Microbial community structure and dynamics in two-stage vs single-stage thermophilic anaerobic digestion of mixed swine slurry and market bio-waste

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two-stage vs single-stage thermophilic anaerobicd swastea a RiAdand Nutronmenspective proved a useful tool for a better understanding and comparison of anaerobic. All rights reserved.organic waste producing energy in the form of biogas of highcalorific value (methane and hydrogen) (Angenent et al., 2004).This technology has been successfully used to producemethane since several decades, and recently its use has raisedenvironmentally-nergy.erated by differentfunctional groups of microorganisms that convert organicmatter to methane through three major steps (hydrolysis/acidogenesis, acetogenesis and methanogenesis). AD iscommonly run in single-stage process, however recently* Corresponding author. Tel.: 39 0250319117; fax: 39 0250319238.Available online at www.sciencedirect.com.e lswat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 5E-mail address: daniele.daffonchio@unimi.it (D. Daffonchio).digestion processes. 2013 Elsevier Ltd1. IntroductionAnaerobic digestion (AD) process is an effective way to treata revived and increased interest as anfriendly alternative to fossil fuel-derived eAD is a complex biological process opBacterial and archaealanaerobic consortiaBio-hydrogenBio-methanePCR-DGGEReal-time PCRfew dominant species associated to stable hydrogen production. The archaeal community,dominated by the acetoclastic Methanosarcinales in both methanogen reactors, showeda significant diversity change in the single-stage process after a period of adaptation to thefeeding conditions, compared to a constant stability in the methanogenic reactor of thetwo-stage process. The more diverse and dynamic bacterial and archaeal community ofsingle-stage process compared to the two-stage process accounted for the best degradationactivity, and consequently the best performance, in this reactor. The microbiological per-Giuseppe Merlino , AurorRoberto Oberti b, FabrizioaDepartment of Food Environmental abDepartment of Agricultural and Envia r t i c l e i n f oArticle history:Received 5 August 2012Received in revised form27 November 2012Accepted 4 January 2013Available online 18 January 2013Keywords:0043-1354/$ e see front matter 2013 Elsevhttp://dx.doi.org/10.1016/j.watres.2013.01.007zzi a, Andrea Schievano b, Alberto Tenca b, Barbara Scaglia b,ni b, Daniele Daffonchio a,*ritional Sciences (DEFENS), University of Milan, Celoria 2, 20133 Milan, Italytal Science (DiSAA), University of Milan, Celoria 2, 20133 Milan, Italya b s t r a c tThe microbial community of a thermophilic two-stage process was monitored during two-months operation and compared to a conventional single-stage process. Qualitative andquantitative microbial dynamics were analysed by Denaturing Gradient Gel Electro-phoresis (DGGE) and real-time PCR techniques, respectively. The bacterial community wasdominated by heat-shock resistant, spore-forming clostridia in the two-stage process,whereas a more diverse and dynamic community (Firmicutes, Bacteroidetes, Synergistes) wasobserved in the single-stage process. A significant evolution of bacterial communityoccurred over time in the acidogenic phase of the two-phase process with the selection ofdigestion of mixe wine slurry and market bio-Microbial community structurejournal homepage: wwwier Ltd. All rights reservedand dynamics inevier .com/locate/watres.wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 51984a two-stage design that splits the overall process in twophases, operated in two reactors in series with production ofhydrogen and methane separately, has been established(Demirel et al., 2010). In the first-stage reactor, bacteria fer-ment organic compounds, generally carbohydrates, directlyto hydrogen, carbon dioxide, organic acids and alcohols(Valdez-Vazquez and Poggi-Varaldo, 2009).In the second-stage reactor, methanogens convert the re-sidual energy contained in the high-volatile fatty acids (VFAs)effluent to bio-methane.Early studies of bio-hydrogen production were focused onpure cultures of Clostridia and Enterobacteria fermenting simplesoluble substrates, like starch, glucose or sucrose. However,pure carbohydrates are very expensive and the use of pureculture system is problematic as they are prone to con-tamination. In following studies, mixed cultures fermentinglow cost solid organic waste were proved to be effective, easilymaintained and controlled. In general, the choice of sub-strates for bio-hydrogen production is based on some majorcriteria such as availability, cost, biodegradability andcarbohydrate-content because of the high bio-hydrogen pro-ducing potential. The use of combined wastewaters has led toa new path for bio-hydrogen production (Guo et al., 2010). Inparticular, co-digestion of nitrogen-rich livestock manuremixed with carbohydrate-rich materials, has been consideredfor bio-hydrogen production (Guo et al., 2010).Hydrogen is an important energy transfer intermediate inAD. In the acidogenic phase of a two-phase system, the keyissue is to enable the accumulation of hydrogen, typicallyconsumed very quickly by microorganisms. Three microbialgroups are key players in hydrogen turnover: H2-producingfermenting bacteria (HPB), H2-consuming methanogens andH2-consuming acetogens. In order to facilitate HPB, whilepreventing H2-consuming microorganisms, pretreatmentsand biokinetic control of parameters, such as, pH and hy-draulic retention time (HRT) are used. Heat-treatment of theinoculum, selecting for spore-forming bacteria, decreasesmethanogen content. Maintaining a low in-reactor pH anda high dilution rate prevent the growth of methanogens andpossibly other H2-consuming microorganisms (Valdez-Vazquez and Poggi-Varaldo, 2009). However, the knowledgeon microbial community structure and dynamics in thetwo-stage processes is still limited. Studies focusedmainly onthe microbial qualitative diversity (Jo et al., 2007; Xing et al.,2008; Luo et al., 2011b) but not on the qualitative and quanti-tative dynamics of the key functional microbial groupsoccurring in the two-stages AD processes. As the microor-ganisms with their biochemical reactions are the key playersof the process, investigating the complexity of the microbialcommunity and its dynamics is a prerequisite to understandthe AD process, control it and improving its efficiency.The aim of this study was to characterize and compare,qualitatively and quantitatively, the bacterial and archaealcommunity of a two-stages and a conventional single-stageprocesses, both fed with the same mixture of swine manureand fruit and vegetable market wastes. In a previous study wecompared the energetic and chemical performances ofa two-stages and a single-stage AD processes run in lab-scalethermophilic intermittent-continuous stirred tank reactors(I-CSTR) (Schievano et al., 2012). We found comparable overall2.2. DNA extractionReactor samples were centrifuged (10,000 g, 30 min, 4 C),the resulting pellet washed twice with sterile water and cen-trifuged again in the same conditions. Variable volumes(2e3 ml) were used for centrifugation to obtain a final pellet of100mg. The pellets were stored at20 C until DNA extractionperformed using the PowerSoil DNA Isolation kit (MoBio Lab-oratories, Inc., Milan, Italy) according to the manufacturersinstructions. All DNA were extracted in duplicate.2.3. PCR-DGGE analysisBacterial and archaeal 16S rRNA gene were amplified by PCRenergy recovery for the two processes, though some organicmatter was left undegraded in two-stage process indicatingpartial inefficiency. Here we report the results of a study onthe structure and dynamics of microbial communities in thetwo processes assessed by PCR-Denaturing Gradient GelElectrophoresis (DGGE). We complemented the study of themicrobial communities dynamics by evaluating the temporalquantitative changes of themajor functional microbial groupsinvolved in the two processes by quantitative Real-Time PCR.Our aim was to give a contribution to the knowledge ofmicrobiological aspects of two types of reactor processesinvestigating, together with previous analytical data, micro-bial signatures associated to the performance of twoprocesses.2. Materials and methods2.1. Bioreactor set up and operationThree previously described (Schievano et al., 2012) anaerobiccompletely stirred tank reactor (CSTR) were operated andused as source of biomass samples. The two-stage processconsisted of a hydrogen-producing reactor (R1) with 2.3 lworking volume and a methane-producing reactor (R2) with14.7 l working volume. The single-stage process was a reactorwith 14.7 l working volume (R3). R1 was inoculated with heat-shocked (100 C for 2 h) anaerobic seeding sludge from a full-scale biogas plant treating household source-separated bio-waste and agro-industrial by-products. The same sludge,without heat-shock, was used as inoculum for both R2 and R3.The feeding substrate, a mixture (4:1 w/w ratio) of swinemanure and fruits and vegetables market waste (a chemicalcharacterization presented in Schievano et al. (2012)) wassupplied intermittently to R1 and R2 by peristaltic pumps. Theoperational hydraulic retention time (HRT) was 3, 22 and 25days in R1, R2 and R3, respectively. Temperature was main-tained at 55 2 C, pH was measured in continuous and notactively controlled. Qualitative and quantitative biogas anal-ysis were performed automatically in each reactor by gasflow-meters (Schievano et al., 2012). The two-stage hydrogen-methane and the single-stage methane AD processes weremonitored for 2 and 1 month, respectively.using the primer sets GC-357-F/907-R and GC-ARC787-F/ARC1059-R, respectively (Sass et al., 2001; Hwang et al., 2008).PCR reactions and thermal programs were performed as pre-viously described (Merlino et al., 2012). PCR products (approx.300 ng) were loaded onto 7% (w/v) polyacrylamide gels(0.75 mm) containing a denaturant gradient of 30e70% or40e60% for Bacteria and Archaea, respectively (100% denatur-ant contained 7 M urea and 40% formamide). Electrophoresiswas run in 1 TAE buffer using a D-Code electrophoresissystem (BioRad) at 90 V and 60 C for 17 h. Gels were stainedwith SYBR(R) Green I Nucleic A (Invitrogen) and documentedwith the GelDoc 2000 apparatus (BioRad) by using the Di-versity Database software (BioRad). Relevant DNA bands wereexcised from the gels and eluted in 50 ml of TriseHCl 10 mM.Five microliters of DNA was PCR reamplified and the obtainedsequences (Macrogen, Seoul, Korea) were compared againstthe NCBI genomic database with the BLAST search alignmenttool. Sequence alignment and phylogenetic trees were carriedout using theMEGA software, version 5.0 (Tamura et al., 2011).The trees were constructed using the Maximum Likelihoodwas calculated based on the Gini value, Dy was determined bythe moving window analysis (Marzorati et al., 2008).2.4. Real-time PCR analysisQuantitative PCR assays were performed using primer setreported in Table 1. Considering that in anaerobic reactormost Archaea are methanogens (Yu et al., 2005), an archaealPCR real time assay was used to estimate quantitativelymethanogens. PCR SYBR green reactions were prepared byusing the Brilliant SYBR Green QPCR Master Mix kit (Stra-tagene) in 96-well plates on the I-Cycler (Biorad). The reactionmix (25 ml) contained: 1 Brilliant SYBR Green (2.5 mMMgCl2),0.12 mM of each primers, and approx. 100 ng of extracted DNA.In the case of primer set Msl812-F/Msl1159-R extra MgCl2 wasadded to a final concentration of 4.0 mM. One real time assaywas carried out per extracted DNA. The thermal cycling pro-ATAAwat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 5 1985algorithm and the Tamura Nei parameter correction and werebootstrapped 2000 times.DGGE gel profiles were analysed using the Quantity Onesoftware (Biorad). Lane background was subtracted by therolling disk tool. Bands were detected automatically andmatched manually. DGGE-based molecular parameters,namely dynamycs (Dy), richness (Rr) and community organi-zation (Co), were calculated as previously described (Marzoratiet al., 2008). Briefly, Dy was calculated from the similaritymatrix (100 e similarity%); Rr was the total number of bandsmultiplied by the percentage of denaturing gradient used; Cowas the percentage of Gini coefficient, a value describing thedegree of evenness within a community by measuring thenormalized area between a given Lorenz curve and the perfectevenness line. The Co parameter informs on the functionalorganization of the microbial community describing the spe-cies abundance distribution within a microbial community interms of degrees of evenness (0e100). Low Co values representa highly even community, whereas high Co values are char-acteristic of uneven communities. Average Co (Co 45e60)values correspond to balanced community, characterized bymost functional stability and resilience. The Co coefficientTable 1 e Real time PCR primer sets used in this study.Target group Name SequenceBacteria Bac357-F CCTACGGGAGGCAGCAGBac907-R CCGTCAATTCCTTTGAGTTTHydrogen-producingbacteria (HPB)hydF1 GCCGACCTKACMATMATGGAhydH ATRCARCCRCCSGGRCAGGCCAcetogens fhs1 GTWTGGGCWAARGGYGGMGFTHFS-r GTATTGDGTYTTRGCCATACASulphate-reducingbacteria (SRB)Drs1-F ACSCACTGGAAGCACGGCGGDsr-R GTGGMRCCGTGCAKRTTGGArchaea Arch 931-F AGGAATTGGCGGGGGAGCAArchM1100-R BGGGTCTCGCTCGTTRCMethanosarcinales Msl812-F GTAAACGATRYTCGCTAGGTMsl1159-R GGTCCCCACAGWGTACCgram consisted of 10 min at 95 C, followed by 40 cycles of 30 sat 95 C, 1 min at X C (X 58 C for Bac357-F/Bac907-R, 49 Cfor hyd-F1/hyd-R1, 55 C for fhs1-F/THFS-R, 59 C for Drs1-F/Dsr-R, 64 C for Arch 931-F/ArchM1100-R, 60 C for Msl812-F/Msl1159-R) and 1min at 72 C. Finally, amelting curve analysiswas performed for verifying the specificity of PCR products.The program was as follows: denaturation of 1 min at 95 C,cooling of 1 min at 55 C and then 95 C again, at a rate of0.5 C per cycle. Cycle threshold (Ct) values were calculatedusing the Biorad real-time software (version 3.0a) according tothe manufacturers instructions. Standard curves were gen-erated by tenfold diluting the standard plasmids to obtaina series of concentrations ranging from 10 to 108 copies ofplasmid DNA. The standard plasmids were constructed aspreviously described (Merlino et al., 2012) by cloning frag-ments obtained from PCR amplification of genomic DNA fromthe bacterium Asaia for bacteria) or from total DNA (forarchaea) from an anaerobic batch digester (Table 1). Conver-sion of 16S rRNA gene copy numbers to cell number was doneconsidering the average 16S rRNA gene copy numbers ofbacteria (4/cell) and methanogens (2.5 copies/cell) reported inthe Ribosomal RNA Database (rrnDB, Lee et al., 2009). In thecase of real-time PCR targeting functional genes, it wasTargetgeneAmpliconsize (bp)Closest relative ofstandard fragment(% similarity)Reference16SrRNA550-585 Asaia sp. AM404260(100%)Favia et al.,2007hydA 700 Uncultured bacteriumEU828435 (75%)Xing et al.,2008GG fhs 250 Clostridium beijerinckiiCP000721 (76%)Xu et al.,2009dsrA 221 DesulfobacteriumautotrophicumCP001087 (98%)Kondo et al.,200416SrRNA169 Methanobrevibactersp. DQ402034 (98%)Einen et al.,200816S 354 Methanosarcina mazeii Yu et al.,rRNA LM5 DQ987528 (98%) 2005assumed that copy number was equivalent to cell numberbased on the premise that the majority of known bacteria inthe database have a single copy of the functional genes con-sidered (Kondo et al., 2004; Xu et al., 2009).3. Results3.1. Operation performance of anaerobic bioreactorsThe two-stage process was monitored over a period of twomonths including the start-up period (days 0e9), a steadystate period (days 9e43) described in Schievano et al. (2012),and a further 17-days long period where some imbalancesoccurred (Fig. 1A and B), whereas the single-stage process wasmonitored only along the steady state period (Fig. 1C). Aspreviously reported (Schievano et al., 2012), at the steady statehydrogen production rate was of 1.5 Ndm3 H2/L d (45% [v/v]content in biogas) in the acidogenic reactors (R1), whereasmethane production rates of 0.53 and 0.54 Ndm3 CH4/L d wereregistered for the methanogenic reactors of the two- (R2) andsingle-stage (R3) processes, respectively (68% and 54% [v/v]content in biogas). In R1 methane was detected between day40 and day 50 with percentages of 1e5% of total biogas. Awat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 51986Fig. 1 e Hydrogen/methane productions in R1 (A), R2 (B) and R3 (start-up period and a 17-long days period after the steady stateC) during the observed operational period. In R1 and R2 theare indicated by shading.serious biogas production failure occurred from days 49e51both in R1 and R2, and was attributed to electric energyshortage that determined a stop of the thermal control anda drop of the reactor temperatures.In R1 the major acidogenic byproducts were hexanoic acid,acetate, butyrate and propionate. Total VFAs concentrationwas approx. 3900 mg acetate/L and acetate accumulated atmore than 2500 mg/L. In R2 the concentration of acetatedecreased and remained at low level (wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 51988The bacterial DGGE pattern was further characterizedusing parameters independent from the DGGE run, namely,microbial richness (Rr), dynamics of change (Dy) and com-munity organization (Co) (Fig. 4). Rr was higher in the start-upFig. 3 e Phylogenetic tree showing the phylogenetic relationshiphylum with reference sequences deposited at the GenBank daindicated with the capital letters F, H, T and S, respectively. Unrepresent bootstrap values. The scale bar represents a sequencperiod (Rr-indices of 30e40), thereafter Rr decreased reachingat the end of the sampling values (6e10) corresponding to lowrange-weighted richness. The Dy values, were generally kepthigh, indicating the adaption of the community during theps of bacterial 16S rRNA sequences affiliated to Firmicutestabase. Sequences from feeding source, R1, R2 and R3 areindicates an uncultered bacterium. Numbers at nodese divergence of 5%.itywat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 5 1989Fig. 4 e Microbial richness (Rr), dynamics (Dy), and communarchaeal (C, D) DGGE profiles of R1 (A), R2 (C) and R3 (B, D).process. Dy decreased after day 43 and was very low the lastday of sampling (rate of 8%). Variable values of Co wereobserved during the start-up period, thereafter stabilizing tovalues (on average approx. 40) representing a relatively mod-erate organized community.In order to account for the methane production recorded,a PCR-DGGE analysis was carried out on Archaea (Fig. 2A0). Aphylogenetic tree of identified sequences is shown in Fig. 5(sequences affiliation in Table S2 of supplemental material).A dominant band (h2) affiliated (96.9%) to Methanosaeta wasfound in the initial days of the process, but after day 36 wasreplaced, at least in term of dominant intensity, by two bands(bands h5 and h6) affiliated (98%) to Metanogenium sp.Quantitative measurements of the bacterial abundance inthe acidogenic reactor are shown in Fig. 6. Bacteria were pre-sent at high concentrations (108e109 bacteria per ml). WithinBacteria, HPB represented only 0.05e1.4% of total Bacteria,whereas acetogens were 0.6e6.2% of total Bacteria. Number ofHPB remained almost constant during the process (106 bac-teria per ml), though a decrease was observed from day 36 today 50 with a ratio HPB to total Bacteria of about 0.05%. Theacetogens, with an abundance similar to that of HPB in thefirst ten days of hydrogen production, thereafter increased ofone order magnitude higher than HPB. Sulfate-reducing bac-teria (SRB) counted one order magnitude lower than HPB(about 105 bacteria per ml). A slight increase was observed atday 43 in correspondence of a declining trend of HPB and alsoof an increase of total Bacteria. Archaea were at low titre afterheat shock treatment (104 bacteria/ml) and were of two-threeorders of magnitude lower than Bacteria. Within Archaea,organization (Co) parameters from bacterial (A, B) andMethanosarcinales represented approx. 1% of total Archaea,declining to 0.1% at day 64. In the influent, Methanosarcinaleswere detected at low concentration too (7%).3.2.2. Methanogenic processThe PCR-DGGE of Archaea is showed in Fig. 2B and the phylo-genetic positions of the identified sequences are indicated inFig. 5 (for sequences affiliation see Table S2 of supplementalmaterial). The PCR-DGGE profiles showed always threestrongly intense bands (t1, t2, t3), closely related to each otherand to Methanosarcina mazeii (>98%). Bands t5 and t6, appear-ing at day 60, were closely related (>99%) to the genus Meth-anothermobacter which depends entirely on H2/CO2 as energyand carbon sources (Schill et al., 1999). The statistical analysisof DGGE profiles evidenced a stable, highly specialized (Rr < 4)community. A notable change (37%) occurred only at day 60 incorrespondence to a partial accumulation of VFAs. The Covalues were around 40e50, usually reported for good perfor-mance reactors (Carballa et al., 2011).PCR-DGGE analysis carried out on Bacteria evidenced in R2,like in R1, a bacterial community dominated by Firmicutes(Fig. 2B0 and Table S2). In R2was found C. cellulosi (band T1) andother microorganisms (bands T3, T7, T8) already identified inR1 and assigned to unclassified Ruminococcaceae. The otheridentified sequences could not be attributed to known species,but were highly similar (>99%) to sequences from thermo-philic reactors (Tang et al., 2011; Sasaki et al., 2011; Gobernaet al., 2009; Shiratori et al., 2006); in particular, bands T10and T11 matched (99e100%) with the unknown DAD cluster 3(Tang et al., 2011). Band T2 was affiliated to the Thermotogaewat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 51990phylum, consisting of anaerobic hyperthermophilic bacteriacapable of using a great variety of carbohydrates, and gen-erating hydrogen (Eriksen et al., 2010).In R2 the abundance of the different populations remainedalmost constant during theprocess (Fig. 6).Archaea and Bacteriawere present at rather similar concentrations. Methanogennumberwas higher than in R1, around 107e108 bacteria perml,a range typical of anaerobic reactors (Yu et al., 2005; Lee et al.,2008).Methanosarcinales were the dominant methanogens rep-resenting approx. 50% of the total Archaea, in agreement withthe PCR-DGGE data. Archaea andMethanosarcinales showed thesame trend during the operation. The number of Meth-anosarcinales decreased in correspondence to the partial inhi-bition of the process, in accordance with PCR-DGGE data.SRB, potential competitors of methanogens, were fourorder magnitude lower than Archaea (103 bacteria per ml),while acetogens and the HPB remained relatively stable dur-ing all the operation (average values of 2 107 and 5 106bacteria per ml, respectively).3.3. Microbial community characterization of single-stage anaerobic processPCR-DGGE bands of Archaea (Fig. 2C0) belonged mainly to theMethanosarcinales (Fig. 5 and Table S2). Bands s1, s2 and s3,Fig. 5 e Phylogenetic tree showing the phylogenetic relationshipdeposited at the GenBank database. Sequences from feeding souf, h, t and s, respectively. Un indicates an uncultered bacterium.represents a sequence divergence of 10%.identical to those detected in R2, were associated to the samesludge used for the start-up of the two processes. Bands s4 ands6 were related (>97.7%) to Methanosarcina spp. Bands s9 ands10 both matched, with 97.3% and 99.5% similarity respec-tively, withMethanosarcina mazeii andMethanosarcina lacustris.Band s5, which showed a strong intensity after 15 day, wasaffiliated to Methanosaeta concilii. The faint bands s7 and s8were affiliated (>98.0%) to Methanothermobacter. The archaealcommunity structure, on the contrary of R2, changed overtime. After two weeks, the community was drastically shifted(rate of 88%) and thereafter stabilized. The community wasricher than in R2 (Rr average value of 6.8). Co values were onslightly higher than those of R2, indicating a moderatelyorganized community.PCR-DGGE analysis of Bacteria indicated a dominance ofFirmicutes, Clostridia and Bacilli classes (Fig. 2C). The bands ofBacilli (S1eS3) were replaced after day 15 by high intensitybands related to Bacteroidetes (S4eS7). Bands S1eS3 werehighly similar (99.1e100%) to Bacillus infernus (96.8e97.8%), ananaerobic species able to ferment glucose and utilize formateand lactate for growth (Boone et al., 1995). Bands S4eS7 wereassigned to unclassified Porphyromonadaceae, bacteria capableof producing VFA from carbohydrates or proteins (Ziganshinet al., 2011). Considering the strong intensity of these bands,probably these bacteria played an important role ins of archaeal 16S rRNA sequences with reference sequencesrce, R1, R2 and R3 are indicated with the lower-case lettersNumbers at nodes represent bootstrap values. The scale barwat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 5 1991hydrolysis and acidogenesis. A microorganism stably detec-ted throughout the process was Anaerobaculum (band S8), ableto ferment mainly peptides and organic acids to acetate,hydrogen and CO2 (Menes and Mux`, 2002). Bands S9 and S10belonged to the Clostridiaceae cluster I. Bands S11 and S12clustered with unknown clones, from thermophilic reactors,grouping in the cluster DAD 1 (Tang et al., 2011) and DAD3,respectively. Band S13 and S14 were correlated to Thermace-togenium (Hattori et al., 2000) and Tepidanaerobacter (SekiguchiFig. 6 e Concentrations of microorganisms in R1 (A), R2 (B) and Rmeasurements. HPB and SRB were not detected at day 0 (after het al., 2006), thermophilic syntrophic acetate-oxidizing bac-teria capable to form methane in association with hydro-genotrophic methanogens. Statistical analysis of bacterialPCR-DGGE profiles indicated a very high diversity (averageRr value of 65), higher than in R1. Dy had a very similar trendto that observed for Archaea, with a notable community shiftat day 15 (rate 66%). Constant Co values of approx. 40 indi-cated a moderately even community that remained stableduring the operations.3 (C) during the observed period. Values are averages of twoeat-shock treatment).dominance of these clostridia has been favoured by a combi-wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 51992nation of various operational parameters (temperaure, feed-ing source, reactor type, TS, pH and HRT) rather than by theinoculum pre-treatment, in agreement with the findings ofLuo et al. (2011).In R2, as well in R1, not only the species diversity was rel-atively low, but a relatively dynamic bacterial communitysimplified over time. A specialized community, however,Quantitativemeasurements of Bacteria, acetogens, SRB andHPB in reactor R3 showed values similar to those in R2 (Fig. 6).On the contrary, both totalArchaea andMethanosarcinaleswereestimated in lower numbers than in R2 (below 107 bacteria perml). The number of Methanosarcinales decreased with thechanging of the community structure, dropping to 32% of totalArchaea at day 15 and increasing again to 58% at day 29.4. DiscussionIn this study the dynamics of microbial community structurein a two- and a single-stage AD reactors have been inves-tigated and compared. As models were used I-CTSR reactorsoperating with some equal working conditions (inocula fromthe same methanogenic sludge, feeding source, temperature,and non-controlled pH) and some operational parameters(HRT, loading rate, heat-shock treatment of acidogenic reac-tor) specifically designed for the two AD processes.Overall, the study showed that the microbial communitystructure and dynamic was different in twoAD processes bothfor Bacteria and Archaea. Resistant spore-formers Firmicutesselected by the heat-shock treatment dominated in R1 and R2,whereas a more diverse community (Firmicutes, Bacteroidetes,Synergistes) was found in R3. In R1 and R2, most of the bacteriawere related to the order Clostridiales, more specifically to theClostridium genus. Clostridium spp. are capable of fermentcellulose and various carbohydrates mainly to acetate, buty-rate and hydrogen (Valdez-Vazquez and Poggi-Varaldo, 2009)and their prevalence in stable H2-producing systems has beenalready documented (Jo et al., 2007). Hence, they can accountfor the hydrogen production recorded in the acidogenic reac-tor. The majority of identified species, however, were notreferable to known cultured species, with the exception of C.cellulosi. Nevertheless, many of the identified microorganismswere phylogenetically related to microorganisms from ther-mophilic acidogenic anaerobic reactors fed with vegetablekitchen waste and, more distantly, to Clostridium species(Clostridium sp. BS-1, Clostridium sp. Z6) included into theRuminococcaceae cluster. The presence in the digester of spe-cies with degrading ability similar to that of Clostridium sp. BS-1 may account for the detection of high hexanoic acid in thereactor. Bioavailable D-galactitol, a reduced form of D-gal-actose, is in fact contained in many fruit and vegetable resi-dues like those used as feeding source. On the other hand, thedominance of uncultured bacteria affiliated to Clostridium sp.Z6 has been previously reported in other hydrogen-producingreactors (Chu et al., 2010; Lee et al., 2010a) which operated at55 C treating food waste without heat treatment of inoculum.Hence, it is likely that in this study the selection and thethough highly functional, is more sensitive to changes since itlacks alternative players when impaired by stresses. This mayexplain the partially inefficient biodegradation observed in R2as deduced by the chemical characterization of reactors ma-terials. The high concentrations of VFAs, alcohols and otherintermediatemetabolites (amines, amino acids, phenols) in R2(Schievano et al., 2012) probably exerted inhibiting effects onmany microorganisms, including methanogens, decreasingthe community diversity and its potential of adaptation. Onthe contrary, in R3 was maintained a more diverse and dy-namic community that probably has a richest network ofmetabolic pathways explaining the most efficient degradingactivity observed in this digester. In particular, in R3 werefound microorganisms related to the Porphyromonadaceaefamily and Anaerobaculum genus. These bacteria, capable offermenting peptides and amino acids, were possibly respon-sible for the low levels of nitrogenous compounds detected inR3 and found instead undegraded in R2. Bacteroidetes are moreefficient than Firmicutes in degrading plant polyphenols andless sensitive to phenols (Rastmanesh, 2011 and referencestherein). It is speculated that a phenol/polyphenols richfeeding promoted the Bacteroidetes growth in R3, but left thesecompounds not degraded in R2.The archaeal community in two methanogenic reactorswas dominated, though at different levels, by the Meth-anosarcinales (average value of 70% and 58% of total Archaea inR2 and R3, respectively), suggesting that acetoclastic meth-anogenesis was the major pathway of methane production inboth systems. In R2, Methanosarcinales were up to 90% ofmethanogens andwere represented at the steady state only bythe genus Methanosarcina. Methanosarcina spp., prevailing athigh acetate concentration (Jetten et al., 1992), were previouslydetected as dominant in other thermophilic methanogenicreactors from two-stage processes (Chu et al., 2010; Luo et al.,2011) and in general from digesters treating manure (Demireland Scherer, 2008). Hence, their abundance in R2 is sustainedby the high levels of acetate (after hexanoic acid) detected ingas and liquidphasesof R1 (Schievanoet al., 2012). Particularly,the identified Methanosarcina-like species were related to M.mazei andM. siciliaewhich are able to utilize various substrates(methanol, methylamines and also H2/CO2 in the case of M.siciliae) other than acetate (Liu et al., 2009; Lee et al., 2010a).In R3, though the high level of Methanosarcinales also pre-sent, the archaeal community was more diverse and dynamicas compared to R2. Methanosarcina, Methanosaeta and Meth-anothermobacter specieswere simultaneously detected, thoughat different density over the course of the operation. Thecontribution of hydrogenotrophic methanogenesis in R3 washighest than in R2 as confirmed by the detection in the latterof thermophilic acetate-oxidizing bacteria (Thermacetogeniumand Tepidanaerobacter) capable of form methane in coopera-tion with hydrogenotrophic methanogens (Hattori, 2008).Such an archaeal community may have been advantageous tothe process performance, promoting an improved adaptationpotential.Data of quantitative determinations of AD functionalgroups indicated that their abundance remained rather con-stant at the steady state despite some variations in biogasproduction during the period.In the two methanogenic reactors the abundance of dif-ferent microbial groups were at the same order of magnitude,except for higher methanogens in R2, probably enriched byAD processes iii) evidence the key role of operational param-eters and of reactors configuration in driving the dominantAcknowledgementsCarballa, M., Smits, M., Etchebehere, C., Boon, N., Verstraete, W.,2011. Correlations between molecular and operationalwat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 9 8 3e1 9 9 5 1993the separation of acidogenic and methanogenic phases. In R1the high abundance of acetogens suggested that theywere themajor competitors of HPB. Acetogens are capable to catalysethe reductive synthesis of acetate from CO2 switching be-tween heterotrophic and autotrophic metabolism dependingon substrate availability (Drake et al., 2002). Their contributionto hydrogen consumption in bioreactors depends on severalchemical and physical factors (acetate concentrations,hydrogen partial pressure, mass transfer phenomena be-tween H2-producers and H2-consumers) and the history of theinocula. The abundance of acetogens in R1 reactor rangedfrom 1.4 108 to 9.3 108 FTHFS (formyltetrahydrofolatesynthetase) genes per gram dry weight, in accordance with Xuet al. (2009) who reported 108-109 FTHFS gene copies per gramdry weight in a sludge under H2/CO2 enrichment conditions.Thus, it is likely that acetogens, even at low percentages(Kraemer and Bagley, 2008), may have somehow contributedto hydrogen consumption in R1, suggesting that heat-treatment is not sufficient to control spore-forming H2-con-sumers. In addition, it was observed that, after some days ofoperation, acetogens prevailed over methanogens. This is inagreement with previous findings indicating that generallymethanogenesis prevails over acetogenesis due to its morefavourable thermodynamics and affinity for H2 (Liu andWhitman, 2008). However, acetogenesis can effectively out-competemethanogenesis in certain conditions, like under lowpH and accumulation of H2 (Drake et al., 2002). The methanedetected in the acidogenic reactor, presumably generated byMetanogenium species, was in biogas in low percentage and fora limited operational time, confirming the efficacy of low pHcondition to inhibit methanogenesis.Quantitative data allowed also to explain some failures inbiogas production of the two-stage process occurred after thesteady state, like the hydrogen production drop occurred fromdays 48e50 accompanied by an almost one order magnitudedecrease of HPB.Partial accumulation of VFAs, particularly acetate andpropionate (370 and 325 mg/L, respectively, at day 59) mayexplain the one order magnitude decrease of methanogensand the acetotrophic methanogen proportion (50%). This VFAaccumulation may also account for the appearance in themethanogen population ofMethanothermobacter, less sensitivethan acetoclastic methanogens to increases in VFAs concen-tration (Hori et al., 2006). All these microbiological evidencessupport the non-optimal condition in general occurring in R2.Overall, the higher diversity and dynamic of prokaryotecommunity, especially the fermentative bacterial one, in thesingle stage process as compared to the two-stage process,may account for the best degradation efficiency observed inR3. The difference in bacterial community and performancebetween the two AD processes is likely a consequence ofdecoupling of acidogenesis from methanogenesis in the twostage stystem and of the different configurations and opera-tional parameters of the two systems. In the two-stage pro-cess, the separation of fermentative and methanogenicenvironments might have affected negatively syntrophic as-sociations among microorganisms and probably reduced thenumber of degradation pathways. Particularly, the enrichedsimplified community established in R2 proved to be unable tocompletely degrade many intermediate metabolites causingparameters in continuous lab-scale anaerobic reactors.Applied Microbiology and Biotechnology 89, 303e314.Chu, C.-F., Ebie, Y., Xu, K.-Q., Li, Y.-Y., Inamori, Y., 2010.Characterization of microbial community in the two-stageprocess for hydrogen and methane production from foodThis study was funded by the project Produzione di bio-idrogeno ed energia rinnovabile da residui agro-zootecnici eAgrIdEn by Regione Lombardia. Partial support comes fromthe project Miniaturizzazione e semplificazione di linee ditrasformazione per piccole produzioni agroalimentari eimpiego di energie rinnovabili e MIERI by Ministero per lePolitiche Agricole Alimentari e Forestali.Appendix A. 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Real-time PCR analysis3. Results3.1. Operation performance of anaerobic bioreactors3.2. Microbial community characterization of two-stage anaerobic process3.2.1. Hydrogenogenic acidogenic process3.2.2. Methanogenic process3.3. Microbial community characterization of single-stage anaerobic process4. Discussion5. ConclusionsAcknowledgementsAppendix A. Supplementary dataReferences

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