Study of microbial community and biodegradation efficiency for single- and two-phase anaerobic co-digestion of brown water and food waste

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grbInstigineed in broties betr for msolids rpredominance of Firmicutes and greater bacterial diversity in two-phase CSTR,and Jansen, 2012). In comparison with landlling or incineration,the anaerobic digestion of food waste was found to be a more suit-able and sustainable treatment method to address the growingability by provid-apacity. Thwas descriigher biogduction and biodegradation efciencies when brown watadded as a co-substrate to the anaerobic degradation of foodProduction of methane via anaerobic digestion of organictants not only provides a cheaper and greener alternative to foodwaste and brown water disposal, it also replaces fossil fuel-derivedenergy and reduces the impact of global warming (Abbasi et al.,2012).Anaerobic digestion of organic matter is carried out syntrophi-cally by microbial communities consisting of both bacterial andarchaeal species. The degradation may be divided into three steps. Corresponding author at: Residues and Resource Reclamation Centre, NanyangEnvironment and Water Research Institute, Nanyang Technological University, 1Cleantech Loop, Singapore 637141, Singapore. Tel.: +65 67904100; fax: +6567927319.E-mail addresses: (J.W. Lim), (C.-L.Chen), (I.J.R. Ho), (J.-Y. Wang).Bioresource Technology 147 (2013) 193201Contents lists availabBioresource Tjournal homepage: www.els1 Tel.: +65 65927760; fax: +65 67927319.waste is a suitable substrate for anaerobic digestion due to its highorganic content. On the other hand, landlling of food waste leadsto uncontrolled emission of methane, and incineration could beinefcient due to the low caloric value of wet food waste (Bernstadcould improve the anaerobic digestion process sting additional nutrients and maintaining buffer cets of co-digesting brown water and food wasteRajagopal et al. (2013). The authors observed h0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved. ben-bed byas pro-er waswaste.pollu-Keywords:Microbial community structureAnaerobic co-digestionBrown waterFood wasteSingle- and two-phase CSTRand the lack of Firmicutes in single-phase CSTR. Methanosaeta was predominant in both CSTRs and thiscorrelated to low levels of acetate in their efuent. Insights gained from this study would enhance theunderstanding of microorganisms involved in co-digestion of brown water and food waste as well asthe complex biochemical interactions promoting digester stability and performance. 2013 Elsevier Ltd. All rights reserved.1. IntroductionAnaerobic digestion is a biochemical process that degradesbiomass biologically and produces biogas consisting mainly ofmethane, which is a valuable source of renewable energy. Foodconcern over large amounts of food waste generated worldwide.Therefore, the treatment of food waste by anaerobic digestion pro-cess has attracted increasing attention in recent years (Wang et al.,2002; Ike et al., 2010).The addition of co-substrate (e.g. brown water) to food wastewas likely due to greater ecould be attributed to theh i g h l i g h t s First study on microorganisms involve Clear differences in bacterial communi Methanosaeta was the main contributo Firmicutes played an important role ina r t i c l e i n f oArticle history:Received 5 June 2013Received in revised form 4 August 2013Accepted 6 August 2013Available online 14 August 2013wn water and food waste degradation.ween single- and two-phase CSTRs.ethane production in both CSTRs.eduction.a b s t r a c tThe objective of this work was to study the microbial community and reactor performance for the anaer-obic co-digestion of brown water and food waste in single- and two-phase continuously stirred tank reac-tors (CSTRs). Bacterial and archaeal communities were analyzed after 150 days of reactor operation. Ascompared to single-phase CSTR, methane production in two-phase CSTR was found to be 23% higher. Thisxtent of solubilization and acidication observed in the latter. These ndingsStudy of microbial community and biodeand two-phase anaerobic co-digestion ofJ.W. Lim a,b,1, C.-L. Chen a, I.J.R. Ho a, J.-Y. Wang a,b,aResidues and Resource Reclamation Centre, Nanyang Environment and Water ResearchbDivision of Environmental and Water Resources, School of Civil and Environmental En639798, Singaporeadation efciency for single-rown water and food wastetute, Nanyang Technological University, 1 Cleantech Loop, Singapore 637141, Singaporering, Nanyang Technological University, 50 Nanyang Avenue, Singaporele at ScienceDirectechnologyevier .com/locate /bior techon this topic. Comprehension of microbial community and its func-tion is necessary to improve the efciency and process stability ofechnanaerobic digesters. 16S rRNA cloning and sequencing is the wellknown method used to characterize microbial community in ananaerobic reactor while uorescent in situ hybridization (FISH) isa useful method to verify cloning ndings and to visualize the dif-ferent cells in anaerobic sludge. Therefore, these two methodswere employed in the current study to determine the microbialpopulations. The objective of this work was to study the microbialcommunity and reactor performance for the anaerobic co-diges-tion of brown water and food waste in single- and two-phase con-tinuously stirred tank reactors (CSTRs). Insights gained from thisstudy would enhance the understanding of microorganisms in-volved in the anaerobic co-digestion of brown water and foodwaste as well as the complex biochemical interactions thatpromote digester stability and performance. These could aid theselection of seeding sludge for rapid startup in future applications.2. Methods2.1. Feedstock and reactor operationFood waste was collected from canteens on campus whilebrown water was collected from a specially designed source-separation toilet, where urine with 0.3 L ush water (as yellowwater) and faeces with 2 L ush water (as brown water) werecollected in separate tanks. The feed for this study consisted of amixture of 300 g blended food waste and 2 L brown water, andhad an average pH of 6.23 0.07. The characteristics of the feedare as shown in Table 1. Anaerobic co-digestion of brown waterand food waste was performed in laboratory scale (5 L) single-and two-phase CSTRs. The co-substrates were prepared daily andfed to the reactors, which included the acidogenic (RA) and metha-nogenic (RM) reactors of the two-phase CSTR system and thesingle-phase CSTR (RS), in batch mode. The working volumes ofRA, RM and RS were 1.2 L, 4.1 L and 5.3 L, respectively, and the con-tents were mixed continuously (mixing time: 5 min ON followedby 5 min OFF) at 80 rpm by an overhead mechanical stirrer asreported previously by Rajagopal et al. (2013). RA, RM and RS wereinitially inoculated with mesophilic anaerobic sludge collectedDuring the rst step, hydrolysis bacteria degrade polymeric organ-ic matter into monomers, such as sugar and amino acid, which arefurther degraded in the second step by acetogenic bacteria intovolatile fatty acids (VFAs), such as acetate. In the last step, metha-nogens produce biogas mainly from formate, hydrogen andacetate.In conventional applications, anaerobic digestion processesusually occur in a single reactor system. However, acid- and meth-ane-forming microorganisms have very different nutritional needs.When kept together in a single reactor system, some of such sys-tems gradually gave rise to reactor instability problems (Demireland Yenigun, 2002). The physical separation of acid- andmethane-forming microorganisms in different reactors was rstproposed by Poland and Ghosh (1971). Such systems providedoptimum environmental conditions for each group of organismsand thus led to enhanced stability and control of the overallprocess.Studies on bacterial and methanogenic archaeal communitystructures in anaerobic digesters treating food waste have been re-ported recently (Ike et al., 2010; Wang et al., 2010). However, theunderstanding of microbial aspects for co-digestion of brownwater and food waste is still limited due to the lack of references194 J.W. Lim et al. / Bioresource Tfrom a local wastewater treatment plant (Ulu Pandan Water Recla-mation Plant, Singapore). Reactor contents were gradually replacedby the brown water and food waste mixture. By the time this studystarted, anaerobic sludge was completely replaced by the brownwater and food waste mixture. The single and two-phase CSTR sys-tems were operated in parallel for 150 days at 35 C with hydraulicretention time (HRT) as shown in Table 1. HRT was reduced byadding increased volumes of the brown water and food waste mix-ture into the reactors of xed working volumes. The organic load-ing rate (OLR) was maintained at around 0.50.8 g-VS L1 d1 inthis study. Both the single- and two-phase CSTRs were operatedin the same way and had the same overall reactor working volumeof 5.3 L. Both RA and RS were fed with brown water and food wastemixtures prepared daily while RM was fed with the acidied efu-ent from RA during the study. The reactor performances for RA, RM,and RS were monitored weekly.2.2. Chemical analysisBiogas production was measured daily using a mass ow meter(McMillan Company, Model 50D-3E), while other parameters suchas pH, total (TS) and volatile (VS) solids, total and soluble chemicaloxygen demand (COD), VFAs and biogas composition were mea-sured weekly. The biogas composition (i.e., methane, carbon diox-ide, nitrogen and hydrogen contents) was analyzed by gaschromatograph (Agilent Technologies 7890A, USA) equipped witha thermal conductivity detector (TCD). pH value was measuredusing a compact titrator (Mettler Toledo) equipped with a pHprobe (Mettler Toledo DGi 115-SC). TS and VS were analyzedaccording to the Standard Methods (APHA, 1998). Total and solubleCOD were measured using COD digestion vials (Hach Chemical)and a spectrophotometer (DR/2800, Hach). Soluble COD was mea-sured using the supernatant of samples after centrifugation(KUBOTA 3700, Japan) at 12,000 rpm for 10 min. The determina-tion of VFAs was carried out using a gas chromatograph (AgilentTechnologies 7890A, USA), equipped with a ame ionization detec-tor (FID) and a DB-FFAP (Agilent Technologies, USA) column(30 m 0.32 mm 0.50 lm) and the samples were lteredthrough 0.45 lm cellulose acetate membrane lters (membranesolutions).2.3. DNA extraction and construction of 16S rRNA gene clone librariesSludge samples were collected on day 150 and genomic DNAwas extracted from sludge using chemical lysis and phenolchloro-formisoamyl alcohol (25:24:1, v:v:v) purication protocol as de-scribed previously (Liu et al., 1997). Primer sets 530F (50-GTGCCAGC(A/C)GCCGCGG-30) and 1490R (50-GGTTACCTTGTTACG-ACTT-30) as well as Ar1F (50-TCYGKTTGATCCYGSCRGAG-30) and1490R were used to amplify 16S rRNA gene from the total-commu-nity DNA, targeting total prokaryotes and Archaea, respectively.The thermal program used for amplication of 16S rRNA genewas as follows: hotstart 94 C for 3 min, 30 cycles of denaturation(30 s at 94 C), annealing (30 s at 54 C) and extension (45 s at72 C) and a nal extension at 72 C for 5 min. TOPO TA cloningkit (Invitrogen, CA) was used for clone library construction accord-ing to the manufacturers instructions. Approximately 100 and 50clones were randomly selected from RA, RM and RS for the membersin the domain Bacteria (amplied by primer set 530F and 1490R),and Archaea (amplied by primer set Ar1F and 1490R), respec-tively. The amplied DNA insert was then PCR amplied with avector-specic primer set (i.e., M13F and M13R). Restriction frag-ment length polymorphism (RFLP) was used to screen the 16SrRNA gene fragments to further remove the possible redundantclones. The M13-PCR products were separately digested to comple-tion with tetramer restriction enzymes MspI and RsaI (Newology 147 (2013) 193201England BioLabs, UK), and separated by electrophoresis in a 3%agarose gel. Gels were visualized using the FireReader gel docu-mentation (UVItec, Cambridge, UK) after staining with GelredTable1Operationalconditionsandreactorperformance.Period1(Days055)2(Days56109)3(Days110150)FeedaR AR MR SFeedaR ARMRSFeedaR ARMRSHRTb[d]10304010304062935BiogasProduction[Lg-VS1d1]n.d.n.d.1.910.370.960.28n.d.n.d.1.150.380.620.27n.d.n.d.1.570.610.650.20TS[gL1]30.409.9923.175.927.930.504.752.2823.306.2512.212.364.831.577.762.4519.6210.3711.141.694.040.937.931.13TSremoval[%]n.d.n.d.72.38.7483.618.26n.d.n.d.76.0715.9863.6718.25n.d.n.d.76.548.9953.0517.07VS[gL1]26.214.8021.565.605.390.433.212.0222.075.9911.372.953.271.405.762.1118.2910.289.631.592.510.845.870.79VSremoval[%]n.d.n.d.79.254.4085.409.20n.d.n.d.76.0715.9863.6718.25n.d.n.d.81.426.0956.506.05SolubleCOD[gL1]15.476.7119.0112.630.700.320.650.2213.013.2615.393.751.041.020.530.098.411.3618.601.110.810.400.490.04SolubleCODremoval[%]n.d.n.d.94.842.3495.102.62n.d.n.d.94.022.3195.591.28n.d.n.d.91.793.3094.190.74TVFA[mg-CODL1]815669353228536218783720016581417333688012564811951234569193677799114640121536n.d.:Notdetermined.a300gfoodwaste+2Lbrownwater.bHydraulicretentiontime.J.W. Lim et al. / Bioresource TechnoSludge samples from RA, RM and RS were collected towards theend of the operational period (on day 150) for FISH analyses. Thesludge samples were pretreated according to the protocol de-scribed previously by Amann et al. (1995), and xed overnightwith 4% paraformaldehyde solution at 4 C. Hybridization wascarried out at 46 C for 3 h with hybridization buffer containing5 ng lL1 of specic uorescent probe. Two oligonucleotideprobes, EUBmix (i.e., EUB338, EUB338-II, EUB338-III) andARC915, were used to target the members of Bacteria and Ar-chaea, respectively (Daims et al., 1999; Amann et al., 1995). FISHhybridization was performed with 35% formamide concentrationfor both probes (EUBmix and ARC915) in the hybridization buffer.An Olympus BX53 epiuorescence microscope equipped with acooled CCD camera DP72 with a 100W halogen bulb and uores-cence lter sets (U-FGW and U-FF-Cy5) under 100 objectivelens (Olympus, Japan) was used to capture FISH-stained images.3. Results and discussion3.1. Reactor performanceThe experimental results of this study were categorized intothree periods as shown in Table 1. The average biogas produc-tions for RM and RS throughout the study were 1.54 and0.74 L g-VS1 d1, and their average CH4 concentrations were60% and 50%, respectively. With no pH control in any of the reac-tors, the pH levels in the three reactors were stable throughoutthe study. RA, RM and RS had average pH levels of 3.72 0.35,6.98 0.14 and 6.98 0.15, respectively. The pH of RM and RSwere not signicantly different and were in the suggested opti-mal pH range suitable for methanogenesis to take place.The overall average concentration of total volatile fatty acid(TVFA) for RA was 11,115 7074 mg-COD L1, representing anaverage of 9-fold increase in TVFA production as compared tothe feed. With average soluble COD values of 12.93 5.67 and17.38 7.66 g L1, the degree of acidication (proportion of VFAin soluble COD) for the feed and RA efuent were 11% and 79%,respectively. This translated to a sevenfold increase in the degreeof acidication after treatment in RA of the two-phase system.Almost 80% of TVFA in R comprised of acetate, propionate and(Invitrogen, CA). Unique RFLP patterns were dened as a uniquesequence type of operational taxonomy unit (OTU).2.4. Sequence analysisThe 16S rRNA gene of the representative clones with differentRFLP patterns were sequenced, by 1st BASE (Singapore), to deter-mine their phylogenetic afliation. Nearly full-length 16S rRNAgene sequences of representative clones were compared to avail-able rRNA gene sequences in GenBank using the NCBI BLASTprogram. Chimeric artifacts were determined using DECIPHER(Wright et al., 2012) and phylogenetic trees were constructedwith MEGA5 program using the remaining clone sequences(approximately 80 for bacterial clones and 45 for archaeal clones)after removing the chimeric sequences. The JukesCantor correc-tion was used for distance matrix analyses and the trees wereconstructed using the Neighbor-joining method. Archaeal andbacterial 16S rRNA partial sequences obtained in this study weredeposited in the nucleotide Genbank database, under the acces-sion numbers: KF169842KF169904.2.5. Fluorescence in situ hybridization (FISH)logy 147 (2013) 193201 195Abutyrate. Acetate was the dominant VFA throughout the studywhere it accounted for 40% of TVFA. The percentages ofpropionate and butyrate in RA were on average 17% and 23%,respectively. The high degree of acidication as well as conversionof longer chain VFA to acetate suggested that the activities of acid-ogens and acetogens were high in RA.In RM, VFAs fed from RA were mostly consumed and their totalcontent in RM was reduced to an average amount of 618 mg-COD L1. Acetate was present in highest concentrations (averageof 101 mg-COD L1) as compared to propionate (average of85 mg-COD L1) and butyrate (average of 114 mg-COD L1). Thelevels of TVFA in RM were approximately 60% and ve times higherthan that in RS for periods 2 and 3 of the study, respectively. RM hadan average NH4 concentration of 689 mg L1, which was almost 2times higher than that in RS (366 mg L1). Despite the higher levelsof TVFA and NH4 in RM, there were no signicant differences in thereductions of total and soluble COD between RM and RS. Bothachieved at least 74% total COD and 91% soluble COD removalrates. In addition, the higher levels of TVFA and NH did not leadtotal, 7 bacterial operational taxonomic units (OTU) were identi-ed. The most detected OTU (BFABac_040), representing 49% ofthe total clones, was afliated to Acetobacter peroxydans strainLMG 1633 (AJ419836) with 99% similarity. BFABac_001 was thesecond most detected OTU accounting for 36% of the clones. To-gether with BFABac_009 and BFABac_111, the second predominantgroup was afliated with Lactobacillus amylovorus GRL 1112(NR_075048) with 99% similarity. BFABac_137 (1% of total clone)was afliated with Lactobacillus fermentum strains IFO 3956(NR_075033) and JCM 8596 (AB690185). The two remaining OTUs(BFABac_065 and BFABac_058) each corresponded to 1% of thetotal clone and were closely related to uncultured Clostridium spe-cies (HQ183766) and Desulfobulbus propionicus DSM 2032(NR_074930), respectively.3.2.2. Bacterial community in methanogenic reactor of two-phase%)vorcillutumtum)diu)anicuscaldFirmicutes196 J.W. Lim et al. / Bioresource Technology 147 (2013) 1932014to any observed inhibition effects by the two-phase system. Asshown in Table 1, the average TS and VS removal efciencies forRM did not vary much throughout the study. On the contrary, TS re-moval efciency for RS dropped from 84% to 53%, while its VS re-moval efciency dropped from 85% to 57% as reactor operationproceeded from period 1 to 3. Overall, the performance of two-phase CSTR system (i.e., RA and RM) was better than that of sin-gle-phase CSTR (i.e., RS) in terms of higher biogas production,methane composition as well as solids reduction.3.2. Microbial community characterizationCloning and subsequent phylogenetic analysis were carried outto characterize the microbial community structures in both single-and two-phase CSTRs. Primer set 530F and 1490R were initiallyused to amplify the prokaryotic sequences from the extractedDNA. However, all the clones sequenced were afliated withinthe domain Bacteria, indicating that bacterial cells were dominantin RA, RM and RS. Similar ndings were also reported previously byTang et al. (2004). Therefore, an additional set of archaeal primersAr1F and 1490R was used in this study to construct the archaealrRNA clone libraries for RM and RS. Neighbor-joining trees showingthe phylogenetic identities of the 16S rRNA gene fragments wereconstructed and are shown in Figs. Bacterial community in acidogenic reactor of two-phase CSTR(RA)As shown in Fig. 1, the bacterial community structure of RA wasexclusively composed of the phyla Firmicutes and Proteobacteria. In BFABac 001_ (36 BFABac 111_ (3%) BFABac 009_ (8%)Lactobacillus amylo Uncultured Lactoba BFABac 137_ (1%)Lactobacillus fermenLactobacillus fermen BFABac 065_ (1% Uncultured Clostri BFABac 040_ (49%Acetobacter peroxyd BFABac 058_ (1%)Desulfobulbus propionSulfolobus acido100100100919282100100 8245321000.05Fig. 1. Phylogenetic tree of 16S rRNA gene sequences constructed for bacterial clones fromas the outgroup. GRL 1112 isolated from po cine faeces (NR 075048)us r _ sp. clone (HM218835)s IFO 3956 (NR 075033)_ JCM 8596 (AB690185) sp. clone (HQ183766)m LMG 1633 (AJ419836)s DSM 2032 (NR 074930)_ (NR 043400)ariusProteobacteria3.2.3. Bacterial community in single-phase CSTR (RS)The bacterial community in RS was slightly less diverse ascompared to RM where a total of 17 bacterial OTU were identi-ed, which could be classied into 8 different phyla. Fig. 3shows that the bacterial OTU were mainly afliated with Fuso-bacteria, Bacteroidetes, Proteobacteria and Chloroexi in propor-tions of 51%, 32%, 4% and 4% of the bacterial clones,respectively. Within the 17 OTU, 2 were classied as Fusobacte-ria, 6 as Bacteroidetes, 3 as Proteobacteria and 2 as Chloroexi.BFSBac_073, the predominant OTU in RS, accounted for 35% oftotal bacterial count and was afliated with uncultured Fusobac-terium species (FM242289). The closest matches for bacterialclones, as shown in Fig. 3, were detected from sources similarto that of RM.CSTR (RM)The bacterial community in RM was found to be more diverseand a total of 28 bacterial OTU were identied and classiedinto 13 different phyla. Fig. 2 shows that the dominant OTU in-cluded members afliated within four different phyla: Bacteroi-detes, Chloroexi, Proteobacteria and Firmicutes in proportions of40%, 13%, 10% and 8% of the bacterial clones, respectively. Withinthe 28 OTU, 5 were classied as Bacteroidetes, 5 as Chloroexi, 5as Proteobacteria and 4 as Firmicutes. As shown in Fig. 2, theclosest matches for bacterial clones were mostly detected fromfood-processing, toluene-degrading and sulfate-rich wastewa-ters, human faeces, human oral cavities, animal waste treatmentand biogas plants, which were all related to anaerobicfermentation.RA. The 16S rRNA gene sequence of Sulfolobus acidocaldarius (NR_043400) was usedssinium echn BFMBac 111_ Uncultured bacterium clone in AD of food proce BFMBac 136_ Uncultured bacterfatty acid-oxidizing syntrophs 66685499100J.W. Lim et al. / Bioresource T3.2.4. Overview of bacterial communities in RA, RM and RSThe reactors in this study were fed daily with mixtures ofbrown water and food waste. Therefore, their bacterial communitystructures showed a close relationship to human sources such asgastrointestinal tract, oral cavity and faeces. Bacteroidetes andFirmicutes are known to be dominant phyla present in the human Uncultured sp. clone (JQ079827)Syntrophobacter strain Tb8106Syntrophobacter sulfatireducens , prop BFMBac 145_ bacterium enrichment culture (KC460Syntrophaceae BFMBac 079_ Uncultured organism clone (EU245608) BFMBac 018_ Uncultured bacterium clone (in sludge pretreatment Uncultured sp. clone (JQ723608)Geothrix BFMBac 007_ strain CB-8 (JF496528)Aeromonas sharmana BFMBac 038_sp. canine oral taxon 223 (JN7133Propionivibrio BFMBac 035_ Uncultured sp. clone Clostridium in Nisargruna bacterium canine oral taxPeptostreptococcaceae BFMBac 106_ sp. strain P2 (AY949856)Clostridium in UASB BFMBac 057_ sp. enrichment culture clone Clostridium in phen sp. (AB4Clostridium isolated from human faeces BFMBac 138_ Uncultured bacterium clone Firmicutes in AD of s BFMBac 028_ Uncultured bacterium clone (FJ437954) strain A4T-83 (NLuteolibacter pohnpeiensis BFMBac 105_ Uncultured bacterium clone (AB744acidogenesis Uncultured bacterium cloneVerrucomicrobia in ABFMBac 032_ Uncultured microorganism clone in sulfur spring strain: JCM 16774 (ALeptotrichia goodfellowii sp. VNs100 (KC800693)Mesotoga BFMBac 091_ Uncultured bacterium clone in swine wast BFMBac 051_ Uncultured bacterium clone (FN436169) str. Buddy (AF357916)Sphaerochaeta globus Uncultured bacterium clone in anaerobic de Uncultured 1 bacterium clone WWE in AD o BFMBac 082_ BFMBac 015_ Uncultured bacterium clBacteroidetes BFMBac 019_ Uncultured bacterium cBacteroidetes BFMBac 143_ strain Macellibacteroides fermentans WB4 strainPaludibacter propionicigenes BFMBac 042_ Uncultured bacterium clone in UASB tre Uncultured sp. clone (JQ8PaludibacterBFMBac 004_ Uncultured bacterium cloBacteroidetes Uncultured Synergistetes bacterium clone OTU-X2-19 BFMBac 139_ Uncultured bacterium clone (JQ624291) Toluene-degrading methanogenic consortium bact BFMBac 008_ Uncultured cloneChloroflexi in acetate-utilizing sy BFMBac 092_ Uncultured bacterium clone (JQ996691) BFMBac 117_ Uncultured sp. clone Bellilinea in Nisargruna bio BFMBac 116_ Uncultured bacthydrogen-producing Uncultured sp. clonDehalococcoides BFMBac 023_ Toluene-degrading methanogenic con (NR 043Sulfolobus acidocaldarius _10010099100821001001001009610010010010099100994753100809899100995910010010010010010010097561001001009910010010010099100100999986798985976248548471255852291016160.05Fig. 2. Phylogenetic tree of 16S rRNA gene sequences constructed for bacterial clones frused as the outgroup. (GU389854)g wasteclone (AF482439)in granular sludgeDeltaproteobacteria (8%)ology 147 (2013) 193201 197gastrointestinal tract and adult faecal microbiota (Harmsen et al.,2002) while Fusobacteria was isolated from human oral cavities(Bennett and Eley, 1993). However, analysis of bacterial communi-ties in this study demonstrated clear differences in both dominantgroups and phylogenetic distribution between single- and two-phase CSTRs. (NR 043073)ionate-oxidizing syntrophs in UASB _267)FJ769440)86) (EU887787) biogas planton 067 (JN713232) (HQ222293)olic biodegradation91207) (CU926291)ludgeR 041625)_095) (AB780948)D of corn straw (JN387540)B558169) (FJ535538)ewater (AB175392)gradation of protein (CU921669)f sludgeone (JX575986)lone (CU918283)in AD of sludge LIND7H (HQ020488) (NR 074577)_ (AB266963)ating food-processing wastewater15605)ne (CU926883)in AD sludge (JQ668564)erium Eub 4 (AF423184) (AB603823)nergistes bacterium (EU887788)gas planterium isolate CMW-14 (FR828730)e (EU887779)in Nisargruna biogas plantsortium bacterium Eub 2 (AF423182)400)Acidobacteria (1%)Gammaproteobacteria (1%)Betaproteobacteria (1%)Firmicutes (8%)Verrucomicrobia (7%)Fusobacteria (8%)Thermotogae (1%)Spirochaetes (5%)WWE1 (4%)Bacteroidetes (40%)Synergistetes (1%)Chloroflexi (13%)om RM. The 16S rRNA gene sequence of Sulfolobus acidocaldarius (NR_043400) wasill wn LI clooceM 1 clonne (Jd9)man(GU (Jt56)cter347 AD211steechnBFSBac 056_ sp. strain Z4Bacteroides in paper mBFSBac 126_ straiMacellibacteroides fermentans sp. canine oral taxon 384Paludibacter BFSBac 087_ Uncultured bacterium clone in food-pr BFSBac 024_ strain: JCBacteroides graminisolvens BFSBac 045_ Uncultured bacteriumBacteroidetes BFSBac 140_ Uncultured bacterium cloBacteroidetes BFSBac 018_ Uncultured clone planctomycete in pit mu Uncultured sp. clone (FM24228Fusobacterium strain F0264 Leptotrichia goodfellowii in hu Uncultured bacterium clone Fusobacteriales Uncultured microorganism clone (JN387540) BFSBac 026_ BFSBac 073_ BFSBac 112_ Uncultured bacterium clone in acetate amendmen Uncultured bacterium clone BXHB128 (GQ4801 Bacterium SUTW 81; D-lactic acid-producing bastrain GPTSA-6 (NR 04Aeromonas sharmana _ BFSBac 129_ BFSBAc 061_ Uncultured organism clone (JN498907) strain G13 (NR 043576)Geobacter pickeringii _ Uncultured bacterium clone Deltaproteobacteria in Uncultured sp. clone (EU9Syntrophus in compost BFSBac 059_ Uncultured bacterium clone in food-processing wa100100978710010010010010093791001009398100769099100506910010010010082978530661518198 J.W. Lim et al. / Bioresource TLactobacillus species and A. peroxydans LMG 1633 representedthe exclusive dominant phylogenetic group (close to 98% by clon-ing analysis) in RA, suggesting a major impact of these bacteria onthe solubilization and acidication of brown water and food waste,at short retention time of 610 days and acidic pH conditions. Inthis study, Lactobacillus species was predominant in RA where thepH was around 4. However, it was not detected in RM and RS wherethe pH was around 7. This is in agreement with other studiesreporting that the presence and dominance of Lactobacillus wasdependent on pH values. According to Ye et al. (2007), Lactobacillusgrew intensively at pH 46, but slowly at pH 78.The predominant Lactobacillus species in RA, L. amylovorus, is alactate-producing organism possessing amylolytic activity. There-fore it is able to metabolize starch directly to produce lactate andsmall amounts of acetate (Zhang and Cheryan, 1991). The otherpredominant species in RA A. peroxydans was reported to containenzymes for the oxidation of lactate, pyruvate, ethanol and acetal-dehyde to acetate, through the transfer of electrons to oxygen. DeLey and Schel (1959) showed that lactate was oxidized to pyruvatefollowed by slow oxidation of pyruvate to acetate. Therefore, theco-existence of Lactobacillus species and A. peroxydans possibly re-sulted in the high levels of solubilization and acidication observedin RA.All the reactors in this study were designed to operate undercompletely anaerobic conditions. Therefore, the predominance ofan obligately aerobic bacteria A. peroxydans in RA was a surprisending. A. peroxydans was able to co-exist with Lactobacillus BFSBac 013_ Uncultured bacteriufatty acid oxidizing syntrophs sp. (KC800693)Mesotoga BFSBac 033_ Uncultured candidate division TM7 bacterium c Uncultured bacterium clone Chloroflexi ; anaerobi BFSBac 060_ Uncultured bacterium clone (JQ996691) Uncultured sp. clone (JX576Bellilinea in pit mud BFSBac 002_ Uncultured bacterium clone ; acetoclastic sulfate (NR 0434Sulfolobus acidocaldarius _70100100100866910010074340.05Fig. 3. Phylogenetic tree of 16S rRNA gene sequences constructed for bacterial clones fromas the outgroup. (AY949860)astewaterND7H (HQ020488) in abattoir wastewaterne (JN713554) (AB266963)ssing wasewater5093 (AB547643)e (AB780930) in corn straw(HQ003606)X575817)(FJ577259) oral microbiome 472735)X224706) (JN035218)ium0)(CU919618) of sludge 78) (GU389881)Bacteroidetes (32%)Planctomycetes (1%)Fusobacteria (51%)Unknown clone (1%)Gammaproteobacteria (1%)Deltaproteobacteria (3%)ology 147 (2013) 193201species in RA since the latter is an aero-tolerant anaerobic bacteria.The exact experimental condition (i.e., anaerobic, microaerobic oraerobic) of RA was not determined by analytical methods such aslevels of dissolved oxygen or oxidation reduction potential. None-theless, the predominance and co-existence of obligately aerobic A.peroxydans and aero-tolerant anaerobic Lactobacillus species sug-gested RA was unintentionally operated at microaerobic conditions.The operation of RA under completely aerobic conditions washighly unlikely since the concentrations of TVFAs and solubleCOD in the efuent of RA were higher than that in the feed mixture(Table 1). However, further investigations are required to verifythat the predominance of A. peroxydans was due to the unintendedoperation at microaerobic conditions.The predominant bacterial group present in RM was Bacteroide-tes (40% by cloning analysis), which is a major microbial compo-nent of anaerobic reactors. Another bacterial group present in RMwas Firmicutes (8% by cloning analysis), which are known to pro-duce cellulases, lipases, proteases and other extracellular enzymes(Levn et al., 2007). Therefore, the presence of Firmicutes reectsthe ability of digesters to metabolize a variety of substrates includ-ing protein, lipids, lignin, cellulose, sugars and amino acids, whichare commonly found in food waste.For RS, the predominant bacterial group present was Fusobacte-rium species (51% by cloning analysis), which are obligate anaerobicgram-negative bacilli found in large numbers in the mouth. It wasreported that Fusobacterium species weakly ferment simple sugarsto produce large amounts of n-butyric acid (Bennett and Eley, 1993).m clone (AF482447)in granular sludgelone (JN656764) (EU266915)c toluene degraders078) (HQ602791)reducers00)Thermotogae (1%)TM 7 (1%)Chloroflexi (4%)RS. The 16S rRNA gene sequence of Sulfolobus acidocaldarius (NR_043400) was used(48aeohan (1)%nosan)noc (2%ryaet4 (uss k (6ryathaeth (3%(3%chathahaeechn BFMArc 004 _ Uncultured arch Uncultured Met BFMArc 039_ BFMArc 177 (_ 2 Uncultured Metha Uncultured Meth BFMArc 103 (7_ % Uncultured Metha BFMArc 183_ Uncultured eu Uncultured M BFMArc 13_MethanoculleMethanopyru7999961005010010072769341720.02 BFSArc 152_ Uncultured eu Uncultured Me Uncultured M BFSArc 034_ BFSArc 157 _ Uncultured ar Uncultured Me Uncultured arc99641003199655675(b)(a)J.W. Lim et al. / Bioresource TIn comparison to RM, the distribution of bacteria within the phy-lum Fusobacteria was higher, that of Bacteroidetes was lower, andFirmicutes was absent in RS. Since Firmicutes contain extracellularenzymes that carry out the solubilization of brown water and foodwaste, its absence could play a part in the poorer performance ofRS, in terms of lower solids reduction. In addition, the lack of Firmi-cutes could also suggest that there were large amounts of longchain fatty acids (LCFAs) in RS since LCFAs were reported to inhibitgram-positive bacteria such as Clostridia (Galbraith and Miller,1973).There is a lack in studies on the microbial diversity of anaerobicdigesters treating brown water or mixtures of brown water andfood waste. On the other hand, several studies had reported thepredominance of Lactobacillus species in the fermentation of foodwaste (Wang et al., 2005; Ye et al., 2007), and that of L. amylovorus-related species in the rst-stage reactor of a two-stage anaerobicdigestion system treating food waste (Shin et al., 2010). However,the diversity of Lactobacillus species in RA was lower as comparedto the above three references. In addition, none of the referencesreported the predominance of A. peroxydans, which represented49% of total clone count in RA. Therefore, the predominance of A.peroxydans was likely due to the unique operation (i.e. unintendedmicroaerobic conditions) of RA in this study. Shin et al. (2010) alsoshowed that the second-stage reactor consisted of membersafliated within four different phyla, Firmicutes, Proteobacteria,Spirochaetes, and Bacteroidetes. This is similar to the bacterialcommunity structure of RM in this study. Comparisons with exist-ing literature showed that the bacterial diversity of reactors treat-ing brown water and food waste shared some similarities with BFSArc 019 (_ 74% Uncultured Metha Uncultured archae sMethanoculleus BFSArc 061 (1_ 4Methanopyrus k346910082Fig. 4. Phylogenetic tree of 16S rRNA gene sequences constructed for archaeal clones fromwas used as the outgroup in both (a) and (b).)%n clone (JN397877) sp. clone (AB479394)osaeta)9% archaeon clone (CU916545)arcinales archaeon clone (AB669270)osaetaceae sp. (GU179438)ulleus)rchaeote clone (AB248621) sp. (JX560560)hanoculleus)21% sp. strain dm2 (AJ550158)(M59932)andleri MethanosaetaceaeMethanomicrobiales )%rchaeote clone (AB248614) sp. clone (AB077211)nosaeta archaeon clone (AB669270)anosaetaceae))eon clone (GU388805) sp. clone (AB479394)nosaetaon clone (JN397877)Methanosaetaceaeology 147 (2013) 193201 199those treating food waste only. They also suggested that the pre-dominance of Lactobacillus species in RA was largely due to the nat-ure of food waste.3.2.5. Archaeal community in RM and RSAs shown in Fig. 4a and b, the diversity of archaeal clones in RMand RS was limited to members of two orders: Methanosarcinalesand Methanomicrobiales with proportions of 69% and 30%, respec-tively for RM and 86% and 14%, respectively for RS. This indicatedthat methanogenesis took place preferentially via acetoclasticmetabolism for both single- and two-phase CSTRs. For RM, the mostdetected archaeal OTU (BFMArc_004), representing 48% of the totalclones, and the second dominant OTU (BFMArc_039), representing19% of total clones, were closely related to Methanosaeta species(AB479394) isolated from beer brewery efuent with 99% similar-ity. 30% of total archaeal count was composed of various speciesand clones within the genus Methanoculleus. They included BFM-Arc_134, BFMArc_103 and BFMArc_183 representing 21%, 7% and2% of total archaeal clones, respectively.The predominant archaeal OTU for RS was BFSArc_019, whichrepresented 74% of total count, and had the same closest matchas BFMArc_004, the predominant archaeal OTU in RM. The seconddominant OTU (BFSArc_061), representing 14% of total clones,was afliated with Methanoculleus species dm2 (AJ550158). Theremaining 12% of archaeal clones were closely related to Methan-osaeta species.Methanogens can be categorized into two major groups accord-ing to the substrate they utilize. Acetoclastic methanogens con-sume acetate while hydrogenotrophic methanogens consume)s sp. clone (JX101963)noculleuon clone (EU369613)p. strain dm2 (AJ550158))% (M59932)andleriMethanomicrobiales(a) RM and (b) RS. The 16S rRNA gene sequence ofMethanopyrus kandleri (M59932)sequencing results.and shorten its recovery time.echnhydrogen and formate for growth. Analysis of archaeal communi-ties in this study showed similar dominant groups and phyloge-netic distribution in single- and two-phase CSTRs. As shown inFig. 4a and b, Methanosarcinales and Methanomicrobiales, whichare acetoclastic and hydrogenotrophic methanogens, respectivelywere found in both RM and RS.The order Methanosarcinales consist of genus Methanosarcinaand Methanosaeta. In this study, only Methanosaeta species weredetected and they formed the predominant archaeal group in bothRM (76% by cloning analysis) and RS (86% by cloning analysis). Theanalysis of archaeal communities in this study well agreed with areview of archaeal populations in anaerobic digesters where Meth-anosarcinales was reported to constitute more than 29% of the se-quences in all the studies, where sequences afliated withMethanosaeta species were most frequently retrieved (Sekiguchiand Kamagata, 2004). The same study also found that the hydro-genotrophic pathway is commonly represented by Methanomicro-biales with proportions in a range of 129%.As compared to Methanosarcina species, Methanosaeta specieshave higher substrate (i.e., acetate) afnity as well as lower maxi-mum specic growth rate (lmax) of 0.20 d1 and half-saturationconstant (Ks) of 1050 mg-COD L1 (Vrieze et al., 2012). Accordingto Vrieze et al. (2012), Methanosaeta dominated at acetateconcentrations not exceeding 100150 mg-COD L1, whereasMethanosarcina became dominant at acetate concentrations above250500 mg-COD L1. In this study, the acetate concentrations inboth RM and RS were low, with average levels of 101 and 46 mg-COD L1, respectively. This correlated with the dominance ofMeth-anosaeta species, which are capable of scavenging acetate at lowacetate concentrations. The results in this study were in agreementwith earlier studies reporting the dominance of Methanosaeta inwell-operated mesophilic methanogenic systems with low efuentsoluble COD (Raskin et al., 1994; Ariesyady et al., 2007). Methan-osaeta was also shown to dominate reactors underfed with foodwaste (Williams et al., 2013). Hence, the predominance of Methan-osaeta and absence of Methanosarcina species suggested that RMwas at steady-state but working at less than optimum OLRs. TheOLR could be further increased to improve biogas production.The analysis of archaeal communities in this study revealed thathigher levels of hydrogen-utilizing methanogens were present inRM (30%) than in RS (14%). This could be attributed to the feed ofRM, which contained higher levels of solubilized organic matteras compared to that of RS. Therefore, the greater extent of fatty acidfermentation in RM possibly led to increased hydrogen productionwhich encouraged the growth of more hydrogen-utilizing metha-nogens. Lerm et al. (2012) also detected a shift of hydrogenotrophicmethanogens due to increased VFA concentrations.It was reported earlier that syntrophic degradation of propio-nate and butyrate is thermodynamically favorable, only when thehydrogen partial pressure is low enough (AcknowledgementsAuthors are grateful to National Research Foundation (NRF),Singapore for nancial support (NRF-CRP5-2009-02) as well asDr. Rajinikanth Rajagopal, for his advice and Ms. Mao Yu, Mr. AshiqAhamed and Mr. Bernard Jia Han Ng for their technical support inthis research.Appendix A. 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Lett. 13 (10), 733738.Study of microbial community and biodegradation efficiency for single- and two-phase anaerobic co-digestion of brown water and food waste1 Introduction2 Methods2.1 Feedstock and reactor operation2.2 Chemical analysis2.3 DNA extraction and construction of 16S rRNA gene clone libraries2.4 Sequence analysis2.5 Fluorescence in situ hybridization (FISH)3 Results and discussion3.1 Reactor performance3.2 Microbial community characterization3.2.1 Bacterial community in acidogenic reactor of two-phase CSTR (RA)3.2.2 Bacterial community in methanogenic reactor of two-phase CSTR (RM)3.2.3 Bacterial community in single-phase CSTR (RS)3.2.4 Overview of bacterial communities in RA, RM and RS3.2.5 Archaeal community in RM and RS3.3 FISH analyses3.4 Relationship between reactor performance and microbial community structure4 ConclusionsAcknowledgementsAppendix A Supplementary dataReferences


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