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Major Pathway of Methane FormationFrom Energy Crops in Agricultural BiogasDigestersBurak Demirel aa Boğaziçi University, Institute of Environmental Sciences , Istanbul ,TurkeyAccepted author version posted online: 01 Mar 2013.Publishedonline: 16 Dec 2013.

To cite this article: Burak Demirel (2014) Major Pathway of Methane Formation From Energy Cropsin Agricultural Biogas Digesters, Critical Reviews in Environmental Science and Technology, 44:3,199-222, DOI: 10.1080/10643389.2012.710452

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Critical Reviews in Environmental Science and Technology, 44:199–222, 2014Copyright © Taylor & Francis Group, LLCISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643389.2012.710452

Major Pathway of Methane Formation FromEnergy Crops in Agricultural Biogas Digesters

BURAK DEMIRELBogazici University, Institute of Environmental Sciences, Istanbul, Turkey

The author reviews the current state of recent microbial commu-nity studies conducted on lab-pilot-full scale agricultural biogasdigesters fed with energy crops operated in mono- and codigestionmodes with or without use of manure as cosubstrate. It is commonlyconcluded by researchers that methane (CH4) formation mostlyresulted from conversion of hydrogen (H2) and carbon dioxide(CO2), rather than aceticlastic methanogenesis. Hydrogenotrophicmethanogenesis seems to be the major pathway for formation ofmethane from energy crops in agricultural biogas digesters.

KEY WORDS: anaerobic digestion, archaea, bacteria, biogas,energy crops

INTRODUCTION

Anaerobic digestion of energy crops has gained significant attention in thelast decade, in order to reduce the greenhouse gas (GHG) emissions, partic-ularly CO2 and CH4, thereby preventing global warming, and to provide asecure and a sustainable source of energy supply.1 The benefits of the anaer-obic digestion process for biogas production have already been summarizedin detail.2,3 Biogas can be used to produce heat and electricity in a combinedheat and power plant (CHP) or as fuel in vehicles or as a substitute of nat-ural gas after being enriched by various techniques commercially available.4

These outstanding advantages have made the anaerobic digestion processan important alternative tool for renewable energy production and such ef-forts have been strongly supported in Europe, particularly in Germany (asthe leading country in biogas technology), through feed-in-tariff policies.5–7

Address correspondence to Burak Demirel, Bogazici University, Institute of Environmen-tal Sciences, Bebek, 34342, Istanbul, Turkey. E-mail: burak.demirel@boun.edu.tr

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FIGURE 1. The stages of the anaerobic digestion process.9 (Color figure available online).

Animal manure, energy crops, agricultural residues, organic fraction of themunicipal solid waste (OFMSW), sewage sludge, and various organic agroin-dustry wastes can all be used as substrates in biogas plants for waste disposaland energy recovery. Among the substrates available for biogas production,energy crops have actually been taken into account since 1980s.8

The biogas production process is complex and sensitive, since severalgroups of microorganisms are involved. The important stages in anaerobicdigestion are hydrolysis, acidogenesis, acetogenesis, and methanogenesis.These steps have been simply illustrated in Figure 1.9 As stated previously,energy crops can be used for feeding anaerobic digesters to produce renew-able energy, and this option is sustainable if it contributes to a net productionof renewable energy and a net reduction of CO2 emissions.10 All types ofbiomass can be employed for biological production of biogas as long as theyare composed of carbohydrates, proteins, fats, cellulose, and hemicellulosesas main components.1 The composition of biogas and methane yields de-pend on the type of biomass, the digestion system and retention time, andonly lignified organic materials, such as wood, are not suitable for anaerobicdecomposition. The net energy yield per hectare makes energy crops favor-able substrates with high biogas yields for the anaerobic digestion process.Biogas yields and methane content of some crops are briefly summarized inTable 1.1 Among the energy crops, particularly maize is reported to present

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TABLE 1. Biogas potential of crops1

Crop Biogas yield (Nm3 tVS–1) Methane content (%)

Sugar beet 730–770 53Fodder beet 750–800 53Maize 560–650 52Grass 530–600 54Sorghum 520–580 55Rye grain 560–780 53Triticale 590–620 54Sun flower 420–540 55Red clover 530–620 56

the best renewable energy productivity and yield.10 In addition, the injec-tion of biomethane produced by monofermentation of crops into the naturalgas grid has a lower contribution to climate change than that of naturalgas importation.11 Detailed consideration of GHG mitigation and energy bal-ance both for crop growth and utilization suggest that perennial crops arefavored over annual crops, where energy balances may be poor.12 In thisstudy, energy crops are solely on focus; therefore, the microbial ecology anddegradation pathways of lignocellulosic materials are not discussed.

A considerable amount of recent literature reports process optimizationand monitoring of laboratory-, pilot-, and full-scale agricultural biogas di-gesters fed with energy crops with or without cosubstrate addition (e.g., ma-nure), such as alfalfa silage,13 sugar beet silage/tops/leaves,14–20 grass/cloversilage,21–25 switchgrass,26 maize,27–30 fodder beet silage,31–32 various grassspecies (cocksfoot, tall fescue, reed canary grass, timothy),33 and hemp.34

The objectives of these studies were mostly aimed at improving biogas andmethane production yields per amount of substrate fed to the digesters. Inaddition, it is well known and documented that the response of the microbialcommunity to the variations in operational (hydraulic retention time [HRT],organic loading rate [OLR]) and environmental parameters (pH, tempera-ture, availability of nutrients, mixing, VFAs, and ammonium concentrations)play a very significant role in establishing a stable and safe biogas digesteroperation, which is actually correlated to establishing a balanced microbialcommunity within the digester.3 Numerous studies investigated the microbialcommunity of anaerobic digesters operated with sewage sludge, manure,biosolids, food waste, and municipal solid waste (MSW) for the last twodecades. For instance, the microbial community structure of anaerobic di-gesters treating solid waste and sewage sludge were analyzed using oligonu-cleotide probe hybridization, and Methanosarcina and Methanosaeta wereobserved to be the most dominant methanogenic species in lab- (3 l volume)and full-scale digesters.35 Methanogenic population dynamics of laboratory-scale anaerobic digesters of 5l volume operated both at mesophilic (37◦C)and thermophilic (55◦C) temperatures were investigated during the start-up

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phase using ribosomal RNA-targeted oligonucleotide probes.36 The digesterswere fed with a mixture of simulated municipal solid waste and biosolids,and were operated at an OLR of 3.1 kg VS m–3 day–1 with a retention timeof 20 days. As a result of increase and decrease in the concentrations ofthe VFAs, large fluctuations were observed in the methanogenic populationlevels. Methanosaeta species were the most abundant in the inoculum, whiletheir levels decreased as the acetate accumulated. Methanosarcina specieswere observed with respect to increase in acetate levels. Methanobacte-riaceae were the most abundant hydrogenotrophic methanogens both inmesophilic and thermophilic digesters. In another previous study, onlyMethanosarcina spp. was identified in biogas reactors operated with ma-nure or mixture of manure with solid organic wastes.37 Influence of op-erational parameters on the microbial community of 15 different full-scalebiogas digesters receiving manure or sludge as substrates have already beenstudied by using the fluorescence in situ hybridization (FISH) technique.38

The volumes of the digesters varied between 945 and 7000 m3, and HRTchanged between 15 and 30 days. It was observed that the manure di-gesters, which contained high levels of ammonia and VFAs, were dominatedby the members of the Methanosarcinaceae, while sludge digesters withlow levels of ammonia and VFA were dominated by the members of theMethanosaetaceae. Full-scale biogas plants treating manure and food wasteswere analyzed using the FISH technique and it was found that acetate oxi-dation was the main pathway for methanogenesis when Methanosaetaceaewas not present.39 Only in the presence of Methanosaetaceae, the aceti-clastic methanogenesis was observed. The microbial community of a ther-mophilic (55◦C) biogas plant handling municipal solid waste, agriculturalresidues, liquid manure, and biowastes was investigated using polymerasechain reaction-DGGE for a period of more than two years.40 The Archaeawere reported to consist of Methanobrevibacter sp., Methanoculleus bour-gensis, Methanosphaera stadtmanae, and Methanomicrococcus blaticolla.Methanosarcinales and Methanomicrobiales were reported to dominate theArchaea community in anaerobic digesters fed with sludge.41

These studies summarized briefly previously all covered the investiga-tion of the biogas digesters receiving substrates such as manure, sludge,MSW, and biosolids. Recently, the investigation of the microbial commu-nity within agricultural biogas digesters has gained more attention. Thus,employment of advanced molecular biology techniques plays an importantrole in understanding and clarifying the complex reactions taking place inbiogas digesters.42 Here I review the current trends in understanding of themicrobial ecology within the agricultural biogas digesters operated with en-ergy crops in mono- and codigestion modes for continuous production ofrenewable energy. Various oligonucleotide probes/primers targeting phylo-genetic markers of methanogens, such as 16S rRNA and the gene for theα-subunit of methyl coenzyme M reductase (mcrA), have been extensively

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developed and experimentally characterized in order to identify and quan-tify the methanogenic Archaea. These oligonucleotides were designed toresolve different groups of methanogens at different taxonomic levels, andhave been widely used as hybridization probes or polymerase chain reaction(PCR) primers for membrane hybridization, fluorescence in situ hybridiza-tion, rRNA cleavage method, gene cloning, DNA microarray, and quantitativepolymerase chain reaction for studies in environmental and determinativemicrobiology.43 However, the scope of this article does not cover a com-parison of the benefits and disadvantages of the various molecular biologytechniques that are commonly used for identification of the microorganismsin agricultural biogas digesters. The main objective is to inform the mainpathways of methane production from renewable biomass, particularly fromenergy crops, by making use of the recent data obtained using advancedmolecular biology techniques and to identify the future research needs inthis particular topic.

MICROBIAL ECOLOGY OF BIOGAS DIGESTERS DURINGMONO-DIGESTION OF ENERGY CROPS

Monodigestion of energy crops without use of manure is a recent trendas the numbers of biogas plants operating without manure are increasing,especially in Germany. More research attention has recently been directedtoward anaerobic monodigestion of energy crops for energy production aswell as the investigation of microorganisms in such systems. A classificationof the microorganims discussed in this article is given in Table 2.42 An overallsummary of recent research studies about the microbial ecology of biogasdigesters for monodigestion of energy crops is also given in Table 3. A moredetailed information about these studies will also be presented in this sectionof the article.

Influence of Operational Parameters

Adjustment of HRT plays a very important role on the overall performanceof the anaerobic digestion process.44 Several studies have recently beenconducted to investigate the effect of HRT on the microbial ecology of thebiogas digesters fed with monoenergy crops using fluorescence microscopy.A study was carried out in order to investigate the mono-fermentationof acidic, low-buffered sugar beet silage in a mesophilic laboratory-scalebiogas digester with a volume of 6l focusing on both process optimizationand identification of the methanogenic community in the digester.45 Afterrunning the reactor for more than six months, it was observed that thechange of HRT from 25 to 15 days did not seem to affect the morphology ofthe methanogens significantly. However, the numbers of the methanogens

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TABLE 2. General characteristics of some methanogens42

Cell Optimal Optimumwidth/(μm) temperature pH

Morphology length (μm) Substrate (◦C) range

Methanobacteriumbryantii

Long rods tofilaments

0.5–1.0/1.5 H2/CO2 37 6.9–7.2

Methanobacteriumformicicum

Long rods tofilaments

0.4–0.8/2–15 H2/CO2,formate

37–45 6.6–7.8

Methanothermobacterwolfeii

Rods 0.4/2.4–2.7 H2/CO2 55–65 7.0–7.5

Methanococcus voltaei Regular toirregularcocci

1.5 (diameter) H2/CO2,formate

35–40 6.0–7.0

Methanosarcinathermophila

Irregularcocci,formingaggregates

— H2/CO2,methanol,methy-lamines,acetate

50 6.0-.70

Methanosarcinabarkeri

Irregularcocci,formingpackets

— H2/CO2,methanol,methy-lamines,acetate

35–40 5.0–7.0

Methanospirillumhungatei

Regularcurved rodsto longspiralfilaments

0.5/7.4 H2/CO2,formate

30–40 —

Methanobrevibactersmithii

Short rods,short chains

0.6–0.7/1.0–1.5 H2/CO2,formate

37–39 —

Methanosarcinaacetivorans

Irregularcocci

- Methanol,acetate

35–40 6.5

Methanosarcinamazeii

Irregularcocci,formingcysts andpackets

- Methanol,methy-lamines,acetate

30–40 6.0–7.0

Methanosaeta concilii(soehngenii)

Rod 0.8 × 2.5–6.0(dimensions)

Acetate 35–40 7.0–7.5

Methanothermobacterthermoautotrophicum

Long rods tofilaments

0.3–0.6/2–7 H2/CO2 65–70 7.0–8.0

Methanococcusvannielii

Regular toirregularcocci

1.3 (diameter) H2/CO2,

formate65 7.0–9.0

was adversely affected by variation in HRT. The specific biogas productionrate also decreased from 0.72 l g VS–1 day–1 at 25 days of HRT to 0.54l g VS–1 day–1 at 15 days of HRT. This decrease in the specific biogasproduction rate could be attributed to the decrease in the numbers of themethanogens in the digester. In a subsequent study, the effect of fuzzy logiccontrol (FLC) technique was investigated on the reactor performance and

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TABLE 3. Summary of literature on the microbial ecology of monodigestion of energy cropsin biogas digesters

Key MolecularTemperature Digester Application organisms technique

Substrate (◦C) volume(l) status present applied Reference

Fodder/sugarbeet silage

41 6 Lab-scale MethanobacterialesMethanomicrobiales

[19]

Beet silage 41 6 Lab-scale MethanobacterialesMethanomicrobiales

FISH,ribosomalRNA generestrictionanalysis

[48]

Sugar beettops &barleycrops

12–33 Lab-scale MethanomicrobialesMethanosarcinaceaeMethanosaeta

FISH [15]

Beet silage 55 6 Lab-scale Methanobacteriales ARDRA,FISH

[54]

Maize silage Meso/Thermo

Methanomicrobiales(Meso)Methanobacteriales(Thermo)

PCR primertargetingmcrA/mrtA

[52]

Maize silage Batch-scale

Methanoculleus sp. [61]

Maize silage Meso 28–30 Lab-scale MethanobacterialesMethanosarcinaceae

[63]

Fodder beetsilage

Meso 8 Lab-scale MethanobacterialesMethanosarcinaceaeMethanosaetaceae

ARDRA [67]

Triticalesilage

Meso 70 Lab-scale Methanosarcinales ARDRA,Real-timePCR

[68]

microbial community of the mesophilic laboratory-scale biogas digester of a6 liters volume fed with sugar beet silage as the mono-substrate.46 Throughemployment of the FLC technique, it was possible to operate the biogasdigester within a HRT range of 8–25 days, and up to an OLR of 7.41 g VSl–1 day–1. In spite of a high loading rate and a HRT of 8–10 days, use ofFLC technique for reactor operation enabled doubling of the number ofthe methanogens. The findings of the microbiological studies carried outon laboratory-scale biogas digesters with 6 l of volume operated using theFLC technique and fed with fodder or sugar beet silages as mono-substrateswere also reported in another recent study.47 The authors concluded that thehydrogenotrophic methanogens using H2+CO2 (Methanobacteriales andMethanomicrobiales) dominated during anaerobic digestion of renewablebiomass, while acetotrophic methanogens (Methanosarcinales) representedonly a minority of the population. A laboratory-scale experiment usingdigesters of 6 l volume was carried out in order to investigate the effect ofHRT on the diversity of methanogens during mesophilic anaerobic digestionof beet silage using ribosomal RNA gene restriction analysis, FISH and

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fluorescence microscopy.48 The authors reported that the hydrogenotrophicmethanogens Methanobacteriales and Methanomicrobiales dominatedthe population. The methanogenic population dynamics of a mesophiliclaboratory-scale biogas digester fed with maize silage was studied duringoverloading and acidification using 16S rDNA-based real-time PCR.49 AtOLR levels above 4.1 g organic dry matter (ODM) l–1 day–1, the aceticlasticmethanogen Methanosaetaceae disappeared along with a dramatic increasein the propionic acid concentration in the digester. Based on these findings,the authors concluded that the absence of Methanosaetaceae might be usedas an indicator of process instability. In a more recent laboratory-scale study,the impacts of HRT and OLR on the diversity of bacteria were reported.50

As persistent groups, Chloroflexi and Clostridia were detected in the biogasdigester.

Influence of Environmental Parameters

The impact of temperature on methanogenesis has been previously reportedin literature.51 The population of thermophilic and mesophilic biogas di-gesters fed with maize silage as monosubstrate was analyzed using a novel,highly degenerated PCR-primer pair targeting mcrA/mrtA coding for the keyenzyme of methanogens.52 In thermophilic digesters, which had a volumeof 28 l, Methanobacteriales were the most important methanogens detected,while Methanomicrobiales dominated in the mesophilic reactor. The authorsconcluded that the hydrogenotrophic methanogens seemed to have a muchmore important role than the aceticlastic methanogens during anaerobic con-version of renewable biomass to methane. Even though the substrates weredifferent, the same conclusion has also been reached in another recent workas a result of the investigation of the diversity of the microbial communityin a mesophilic biogas digester fed with swine feces as monosubstrate usingthe mcrA analysis.53 The temperature decrease from 60 to 55◦C was reportedto influence the morphology of the methanogens during anaerobic digestionof beet silage in a lab-scale study conducted with reactors having a volumeof 6 l.54 The amplified rDNA restriction analysis (ARDRA) and FISH resultsalso indicated that the hydrogenotrophic Methanobacteriales dominated inthe thermophilic biogas digester.

Krakat et al.55 reported that the methanogenesis of energy crops wasinitiated by a conversion to H2+CO2 involving bacteria, which was thenfollowed by a conversion to CH4+CO2 mainly carried out by the hy-drogenotrophic methanogens. The authors concluded that running the bio-gas digesters within the thermophilic temperature range of 55–60◦C, insteadof the commonly employed mesophilic range, may be an effective measurein order to increase the stability of the anaerobic digestion process by ex-cluding the acetotrophic methanogens that are more susceptible to stress

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conditions such as high ammonium and hydrogen sulfide (H2S) concentra-tions. At temperatures between 55 and 60◦C, Methanosaeta seemed to becompletely absent.

Changes in VFA levels within an anaerobic digester were shown to bea good parameter to indicate process stability.56 In a previous study, the dy-namics of bacterial and archaeal populations in a lab-scale anaerobic digesterfed with glucose as the sole source of substrate were investigated.57 The di-gester had a volume of 10 l, and it was operated at 36◦C and pH of 7.2. Thechanges in acetate levels affected the archaeal population shift significantly.It was also reported that the archaeal community structure in a thermophilicanaerobic digester was closely correlated with the VFA concentration, whilethe bacterial population was influenced by pH.58 Regarding the changes inVFA profiles and their potential impacts on the microbial community withinagricultural biogas digesters fed with energy crops without manure addition,no study was encountered in the literature, even though the concentrationlevels of VFAs play a significant role on microbial population shifts.

These studies indicate that, among the environmental parameters, tem-perature and pH (with regard to VFA concentration) can influence the micro-bial shifts in biogas digesters significantly. Particularly, thermophilic opera-tions tend to exclude aceticlastic methanogens and prevail hydrogenotrophicmethanogens.

Influence of Trace Metals

Trace element requirements of agricultural biogas digesters fed with mono-substrates without manure addition and the impacts of trace elements onthe microbial community in such digesters is another area, where furtherresearch activity is required. It is already known that various methanogensneed iron (Fe), nickel (Ni), cobalt (Co), molybdenum (Mo), selenium (Se),and tungsten (W), so that they can carry out their metabolic activities, whichis essential for a stable digester performance to achieve high biogas andmethane yields. Supplementation of trace metals can be of paramount im-portance during anaerobic digestion of energy crops lacking availability ofnutrients, such as sugar beet silage. More details about the effects of tracemetals on agricultural biogas digesters can be found elsewhere.59,60

Among the studies conducted regarding the influence of trace metals onthe microbial ecology of biogas digesters running with monoenergy crops,addition of Ni was reported to increase methane formation by up to 20% dur-ing anaerobic batch fermentation of a synthetic model substrate representingmaize silage and the hydrogenotrophic Methanoculleus sp. were detected tobe the dominant genus using ARDRA.61 In a later study, the impacts of Coand Ni addition on biogas production of maize silage was also investigated,however, there was no microbiological investigation conducted.62

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Methanomicrobiales were reported to be dominating during acid-ification of mesophilic biogas digesters fed with maize silage as amonosubstrate.63 Without acidification conditions, Methanobacteriales andMethanosarcinaceae were reported to be dominating. It was also concludedby the authors that without addition of manure, primarily Co and Na can be-come limiting during long-term anaerobic digestion of maize silage leadingto process acidification. The authors reported the threshold concentrationsof Co and Na as 0.03 and 10 mg l–1, respectively, without supplementation.

Investigation of the Microbial Community During the HydrolysisStage

The microbial ecology of biogas digesters during hydrolysis has particu-larly been investigated in detail, since hydrolysis can be the rate limitingstep in the overall process when a complex type of substrate is used.Mesophilic laboratory-scale two-phase anaerobic digestion system fed withsugar beet and grass/clover silage was operated in order to analyze therole of microbial community responsible for the conversion process usingthe FISH technique.64 The volumes of hydrolysis and methanogenic reactorswere 0.75 and 1.5 l, respectively. The Archaea appeared in the hydrolyticstage and their presence was due to the inoculation from the methanogenicreactor. The microbial populations developed in the hydrolytic stages ofsugar beet and grass/clover silage were not very similar as expected due tothe different characteristics of the substrates (e.g., buffering capacity, avail-ability of nutrients) that would eventually influence the microbial commu-nity. The investigation of the microbial community in the hydrolysis stageof plant biomass digestion has also been conducted in another work.65

It was reported that, during investigation of the composition of the ther-mophilic bacterial consortium, cultures of Thermocellum and Stercorariumwere determined to be good degraders of maize silage. The developmentof the microbial community during the hydrolysis phase of a two-phaseanaerobic digestion system fed with grass silage was evaluated and it wasreported that the Clostridium-like species clearly dominated the bacterialcommunity.66

The Role of Hydrogenotrophic Methanogens

In addition to the studies reported above, an analysis of the biogas pro-cess within a 8 l volume laboratory-scale mesophilic continuously stirredtank reactor (CSTR) fed with fodder beet silage as mono-substrate wasconducted through constructing a 16S rDNA library and analyzing the 16SrDNA library by ARDRA.67 The authors reported that the major bacterialgroups represented in the clone library belonged to the class Clostridiaof the phylum Firmicutes, the class Deltaproteobacteria of the phylum

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Major Pathway of Methane Formation 209

Proteobacteria, the class Bacilli of the phylum Firmicutes, and mem-bers of the phylum Bacteroidetes. Within the domain Archaea, all of theclones were affiliated to the phylum Euryarchaeota, and all clones wereclosely related to the methanogenic species. Molecular analysis revealedthe presence of H2/CO2/formate-oxidizing Methanobacteriales, the H2/CO2-oxidizing Methanosarcinaceae or the acetate-splitting Methanosaetaceae.

The methanogenic microbial community of a mesophilic laboratory-scale two-phase anaerobic digestion system fed with triticale silage as the solesubstrate using ARDRA and real-time PCR was also studied.68 The reactorshad a volume of 70 l and they were operated at 38 ± 1◦C. The authorsreported that the most abundant operational taxonomic units (OTUs) withinthe 16S rDNA libraries derived from the laboratory system belonged to theorder Methanosarcinales.

An important finding of these recent works summarized above is that themethanogenesis of renewable biomass, namely energy crops, was initiatedby a conversion to H2+CO2 involving bacteria, which was then followedby a conversion to CH4+CO2 mainly carried out by the hydrogenotrophicmethanogens.55 Different types of substrates such as fodder an sugar beetsilage, maize silage, and triticale silage were used, and the common conclu-sion reached by the authors agreed with each other, all stating the dominanceof the hydrogenotrophic methanogens during methane formation stage inbiogas digesters fed with monoenergy crops. However, previous studies re-ported that the main pathway of methane formation was the aceticlasticmethanogenesis during anaerobic digestion of animal waste.69 About 70% ofthe methane produced in anaerobic digestion was reported to originate fromacetate by aceticlastic methanogenesis.70–72

When these recent studies covering the anaerobic digestion of energycrops as monosubstrate are evaluated as a whole, it is easily observed thatthere has been an increase in the amount of experimental work conductedparticularly on the investigation of the microbial community present in theagricultural biogas digesters fed with energy crops using several advancedmolecular biology techniques such as FISH, 16S rDNA-based quantitativereal-time PCR, and ARDRA. These activities helped the scientists to betterunderstand the pathway of methane formation in biomass digesters. How-ever, these studies mostly covered the operation of laboratory-scale biogasdigesters and data covering the microbial ecology of pilot- and full-scalecommercial biogas plants fed with monoenergy crops without manure arescarce. Therefore, in order to fill this gap in current knowledge, microbialecology of pilot- and full-scale digesters must also be studied using molecu-lar biology techniques available. These molecular tools may be sufficient todetermine the major pathway of methane production in pilot- and full-scaledigesters. In addition, biogas plant operators need more practical informa-tion about how they can benefit from understanding the main pathway ofmethane formation from energy crops during monodigestion. More research

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attention should be directed toward the microbial investigation of pilot-and full-scale commercial agricultural biogas digesters operated with energycrops as monosubstrate without any cosubstrate addition.

MICROBIAL ECOLOGY OF BIOGAS DIGESTERS DURINGCODIGESTION OF ENERGY CROPS

Manure is the most common substrate used in anaerobic codigestion of en-ergy crops.27,73–76 Manure is also used for codigestion of organic wastesin centralized biogas plants, for instance in biogas plants operated inDenmark.77 Although many agricultural biogas plants use manure, until re-cently not much research attention has been directed toward studying themicrobial ecology of such agricultural biogas digesters fed with renewablebiomass. An overall summary of recent research studies about the micro-bial ecology of biogas digesters for codigestion of energy crops is given inTable 4.

Influence of Operational Parameters

A molecular study of the archaeal community of a full-scale commercial agri-cultural biogas reactor fed with cattle liquid manure and maize silage wasconducted using 16S rDNA and mcrA analyses.78 The biogas digester, whichhad a volume of 2330 m3, was operated at a mesophilic temperature range(approximately at 39.5◦C) and pH of 7.8. The HRT and OLR were 54 daysand 2.86 kg m–3day–1, respectively. According to the findings reported bythe authors, Methanomicrobiales dominated the methanogenic population.The authors also concluded that the methane formation mostly resulted fromconversion of H2+CO2 and the hydrogenotrophic methanogenesis seemedto be the major pathway for formation of methane. In a similar work, thesamples were taken from a full-scale agricultural biogas plant fed continu-ously with a mixture of maize (63%), green rye (35%), and chicken manure(2%).79 The digester was operated at temperature and pH levels of 41◦C and7.7, respectively. The authors observed that the Clostridiales from the phylumFirmicutes were the most prevalent phylogenetic order, while Methanomi-crobiales were dominant among the Archaea. It was concluded that thebiomethane production was carried out via the hydrogenotrophic pathway.Schluter et al.80 also conducted a study to investigate the composition andgene content of biogas digester of a full-scale biogas plant fed with renew-able biomass (maize silage + green rye + chicken manure) using a metage-nomic approach applying the ultrafast 454-pyrosequencing technology. Thebiogas digester was operated within a HRT range of 40–60 days, and thetemperature and pH were 41◦C and 7.7, respectively. It was reported by theauthors that the species related to those of the genus Methanoculleus played

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TABLE 4. Summary of literature on the microbial ecology of codigestion of energy crops inbiogas digesters

Digester Key MolecularTemperature volume Application organisms technique

Substrate (◦C) (m3) status present applied Reference

Cattle manure +maize silage

39.5 2330 Full-scale Methanomicrobiales 16S rDNA,mcrA

[78]

Maize silage +green rye +manure

41 Full-scale MethanomicrobialesClostridiales

[79]

Maize silage +green rye +manure

41 Full-scale Methanoculleus 454-pyroseq-uencing

[80]

Maize silage +green rye +manure

41 Full-scale Methanomicrobiales 454-pyroseq-uencing

[81]

Grass silage +cow manure

35 Lab-scale Bacteriodetes 16S rRNAgene-basedfinger-prints

[82]

Manure + maizesilage + cattledung + grasssilage

Meso Full-scale MethanomicrobialesMethanosaetaceae

FISH,real-timePCR

[85]

Manure + maizesilage +grains

Meso Full-scale Methanomicrobiaceae PCR-RFLP,real-timePCR

[89]

Manure Meso 600 Full-scale MethanoculleusbourgensisMethanosarcinabarkeriMethanospirillumhungatei

DGGE, 16SrDNA se-quencing

[87]

Manure + grasssilage + oatstraw + sugarbeet tops

35 Lab-scale Methanobacterium 16S rRNAgene-based

[88]

a dominant role in methanogenesis. According to Krober et al.,81 the abun-dance of the phyla Firmicutes, Bacteroidetes, and Euryarchaeota, and theorders Clostridiales, Bacteroidales, and Methanomicrobiales were detectedin samples obtained from a full-scale biogas digester fed with a mixture ofrenewable biomass. The digester was fed with maize silage, green rye, andliquid manure, and it was operated within the mesophilic temperature range.

The impacts of feeding ratio and loading rate on the microbial com-munity during mesophilic codigestion of grass silage with cow manure inan anaerobic laboratory semi-continuous CSTR were investigated using 16SrRNA gene-based fingerprints and clone libraries.82 The HRT and tempera-ture were 20 days and 35◦C, respectively. The major groups represented indigesters were Clostridia and Bacteroidetes

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Influence of Environmental Parameters

There exists very little information about the impacts of changes in en-vironmental parameters on the microbial ecology of biogas digesters fedwith manure and energy crops. Ammonia may play an important role inanaerobic digestion processes, particularly during biogasification of manureand animal wastes.83,84 The free ammonia concentration causes problems,depending on the total ammonia concentration, pH, and temperature. Par-ticularly, when manure is used as cosubstrate in agricultural biogas plants,which are operated within the thermophilic range, ammonia concentrationsshould be carefully monitored by the biogas plant operators.

The microbial community of six different mesophilic full-scale agricul-tural biogas plants receiving renewable biomass (maize silage, grass silage)and liquid manure (pig manure, cattle manure) as substrates were investi-gated using the FISH technique and real-time PCR.85 Among the six reac-tors analyzed, five of the reactors were dominated by the hydrogenotrophicMethanomicrobiales, while only one reactor was dominated by the aceticlas-tic Methanosaetaceae. This difference might have been attributed to digesteroperational (e.g., HRT) and/or environmental (pH, temperature, VFA, andammonia concentrations in the digester) parameters. The authors also ob-served a correlation between the absence of Methanosaetaceae and highconcentrations of total ammonia (NH3) in biogas reactors. The calculatedNH3 concentrations ranged between 0.37 and 1.15 g l–1 resulting in the ab-sence of Methanosaetaceae in three of the analyzed biogas plants. It waspreviously reported that the members of the Methanosaetaceae dominatedthe anaerobic biogas reactors fed with sludge that contained low levels ofammonia.86 The aceticlastic methanogens seem to favor low ammonia con-centrations in biogas digesters. This finding was later confirmed in anotherstudy.37 According to Angenent et al.,86 at a total ammonia concentrationof around 3600 g m–3, the levels of the acetate-utilizing methanogens ofthe genus Methanosarcina decreased, while the order Methanomicrobialesincreased. Therefore the authors concluded that at high ammonia levels,the hydrogen-utilizing methanogens played an important role for methaneproduction. This observation actually agrees with the hypothesis that themanure containing biogas digesters have higher ammonia levels leading tothe dominance of hydrogenotrophic methanogens.

The Role of Hydrogenotrophic Methanogens

In addition to the studies reported previously, Liu et al.87 investigatedthe structure of bacterial and archaeal community of a mesophilic full-scale biogas digester with a volume of 600 m3 using DGGE and 16SrDNA sequencing analyses. It was reported by the authors that most bac-terial species in the digester belonged to Firmicutes, Bacteroides, and

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Major Pathway of Methane Formation 213

Spirochaetes, and the most methanogens were typical hydrogenotrophic orhydrogenotrophic/aceticlastic. Methanoculleusbourgensis, Methanosarcinabarkeri, and Methanospirillum hungatei were the dominant methanogensdetected. Wang et al.88 investigated the bacterial communities in mesophilic(35◦C) two-phase CSTRs codigesting cow manure with grass silage, oat straw,and sugar beet tops, using 16S rRNA gene-based fingerprints and clone li-braries. The hydrolytic reactors had a volume of 1 l. The major membersof the phyla represented in the reactors were Clostridia and Bacteroidetes.Phylotypes affiliated with Bacilli or Deltaproteobacteria were unique to thesugar beet and straw reactors, respectively. It was also reported that the ma-jority of the Archaea fell within the hydrogenotrophic genus Methanobac-terium. Bergmann et al.90 studied the microbial community of a full-scalemesophilic biogas digester fed with pig manure, maize silage and grainsthrough constructing of a methanogenic Archaea specific 16S rRNA geneclone library combined with PCR-RFLP analysis and group-specific real-timePCR. The authors reported that the hydrogenotrophic Methanomicrobiaceaewere predominant in the biogas digester.

The findings of these studies based on the samples obtained mostlyfrom commercial full-scale agricultural biogas plants fed with manure andvarious types of energy crops indicated that the methane formation mostlyresulted from conversion of H2+CO2, and the hydrogenotrophic methano-genesis seemed to be the major pathway for formation of methane, ratherthan the aceticlastic methanogenesis. This conclusion is in agreement withthe results reported both by Scherer et al.47 and Krakat et al.,55 contradictingthe common theory that the acetate is the primary substrate for methanogen-esis in anaerobic treatment systems.72,90–91 However, Lubken et al.92 reportedthe application and modification of ADM1 to simulate energy production ofthe digestion of cattle manure and renewable energy crops, and the au-thors concluded that the simulation of biogas production and compositionwas possible by using the ADM1, a contradictory assumption to the find-ings of the studies that have been reviewed previously. The ADM1 wasreported to focus on sewage sludge, while other substrates such as agrowastes were not taken into account.93 Monodigestion of grass silage with-out manure addition was carried out using lab-scale biogas digesters op-erated at 38◦C and a simulation study was conducted using the ADM1.94

The authors concluded that the model could be used to model anaerobicdigestion of grass silage. Finally, a modified ADM1 was employed to modelthe anaerobic digestion of cow manure, corn silage, grass silage and rapeoil.95 The model was reported to show a good performance on predictingthe stable anaerobic digestion of these agricultural resources. These mod-eling studies all agreed that the ADM1 could be used to model anaerobicdigestion of agricultural resources (renewable biomass) for production ofbiogas.

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ADVANCED MOLECULAR BIOLOGY TECHNIQUES

It seems obvious that the advanced molecular biology techniques will helpthe researchers to explain the complex behavior of the microbial communityin anaerobic biogas digesters fed with renewable biomass. Improvementsto overcome the difficulties associated in application of the molecular biol-ogy techniques for investigating the microbial ecology of agricultural biogasdigesters are also underway. DNA-SIP (stable isotope probing) was con-ducted on anaerobic MSW samples incubated with 13C-cellulose, 13C-glucose,and 13C-acetate under mesophilic conditions.96 In the clone libraries derivedfrom DNA fractions, the most abundant sequences were affiliated with thephyla Firmicutes, Bacteroidetes, the gamma-subclass of Proteobacteria, andmethanogenic orders Methanomicrobiales and Methanosarcinales. The au-thors stated that the combination of DNA-SIP and FISH applied with a se-ries of functionally connected substrates can shed light on the networks ofuncultured microbes catalyzing the methanization. In a recent study, themicrobial community of a CSTR fed with garbage slurry was investigated us-ing 13C-labeled acetate analysis.97 The authors reported that around 80% ofmethane formation resulted from the nonaceticlastic methanogenic pathway.The 16S rRNA analyses also showed that the hydrogenotrophic methanogensMethanoculleus sp. dominated, while the aceticlastic Methanosarcina sp.were minor. Even though the substrates were not energy crops, but anotherrenewable biomass, namely solid waste, these two studies also confirmedthat the hydrogenotrophic methanogens played the significant role duringconversion of renewable biomass to methane.

Furthermore, Franke-Whittle et al.98 reported about a microarray tar-geting methanogens found in anaerobic digesters, and to apply this chiptogether with a cloning approach to investigate the methanogenic commu-nity present in an anaerobic digester. Bergmann et al.99 proposed a com-bined lysozyme/SDS-based cell lysis followed by a purification step withsephacryl columns for extraction of high amounts of microbial DNA withhigh purity from samples of biogas plants, in order to reduce the effectsof PCR-interfering substances when quantitative PCR was applied to detectmethanogenic Archaea in agricultural biogas fermenters.

CONCLUSIONS

Because Bacteria and Archaea carry out the anaerobic conversion of re-newable biomass, namely energy crops such as maize and sugar beet intomethane, it is very crucial to understand how the microbes perform withinthe biogas digester to accomplish this task. Application of advanced molecu-lar biology techniques will play an important role in understanding the mainreaction pathways during conversion of renewable biomass to methane. The

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Major Pathway of Methane Formation 215

common theory regarding the main pathway for methane formation in anaer-obic systems treating simple-soluble types of substrates, various types of in-dustrial wastewaters and sewage sludge was the aceticlastic methanogenesis,which contributed to 70% of methane formation. However, for anaerobic di-gestion of renewable biomass, with or without manure addition, the recentmicrobiological studies report that the methane formation mostly results fromconversion of H2+CO2 and the hydrogenotrophic methanogenesis seems tobe the major pathway for formation of methane. There is still further researchrequired, at lab-, pilot-, and full-scale applications, in order to identify the im-pacts of changes in significant operational variables such as pH, temperature,VFA, and ammonia concentration profiles on the microbial community shiftsin agricultural biogas digesters running with energy crops with or withoutmanure addition. Even though I did not cover a comparison of molecularbiology techniques currently available, it should be mentioned that moreresearch attention must be directed toward developing more rapid and sim-ple techniques for microbial detection, identification and quantification. Themicrobial ecology and degradation pathways of lignocellulosic materials alsoneed to be studied in more detail in the future.

ACKNOWLEDGMENT

The author wishes to thank to Bogazici University Research Fund for supportby project numbers 10Y00P3, 12Y00P1, and 10Y00D9.

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