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Journal of Environmental Management 143 (2014) 54e60
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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. Lhde b, M. Kymlinen 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 Hmeenlinna, 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.: 358 9 191 20334.E-mail address: email@example.com (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 m3 d1), 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.
1.1. 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
substitute for fossil fuels in a myriad of technical applications, theactual application determining the quality requirements of the gasproduced (Bagge et al., 2005; Kymlinen 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 m3 d1 (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
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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; Levn 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; Levn 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 (Levn 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; Fernndez-Rodrguez et al., 2013), more total biogas produced (McHughet al., 2004; Levn 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 m3 d1 (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 (
Table 1Mean biogas production (SD) and methane content from OLRs of 3e10 kg VS m3 d1 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 m3 d1.b litres kg1 VS1.
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 threet