lable at ScienceDirect

Anaerobe xxx (2014) 1e7

Contents lists avai

Anaerobe

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

Molecular biology, genetics and biotechnology

Dynamics of microbial communities in untreated and autoclaved foodwaste anaerobic digesters

Lucia Blasco a,*, Minna Kahala a, Elina Tampio a, Satu Ervasti a, Teija Paavola a,1,Jukka Rintala a,b, Vesa Joutsjoki a

aMTT Agrifood Research, Finlandb Tampere University of Technology, Finland

a r t i c l e i n f o

Article history:Received 20 May 2013Received in revised form17 April 2014Accepted 18 April 2014Available online xxx

Keywords:BiogasAnaerobic digestionMicrobial communitiesT-RFLP

* Corresponding author. Tel.: þ358 295317133.E-mail address: lucia.blasco@mtt.fi (L. Blasco).

1 Present address: Biovakka Suomi Ltd, Finland.

http://dx.doi.org/10.1016/j.anaerobe.2014.04.0111075-9964/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Blasco Ldigesters, Anaerobe (2014), http://dx.doi.org

a b s t r a c t

This study describes the microbial community richness and dynamics of two semi-continuously stirredbiogas reactors during a time-course study of 120 days. The reactors were fed with untreated andautoclaved (160 �C, 6.2 bar) food waste. The microbial community was analysed using a bacteria- andarchaea-targeting 16S rRNA gene-based Terminal-Restriction Fragment Length Polymorphism (T-RFLP)approach. Compared with the archaeal community, the structures and functions of the bacterial com-munity were found to be more complex and diverse. With the principal coordinates analysis it waspossible to separate both microbial communities with 75 and 50% difference for bacteria and archaea,respectively, in the two reactors fed with the same waste but with different pretreatment. Despite theuse of the same feeding material, anaerobic reactors showed a distinct community profile which couldexplain the differences in methane yield (2e17%). The community composition was highly dynamic forbacteria and archaea during the entire studied period. This study illustrates that microbial communitiesare dependent on feeding material and that correlations among specific bacterial and archaeal T-RFs canbe established.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion (AD) is a sustainable waste managementapproach for a wide range of organic waste types in agriculture andindustry [1]. The AD of organic waste is an environmentally usefultechnology. This treatment is advantageous because it producesvaluable energy in the form of biogas. During the process, organicmatter is converted to methane, carbon dioxide, water, inorganicnutrients and humus through sequential metabolic processes: hy-drolysis, acidogenesis, acetogenesis mainly carried out by bacteria,and methanogenesis performed exclusively by archaeal species[2,3].

Consequently, anaerobic digesters are characterized by complexmicrobial communities [4]. Regardless of the growth of AD use, therelationship between these microbial communities and processefficiency is not thoroughly understood. Currently, the anaerobicdigestion process can only be empirically optimized by adjusting

, et al., Dynamics of microb/10.1016/j.anaerobe.2014.04.

digester conditions such as: temperature, pH, redox potential,organic loading rate (OLR) and solid and hydraulic retention time(SRT, HRT). Usually, parameters such as pH, volatile fatty acid (VFA)concentration, the total alkalinity in the digester and biogascomposition are monitored and used for operational control [5].

Domestic food waste is an energy-rich substrate for anaerobicdigestion. With pre-treatment such as autoclaving, the materialhydrolyses. This process is followed by easier degradation duringdigestion. However, the treatment temperature and pressure aswell as the characteristics of the treated material affect the anaer-obic digestion process and biogas production. Autoclaving acts as ahygienisation step prior to digestion, but at temperatures 130e180 �C methane production has been reported to decrease [6e8]due to formation of hardly biodegradable Maillard compounds [9].

Generic tools and approaches to relate microbial communitystructure and dynamics to reactor performance include finger-printing techniques such as: amplified ribosomal DNA restrictionanalysis (ARDRA), single strand conformation polymorphism(SSCP), denaturing gradient gel electrophoresis (DGGE), tempera-ture gradient gel electrophoresis (TGGE), terminal restrictionfragment length polymorphism (T-RFLP) and ribosomal intergenicspacer analysis (RISA) [10]. Furthermore, microbial identification

ial communities in untreated and autoclaved food waste anaerobic011

Table 1Feed material characteristics with standard deviations.

Untreated FW Autoclaved FW

pH 5.02 � 0.13 5.01 � 0.13TS (%) 24.65 � 0.48 20.51 � 0.83VS (%) 22.90 � 0.44 18.91 � 0.72VS/TS (%) 92.90 92.18TKN (g/kg) 7.40 � 0.34 6.78 � 0.28NH4eN (g/kg) 0.35 � 0.13 0.43 � 0.12

Table 2Reactor characteristics.

Days (d) OLR(kg VS/m3 day)

HRT (d) CH4 (m3/kg VS)a H2S (ppm) pH

R1M R3A R1M R3A R1M R3A R1M R3A

230 3 78 63 0.511 0.457 25 N/A 7.8 7.5251 3 78 63 0.505 0.452 60 35 7.8 7.4265 4 58 47 0.490 0.453 110 50 7.8 7.5286 4 58 47 0.508 0.392 200 30 7.7 7.5294 4 58 47 0.552 0.515 255 0 7.8 7.5307 4 58 47 0.512 0.469 370 20 7.8 7.5321 4 58 47 0.489 0.480 N/A N/A 7.7 7.4328 4 58 47 N/A N/A N/A N/A 7.8 7.5

N/A, not available.a CH4 concentration calculated as a weekly average.

L. Blasco et al. / Anaerobe xxx (2014) 1e72

can be done using fluorescence in situ hybridization (FISH), DNAmicroarrays or sequencing [10].

T-RFLP is a molecular tool for monitoring microbial communitycompositions and their relative abundances [11]. It has been suc-cessfully applied in studying different ecosystems, includingmethanogenic reactors [12e14]. In previous studies, T-RFLP tech-nique results were found to reflect the change in the microbialpopulations and agree with other techniques based on the 16SrDNA gene but showingmore accurate information [15e20]. T-RFLPis currently recognized as one of the most powerful fingerprintingmethods in microbial ecology, because it allows high samplethroughput and precise fragment length determination. Therefore,although T-RFLP gives information about the diversity, structureand dynamic of the complex microbial community in anaerobicreactors, its main disadvantages are PCR bias and low resolution. Onthe other hand, it is fast, cheap and semi-quantitative.

The present study monitored microbial community structureand dynamics from two laboratory-scale methanogenic reactors,based on their bacterial and archaeal 16S rRNA gene-targeted T-RFLP profiles. The study evaluated the impact of pretreatment(autoclaving) and OLR on the dynamics of themicrobial communityin an STR. This study connects microbial community structure anddynamics with operational conditions in different anaerobicdigesters.

2. Materials and methods

2.1. Feed and STR operation

The stirred tank reactors (STRs) used in the study were fed withsource-segregated domestic food waste (FW) collected from theSouth Shropshire Biowaste digestion plant in Ludlow, UK. Oneportion was pre-treated with a novel double-auger autoclave(AeroThermal Group Ltd, UK) at 160 �C and 6.2 bars (autoclavedFW); the other portion was left untreated (untreated FW). Bothportions were then passed through a macerating grinder (S52/010Waste Disposer, IMC Limited, UK), frozen, shipped to Finland andthawed prior to use.

The STRs (Metener Ltd, Finland) were 11-l stainless-steel re-actors with semi-continuous stirring. The operating temperaturewas mesophilic (37 �C). Reactors were fed five times a week andsamples for analyses were collected through the feeding inlet. Thebiogas produced was measured with a volume-calibrated cylin-drical gas collector based on water displacement, after which thegas was collected in gas bags. The gas composition (CH4, H2S) fromthe bags was analysed Combimass GA-m gas analyser (Binder En-gineering, Germany).

At the beginning of the experimental run, both reactors (R1Muntreated and R3A autoclaved) were inoculated with sewagesludge digestate from the Biovakka Suomi Ltd Turku plant. Thereactors started off with an organic loading rate (OLR) of 2 kg VS/m3 day and a hydraulic retention time (HRT) of 117 and 94 days foruntreated R1M and autoclaved R3A respectively. On day 151 theOLR was raised to3 kg VS/m3 day and on day 256, to 4 kg VS/m3 dayshortening HRTs from 78 d to 58 d in R1M and from 63 d to 47 d inR3A. Microbial sampling was done in OLRs of 3 and 4 kg VS/m3. Thereactors were supplemented with trace element solutions con-taining the cationic elements Al (0.1 mg/l), B (0.1 mg/l), Co (1.0 mg/l), Cu (0.1 mg/l), Fe (5.0 mg/l), Mn (1.0 mg/l), Ni (1.0 mg/l), Zn(0.2 mg/l) and oxyanions Mo (0.2 mg/l), Se (0.2 mg/l) and W(0.2 mg/l) [21]. More detailed feed handling and STR operation aredescribed in Ref. [8]. Digestate samples for chemical analyses andmicrobial samples during the sampling period (days 231e328)were collected routinely once a week. The characteristics of thefeed and biogas reactors are provided in Tables 1 and 2.

Please cite this article in press as: Blasco L, et al., Dynamics of microbdigesters, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.

2.2. Analytics

The total solids (TS) and Volatile solids (VS) were determinedaccording to SFS 3008 [22] (Finnish Standard Association, 1990).The total Kjeldahl nitrogen (TKN) was analysed with a standardmethod [23] using a Foss Kjeltec 2400 Analyser Unit (Foss TecatorAB, Höganäs, Sweden) with Cu as a catalyst and ammonium ni-trogen (NH4eN) according to Ref. [24], pH was determined using aVWR pH100 pH-analyser (VWR International).

2.3. Microbial samples and total genomic DNA extraction

Samples of 5 ml were obtained from each STR every week andaliquots of 2 ml were prepared (mixing with glycerol 1:1) andstored at �80 �C until DNA extraction was performed. A sample ofthe untreated feeding material was also analysed.

The total community DNA was extracted from selected samplesbased on CH4, pH and NH4eN changes. Approximately 0.25 g ofeach sample was used for extraction using FastDNA� SPIN Kit forSoil (MP Biomedicals, US) according to the manufacturer protocol.DNA extractions were visualized by ethidium bromide stainingfollowing gel electrophoresis in 1% (w/v) agarose and 1� TBEbuffer. Concentration measurement of genomic DNA was per-formed using a NanoDrop ND1000 (NanoDrop Technologies, Wil-mington, DE, USA).

2.3.1. T-RFLP analysisFor T-RFLP analyses, 16S rRNA genes were amplified in duplicate

for each sample using the bacterial primers pA and pH [25] and thearchaeal primers 2AF and 915R [26,27]. Both forward primers werelabelled at the 50-end with the phosphoramidite dye 6-FAM and thereverse primers with VIC� (Applied Biosystems). 1 ml of DNA extractwas applied in the PCR mix for bacteria and 2 ml for archaea. Thecycle profiles used were: an initial denaturation at 95 �C for 3 minfollowed by denaturation at 95 �C for 1 min, annealing at 55 �C(archaea) or 52 �C (bacteria) for 1 min, extension at 72 �C for 3 min;the number of cycles was 35, and a final extension of 20 min wasperformed at 72 �C. The amplicons were purified using a PCR pu-rification kit (Qiagen, Venlo, Netherlands) and quantified using the

ial communities in untreated and autoclaved food waste anaerobic011

Fig. 1. Relative abundance of bacterial 16S rRNA gene fragments retrieved from the anaerobic reactors R1M and R3A during the sampling period based on T-RFLP analyses with HhaIenzyme forward and reverse labelled primers for bacterial (a) and archaeal (b) populations. The length of T-RFs in base pairs (bp) was indicated.

L. Blasco et al. / Anaerobe xxx (2014) 1e7 3

Please cite this article in press as: Blasco L, et al., Dynamics of microbial communities in untreated and autoclaved food waste anaerobicdigesters, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.011

L. Blasco et al. / Anaerobe xxx (2014) 1e74

NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE,USA). The purified PCR product (100 ng) was digested with theHhaIand HpyAV restriction enzymes for bacteria and HhaI and TaqI forarchaea (Fermentas, St. Leon-Rot, Germany). DNA fragments wereprecipitated using 95% ethanol and washed with 70% ethanol, thenvacuum-dried and resuspended in 15 ml of distilled water. 1 ml ofthis suspension was mixed with 9 ml of formamide containingGeneScan 1200 LIZ Size Standard (Applied Biosystems, Halle,Belgium) and separated on a 3500�l Genetic Analyser (AppliedBiosystems, Halle, Belgium).

2.3.2. Statistical analysis of the T-RFLP dataThe T-RFLP electropherograms were analysed using Peak Scan-

ner Software v. 1.0 (Applied Biosystems, Halle, Belgium). The rela-tive abundances of T-RFs were determined by calculating the ratiobetween the height of each peak and the total peak height of allpeaks within one sample. The cut off point for fragment sizesincluded in further analysis was >20 bp. Terminal restrictionfragment length polymorphism (T-RFLP) analysis was done intriplicate for each sample. The restriction endonucleases selectedfor further analyses were those producing the highest numbers andbest size distribution of T-RFs in silico using the online programMiCA ISPaR [28]. After testing these enzymes on the extracted DNAone enzyme seemed themost suitable. Thus HhaI using reverse andforward primers was chosen for further analyses of archaea andbacteria, respectively. MiCA APLAUS [28] was used to infer theplausible community structure data.

Only peaks with more than 2% abundance were considered. Therange-weighted richness was determined as the number of peaksin each electropherogram. The T-RFLP profile was interpreted usingprincipal coordinates analysis (PCoA) with the Bray Curtis similar-ity index, using the software PAST v.2.15 (PAleontological STatistic[29] and Qiime [30]). Shannon’s diversity index (H) was calculatedon T-RFLP data using PAST software as: H¼ Sri ln ri [31]. A one-wayanalysis of variance (ANOVA) was performed (SAS� softwarepackage, Version 9.2.) to statistically evaluate whether microbialassemblages varied between bioreactor treatments, and Spear-man’s rank correlation test was used to assess possible correlationsbetween abundance and presence of archaeal and bacterial T-RFs.

3. Results

Microbial community structure was investigated during thesampling period (days 230e328) in both reactors by performing aPCR amplification of 16S rDNA. Using the same amount of DNAsolution prepared from 250 mg of each sample, T-RFLP analysis ofthe samples was carried out under the same conditions.

The T-RFLP fingerprints of bacterial 16S rRNA gene fragmentsrevealed a total of 33 terminal restriction fragments (T-RFs) on the

Fig. 2. TKN and NH4eN during the studied period on both reactors.

Please cite this article in press as: Blasco L, et al., Dynamics of microbdigesters, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.

forward fragment and 19 T-RFs on the reverse fragment of theenzyme HhaI for the bacterial population (Fig. 1). For archaea, 17 T-RFs on the forward fragment and 18 T-RFs on the reverse fragmentwere generated with the same enzyme. This result implies a lowerdiversity of the archaeal community than of the bacterial popula-tion in both reactors (the tested enzymes HpyAV for bacteria andTaqI for archaea showed a lower number of T-RFs). Some T-RFs weredetected in high abundance; thereby, on bacteria the T-RFs 377,555, 560 and 1084 accounted for an average of 70% of the totalpopulation and the reverse fragments 431, 435 and 436 an averageof 61%. At the same time the archaea HhaI forward fragments T-RFs321 and 326 accounted for a range of abundance from 34 to 94%(Fig. 1). It was encountered that for fragments 182 and 376, TaqIenzyme accounted for 91% of total abundance of population (datanot shown). The change of OLR did not significantly influence theappearance or disappearance of specific T-RFs but it did reveal aclear change of the abundance of some T-RFs on bacterial andarchaeal populations. The presence of T-RFs within STRs was verysimilar during the sampling period, primarily displaying differencein abundances between the reactors. The methane production wasnotably higher (2e17%) in the untreated reactor (R1M) comparedwith the autoclaved (R3A) (Table 2). When comparing ShannoneWiener diversity indexes, the reactor R1M showed a higher di-versity for bacterial communities than the R3A during the course ofthe process, whereas the correlation of indexes on archaeal pop-ulations appeared to be more or less random (Fig. 3). PCoAwith theBray Curtis similarity index was used to visualize the relationshipsamong bacterial and archaeal communities. PCoA of 16S rDNA T-RFLP data revealed clustering related to the type of reactor (forbacterial communities, variance between reactors was as high as74%) but not so clearly related to date (13%) although there was asegregation based on the change of OLR on day 259 (see Fig. 4a).Associations between the community structure and pretreatmentsor OLR change were observed but not as evidently on the archaealpopulation compared to that of bacterial population (Fig. 4b).

When archaeal T-RFLP data was analysed using the APLAUSapplication, we found out that the genus Methanosarcina waspresent during the entire period in both reactors. Meanwhile thegenera Methanocalculus and Methanoculleus (both belonging to theorder Methanomicrobiales) and the genus Methanococcus (familyMethanococcaceae) were present in the R1M during the entireperiod but were undetectable in R3A after day 251. Moreover, alarge fraction of 16S-rDNA T-RFs could only be assigned to uncul-tured archaeon, demonstrating that numerous microorganisms arestill unclassified or unknown. When the bacterial population wasanalysed it was found that the genus Clostridia as well as Bacilluswas found in both reactors, allocated in several sizes of T-RFs.

Fig. 3. ShannoneWiener diversity indexes of archaea and bacteria.

ial communities in untreated and autoclaved food waste anaerobic011

Fig. 4. Differences in microbial community structure based on principal coordinatesanalysis of T-RFLP peak patterns in bacteria with the forward fragment obtained withthe enzyme HhaI (a) and archaea along with the reverse fragment obtained with theenzyme HhaI (b). Circles denote the communities before and after the change in OLR.

L. Blasco et al. / Anaerobe xxx (2014) 1e7 5

Regarding the archaeal community structure only 6.25% of theforward fragments and 27.78% of the reverse for the enzyme HhaIshowed significant differences (p < 0.01) between reactors (auto-claved vs untreated). At the same time, for the bacterial populationthe significance was 48.48% and 47.36% of the T-RFs respectively forthe forward and reverse fragment of the same enzyme (the otherenzymes showed lower differences; data not shown). Consideringthe common bands among reactors as a core microbiota, it wasfound a good representative core achaea, for the forward HhaIfragments 83.65% and 98.42% of the total community in reactorsR1M and R3A respectively, meanwhile the bacterial core was notvery well represented in reactor R1M showing only 17.74% of thetotal bacterial community, as opposed to 73.76% in reactor R3A. The

Please cite this article in press as: Blasco L, et al., Dynamics of microbdigesters, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.

microbial population composition in the untreated feeding mate-rial differed totally from that observed in the reactors (data notshowed). Fig. 5 marks the common T-RFs and T-RFs which showedsignificant differences between reactors and illustrates the Spear-man’s rank correlation coefficient between different T-RFs ofarchaeal and bacterial population for the enzyme HhaI.

TKN, NH4eN and pH levels remained relatively stable during theperiod of study; however, in R3M the levels were lower (Fig. 2,Table 2). H2S content increased from <60 ppm to over 200 ppm inR1M after the OLR increase but remained under 50 ppm in R3A.

4. Discussion

In this study, we examined the effects of pretreatment and OLRincrease on the microbiota composition of anaerobic reactors, aswell as their influence on CH4 production. Other products such asH2S, TKN and NH4eN were analysed in both reactors in order torelate them to possible changes in microbial dynamics. H2S con-centration in the R1M reactor increased rapidly after the OLR in-crease, indicating change in the microbial community structure.This change was not observed in the autoclaved R3A reactor,where the pretreatment had likely reduced the availability ofsulphur.

The CH4 production varied between reactors, being 2e17% lowerin the R3A due to the effect on microbial population after auto-claving the food waste. In addition, the NH4eN concentration waslower in the R3A compared to the R1M. This effect was most likelycaused by the transformation of proteins during autoclaving, whichwould also explain why CH4 production was lower in the R3A.During autoclaving some properties and conformations of sugarsand proteins could have changed by formation of, for example,Maillard compounds which are reported to be hardly biodegrad-able [7,9] making them less available for the growing microbes.Pretreatment induced differences in community composition,showing significant differences (p < 0.01) in certain T-RF. Some ofthe bacterial T-RFs present in both reactors showed strong differ-ences especially in correlation with archaeal T-RFs. This fact wasobserved in the microbeecore interaction, which would be indic-ative of the existence of different metabolic routes used by micro-organisms (sharing the same T-RF). The fact that the microbialpopulation of the feeding material showed no similarities to themicrobial community of the reactors reinforced the idea that mi-crobial changes occurring during the biogas production would beinduced by nutrient availability and not because of the addition ofexternal microbial sources. The higher methane production in R1Mcould also be related to the easier availability of nutrients ratherthan the microbial composition of the added feed.

PCoA showed a significant influence on the type of food wastetreatment on bacterial populations but archaeal populations werenot as clearly distinguishable between treatments. The bacterialpopulation composition seemed to be more sensitive to pretreat-ment than the archaeal population; this could be due to theirsensitivity to availability of nutrients. The ShannoneWiener di-versity index for bacteria showed higher levels in R1M and thiscould be related to the constant input of new microbes due to theunautoclaved feed, In contrast, the archaeal population in R1Mshowed no constantly higher values in the course of the process.

Our result agreed with previous studies in which acetate-oxidizing bacteria and hydrogenotrophic methanogens dominatedin mesophilic conditions under high ammonia levels [32]. MiCaanalysis of the dominant archaeal T-RFs obtained matched withdifferent hydrogenotrophic methanogens and the acetoclasticmethanogens Methanosarcina in all samples, indicating that bothacetoclastic and hydrogenotrophic pathways were used formethane production in both of the reactors. The constant presence

ial communities in untreated and autoclaved food waste anaerobic011

Fig. 5. Spearman’s rank correlation coefficient between T-RFs obtained by HhaI digestion of archaeal and bacterial populations on both reactors. The arrows mark the T-RF orarchaea and bacteria with significant differences between reactors (p < 0.01) and the asterisk (*) marks all common bands between reactors. NOTE: ARC_f: archaea forwardfragment; ARC_r: archaea reverse fragment; BAC_f: bacteria forward fragment; BAC_r: bacteria reverse fragment.

L. Blasco et al. / Anaerobe xxx (2014) 1e76

of Methanosarcina in the course of the process has been reportedpreviously in other studies using for example DGGE and ANAE-ROCHIP [33,34]. Typically, restriction fragments generated from asingle primer are not sufficiently accurate for species identification.In this work, multiple T-RFLP restriction digests combined withforward and reverse labelled fragments were used and had thepotential to resolve complex communities. However, this techniqueis based on 16S rDNA restriction, and many species of differentgenera or, even families or orders, can share the same length of T-RFs. Therefore it is not a straightforward process to match the ob-tained fragments with a specific microorganism. In addition, thearchaea domain in particular has a high number of sequenced un-cultured members which make identification even more difficult.Therefore, we conclude that use of T-RFLP for screening the changesoccurring globally in the reactors is easy and economically afford-able. It can be used for monitoring the process, but it is not specificenough when detailed information about microorganisms isrequired.

In summary, it can be stated that autoclaving as a pretreatmentas well as change of OLR influenced microbial community struc-tures, especially the bacterial one. The untreated reactor showedhigher specific methane production than autoclaved one at bothOLRs studied. The effects caused by pretreatment at high temper-ature modified the availability of substrates; different groups ofbacteria adapt to these conditions. Since archaea use metabolitesproduced by these bacteria, pretreatment probably had less effecton the archaea community. The study obtained evidence of certainbacterial T-RFs correlating with archaeal T-RFs; however, furtherresearch with species level identification techniques, such as next-generation sequencing, will be needed to better understand syn-trophisms between bacteria and archaea in this complex microbialprocess.

Please cite this article in press as: Blasco L, et al., Dynamics of microbdigesters, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.

Acknowledgements

Funding from the EU FP7 Valorisation of Food Waste to Biogas(VALORGAS) project (241334) is gratefully acknowledged.

References

[1] Verstraete W, Morgan-Sagastume F, Aiyuk S, Waweru M, Rabaey K, Lissens G.Anaerobic digestion as a core technology in sustainable management oforganic matter. Water Sci Technol 2005;52:59e66.

[2] Zinder SH, Cardwell SC, Anguish T, Lee M, Koch M. Methanogenesis in athermophilic (581C) anaerobic digester: Methanothrix sp. as an importantacetoclastic methanogen. Water Sci Technol 1984;52:59e66.

[3] Raskin L, Poulsen LK, Noguera DR, Rittmann BE, Stahl DA. Quantification ofmethanogenic groups in anaerobic biological reactors by oligonucleotideprobe hybridization. Appl Environ Microbiol 1994;60:1241e8.

[4] Rivière D, Desvignes V, Pelletier E, Chaussonnerie S, Guermazi S,Weissenbach J, et al. Towards the definition of a core of microorganismsinvolved in anaerobic digestion of sludge. ISME J 2009;3:700e14.

[5] Ahring BK, Sandberg M, Angelidaki I. Volatile fatty acids as indicators ofprocess imbalance in anaerobic digesters. Appl Microbiol Biotechnol 1995;43:559e65.

[6] Cuetos MJ, Gómez X, Otero M, Morán A. Anaerobic digestion and co-digestionof slaughterhouse waste (SHW): influence of heat and pressure pre-treatmentin biogas yield. Waste Manage 2010;30:1780e9.

[7] Pinnekamp J. Effect of thermal pretreatment of sewage sludge on anaerobicdigestion. Water Sci Technol 1989;21:97e108.

[8] Tampio E, Ervasti S, Paavola T, Heaven S, Banks C, Rintala J. Anaerobic digestionof autoclaved and untreated food waste. Waste Manag 2014;34:370e7.

[9] Bougrier C, Delgenès JP, Carrère H. Effects of thermal treatments on fivedifferent waste activated sludge samples solubilisation, physical propertiesand anaerobic digestion. Chem Eng J 2008;139:236e44.

[10] �Cater M, Fanedl L, Marinsec Logar R. Microbial community analyses in biogasreactors by molecular methods. Acta Chim Slov 2013;60:243e55.

[11] Liu WT, Marsh TL, Cheng H, Forney LJ. Characterization of microbial diversityby determining terminal restriction fragment length polymorphisms of genesencoding 16S rRNA. Appl Environ Microbiol 1997;63:4516e22.

[12] Collins G, Woods A, McHugh S, Carton MW, O’Flaherty V. Microbial commu-nity structure and methanogenic activity during start-up of psychrophilic

ial communities in untreated and autoclaved food waste anaerobic011

L. Blasco et al. / Anaerobe xxx (2014) 1e7 7

anaerobic digesters treating synthetic industrial wastewaters. FEMS MicrobiolEcol 2003;46:159e70.

[13] Scully C, Collins G, O’Flaherty V. Assessment of anaerobic wastewater treat-ment failure using terminal restriction fragment length polymorphism anal-ysis. J Appl Microbiol 2005;99:1463e71.

[14] McHugh S, Collins G, O’Flaherty V. Long-term, high rate anaerobic biologicaltreatment of whey wastewaters at psychrophilic temperatures. BioresourTechnol 2006;97:1669e78.

[15] Wang H, Tolvanen K, Lehtomäki A, Puhakka J, Rintala J. Microbial communitystructure in anaerobic co-digestion of grass silage and cow manure in a lab-oratory continuously stirred tank reactor. Biodegradation 2010;21:135e46.

[16] McMahon KD, Stroot PG, Mackie RI, Raskin L. Anaerobic codigestion ofmunicipal solid waste and biosolids under various mixing conditions e II:microbial population dynamics. Water Res 2001;35:1817e27.

[17] Mladenovska Z, Hartmann H, Kvist T, Sales-Cruz M, Gani R, Ahring BK. Thermalpretreatment of the solid fraction of manure: impact on the biogas reactorperformance and microbial community. Water Sci Technol 2006;53:59e67.

[18] Moesender MM, Arrieta JM, Muyzer G, Winter C, Herndl GJ. Optimization ofterminal-restriction fragment length polymorphism analysis for complexmarine bacterioplankton communities and comparison with denaturinggradient gel electrophoresis. Appl Environ Microbiol 1999;65:3518e25.

[19] Koizumi Y, Kojima H, Fukui M. Characterization of depth-related microbialcommunity structure in lake sediment by denaturing gradient gel electro-phoresis of amplified 16S rDNA and reversely transcribed 16S rRNA frag-ments. FEMS Microbiol Ecol 2003;46:147e57.

[20] Schwarz JI, Eckert W, Conrad R. Community structure of archaea and bacteriain a profundal lake sediment Lake Kinneret (Israel). Syst Appl Microbiol2007;30:239e54.

[21] Banks CJ, Zhang Y, Jiang Y, Heaven S. Trace element requirements for stablefood waste digestion at elevated ammonia concentrations. Bioresour Technol2012;104:127e35.

[22] Finnish Standard Association. SFS 3008, Determination of total residue andtotal fixed residue in water, sludge and sediment. Helsinki, Finland: FinnishStandard Association; 1990.

Please cite this article in press as: Blasco L, et al., Dynamics of microbdigesters, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.

[23] AOAC. Official methods of analysis. Arlington, VA: Association of OfficialAnalytical Chemists, Inc.; 1990. p. 1298.

[24] McCullough H. The determination of ammonia in whole blood by directcolorimetric method. Clin Chim Acta 1967;17:297e304.

[25] Edwards U, Rogall T, Blocker H, Emde M, Bottger EC. Isolation and directcomplete nucleotide determination of entire genes, characterization of a genecoding for 16S ribosomal RNA. Nucleic Acids Res 1989;17:7843e53.

[26] Stahl DA, Amann R. Development and application of nucleic acid probes inbacterial systematic. In: Stackebrandt E, Goodfellow M, editors. Sequencingand hybridization techniques in bacterial systematics. Chichester, England:John Wiley & Sons, Inc.; 1991. pp. 205e48.

[27] DeLong EF. Archaea in coastal marine environments. Proc Natl Acad Sci USA1992;89:5685e9.

[28] Shyu C, Soule T, Bent SJ, Foster JA, Forney LJ. MiCA: a web-based tool for theanalysis of microbial communities based on terminal-restriction fragmentlength polymorphisms of 16S and 18S rRNA genes. J Microb. Ecol 2007;53:562e70.

[29] Hammer Ø, Harper DAT, Ryan PD. PAST: paleontological statistics softwarepackage for education and data analysis. Palaeontol Electron 2001;4.

[30] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK,et al. Nat Methods; 2010. http://dx.doi.org/10.1038/nmeth.f.303.

[31] Magurran AE. Ecological diversity and its measurement. London: Chapmanand Hall; 1998.

[32] Schnurer A, Nordberg Å. Ammonia, a selective agent for methane productionby syntrophic acetate oxidation at mesophilic temperature. Water Sci Technol2008;57:735e40.

[33] Goberna M, Gadermaier M, García C, Wett B, Insam H. Methanogenic com-munities fermenting cattle excreta and olive mill wastes into biogas moni-tored using the AnaeroChip and quantitative PCR. Appl Environ Microbiol2010;76:6564e71.

[34] Walter A, Knapp BA, Farbmacher T, Ebner C, Insam H, Franke-Whittle IH.Searching for links in the biotic characteristics and abiotic process parametersof nine different biogas plants. Microb Biotechnol 2012;5:717e30.

ial communities in untreated and autoclaved food waste anaerobic011