Introduction

Aims Methods

Results

Conclusions and future plans

Biogas is a promising renewable energy source due to its methane content. Methane itself

seems to have high energy and heating potential. Its heating value was estimated as

higher (55.5 MJ/kg) compared to diesel and wood (Fountoulakis and Manios 2009).

Another advantage of biogas production is the possibility of the use of many different

organic wastes as a substrate in the process (Amon et al. 2007; Li et al. 2011).

Biogas is produced in the anaerobic digestion process that consists of 4 stages:

hydrolysis, acidogenesis, acetogenesis and methanogenesis (fig. 1) (Li et al. 2011). Each

stage is conducted by specific microbial community able to interact with each other in

syntrophic manner (Weiland 2010; Worm et al. 2010). The last step can be carried out by

hydrogenotrophic (CH4 production from H2 and CO2) or acetotrophic (CH4 production from

acetate) methanogens (Garcia et al. 2000). It has been already proved that there is

syntrophy between some particular methanogens and some groups of bacteria (Stams et

al. 1992).

Polymers (lipids, proteins,

polysaccharides)

Mono- and oligomers (long

chain fatty acids, sugars, amino

acids)

Volatile fatty acids

Short-chain volatile fatty

acids

H2 and CO2 Acetate

Biogas –

mainly

methane

H2S H2 and

ammonia

The development of robust technique

(targeting the biomarker – gene of candidate division

bacteria) to monitor methane yields in

anaerobic digesters

The investigation of possible syntrophy between candidate division hydrogen-producing bacteria

and hydrogenotrophic methanogens and its influence on methane

yields by using metagenomic tools

The study on the possible shifts in

microbial community as a result of various

reactor performance – especially in terms of

feedstock

Collection of a sample

– high methane-

yielded two-stage

pilot-scale mesophilic

anaerobic digester

(AFBI, Hillsborough);

from some lab-scale

digesters

DNA

extraction –

Power Soil

DNA Isolation

Kit (Thermo

Scientific)

454-pyrosequencing (bacterial and

archaeal 16S investigation) and

processing results in QIIME

454-pyrosequencing of the sample revealed that the most abundant

group of bacteria was Firmicutes (especially order Clostridiales). The most

dominant Archaea were Euryarchaeota (especially order

Methanosarcinales). More details on fig. 2 and fig. 3.

qPCR optimization for primers mcrA_1035F and mcrA_1538R

was done (SYBR Green approach; fig. 4). Optimization of qPCR

of some sets of primers (targeting different groups of

methanogens) is conducted (TaqMan probes usage) (Steinberg

and Regan 2009) (tab. 1.; fig. 5).

Lab-scale 1 L digesters are being designed and constructed at

the moment (fig. 6).

Joanna Grebosz, Michael Larkin, Christopher Allen, Leonid Kulakov; Queen’s University Belfast, School of Biological Sciences

97 Lisburn Road, Belfast BT9 7BL , E-mail address: jgrebosz01@qub.ac.uk

k_Bacteria;p_WWE1

k_Archaea;p_Euryarchaeota;c_Methanobacteria;

o_Methanobacteriales;f_Methanobacteriaceae;

g_Methanobrevibacter k_Bacteria;p_Bacteroidetes

k_Bacteria;p_Fibrobacteres

k_Bacteria;p_Firmicutes

k_Bacteria;p_Proteobacteria

k_Archaea;p_Crenarchaeota;c_MCG;o_pGrfC26

k_Archaea;p_Euryarchaeota;c_Methanobacteria;

o_Methanobacteriales;f_Methanobacteriaceae;

g_Methanobacterium

k_Archaea;p_Euryarchaeota;c_Methanomicrobia;

o_Methanosarcinales;f_Methanosarcinaceae;

g_Methanosarcina

Fig. 2. The chart showing the

abundancy of bacterial phyla in the

sample from anaerobic digester

Fig. 3. Pie chart indicating the proportion between

different species of Archaea in anaerobic digester

qPCR programme:

95°C 10min

95°C 40sec

56°C 30sec

72°C 30sec

36

cycles

qPCR MIX:

•0.2 µM of each primer

•5 µL 2xSYBR Green/ROX

MIX (Thermo Scientific)

•1 µL DNA

•H2O up to 10 µL

Fig. 4. The optimized qPCR programme and mix for

mcrA_1035 and mcrA_1538 primers (Pereyra et al. 2010)

The pyrosequencing results indicate that the most abundant Archaea in the AFBI digester is Methanosarcina which is classified as aceticlastic methanogen group.

The predominance of such methanogens with simultaneous absence of particular candidate division bacteria in the digester characterized by high methane

production is in agreement with our hypothesis. In contrast, hydrogenotrophic methanogens predominance with the presence of particular candidate division

hydrogen-producing bacteria would be connected with non-optimal digester performance. However, the hypothesis still needs confirmation by investigating

samples (by 454-pyrosequencing and qPCR) from other full- and lab-scale (under construction) reactors.

Fig. 1. Diagram showing the biogas production process. Designation: 1 – hydrolysis, 2 –

acidogenesis, 3 – acetogenesis, 4 - methanogenesis

3 2

4

References: Amon T, Amon B, Kryvoruchko V, Zollitsch W, Mayer K, Gruber L (2007) Biogas production from maize and dairy cattle manure – influence of biomass composition on the methane yield. Agriculture, Ecosystems and Environment 118:173-182

Fountoulakis MS, Manios T (2009) Enhanced methane and hydrogen production from municipal solid waste and agro-industrial by-products co-digested with crude glycerol. Bioresource Technology 100:3043-3047

Garcia JL, Patel BKC, Ollivier B (2000) Taxonomic, phylogenetic and ecological diversity of methanogenic Archaea. Anaerobe 6:205-226

Li Y, Park SY, Zhu J (2011) Solid-state anaerobic digestion for methane production from organic waste. Renewable and Sustainable Energy Reviews 15:821-826

Pereyra LP, Hiibel SR, Riquelme MVP, Reardon KF, Pruden A (2010) Detection and quantification of functional genes of cellulose-degrading, fermentative and sulfate-reducing bacteria and methanogenic Archaea. Applied and Environmental Microbiology 76:2192-2202

Stams AJM, Grolle KCF, Frijters CTMJ, Van Lier JB (1992) Enrichment of thermophilic propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum or Methanobacterium themoformicicum. Applied and Environmental Microbiology 58:346-352

Steinberg LM, Regan JM (2009) mcrA-targeted real-time quantitative PCR method to examine methanogen communities. Applied and Environmental Microbiology 75:4435-4442

Weiland P (2010) Biogas production: current state and perspectives. Appl Microbiol Biotechnol 85:849-860

Worm P, Muller N, Plugge CM, Stams AJM, Schink B (2010) Syntrophy in methanogenic degradation. In: Hackstein JHP (ed.), (Endo)symbiotic methanogenic Archaea. Berlin, Springer Berlin Heidelberg, p. 143-173

Ziganshin AM, Schmidt T, Scholwin F, Il’inskaya ON, Harms H, Kleinsteuber S (2011) Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles. Applied Microbiology and Biotechnology 89:2039-2052

qPCR optimization

Fig. 6. Lab-scale 1L reactor

The project assumption is that there might be a syntrophic relationship (based on H2 partial pressure) between some fermentative hydrogen-producing bacteria and

hydrogenotrophic methanogens. In addition, hydrogenotrophic methanogens were proposed by Ziganshin et al. (2011) to give lower methane yields compared to

acetoclastic methanogens. Thus, gene-monitoring specific for the particular hydrogen-producing bacteria (being in correlation with hydrogenotrophic Archaea) will

bring the information about the methane yield (low specific gene abundancy – high methane yield). The project will bring a robust molecular biology technique of

monitoring the methane production in anaerobic digesters.

The sequenced plasmids (with

a mcrA insert):

Methanosarcina thermophila,

Methanobrevibacter sp. D5

and Methanobacterium

petrolearium

The plasmids - used as a

standards in PCR – non-

specific amplification of

negative control (containing

DNA from Methanosarcina or

Methanobacteriales)

More standard plasmids will be

tested in order to check the

specificity of the amplification

of different sets of primers

PCR with Mlas and

mcrA-rev primers Amplicon

purification –

GeneJET

Gel

Extraction

Kit (Thermo

Scientific)

CloneJET

PCR Cloning

Kit (Thermo

Scientific)

Cloning the plasmid with an

insert into E.coli competent cells Sanger sequencing of plasmids PCR with plasmids and some

sets of primers

PCR with mbac-

mcrA and mcrA-

rev; msar and

mcrA-rev (SYBR

Green approach)

PCR with Mlas,

mbac-mcrA

(TaqMan probe)

and mcrA-rev;

Mlas, msar

(TaqMan probe)

and mcrA-rev

(TaqMan rpobe-

based approach)

2 1

Name (for

primer,

TaqMan

probe, rev

primer)

Target group

Size of

product

(bp)

Mlas

mbac-mcrA

mcrA-rev

Methanobacteriaceae mcrA 466-472

Mlas

msar

mcrA-rev

Methanosarcina 490

Tab. 1. Some of used sets of

primers

Fig. 5. The results after PCR with

plasmid standards and two sets of

primers (Steinberg and Regan 2009)