. 2004; Sipma et al.

2004; Tada et al. 2005; Tang et al. 2005; Roest et al.

2005; Sawayama et al. 2006; Shigematsu et al.

2006). On the contrary, the role and activity of

hydrogenotrophic methanogens in anaerobic conver-

sion of biomass/complex organic materials to

methane has not been regularly discussed, and there

exists relatively less amount of data during the

previous decade (Blotevogel et al. 1985; Jarvis et al.

1995; Zhu et al. 1997; Schnurer et al. 1999). Fur-

thermore, the amount of literature on this particular

topic also seems scarce at the moment as well. An

outline of these recent research activities is presented

in this section of the paper.

4.1 The influence of operational and

environmental parameters

Degradation of the organic fraction of the municipal

grey waste (the residual counterpart of the separately

collected biowaste, paper and glass) was investigated

under thermophilic and hyperthermophilic (up to

max. 70C) conditions, using lab-scale continuousreactors for biogas production (Scherer et al. 2000).

Analysis of bacterial populations in anaerobic reac-

tors by MPN technique indicated that the H2CO2utilizers, ranging from 108 to 1010/g TS, obviously

dominated the acetotrophic methanogens by a factor

of 10 to 10,000, presumably due to short HRTs

(between 14.2 and 1.25 days) employed. However,

the laboratory-scale reactors were inoculated by a

mixture of mesophilic sewage sludge, swine manure


123

and compost of the hot rot phase. Compost of the hot

rot phase was found to contain up to 108/ml of

thermophilic hydrogenotrophic methanogens (Derikx

et al. 1989; Jackel et al. 2005). Therefore compost

could be a stimulating agent for methanogenesis of

biomass (Scherer et al. personal communication).

The effect of a temperature increase, from 55 to

65C, on process performance and microbial popu-lation dynamics of anaerobic CSTRs treating cattle

manure were investigated in a laboratory-scale work

(Ahring et al. 2001). Hydrogenotrophic methanogens

were the only microbial group, which exhibited

higher specific methanogenic activity (SMA) and

unchanged MPN (most probable number) at 65C,compared to 55C, while the activity and the amountsof other methanogens were significantly reduced. The

authors concluded that the hydrogenotrophic Archaea

seemed to play a more important role during anaer-

obic digestion at 65C. Actually, it was previouslymentioned that several of the isolated thermophilic

hydrogen-utilizing methanogens of the family Meth-

anobacteriaceae were capable of using both

hydrogen/carbon dioxide and formate as substrates

(Boone et al. 1993a). The authors also reported that,

at 65C, only hydrogen utilizers were favored, whilethe activity of formate utilizers were reduced.

Cattle manure is a complex type of substrate,

composed of carbohydrates, proteins and fats, and

thermophilic anaerobic digestion process can success-

fully be applied to treat cattle manure (McInerney 1988).

Characterization of bacterial and archaeal communities

were investigated during start-up of anaerobic thermo-

philic digestion system for treating cattle manure

(Chachkiani et al. 2004). A laboratory-scale continu-

ously stirred anaerobic thermophilic batch digester was

used in this experimental work, inoculated with cattle

manure. Two dominant archaeal species were observed

to be Methanoculleus thermophilicus and the acetate-

utilizing Methanosarcina thermophila.

A comparison of anaerobic digestion of cattle

manure was carried out, using a single-stage thermo-

philic (55C) and a two-stage thermophilic (68/55C)anaerobic digestion system, in a laboratory-scale

study (Nielsen et al. 2004). In two-stage anaerobic

digestion system, the first digester was operated at a

HRT of 3 days and a temperature of 68C. Thisdigester was connected to another digester, operated

at a HRT and temperature of 12 days and 55C,respectively. The conventional single-stage digester

was operated at a HRT and temperature of 15 days

and 55C, respectively. The density levels of hydro-gen utilizing methanogens were found out to be in the

same range for the both digestion systems.

The microbial community of a laboratory-scale

mesophilic two-phase anaerobic digestion system

treating fruit and vegetable wastes was studied (Boua-

llagui et al. 2004). The species composition seemed to

change significantly during the entire study. In the first-

phase/acidogenic reactor, Methanosphaera stadtmanii

and Methanobrevibacter wolinii were observed to be

the major hydrogenotrophic methanogens. Recently,

the bacterial population of mesophilic and thermo-

philic laboratory-scale anaerobic biogas digesters fed

with beet silages was investigated (Scherer et al, pers.

comm.). The anaerobic digesters were fed with fodder

and sugar beet silage, and were automatically operated

by a Fuzzy logic control (Scherer and Lehmann 2004).

The morphology of the bacterial population is shown in

Fig. 4 Rod-like methanogens known to grow on H2/

CO2 or formate were found to be dominant only at short

HRTs between 6.5 and 7.5 days. For a thermophilic

reactor at 60C receiving fodder beet silage as the solesubstrate, only Methanobacteriales were dominant.

About 50% of the clones were found to be Methano-

bacterium thermoautotrophicum (Scherer et al.

2005). In a previous study, the number of aceticlastic

methanogens were reported to be 10 times higher

than those of H2CO2 using methanogens during

anaerobic digestion of sugar beet pulp (Labat and

Garcia 1986). The differences in these results obtained

can probably be attributed to the seed sludge

(inoculum) and the reactor operating conditions

(higher retention times).

4.2 The effect of reactor configuration

A completely mixed anaerobic bioreactor coupled

with an external ultrafiltration membrane module was

operated to investigate the start-up phase and the

performance during digestion of swine manure (Pad-

masiri et al. 2007). The methanogenic population

dynamics of the anaerobic bioreactor was monitored

with the terminal restriction fragment length poly-

morphism (T-RFLP). The authors reported that the

levels of hydrogenotrophic methanogens of the order

of Methanomicrobiales increased during decreased

reactor performance, suggesting that the syntrophic


123

interactions involving hydrogenotrophic methano-

gens remained intact regardless of the degree of

shear in the bioreactor.

The microbial population dynamics were investi-

gated during the start-up phase and stabilization

period of thermophilic-dry anaerobic digestion of

municipal solid waste using lab-scale CSTR without

solids recycling (Montero et al. 2008). The experi-

mental protocol was defined to quantify Archaea

using the FISH technique. The authors reported that

the hydrogenotrophic methanogens, which had an

important role during the start-up phase, were later

replaced by the acetotrophic methanogens, when the

reactor reached the steady-state conditions.

Fig. 3 (ad). (a) Thefluorescence picture taken

at steady-state conditions

from the biogas digester at

25 days of HRT with the

substrate charge-1; (b) Thefluorescence picture taken

at steady-state conditions


25 days of HRT with

substrate charge-2; (c) Thefluorescence picture taken

after the reactor failure

(souring of the digester due

to high VFA

concentrations) at 15 days

of HRT with substrate

charge-2; (d) Thefluorescence picture taken

for steady-state conditions


15 days of HRT with

substrate charge-2

Fig. 4 Morphology oflaboratory-scale anaerobic

biogas digesters; (a)Mesophilic reactor

receiving fodder beet silage,

(b) Mesophilic reactorreceiving sugar beet silage,

(c) Thermophilic reactorreceiving fodder beet silage

(pictures by the authors)


123

In a recent study, lab-scale experiments were

carried out in a CSTR, for mesophilic digestion of

fodder beet silage as a mono-substrate to produce

methane (Klocke et al. 2007). The OLR applied

ranged from 1.23 to 2.32 kg/m3/day, and the HRT

ranged between 56 and 106 days. The molecular

analysis revealed the presence of H2/CO2/formate

oxidizing Methanobacteriales and the H2/CO2-oxi-

dizing Methanosarcinaceae. Besides, the presence of

Methanosaetaceae (acetate-splitting) was also

confirmed.

4.3 Microbial community characteristics of solid

waste landfills

Utilization of biogas (methane) generated from

municipal or industrial solid waste landfills is nowa-

days also a common procedure for energy production,

in addition to production of biogas from biomass. In a

relatively recent study, molecular detection and direct

enumeration of methanogenic Archaea and methano-

trophic bacteria in domestic solid waste landfill was

studied, in order to evaluate methane oxidizing and

producing activities of landfill soil cover and burial

waste (Chen et al. 2003a). Buried waste samples

showed the highest methane production capability.

All the sequences of the burial waste samples were

found to be closely related to 16S rDNAs of mainly

hydrogenotrophic methanogens known, such as genera

Methanoculleus and Methanobacterium, and Methan-

osarcina (typically acetotrophic). Thermophilic areas

of a 1030 m depth of municipal solid waste landfill

revealed the dominance of thermophilic methanogens

being closely related to Methanothermobacter ther-

moautotrophicus (Chen et al. 2003b). Another related

study also included the investigation of the phyloge-

netic composition of Archaea and the relative

abundance of phylogenetically defined groups of

methanogens in the leachate of a closed municipal

solid waste landfill (Huang et al. 2003). The authors

reported that the most of the methanogen-like clones

were affiliated with the hydrogenotrophic Methano-

microbiales and the methylotrophic and aceticlastic

Methanosarcinales.

The availability of hydrogen may be a limiting

factor for hydrogenotrophic methanogens in biogas

production (Bagi et al. 2007). Furthermore, during

investigation of the activity of the hydrogenotrophic

methanogens in biogas digesters, hydraulic retention

time and thermophilic temperature ranges were

particularly emphasized in literature, in addition to

the effect of the type of substrate to be digested. The

effect of the inoculum material used (like in the case

of using a mixture of sewage sludge, compost and

manure) also has an influence. Selection of the

appropriate seed material will obviously lead to better

digester performances. However, it is difficult to

establish some general rules about the selection of the

appropriate type of seed material for the each type of

substrate to be digested. On the other hand, biogas

plant operators with previous experience can possibly

cope with this type of a problem.

5 Advanced molecular biology techniques

The use of advanced molecular biology techniques

are essential in order to understand and to clarify the

complex reactions taking place in a biogas digester.

Particularly, the response of the Archaea to the

reactor operating conditions should clearly be under-

stood for a stable and efficient reactor management.

Therefore, new molecular techniques have been

developed in the last decade, to improve the micro-

bial ecology research. Among these techniques,

cloning and creation of a gene library, denaturant

gradient gel electrophoresis (DGGE) and fluorescent

in situ hybridization with DNA probes (FISH) are

most commonly used (Sanz and Kochling 2007).

The fluorescence in situ hybridization (FISH)

technique makes it possible to identify microorgan-

isms at any desired taxonomical level, depending on

the specificity of the probe employed. The FISH

technique was used to identify the dominant meth-

anogenic members of the Archaea in full-scale biogas

reactors (Karakashev et al. 2005). Dominance could

be identified by a positive response by more than

90% of the total members of the Archaea to a specific

group order-level probe. The use of FISH technique

has recently been reported to identify the microbial

population dynamics of anaerobic bioreactors run-

ning on municipal solid waste (Montero et al. 2008),

distillery wastewater (Fernandez et al. 2007a), brew-

ery wastewater (Fernandez et al. 2007b), and sludge

(Ariesyady et al. 2007a). In the last study reported,

the Archaea community structure of sludge digesting

reactors were investigated using a full-cycle 16S


123

rRNA approach followed by microautoradiography

(MAR)-FISH technique and micromanipulation. In

another work, the same authors also investigated the

phylogenetic and functional diversity of syntrophic

propionate-oxidizing bacteria present in anaerobic

digester using microautoradiography combined with

MAR-FISH (Ariesyady et al. 2007b).

A new method of quantification for methanogens was

studied, based on the measurement of specific binding

(hybridization) of 16S rRNA-targeted oligonucleotide

probe Arc915, performed by fluorescence in situ

hybridization (FISH) and quantified by fluorescence

spectrometry (Stabnikova et al. 2006). The authors have

reported that this technique could quantitatively deter-

mine the methanogens in attached (biofilms) or

suspended (biomass) microbial aggregates.

The effects of volatile fatty concentrations on the

fate and behaviour of a thermophilic methanogenic

population was studied using PCR-mediated single-

strand conformation polymorphism (SSCP) based on

the 16S rRNA gene, quantitative PCR and the FISH

technique (Hori et al. 2006). The authors have

reported the close relation between the VFA concen-

trations and the structure of the archaeal community.

The results of the phylogenetic analysis of 16S

rRNA gene provided useful information during

identification of the methanogenic community in a

recently developed anaerobic rotating disk reactor

packed with polyurethane, which could be used for

thermophilic continuous anaerobic digestion of

organic waste (Yang et al. 2007). Another recent

study reported 16S rRNA analysis of reactor material

in an anaerobic packed-bed reactor (using carbon

fiber textiles as the support material) running on

artificial garbage slurry (Sasaki et al. 2007).

The concentration of hydrogen influences the

structure of a methanogenic community. Terminal

restriction fragment length polymorphism (T-RFLP)

analysis of 16S rRNA genes was proposed to monitor

the changes within the composition of the population

of methanogens (Leybo et al. 2006). More studies

have also been recently reported for using the T-

RFLP technique for the investigation of the metha-

nogenic population dynamics in anaerobic reactors

(Padmasiri et al. 2007; Enright et al. 2007).

The microbial community analysis of a CSTR

operating with fodder beet silage as mono-substrate

was investigated in a lab-scale work (Klocke et al.

2007). A 16S rDNA clone library was constructed by

PCR amplification applying a prokaryote-specific

primer set, in order to identify the microorganisms

in the CSTR.

The methanogenic community in a biogas reactor

running on cattle dung was investigated by using a

molecular characterization of methyl-coenzyme M

reductase A (mcr A) genes (Rastogi et al. 2007).

6 Conclusions

The roles of acetotrophic and hydrogenotrophic

methanogens are obviously of paramount importance

in anaerobic conversion process, no matter what type

of substrate is going to be digested. The activity and

performance of both types of methane-forming

bacteria have been reviewed, especially during the

last two decades. Studies covering particularly the

specific roles of acetotrophic and hydrogenotrophic

methanogens in anaerobic digestion of biomass or

complex types of particulate materials for biogas

production, such as methane and hydrogen, are

relatively scarce in literature. In spite of various

research works carried out to determine the optimum

environmental and operational parameters during

anaerobic conversion of biomass to methane, more

comprehensive research work is essentially required

for determination of the specific activity and the

behaviour of both groups of methanogens during

anaerobic conversion of various feedstock substrates

(biomass). However, some conclusions can still be

drawn from the cited literature; Typical filamentous

acetotrophic methanogens are favoured at low acetate

concentrations. On the contrary, they disappear at

high concentrations of ammonium and sulfide, like in

cattle and swine manure. High acetate concentrations

favour Methanosarcineae consisting of many irreg-

ular cell clumps. Apparently, these natural flocs

protect them against harmful chemical agents. Under

thermophilic conditions, mostly rodlike or coccoid

hydrogenotrophic methanogens are favoured. Some-

times, thermophilic Methanosarcinae are observed,

but not thermophilic Methanosaetae.

Recent developments in molecular biology tech-

niques today enable more specific determination of

the methanogenic species. Microbial population

selection according to the type of the substrate to be

anaerobically digested, the main reaction pathways

for conversion of different types of biomass substrates


123

to ultimate products, and the potential inhibitory

effects of different compounds on acetotrophic and

hydrogenotrophic methanogens currently seem the

main research themes that should be investigated

more in detail in near future, so that determination and

subsequent manipulation of the appropriate species in

anaerobic digesters for a more stable and satisfactory

process can perhaps be achieved in future.

Acknowledgements The authors would like to express theirgratitude to Lukas Neumann, Olaf Schmidt, Karsten Lehmann

and Monika Unbehauen for their help.

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The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a reviewAbstractIntroductionTheor

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