lable at ScienceDirect

Journal of Environmental Management 143 (2014) 54e60

Contents lists avai

Journal of Environmental Management

journal homepage: www.elsevier .com/locate/ jenvman

Biogas production and methanogenic archaeal community inmesophilic and thermophilic anaerobic co-digestion processes

D. Yu a, J.M. Kurola a, K. Lähde b, M. Kymäläinen b, A. Sinkkonen a, M. Romantschuk a,*

aUniversity of Helsinki, Department of Environmental Sciences, Niemenkatu 73, 15140 Lahti, FinlandbHAMK University of Applied Sciences, P.O. Box 230, 13101 Hämeenlinna, Finland

a r t i c l e i n f o

Article history:Received 6 November 2013Received in revised form5 March 2014Accepted 23 April 2014Available online

Keywords:Anaerobic digestionOrganic loading rateMesophilicThermophilicMethanogenic archaeal communityBiogas production

* Corresponding author. Tel.: þ358 9 191 20334.E-mail address: martin.romantschuk@helsinki.fi (M

http://dx.doi.org/10.1016/j.jenvman.2014.04.0250301-4797/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Over 258 Mt of solid waste are generated annually in Europe, a large fraction of which is biowaste.Sewage sludge is another major waste fraction. In this study, biowaste and sewage sludge were co-digested in an anaerobic digestion reactor (30% and 70% of total wet weight, respectively). The pur-pose was to investigate the biogas production and methanogenic archaeal community composition in theanaerobic digestion reactor under meso- (35e37 �C) and thermophilic (55e57 �C) processes and anincreasing organic loading rate (OLR, 1e10 kg VS m�3 d�1), and also to find a feasible compromise be-tween waste treatment capacity and biogas production without causing process instability. In summary,more biogas was produced with all OLRs by the thermophilic process. Both processes showed a limiteddiversity of the methanogenic archaeal community which was dominated by Methanobacteriales andMethanosarcinales (e.g.Methanosarcina) in both meso- and thermophilic processes.Methanothermobacterwas detected as an additional dominant genus in the thermophilic process. In addition to operatingtemperatures, the OLRs, the acetate concentration, and the presence of key substrates like propionatealso affected the methanogenic archaeal community composition. A bacterial cell count 6.25 timeshigher than archaeal cell count was observed throughout the thermophilic process, while the cell countratio varied between 0.2 and 8.5 in the mesophilic process. This suggests that the thermophilic process ismore stable, but also that the relative abundance between bacteria and archaea can vary without seri-ously affecting biogas production.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

1.1. Anaerobic digestion

In Europe, municipalities produce in excess of 258 Mt of solidwaste annually (Montejo et al., 2010), a large fraction of which isbiowaste. Sewage sludge, an insoluble residue produced duringwastewater treatment and subsequent sludge stabilization, isanother major waste fraction (Arthurson, 2008). Anaerobic diges-tion is an established and sustainable treatment option for bio-waste and sewage sludge, giving that according to the EuropeanCouncil Regulation (EC) No. 1774/2002 the process residues canpotentially be used as a biofertiliser in agriculture (Bagge et al.,2005; Arthurson, 2008; Lozano et al., 2009; Goberna et al., 2010).The biogas produced by anaerobic digestion processes is a valid

. Romantschuk).

substitute for fossil fuels in a myriad of technical applications, theactual application determining the quality requirements of the gasproduced (Bagge et al., 2005; Kymäläinen et al., 2012). Anaerobicdigestion produces methane, carbon dioxide, a number of tracegases, some heat, and an end product of stabilised sludge. A typicalorganic loading rate (OLR) for fully mixed anaerobic digesters liesbetween 1 and 5 kg COD m�3 d�1 (Tchobanoglous et al., 2003).There are four stages in anaerobic digestion d hydrolysis, acido-genesis, acetogenesis and methanogenesis. Bacterial groups areresponsible for acetate, hydrogen and carbon dioxide production inthe first three stages. In the last stage, methanogenic archaea pro-duce methane from acetate, or alternatively from hydrogen andcarbon dioxide (Griffin et al., 1998; Liu et al., 2004; Bouallagui et al.,2005; Kotsyurbenko, 2005; Lozano et al., 2009; Pycke et al., 2011;Ritari et al., 2012).

The most common problematic organic wastes are those thatare rich in lipids, cellulose and proteins. Previous studies havedemonstrated that combining different organic wastes for anaer-obic co-digestion results in a substrate better balanced and more

D. Yu et al. / Journal of Environmental Management 143 (2014) 54e60 55

efficiently degradable, leading to a significant increase in biogasproduction (Esposito et al., 2012). Wang (2009) andWu et al. (2010)reported significant biogas production increases in the co-digestionprocess by combining carbon rich agricultural residues with swinemanure.

1.2. Microorganisms in anaerobic digestion

Microbial communities in anaerobic co-digestion processesrespond easily to changes in substrate composition, OLR, reactordesign and operating temperatures (Tang et al., 2011; Dohrmannet al., 2011; Levén et al., 2007; McHugh et al., 2004). Previously,only a few studies have focused on the effects of temperature onbacterial and methanogenic archaeal communities in anaerobicbioreactors (Pycke et al., 2011; Levén et al., 2007; Pender et al.,2004; Hernon et al., 2006; Sekiguchi et al., 1998, 2002). Anaer-obic digestion reactors have commonly been operated at meso-philic (30e40 �C) and thermophilic (50e60 �C) temperatures. Ingeneral, higher bacterial and archaeal diversities are found atmesophilic temperatures (Levén et al., 2007; Pycke et al., 2011).Bacterial communities appear to be considerably more diverse anddynamic than archaeal communities at any temperature (McHughet al., 2004; Ritari et al., 2012). Despite lower diversity, digestionat thermophilic temperatures results in higher organic matterdegradation efficiency (Zabranska et al., 2000; Fernández-Rodríguez et al., 2013), more total biogas produced (McHughet al., 2004; Levén et al., 2007; Goberna et al., 2010; Siddiqueet al., 2014), and superior feed substrate hygienization (Zabranskaet al., 2000; Bagge et al., 2005; Arthurson, 2008).

The aim of the research was to understand the link between themicrobial communities co-digesting biowaste and sewage sludgeand the key methanogenesis intermediates at both meso- andthermophilic temperatures. The aim was also to find a functionalcompromise betweenwaste treatment capacity, biogas production,and a stable microbial community. To the best of our knowledgethis concept has not been previously documented. Specifically theobjectives were a) to identify major methanogens in themesophilic(35e37 �C) and thermophilic (55e57 �C) anaerobic co-digestionprocesses, b) to study the effects of incrementally rising OLRs onbiogas production and methanogenic archaeal communitycomposition, and c) to study the effects of elevated loading rates onthe relative abundance of microbial types and production of keymethanogenesis intermediates. The hypothesis was that clearchanges in dominating methanogenic groups would be observedwith increasing temperatures and OLRs.

2. Material and methods

2.1. Anaerobic digester and gas analysis

A semi-continuously operated anaerobic digestion reactor (fedonce per day) with an operating volume of 150 L was used for twoconsecutive production cycles under differing temperature condi-tions; the mesophilic digestion process was held at 35e37 �C for 19weeks (September 2007eFebruary 2008), and the following ther-mophilic digestion process was held at 55e57 �C for 20 weeks(AprileSeptember 2008). The feed mixture of finely minced,homogenised, and hygienized biowaste and sewage sludge (30%and 70% of total wet weight, respectively) was diluted with waterbefore loading into the anaerobic digester. The reactor was stirred(ca. 160 rpm) for 30 min every 2 h and the OLR was increasedincrementally from 1 to 10 kg VS m�3 d�1 (kg volatile solids perreactor volume per day). The dry solids content of the feed mixturewas kept constant (ca. 8%) and increased amount of this mixture

was fed. Thus, the hydraulic retention time was decreased stepwisefrom 58 days to 8 days.

Online reaction monitoring of the total volume of producedbiogas was measured with a KIMMON SK35 gas metre, and themethane fraction was measured with a Simrad GD10 IR gas de-tector. The biogas flowed out freely from the reactor to the gasmetre. The overpressure in the digestion reactor was continuouslymeasured (<5 mbar), therefore the pressure in the gas metre wasexpected to be close to 1 bar. Major gas components d methaneand carbon dioxide d as well as those of ammonia and nitrousoxides, were measured by FT-IR analysis (Gasmet, Temet In-struments), while the quantity of key trace compounds such assiloxanes, sulphur compounds, and volatile organic compounds(VOCs) were measured with gas chromatography (Voyager PerkinElmer) (Arnold and Kajolinna, 2008). Biogas production (meso-philic versus thermophilic process at each OLR) was analysed withpaired-samples t-tests at each time point (IBM SPSS 21, IBM Inc,Armonk, NY). The assumptions of the analyses were met.

2.2. DNA extraction and quantification

In order to study the microbial communities, total DNA wasextracted from 0.25 ml of the reactor’s output sludge at OLRs of 1e10 kg VS m�3 d�1 in both meso- and thermophilic treatments usinga FastDNA� SPIN Kit for Soil (Qbiogene Inc., Carlsbad, USA) ac-cording to the manufacturer’s instructions. The DNA concentrationwas measured fluorometrically using PicoGreen� dsDNA Quanti-tation Reagent and Kits (Molecular Probes Inc., Eugene, OR, USA).

2.3. PCR, DGGE and cloning analyses

Methyl-coenzyme M reductase (MCR) is the catalyst for themethane-forming step in methanogenic archaea metabolism, andthe mcrA gene is a functional marker present in all methanogens(Friedrich, 2005). The methanogen-specific primers were obtainedfrom TAGC (Copenhagen, Denmark). Primer sets of mcrA-F (50-GGTGGT GTM GGA TTC ACA CAR TAY GCW ACA GC-30) and mcrA-R (50-TTC ATT GCR TAG TTW GGR TAG TT-30) by Luton et al. (2002) wereused for PCR amplification. A 41-bp GC-rich sequence (50-CGC CCGCCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GC-30) wasattached to the 50 end of the mcrA-R for DGGE analysis. The PCRreaction mixture was composed of 3 ml of crude DNA extract, 1 ml ofeach primer (10 mM); 5 ml of 10� DyNAzyme Buffer, 1 ml of dNTP(10 mM), 1 ml of DyNAzymes II DNA polymerase (2 U ml�1) fromFinnzymes, Thermo Scientific, Finland; and 1 ml of Bovine SerumAlbumin (BSA) (20 mg ml�1) from Fermentas, Thermo Scientific,Finland. Sterile water was added to reach a final volume of 50 ml.The PCR amplification of 3 ml of crude DNA extract was done in aPTC-100 Thermo Cycler (MJ Research Inc., Waltham, MA, USA). Theinitial denaturation step was set to 94 �C for 7 min followed by 40cycles of 45 s at 94 �C, 45 s at 55 �C, 1 min at 72 �C, and a finalelongation step of 10 min at 72 �C. The yield of PCR products wasestimated with agarose gel electrophoresis stained with EtBr.

The methanogen community composition was examined byutilising DGGE and direct clone library analysis on PCR productsobtained withmcrA-GC andmcrA primers. The PCR products with aGC-clamp (20e25 ml) were separated using DGGE as described byKurola et al. (2005) with modifications in the denaturant gradient(30e60%) and acrylamide-bisacrylamide concentration (9%). Thegels were run for 17.5 h at 80 V. After electrophoresis, the gels werestainedwith SYBR�Gold nucleic acid gel stain and photographed asdescribed by Kurola et al. (2005). The PCR fragments without a GC-clamp were cloned into commercial plasmid vectors as describedby Partanen et al. (2010).

Table 1Mean biogas production (�SD) and methane content from OLRs of 3e10 kg VS m�3 d�1 at mesophilic and thermophilic temperatures. The maximumbiogas productions at both runs are indicated by the bold numbers.

OLRa Mesophilic Thermophilic

Biogasb Methane (%) Biogasb Methane (%)

3 628.80 � 78 51.2e62.2 749.69 ± 48 54.5e61.95 689.44 ± 51 44.4e62.6 679.04 � 33 49.6e60.18 628.00 50.6e65.1 536.00 ND10 531.20 ND 540.80 ND

a kg VS m�3 d�1.b litres kg�1 VS�1.

D. Yu et al. / Journal of Environmental Management 143 (2014) 54e6056

2.4. PCR amplification after DGGE, DNA sequencing andphylogenetic analyses

Distinct bands from the DGGE gels were excised, crushed threetimes in eppendorf tubes containing 25 ml of sterilized distilledwater, and stored overnight at 4 �C. The eluted DNA was re-amplified with mcrA primers (without a GC-clamp) as describedin Section 2.3. Prior to sequencing, 45 ml of each PCR product waspurified with a QIAquick PCR purification kit (QIAGEN GmbH, Hil-den, Germany) as per manufacturer’s instructions. ThemcrA clonesand PCR products from the DGGE bands were sequenced using anABI PRISM� BigDye� Terminator Cycle Sequencing Ready Reactionkit and analysed on an ABI Prism 3700 DNA sequencer (AppliedBiosystems, Foster City, CA, USA). All sequence chromatogramswere analysed with the Staden Package (University of Cambridge,UK). Sequences were compared with those available in the EMBLdatabase using a BLAST server, and aligned using the CLUSTAL Wpackage hosted by the European Bioinformatics Institute (http://www.ebi.ac.uk/). Sequence alignment was examined manuallyusing the program GENEDOC (v.2.6.002), and phylogenetic analysiswas accomplished with the PHYLIP package (v.3.57C). Geneticdistances were calculated utilising the Kimura-2 model within theprogram DNADIST (Kurola et al., 2005). Finally, partial mcrA genesequences from the DGGE excised bands were assigned GenBankaccession numbers from HF536720 to HF536726.

2.5. Quantitative PCR amplification

To determine the 16S rDNA ratio of archaea and bacteria at bothmeso- and thermophilic temperatures, quantitative PCR (qPCR)was done in a DNA Engine OPTICON 2 (MJ Research Inc., Waltham,MA, USA) using specific primer sets in a final volume of 20 mlcontaining 1e10 ng of total DNA. A DyNAmo� HS SYBR� GreenqPCR kit (Finnzymes, Thermo Scientific, Finland) was used in allPCR runs. The archaeal 16S rDNA was amplified using primersArch1369F (50-CGG TGA ATA YGY CCC TGC-30) and Prok1541R (50-AAG GAG GTC ATC CRG CCG CA-30) (Suzuki et al., 2000). The PCRmixtures consisted of 2 ml of diluted DNA,10 ml of Master Mix, 0.8 mlof each primer in a concentration of 10 mM, 0.4 ml of BSA(20 mg ml�1), and 6 ml of sterile water. Cycling conditions were15 min at 94 �C followed by 40 cycles of 10 s at 94 �C, 20 s at 58 �C,30 s at 72 �C, and a final elongation of 5 min at 72 �C. Sulfolobusacidocaldarius DSM 639 genomic DNA (DSMZ, Germany) was usedas a positive control to create a standard curve. Bacterial 16S rDNAwas amplified using primers pE (50-AAA CTC AAAGGA ATT GAC GG-

0

50

100

150

200

250

300

350

400

450

0 1 2 3 4 5 6 7 8

Biog

aspr

oduc

tion,

liter

s

Time from feeding, hours

33 kkggVSVS - MMEESOSO

3 kgVS - THERMO

5 kgVS - MESO

55 kkggVSVS - TTHHEERRMOMO

8 kgVS - MESO

8 kgVS - THERMO

1100 kkggVVSS - TTHHEERRMOMO

Fig. 1. Cumulative biogas production in litres from organic loading rates of 3e10 kg VS m�3 d�1 at mesophilic and thermophilic temperatures.

30) and pF (50-ACG AGC TGA CGA CAG CCA TG-30) (Ekman et al.,2007; Kanto Öqvist et al., 2008). PCR mixtures included 2 ml ofdiluted DNA, 10 ml of Master Mix, 0.5 ml of each primer (10 mM),0.4 ml of BSA (20 mg ml�1), and 6.6 ml of sterile water. Cyclingconditions were set at 10 min at 94 �C, followed by 30 cycles of 10 sat 94 �C, 20 s at 57 �C, 30 s at 72 �C, and a final elongation step of5 min at 72 �C. Nitrosomonas europea ATCC 19178 genomic DNA(ATCC-LGC Standards, USA) was used as a positive control to createa standard curve.

Quantitative changes in the bacterial versus archaeal ratio of 16Sribosomal genes were analysed with a ManneWhitney U test. Thebacterial versus archaeal ratios (ca. 15 d intervals) were standard-ized before the analysis, and the absolute difference from treatmentmean was calculated in each case. This facilitated testing the nullhypothesis that variation in the relative abundance of bacteria andarchaea was similar in both meso- and thermophilic processes.

3. Results and discussion

3.1. Gas production in anaerobic digestion

Cumulative biogas production at the meso- and thermophilicproduction cycles increased steadily over time, corresponding withincreases in OLRs up to 8 kg VS m�3 d�1. Biogas production wasmore efficient at all OLRs within the thermophilic process (t � 5.8,df ¼ 3, p � 0.01; Fig. 1). Biogas accumulation was monitored foreight hours, at the end of which the production rate started todecline (data not shown). These findings agree with those of Levénet al. (2007), who found that digestion at thermophilic tempera-tures is associated with higher total biogas production. Theapparent reason for this result is that the thermophilic microbesexpress higher organic matter processing rate at their optimaltemperature than mesophilic microbes at their optimumtemperature.

Data obtained from metering the production capacity of theanaerobic processes per kgVS at both temperatures were as fol-lows: At an OLR of 3 kg VS m�3 d�1, the thermophilic processproduced 750 L kg VS�1 of biogas, whereas the mesophilic processproduced 629 L kg VS�1. When the OLR was increased to5 kg VS m�3 d�1, biogas production continued to increase in themesophilic process, but began to decrease in the thermophilicprocess (Table 1). The biogas production peak of 750 L kg VS�1 wasreached at an OLR of 3 kg VS m�3 d�1 in the thermophilic process.The mesophilic process produced the maximum volume of biogas(689 L kg VS�1) at an OLR of 5 kg VS m�3 d�1. Above this OLR, theyield of biogas per kg of volatile solids decreased at both temper-atures, although the cumulative generation of biogas continued toincrease up to an OLR of 10 kg VS m�3 d�1. Thus, an OLR of5 kg VS m�3 d�1 was found to be optimal for maximal wastetreatment capacity while still maximising biogas output from theprocessed biowaste. Up to the optimum OLR, the methane fraction

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

0 20 40 60 80 100 120

Day

a. Mesophilic

copi

esm

l-1

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

0 20 40 60 80 100 120 140 160

Day

b. Thermophilic

copi

esm

l-1

Fig. 2. Quantitative changes in bacterial and archaeal 16S ribosomal genes using qPCRwere higher in mesophilic (a) than in thermophilic (b) process (U ¼ 0, Z ¼ �3.24,n ¼ 15, p < 0.001).

D. Yu et al. / Journal of Environmental Management 143 (2014) 54e60 57

varied in the range of 44.4% and 62.6% in the mesophilic process,and 49.6e61.9% in the thermophilic process, the rest being mainlyCO2. Trace gases such as ammonia, hydrogen sulphide, and the totalsiloxane were also detected at the rather low maximum levels of83 ppm, 10 ppm, and 1 ppm, respectively. The concentration ofethanol was found to vary significantly (10e2380 ppm) at differentOLRs, indicating that formation of ethanol was related to processchanges (Kymäläinen et al., 2012; Ritari et al., 2012). In addition, theconcentrations of all VOCs detected (i.e. toluene, ethyl benzene,nonane, p-xylene and a-pinene) remained low in all measurements(Kymäläinen et al., 2012).

3.2. Abundance analysis during the meso- and thermophilicprocesses

In order to examine the effects of temperature and OLR vari-ances on archaea to bacteria ratio, we quantified the gene copynumbers of bacterial and archaeal 16S ribosomal genes. Resultscalculated from qPCR standard curves indicated that the copynumber ratios of bacterial and archaeal 16S rDNA were not stable,varying from 0.32 to 13.62 in the mesophilic process (Fig. 2a). Incontrast, the copy numbers of bacterial 16S rDNA in all samplereplicates were roughly 10 times greater than that of archaeal 16SrDNA all through the thermophilic process cycle, staying within theranges of 108e109 and 107e108 per ml, respectively (Fig. 2b).Klappenbach et al. (2001) suggested that the copy number of the16S rRNA gene is 4 per cell for bacteria, and 2.5 per cell for(methanogenic) archaea. Using these figures to calculate cell countsfrom absolute 16S rRNA gene copy numbers, the bacterial cell countin the current study was estimated to be circa 6.25 times that of thearchaeal cell count throughout the monitoring period in the ther-mophilic process, and 0.2 to 8.5 times that of archaeal cell count inthemesophilic process. These findings are in rough agreement withthose of Narihiro et al. (2009) who reported a ratio range of 0.37e2.5 of 16S rRNA gene clones of bacteria to archaea at mesophilic

temperatures, and close to 1 at thermophilic temperatures. On theother hand, Cardinali-Rezende et al. (2012) reported that comparedto archaea, they found about 360 times more of bacterial 16S rRNAgene copies (and 225 times the cell count) per gram of sludge in aswine-waste-sludge anaerobic lagoon. The differences in theseobservations could be explained by different feed materials andconditions (e.g. OLRs, temperatures) used in the abovementionedstudies. In all, the bacteria to archaea ratio appears to be morestable in thermophilic processes, but still case specific and prone tolarge variations.

It has previously been shown that most archaea in anaerobicdigesters are methanogens (Coats et al., 2012). Our findings supportthis in that we found the archaeal 16S rDNA gene copy numbers tohave a positive correlation with methane production. The highestarchaeal 16S ribosomal gene copy number for the mesophilic pro-cess was recorded at the OLR of 5 kg VS m�3 d�1. This correspondedwith peak methane production of 306e432 L kg VS�1. An OLR of3 kg VS m�3 d�1 resulted in the highest observed archaeal 16S ri-bosomal gene copy number, and a methane production of 408e464 L kg VS�1 in the thermophilic process.

3.3. Methanogenic archaeal compositions in anaerobic digesters

3.3.1. Effects of temperature and acetate concentration on themethanogenic community

Target gene PCR combined with clone library sequencing arecommon methods for acquiring an overview of the dominantgroups in a given microbial community. Here, the clone library andDGGE band sequencing results suggest that the methanogeniccommunity diversity in the anaerobic digester used was ratherlimited. Among five domain methanogenic archaeal orders (Meth-anobacteriales, Methanococcales, Methanomicrobiales, Meth-anosarcinales, and Methanopyrales), only Methanobacteriales andMethanosarcinales were found to be abundant in both processes,while also Methanothermobacter was present in the thermophilicprocess (Fig. 3). Additionally, cloning revealed representatives ofMethanomicrobiales (e.g. Methanospirillum) in the mesophilic cycle.This agrees with previous studies (McHugh et al., 2004; Conrad andWetter, 1990), which have recorded the positive effects of lowtemperatures on hydrogenotrophic methanogens, e.g. Meth-anomicrobiales. Narihiro et al. (2009) reported similar results:Methanomicrobiales and Methanosaetaceae (e.g. Methanosaeta)were detected only in the mesophilic cycles.

Methanosarcina is the only well-known methanogen that canuse more than one substrate to produce methane, e.g. acetate, H2e

CO2, methanol, mono-, di-, and tri-methylamines, and CO (Bryantand Boone, 1987). This is a probable explanation for its predomi-nance in the current study, which included highly variable condi-tions. The results from mcrA clone and DGGE band sequencingshowed that Methanosarcina was very abundant in all samples.Notably, of the two commonly known acetotrophic methanogengenera Methanosaeta and Methanosarcina (alternatively called‘acetoclastic’, or ‘acetate-utilizing’ methanogens), only Meth-anosarcinawas detected in the present study. It is well documentedthat acetate concentration has a crucial impact on the presence andrelative abundance of acetotrophic methanogens (Westermannet al., 1989). Methanosaeta can only use acetate for methane pro-duction, but its growth is favoured by low concentrations of thissubstrate (Schmidt and Ahring, 1996). A possible explanation forMethanosaeta not having been detected is therefore that mostrecorded acetate concentrations (0.17e4.44 mM) were above athreshold beyond which the fast growing Methanosarcina wouldoutcompete Methanosaeta. Methanothermobacter has previouslybeen reported as the dominant genus in a thermophilic anaerobicprocess (Sekiguchi et al., 1998; Chen et al., 2008). In contrast to

Fig. 3. Phylogenetic tree of archaealmcrA gene retrieved from clones, DGGE separated PCR products and recorded methanogenic archaea at mesophilic and thermophilic processes.Methanocella arvoryzae was used as an outgroup to root the tree. The bar indicates 10% base substitution. The symbols at bodes indicate bootstrap values with 1000 resamplinganalyses.

D. Yu et al. / Journal of Environmental Management 143 (2014) 54e6058

D. Yu et al. / Journal of Environmental Management 143 (2014) 54e60 59

studies that have reported a more limited methanogen diversity inthermophilic processes (Hernon et al., 2006; Levén et al., 2007; Liuet al., 2009; Pender et al., 2004; Pycke et al., 2011; Sekiguchi et al.,1998, 2002; Tang et al., 2004), here both the meso- and thermo-philic processes presented a very limited methanogen diversity.The reason for the low observed diversity of dominant metha-nogens is not lack of inoculum, since Ritari et al. (2012) did detectthe presence of also additional methanogens (i.e. Methano-brevibacter, Methanosphaera and Methanophaerula) by 454sequencing of a subset of samples of the present study. Instead thereason is likely to be the range of conditions favouring robustarchaea that adapt to various conditions.

3.3.2. Effects of substrate composition, and OLR on acetotrophicMethanosaetaceae

Methanosaeta was not detected in either treatment where pro-pionate concentrations between 16 and 168 ppm were measured.McHugh et al. (2004) reported a clear shift with a proliferation ofMethanomicrobiales and a simultaneous decrease in Meth-anosarcina and Methanosaeta. They proposed that this could havebeen due to the stressed state of the digestion process in the re-actors. They also reported that Methanomicrobiales promptlydominated Methanosarcina in the reactor that was fed withwastewater containing butyrate and propionate, while Meth-anosaeta was predominant in the reactor fed with sugar wastebefore being replaced by Methanomicrobiales. Key substrates likepropionate could therefore be an element regulating the presenceand predominance of acetotrophic methanogens d such as Meth-anosaeta d in the anaerobic digestion process.

We found that neither the meso- nor thermophilic methano-genic communities were influenced significantly by OLRs in con-centrations of 1e10 kg VS m�3 d�1. A recent study treatingpharmaceutical wastewater (Chelliapan et al., 2011) concluded thatMethanosaeta generally dominated at low OLRs of 0.86e1.86 kg COD m�3 d�1, while Methanosarcina dominated at highOLRs of 2.98e3.73 kg COD m�3 d�1. The loading rates used in ourstudy may therefore have been too high for Methanosaeta to bepresent.

4. Conclusions

In general, the biogas output was greater in the thermophilicprocess. At an OLR of 5 kg VS m�3 d�1, a production efficiencymaximum was reached. Beyond this, efficiency declined at bothtemperature ranges. However, the production rate per hourremained high; the methanogenic process did not collapse even at10 kg VS m�3 d�1, and at no OLR was the methanogenic communityflushed out. The OLR does not seem to have a strong impact on themethanogen community in the anaerobic digester studied. Processtemperature, on the other hand, does. The methanogenic archaealcommunity was dominated by Methanobacteriales and Meth-anosarcina at both the meso- and thermophilic cycles, signifyinglow genus diversity at community level, even with Meth-anothermobacter as an additional abundant genus in the thermo-philic production cycle. The prevalent over-threshold acetateconcentration, high OLRs, and the presence of key substrates likepropionate were potentially responsible for the dominance ofMethanosarcina.

Acknowledgements

The research was mainly funded by Tekes (the Finnish FundingAgency for Technology and Innovation) project (40080/07). Yu’swork was partially supported by the Finnish Cultural Foundation(70101153, 00111069) and the ENSTE graduate school. Technical

support from Preseco Oy, Kiertokapula Oy is greatly acknowledged.MSc. RiikkaMäkelä and Jon Thompson Coon are greatly appreciatedfor assisting in English language revision.

References

Arnold, M., Kajolinna, T., 2008. On-line measurement and removal of biogas tracecompounds. In: 2nd International Symposium on Energy from Biomass andWaste. Venice, Italy, 17e20 Nov. 2008. Euro Waste S.r.l.

Arthurson, V., 2008. Proper sanitization of sewage sludge: a critical issue for asustainable society. Appl. Environ. Microbiol. 74, 5267e5275.

Bagge, E., Sahlström, L., Albihn, A., 2005. The effect of hygienic treatment on themicrobial flora of biowaste at biogas plants. Water Res. 39, 4879e4886.

Bouallagui, H., Touhami, Y., Cheikh, R.B., Hamdi, M., 2005. Bioreactor performancein anaerobic digestion of fruit and vegetable wastes. Process Biochem. 40, 989e995.

Bryant, M.P., Boone, D.R., 1987. Emended description of strain MST (DSM 800T), thetype strain of Methanosarcina barkeri. Int. J. Syst. Bacteriol. 37, 169e170.

Cardinali-Rezende, J., Pereira, Z.L., Sanz, J.L., Chartone-Souza, E., Nascimento, A.M.A.,2012. Bacterial and archaeal phylogenetic diversity associated with swinesludge from an anaerobic treatment lagoon. World J. Microbiol. Biotechnol. 28,3187e3195.

Chelliapan, S., Wilby, T., Yuzir, A., Sallis, P.J., 2011. Influence of prganic loading on theperformance and microbial community structure of an anaerobic stage reactortreating pharmaceutical wastewater. Desalination 271, 257e264.

Chen, C.L., Wu, J.H., Liu, W.T., 2008. Identification of important microbial pop-ulations in the mesophilic and thermophilic phenol-degrading methanogenicconsortia. Water Res. 42, 1963e1976.

Coats, E.R., Ibrahim, I., Briones, A., Brinkman, C.K., 2012. Methane production onthickened, pre-fermented manure. Bioresour. Technol. 107, 205e212.

Conrad, R., Wetter, B., 1990. Influence of temperature on energetic of hydrogenmetabolism in homoacetogenic, methanogenic, and other anaerobic bacteria.Arch. Microbiol. 155, 94e98.

Dohrmann, A.B., Baumert, S., Klingebiel, L., Weiland, P., Tebbe, C.C., 2011. Bacterialcommunity structure in experimental methanogenic bioreactors and search forpathogenic clostridia as community members. Appl. Microbiol. Biotechnol. 89,1991e2004.

Ekman, J., Kosonen, M., Jokela, S., Kolari, M., Korhonen, P., Salkinoja-Salonen, M.,2007. Detection and quantitation of colored deposit-forming Meiothermus spp.in paper industry processes and end products. J. Industrial Microbiol. Bio-technol. 34, 203e211.

Esposito, G., Frunzo, L., Giordano, A., Liotta, F., Panico, A., Pirozzi, F., 2012. Anaerobicco-digestion of organic wastes. Rev. Environ. Sci. Bio/Technol. 11, 325e341.

Fernández-Rodríguez, J., Pérez, M., Romero, L.I., 2013. Comparison of mesophilicand thermophilic dry anaerobic digestion of OFMSW: kinetic analysis. Chem.Eng. J. 232, 59e64.

Friedrich, M.W., 2005. Methyl-coenzyme M reductase genes: unique functionalmarker for methanogenic and anaerobic methane-oxidising Archaea. Methodsin Enzymology. Environ. Microbiol. 397, 428e442.

Goberna, M., Schoen, M.A., Sperl, D., Wett, B., Insam, H., 2010. Mesophilic andthermophilic co-fermentation of cattle excreta and olive mill wastes in pilotanaerobic digesters. Biomass Bioenergy 34, 340e346.

Griffin, M.E., McMahon, K.D., Mackie, R.I., Raskin, I., 1998. Methanogenic populationdynamics during start-up anaerobic digesters treating municipal solid wasteand biosolids. Biotechnol. Bioeng. 57, 342e355.

Hernon, F., Forbes, C., Colleran, E., 2006. Identification of mesophilic and thermo-philic fermentative species in anaerobic granular sludge. Water Sci. Technol. 54,19e24.

Kanto Öqvist, C., Kurola, J., Pakarinen, J., Ekman, J., Ikävalko, S., Simell, J., Salkinoja-Salonen, M., 2008. Prokaryotic microbiota of recycled paper mills with low orzero effluent. J. Indus. Microbiol. Biotechnol. 35, 1165e1173.

Klappenbach, J.A., Saxman, P.R., Cole, J.R., Schmidt, T.M., 2001. rrndb: the RibosomalRNA operon copy number database. Nucleic Acids Res. 29, 181e184.

Kotsyurbenko, O.R., 2005. Trophic interactions in the methanogenic microbialcommunity of low-temperature terrestrial ecosystems. FEMS Microbiol. Ecol.53, 3e13.

Kurola, J., Wittmann, C., Salkinoja-Salonen, M., Aarnio, T., Romantschuk, M., 2005.Application of cation-exchange membranes for characterisation and imagingammonia-oxidising bacteria in soils. FEMS Microbiol. Ecol. 53, 463e472.

Kymäläinen, M., Lähde, K., Arnold, M., Kurola, J.M., Romantschuk, M., Kautola, H.,2012. Biogasification of biowaste and sewage sludge e measurement of biogasquality. Environ. Manag. 95, S122eS127.

Levén, L., Eriksson, A.R.B., Schnürer, A., 2007. Effects of process temperature onbacterial and archaeal communities in two methanogenic bioreactors treatingorganic household waste. FEMS Microbiol. Ecol. 59, 683e693.

Liu, J., Olsson, G., Mattiasson, B., 2004. On-line monitoring of a two-stage anaerobicdigestion process using a BOD analyzer. J. Biotechnol. 109, 263e275.

Liu, K., Tang, Y.Q., Matsui, T., Morimura, S., Wu, X.L., Kida, K., 2009. Thermophilicanaerobic co-digestion of garbage, screened swine and dairy cattle manure.J. Biosci. Bioeng. 107, 56e60.

Lozano, C.J.S., Mendoza, M.V., de Arango, M.C., Monroy, E.F.C., 2009. Microbiologicalcharacterization and specific methanogenic activity of anaerobe sludges used inurban solid waste treatment. Waste Manag. 29, 704e711.

D. Yu et al. / Journal of Environmental Management 143 (2014) 54e6060

Luton, P.E., Wayne, J.M., Sharp, R.J., Riley, P.W., 2002. ThemcrA gene as an alternativeto 16S rRNA in the phylogenetic analysis of methanogen populations in landfill.Microbiology 148, 3521e3530.

McHugh, S., Carton, M., Collins, G., O’Flaherty, V., 2004. Reactors performance andmicrobial community dynamics during anaerobic biological treatment ofwastewaters at 16e37 �C. FEMS Microbiol. Ecol. 48, 369e378.

Montejo, C., Martín, R., Costa, C., Ramos, P., Márquez, M.-C., 2010. Energy recovery ofreject fraction of municipal solid waste resulting from the mechanical-biological treatment plants. Chem. Eng. Trans. 21, 751e756.

Narihiro, T., Terada, T., Ohashi, A., Wu, J.H., Liu, W.T., Araki, N., Kamagata, Y.,Nakamura, K., Sekiguchi, Y., 2009. Quantitative detection of culturable meth-anogenic archaea abundance in anaerobic treatment systems using thesequence-specific rRNA cleavage method. ISME J. 3, 522e535.

Partanen, P., Hultman, J., Paulin, L., Auvinen, P., Romantchuk, M., 2010. Bacterialdiversity at different stages of the composting process. BMC Microbiol. 10, 94.

Pender, S., Toomey, M., Carton, M., Eardly, D., Patching, J.W., Colleran, E.,O’Flaherty, V., 2004. Long-term effects of operating temperature and sulphateaddition on the methanogenic community structure of anaerobic hybrid re-actors. Water Res. 38, 619e630.

Pycke, B.F.G., Etchebehere, C., Van de Caveye, P., Negroni, A., Verstraete, W., Boon, N.,2011. A time-course analysis of four full-scale anaerobic digesters in relation tothe dynamics of change of their microbial communities. Water Sci. Technol. 63,769e775.

Ritari, J., Koskinen, K., Hultman, J., Kurola, J.M., Kymäläinen, M., Romantschuk, M.,Paulin, L., Auvinen, P., 2012. Molecular analysis of meso- and thermophilicmicrobiota associated with anaerobic biowaste degradation. BMC Microbiol. 12,121.

Schmidt, J.E., Ahring, B.K., 1996. Review: granular sludge formation in upflowanaerobic sludge blanket (UASB) reactors. Biotechnol. Bioeng. 49, 229e246.

Sekiguchi, Y., Kamagata, Y., Syutsubo, K., Ohashi, A., Harada, H., Nakamura, K., 1998.Phylogenetic diversity of mesophilic and thermophilic granular sludges deter-mined by 16S rRNA gene analysis. Microbiology-UK 144, 2655e2665.

Sekiguchi, Y., Kamagata, Y., Ohashi, A., Harada, H., 2002. Molecular and conventionalanalyses of microbial diversity in mesophilic and thermophilic upflow anaer-obic sludge blanket granular sludges. Water Sci. Technol. 45, 19e25.

Siddique, M.N.I., Abd Munaim, M.S., Zularisam, A.W., 2014. Mesophilic and ther-mophilic biomethane production by co-digesting pretreated petrochemicalwastewater with beef and dairy cattle manure. J. Indus. Eng. Chem. 20, 331e337.

Suzuki, M.T., Taylor, L.T., DeLong, E.F., 2000. Quantitative analysis of small-subunitrRNA genes in mixed microbial populations via 50enuclease assays. Appl. En-viron. Microbiol. 66, 4605e4614.

Tang, Y.Q., Ji, Pan, Hayashi, J., Koike, Y., Wu, X.L., Kida, K., 2011. Characteristic mi-crobial community of a dry thermophilic methanogenic digester: its long-termstability and change with feeding. Appl. Microbiol. Biotechnol. 91, 1447e1461.

Tang, Y.Q., Shigematsu, T., Ikbal, Morimura, S., Kida, K., 2004. The effects of micro-aeration on the phylogenetic diversity of microorganisms in a thermophilicanaerobic municipal solid-waste digester. Water Res. 38, 2537e2550.

Tchobanoglous, G., Burton, F.L., Stensel, H.D., 2003. Wastewater EngineeringTreatment and Reuse, fourth ed./Revised. Metcalf and Eddy, Inc./Mc-Graw-Hill,New York, USA, ISBN 0-07-041878-0.

Wang, G., 2009. Biogas Production from Energy Crops and Agriculture Residues.Dissertation. Technical University of Denmark.

Westermann, P., Ahring, B.K., Mah, R.A., 1989. Threshold acetate concentrations foracetate catabolism by acetoclastic methanogenic bacteria. Appl. Environ.Microbiol. 55, 514e515.

Wu, X., Yao, W., Zhu, J., Miller, C., 2010. Biogas and CH4 productivity by co-digestingswine manure with three crop residues as an external carbon source. Bioresour.Technol. 101, 4042e4047.

Zabranska, J., Stepova, J., Wachti, R., Jenicek, P., Dohanyos, M., 2000. The activity ofanaerobic biomass in thermophilic and mesophilic digesters at different loadingrates. Water Sci. Technol. 42, 49e56.