ENVIRONMENTAL BIOTECHNOLOGY

Microbial community structure of a pilot-scale thermophilicanaerobic digester treating poultry litter

Ami M. Smith & Deepak Sharma & Hilary Lappin-Scott &Sara Burton & David H. Huber

Received: 2 May 2013 /Revised: 17 July 2013 /Accepted: 19 July 2013 /Published online: 30 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The microbial community structure of a stable pilot-scale thermophilic continuous stirred tank reactor digester sta-bilized on poultry litter was investigated. This 40-m3 digesterproduced biogas with 57 % methane, and chemical oxygendemand removal of 54 %. Bacterial and archaeal diversity wereexamined using both cloning and pyrosequencing that targeted16S rRNA genes. The bacterial community was dominated byphylum Firmicutes , constituting 93 % of the clones and 76 %of the pyrotags. Of the Firmicutes , class Clostridia (52 %pyrotags) was most abundant followed by class Bacilli (13 %pyrotags). The bacterial libraries identified 94 operational tax-onomic units (OTUs) and pyrosequencing identified 577 OTUsat the 97 % minimum similarity level. Fifteen OTUs weredominant (≥2 % abundance), and nine of these were novelunclassified Firmicutes . Several of the dominant OTUs couldnot be classified more specifically than Clostridiales , but weremost similar to plant biomass degraders, including Clostridiumthermocellum . Of the rare pyrotag OTUs (<0.5 % abundance),75 % were Firmicutes . The dominant methanogen was

Methanothermobacter which has hydrogenotrophic metabo-lism, and accounted for >99 % of the archaeal clones. Basedon the primary methanogen, as well as digester chemistry (highVA and ammonia levels), we propose that bacterial acetateoxidation is the primary pathway in this digester for the controlof acetate levels.

Keywords Thermophilic digester . Microbial diversity .

Poultry waste .Methane

Introduction

Anaerobic digestion (AD) is a widely used sustainable methodfor organic waste treatment and bioenergy production (Speece1996). AD can be coupled to large multistage waste treatmentsystems such as found in cities, or it can be a stand-alonesystem for smaller applications, such as animal farms andhouseholds. AD is becoming an increasingly important com-ponent of the portfolio of technologies for environmentalsustainability. The US Environmental Protection Agency re-ports that electricity generation from US digesters has in-creased almost 25-fold since 2000 (US EPA 2010). AD tech-nologies also help to reduce global methane emissions bycontrolling the anaerobic decomposition of waste organicmatter (Tilche and Galatola 2008).

In the USA, the majority of anaerobic digesters are used totreat cattle and piggery wastes, while poultry farms have gen-erally not adopted this technology (US EPA 2010). However,the quantity of poultry litter produced in the USA is consider-able: more than ten million tons per year (Perera et al. 2010).Poultry litter, the waste from broiler houses, contains a largefraction of wood chips and feed residue as well as animal waste.This waste has been considered to be less amenable to ADwhich may be one reason for the apparent reticence in adoptingthe technology. Problems associated with the digestion of poul-try litter include high ammonia levels, high lignocellulosic

Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-5144-y) contains supplementary material,which is available to authorized users.

A. M. Smith :D. Sharma :D. H. Huber (*)Department of Biology, West Virginia State University,Hamblin Hall, Institute, WV 25112-1000, USAe-mail: huberdh@wvstateu.edu

D. Sharma :D. H. HuberGus R. Douglass Institute, West Virginia State University,Institute, WV 25112, USA

H. Lappin-ScottDepartment of Biosciences, Swansea University, Wallace Building,Singleton Park, Swansea SA2 8PP, Wales, UK

A. M. Smith : S. BurtonBiosciences, College of Life and Environmental Sciences,University of Exeter, Geoffrey Pope Building, Stocker Road,Exeter EX4 4QD, UK

Appl Microbiol Biotechnol (2014) 98:2321–2334DOI 10.1007/s00253-013-5144-y

content, and low C:N ratio (Costa et al. 2012). Recent researchhas explored new methods for digesting poultry litter, forexample, enhancement of hydrolysis through thermochemicalpre-treatment and bioaugmentation (Costa et al. 2012), multi-stage digestion (Rao et al. 2011), and the reduction of ammonialevels (Abouelenien et al. 2010).

The general metabolic pathways in AD are well known andcan be characterized as having three principal trophic levels(Speece 1996). Briefly, large polymers are first hydrolyzedand then consumed by primary fermentative bacteria which, inturn, produce fatty acids, H2, and CO2. The second trophiclevel oxidizes the fatty acids, while the third level, themethanogens, utilize either acetate or H2/CO2 to synthesizemethane. Although this general food web is well known,many details remain to be uncovered, particularly thediversity/function relationships of the first two levels.

The anaerobic food web in digesters requires an integratedgroup of specialized populations that function as an ecologicalcommunity. Elucidating the relationship between microbial di-versity and functions in these communities has been challengingbecause of the many different bioreactor designs, operationalparameters, and feedstocks used in AD. In order to search forunifying principles governing the assembly and functions ofdigester microbial communities, biodiversity surveys have beenundertaken representing different types of AD (e.g., Godon et al.1997; Chouari et al. 2005; Weiss et al. 2008; Kröber et al. 2009;Riviere et al. 2009; Werner et al. 2011; Sasaki et al. 2011).

West Virginia State University operates a model pilot-scalethermophilic digester that has been stabilized exclusively onpoultry litter. Previous research from this facility has investi-gated process optimization methods, a variety of control pa-rameters, and the energy economy of the system (Bombardiereet al. 2007; Espinosa-Solares et al. 2006, 2009; Sharma et al.2013). In addition, Bombardiere et al. (2005) found that thecontent of cellulose, hemicellulose, and lignin in the poultrylitter feedstock was reduced. Therefore, the metabolic proper-ties, stable performance, and rather harsh environment of thispoultry litter digester indicate that this microbial communitypossesses properties that may contribute to our emerging con-cept of microbial bioenergy production from waste biomass.We are not aware of previous studies investigating the micro-bial diversity of digesters treating poultry litter as the solesubstrate. The objective of this study was to analyze the eco-logical structure of the bacterial and archaeal community of thisunique digester.

Materials and methods

Pilot plant digester operation

A 40-m3 pilot-scale thermophilic (56 °C) anaerobic digesterlocated at West Virginia State University (WVSU, USA) was

sampled. The digester is a continuous stirred tank reactor(CSTR) and has been previously described in detail(Bombardiere et al. 2007; Espinosa-Solares et al. 2006). Thedigester was fed 568–908 l poultry litter per day for 3 monthsprior to sample collection. The chemical characteristics of thefeedstock have been described in detail (Bombardiere et al.2005). Temperature, pH, biogas volume, and methane wereautomatically measured as previously described (Bombardiereet al. 2007; Espinosa-Solares et al. 2006). Volatile acids (VA),chemical oxygen demand (COD), and ammonia were analyzedwithmethods 8196, 8000, and 10031, respectively, found in theWater Analysis Handbook (Hach 2004). Volatile solids (VS)were determined using the standard methods for wastewater(APHA1998).Methanogenic activity calculations followed themethod of Bombardiere et al. (2007).

Sample collection and clone library construction

Liquid samples for chemical analysis were collected each dayfrom a port located mid-way on the side of the digester.Samples were transported to the laboratory on ice. The sampleused for microbe analysis was centrifuged at 10,000 rpm for30 min to obtain a pellet of cells which was stored at −80 °C.DNAwas extracted from the pellet with the MoBio PowerSoilDNA kit.

One bacterial and two archaeal 16S rRNA clone librarieswere constructed using the “reconditioning” PCR method ofThompson et al. (2002) to minimize PCR artifacts. PCRamplification of bacterial 16S rRNA genes was done withbacteria-specific primers 27F and 1492R (Dojka et al. 1998).The first set of reactions consisted of five duplicate 25-μlreactions which contained 400 mM of each primer, 10XPCR buffer, 1 U of Accuprime Taq polymerase (Invitrogen),and 1.25 ng of DNA. The reaction conditions were initialdenaturation of 94 °C for 2 min, followed by 20 cycles at94 °C for 1 min, 59 °C for 90 s, and 72 °C for 3 min, and afinal extension of 72 °C for 7 min. PCR of archaeal 16S rRNAgenes was done with two primer sets: 109F/915R and 344F/915R (Raskin et al. 1994). Reactions were done as follows:initial denaturation of 94 °C for 2 min, followed by 20 cyclesof 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 90 s, and afinal extension of 72 °C for 3 min. For the second set ofreconditioning PCR reactions, 2.5 μl of amplicons from eachof the first reactions were used in five-cycle PCR reactionswith the same conditions. The replicate five-cycle PCR reac-tions were then pooled and clonedwith the TOPOTACloningkit (Invitrogen).

DNA sequencing and phylogenetic analysis

Plasmid sequencing was done with the Applied BiosystemsBig Dye terminator mix v.3.1.0. Extension products were se-quenced at either the Nevada Genomics Center or on site with

2322 Appl Microbiol Biotechnol (2014) 98:2321–2334

an Applied Biosystems 3130xl Genetic Analyzer. The DNAsequences of the clones were checked for chimeras with Pintailv 1.0 (Ashelford et al. 2005). Anomalous sequences wereexcluded from further analysis. Similarity searches were doneusing the Seqmatch function of the Ribosomal Database Project(RDP). Alignment of the 16S rRNA sequences was performedusing an implementation of Clustal W by MEGA 4.0 (Tamuraet al. 2007). The resulting alignments of 470 nucleotides (whichinclude the V2 variable region) were manually checked andcorrected as needed.

Pyrosequencing of the V4 region of 16S rRNAwas accom-plished using the forward primer 5′-AYTGGGYDTAAAGNG-3′ (Escherichia coli nucleotide position 563–577) and reverseprimers 01, 5′-TACNVGGGTATCTAATCC-3′; 03, 5′-TACCRGGGTHTCTAATCC-3′; 04, 5′-TACCAGAGTATCTAATTC-3′; and 05, 5′-CTACDSRGGTMTCTAATC-3′(E. coli position 785–802) presented in the RDPs Pyrose-quencing Pipeline (http://pyro.cme.msu.edu/pyro/help.jsp). Anadapter sequence (Adapter A; 5′-GCCTCCCTCGCGCCATCAG-3′) as well as a unique identifier tag sequencewas fused upstream of the forward primer. The reverseprimers were designed with an adapter sequence (Adapter B;5′-GCCTTGCCAGCCCGCTCAG-3′) fused upstream as well.The primers were constructed and dual HPLC purified byIntegrated DNA Technologies, Inc. The reverse primers weremixed at a ratio of 12:6:1:2 as recommended (Sul et al. 2011).The 25-μl amplification reaction mixture consisted of 0.5 μlAccuprime Taq polymerase (Invitrogen), 10X PCR buffer, 1.25 ng DNA, forward primer (800 mM), and reverse primer mix(800 mM). The reactions were done as follows: initial denatur-ation of 94 °C for 2 min, followed by 30 cycles of 94 °C for45 s, 57 °C for 45 s, and 72 °C for 1 min, and a final extensionof 72 °C for 4 min. The reactions were run in triplicate andanalyzed with a 2 % agarose gel (1X TAE). The ampliconswere excised from the gel, triplicates combined, and purifiedwith the QIAquick Gel Extraction Kit (Qiagen). The eluateswere further purified using the QIAquick PCR Purification Kit(Qiagen). The purified amplicons were sent to the W. M. KeckCenter for Comparative and Functional Genomics (Universityof Illinois) for pyrosequencing. The resulting sequences weresorted, trimmed, and aligned using the RDP's PyrosequencingPipeline (Cole et al. 2009). A total of 5,054 partial 16S rRNAsequences were obtained, and 4,463 were retained after pro-cessing through the RDP quality control. The average length ofthe pyrosequences (pyrotags) was 167 bases, which exceeds theminimum length quality control cut-off for the RDP Pipeline.The sequences were phylogenetically assigned using the RDPClassifier.

The rarefaction and Chao 1 analyses and the biodiversitystatistics (Shannon H index, evenness E index) were deter-mined using the Analysis Tools applications of the RDP.Although the 27F/1492R primer set spans the V4 regioncovered by the pyrosequence primers, the 470-bp sequences

generated from the library did not overlap the V4 regionwhich is located between nucleotides 576 and 682. In orderto compare the operational taxonomic units (OTUs) betweenthe two data sets (clone library and pyrotags), the data setswere first aligned separately using the RDP aligner. Alignedsequences were clustered at 99 % and 97 % sequence simi-larity by the complete-linkage clustering method (RDP). Themost abundant clusters (OTUs) from each data set weretracked manually by selecting a representative sequence fromeach cluster and assigning taxonomywith the RDP Seqmatch.Sequences thus assigned to the same taxonomy from eachdataset were represented as the same OTU.

Nucleotide sequence accession numbers

The accession numbers for the 16S rRNA gene clones areJN708623–JN709033 (bacteria) and JN812092–JN812210(archaea). The accession number for the pyrosequences isSRP026661.

Results

Digester performance

The performance of the digester was measured daily for a 1-month period bracketing the microbe sampling date (Fig. 1).The average methane production was 57.7±2.2 % of the totalbiogas, and 7,088±1,694 l/day. The average methanogenicactivity during this time period was 0.34±0.19 m3 kg−1 day−1.The VA concentration was 2,160±417 mg/l. Chemical oxy-gen demand removal (CODr) was 34,157±12,025 mg/l, thatis, 54.4±10.3 %. The VS content was 58% of total solids, andthe free ammonia concentration was 2,000 mg/l.

Biodiversity metrics

The bacterial diversity of the digester was examined usingclone libraries and pyrosequencing that targeted the 16SrRNA genes. A total of 412 clones and 4,463 pyrotag se-quences were utilized for this analysis. The coverage of thedigester biodiversity by the two molecular methods was com-pared. As expected, the larger pyrosequencing dataset re-vealed greater bacterial diversity for each biodiversity metricevaluated (Table 1). The number of observed OTUs at the99 % 16S rRNA similarity level was 114 for the clone libraryand 1,444 for the pyrotags; the number of OTUs at the 97 %level was 94 (library) versus 577 (pyrotags); and the numberof OTUs at the 80 % level was 14 (library) versus 97(pyrotags). The Chao 1 estimates of bacterial diversity werealso much higher for the pyrotag dataset: 184 (library) versus3,045 (pyrotags) OTUs at 99 % similarity, and 148 (library)versus 949 (pyrotags) OTUs at 97 % similarity. Rarefaction

Appl Microbiol Biotechnol (2014) 98:2321–2334 2323

curves for both data sets are found in Fig. 2. The pyrotags at≤97 % similarity levels were asymptotic, indicating that thepyrosequence dataset had thoroughly sampled diversity atthese levels. However, the number of pyrotag OTUs at the99 % level was still rising, indicating much more unsampleddiversity at this level (Fig. 2).

A comparison of rank-abundance distributions for the com-munity is shown in Fig. 3 and Fig. S1. Both datasets showed asimilar distribution for the relative abundance of the dominantOTUs, except the most abundant OTUs in the pyrotag datasetshowed an abundance of 4–6 %, whereas those same OTUs inthe library reached 10–12 % abundance. We also analyzed thepyrotags in terms of three categories of abundance: dominantOTUs with ≥2 % abundance, mid-range OTUs with <2–0.5 %abundance, and rare OTUs with <0.5 % abundance (Table 2).The rank-abundance curve for the pyrotag data set showed along tail of rare organisms at the 97 % and 99 % similaritylevels. While the rare phylotypes accounted for some highertaxa, the composition of the low abundance members washighly skewed toward Firmicutes (74.7 %). The rareFirmicutes were primarily composed of classes Clostridia(45.8 %) and Bacilli (17.3 %). In addition, the percentage ofsingletons (99 % similarity) in the pyrotags exceeded the per-centage in the library (19.1 % versus 15 %), but the doubletonswere more abundant in the library (6.3 % versus 5.06 %).

The capacity of pyrosequencing to capture communitystructure relative to the PCR/cloning method was alsoassessed using standard biodiversity indices. The Shannon H

index weights richness more heavily than evenness and washigher for the pyrotag data set at every level of similaritymeasured (Table 1). The Shannon evenness index (E) showedthat the pyrotag data set was also more sensitive to changes inthe evenness of the community at each level of similarity(100–95 %) compared to the library (Table 1).

Taxonomic composition

Figure 4 compares the relative abundances of phyla, classes,and some orders for both molecular methods. A complete listof higher taxa identified with each method can be found inTable S1. These classifications are based on the RDPClassifier default confidence threshold of 80 % for the clonesequences and 50 % for the pyrosequences. The number ofclassifiable taxonomic groups was higher in the pyrosequencedataset which revealed greater numbers of higher level taxa(Table 3 and Table S1). To test whether the pyrosequenceprimer set provided greater taxonomic coverage than thecloning primer set, we also randomly selected 412 pyrotags,matching the number of clones, and classified these. Again,the pyrosequencing method showed greater coverage of tax-onomic diversity, even picking up two new phyla and six newfamilies (Table 3). The total number of classifiable higher-level taxa in the complete pyrotag data set was 9 phyla, 18classes, 28 orders, and 51 families (Table 3).

At the phylum level, both methods showed that the digesterwas composed primarily of Firmicutes : 93 % in the library

Fig. 1 Digester performanceduring 1 month of operation. Theday of microbe sampling isindicated by the arrow

Table 1 Comparison of biodiversity statistics and indices for the 16S rRNA gene library and pyrotag data sets from the thermophilic anaerobic digester

% Nucleotide similarity

Clone Pyrotag

100 % 99 % 98 % 97 % 96 % 95 % 80 % 100 % 99 % 98 % 97 % 96 % 95 % 80 %

Observed OTU 227 114 98 94 89 85 14 2,450 1,444 830 577 446 372 87

Chao1 estimate 795 184 153 148 143 144 16 9,720 3,045 1,441 949 694 563 98

Shannon H index 4.8 3.8 3.6 3.6 3.5 3.5 1.6 7.1 6.4 5.6 5.1 4.8 4.5 3.4

Evenness E index 0.89 0.81 0.79 0.79 0.79 0.79 0.61 0.92 0.88 0.84 0.8 0.78 0.76 0.75

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and 76 % in the pyrotags. The unclassified Bacteria were thesecond largest group represented by 4 % of the library and16 % of the pyrotags. Pyrosequencing also identified threephyla not found in the clone library, including one taxon(Thermotogae ) which was present as a significant fraction(3 %), but completely absent in the library. At the class level,the most abundant group was Clostridia with 64 % of thelibrary and 52 % of the pyrotags. The second most abundantclass was Bacilli (22 % library and 13 % pyrotags). Theunclassified Firmicutes were also a major component of theclass-level diversity: 7 % of the library and 10 % of thepyrotags. In addition, pyrosequencing picked up eight classesof bacteria not present in the library.

A comparison of the taxonomic coverage of the methodswas also done for the two most abundant classes: Clostridiaand Bacilli (Fig. 4 and Table S1). Both methods revealed thatclass Clostridia was dominated by order Clostridiales (57 %library versus 64 % pyrotags), and also included a large com-ponent of unclassified Clostridia (40 % library versus 20 %pyrotags). The third most abundant order in both data sets wasThermoanaerobacterales (3 % library and 11 % pyrotags). Theclass Bacilli was dominated by Lactobacillales (60 % library

versus 53 % pyrotags). The second most abundant class inBacilli was Bacillales followed by unclassified Bacilli .Interestingly, the number of unclassified sequences at the classlevel was higher in the library than in the pyrotags for bothClostridia and Bacilli even though the sequences in the librarywere considerably longer. This may be due to the lower confi-dence limit (50 %) used to classify pyrosequences by the RDP.It should also be noted that the library did detect two orders andfour families not specifically identified in the ten-fold largerpyrosequence data set. It may be that these taxa are present inthe “unclassified” categories of pyrosequences.

Dominant bacterial populations

The dominant phylotypes in terms of abundance were identi-fied in both data sets and compared. Table 4 shows all of theOTUs that had an abundance of 2 % or greater at the 99% and97 % sequence similarity levels in both data sets. FifteenOTUs met this criterion, and there was broad agreementbetween the two methods as to the relative abundances ofthe dominant OTUs. The same three most abundant OTUswere identified in both data sets although the percent abun-dances of the top ten were lower in the pyrotags. One notabledifference between the methods was the abundances of OTU 4and OTU C. OTU 4 was the fourth most abundant OTU in thelibrary (5–6 %) but was <1 % of the pyrotags. OTU C(Thermotogae ) was present at 3 % abundance in the pyrotagsbut completely absent in the library. We also tested whetherthe abundance and identity of the dominant OTUs changedwhen measured by either 97 % or 99 % minimum similaritycriteria. We found that the same OTUs were identified at bothsimilarity levels; that is, no two OTUs identified at the 99 %level were merged as a single phylotype at the 97 % level.Furthermore, the size of the OTUs only increased by 1 % in afew cases (for example, OTU 2) as the similarity level waslowered. This indicates that the dominant OTUs primarilyconsisted of 16S rRNA sequences that diverged by 1 % orless (within the region amplified), are separated from eachother by >3 % 16S rRNA divergence, and probably representeither single populations or clusters of similar populations thatcannot be resolved with these sequences.

Of the top 15 OTUs from the combined data sets, ten fall inclass Clostridia , three in class Bacilli , one in class Bacteroidea ,and one in Thermotogae . Four of these OTUs cannot be classi-fied at the family level. The three most abundant OTUs in bothdata sets (OTU 1, 2, and 3) accounted for 34% of the library and16 % of the pyrotags (Table 4). OTUs 1 and 3 cannot beclassified more specifically than Clostridiales by the RDP. Theclosest cultured representatives to these two OTUs areMoorellaglycerini (U82377) andMoorella thermoacetica (CP000232) at91 % similarity. OTU 1 is highly similar (≥99 %) to unculturedbacterial clone 3wk_1LB13 (AM947554), which was previous-ly found in a thermophilic anaerobic plug-flow digester in

Fig. 2 Comparison of rarefaction curves for 16S rRNA genes at differentsequence similarities from (a) clone library and (b) pyrotags

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Austria (Goberna et al. 2009). OTU 1 is also similar touncultured representatives found in a thermophilic biogas plantin Germany (Weiss et al. 2009), other unpublished digesterstudies (e.g., EF558949, EF586033, and DQ887962), and thegut of earthworms (FJ716076; Knapp et al. 2009). OTU 3 ishighly similar (≥99 %) to uncultured bacterial clones MBN06(AB114316) and MBA02 (AB114312). MBA02, classified asMSW Cluster I, was retrieved from a 5-L synthetic municipalsolid waste digester in Japan (Tang et al. 2004). In addition,sequences 99 % similar to both OTUs 1 and 3 have beenrecovered from a mesophilic biogas plant in Germany (Kröberet al. 2009).

Order Clostridiales also includes several other dominantphylotypes (Table 4). OTU 2, one of the top three most abun-dant phylotypes, contains 12 % (library) and 5 % (pyrotags) ofthe total bacterial sequences, and shares 94 % rRNA identitywith Clostridium thermocellum (CP000568) which is a modelthermophilic cellulose degrader (Zverlov and Schwarz 2008).This OTU is also nearly identical (99 % similarity) touncultured bacterial clones 3wk_1LB26 (AM947536) andCFB-7 (AB274496). Both of these environmental sequenceswere collected from thermophilic digesters (Goberna et al.2009; Sasaki et al. 2007). OTU 8 is also 95 % similar to C.

thermocellum (CP000568) and CFB-7, and is ≥98 % similar tosequences from bacteria that utilized cellulose in a mesophilicmunicipal solid waste digester (Li et al. 2009). OTU 7 falls in agroup of unclassified Lachnospiraceae ; an identical sequencehas been found in digested sludge (AM947511; Goberna et al.2009). OTU 4 contains 6 % (library) and 1 % (pyrotags) of thetotal diversity, and is 99 % similar to some uncultured thermo-philic digester bacteria (e.g., DQ88836 and EF558960). Theclosest cultured relative is M. thermoacetica (CP000232) at92 % similarity, but the RDP designates OTU 4 as unclassifiedClostridiales .

Three of the 15 dominant OTUs (5, 6, and 9) fall in classBacilli and comprise 10 % of the library and 6 % of thepyrotags. OTU 5 is 99 % similar to Atopostipes suicloacalis ,a member of the familyCarnobacteriaceae , whichwas isolatedfrom a swine manure storage pit with a maximum growthtemperature of 32 °C (Cotta et al. 2003, 2004). OTU 6 com-prises 4 % of the total bacterial abundance and is designated bythe RDP as family Bacillaceae 2 . The most similar organism isan uncultured bacterium (FJ380168) found in ornithogenicsoils in Antarctica (Aislabie et al. 2009). OTU 9 represents3% of the library and 1% of the pyrotags and belongs to familyEnterococcaceae . Members of this OTU share ≥96 %

Fig. 3 Comparison of rank abundance curves for 16S rRNA genes from pyrotag and library data sets at different percentage sequence similarities: (a)99 % library sequences, (b) 97 % library sequences, (c) 99 % pyrotag sequences, and (d) 97 % pyrotag sequences

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nucleotide identity with Enterococcus inusitatus (AM050563)and are ≥99 % similar to uncultured bacteria from an anaerobicswine lagoon (AY953149) and a swine wastewater UASBreactor (FJ535552).

The presence of syntrophic bacteria can be inferred from thefamilies Thermoanaerobacteriaceae , Syntrophomonadaceae ,and Synergistaceae , which have known syntrophic members(Liu and Conrad 2010; Schink and Stams 2006; Werner et al.2011). OTU 10, present at 2 % abundance in both data sets,is >98 % similar to a new genus, Syntrophaceticus(Thermoanaerobacteriaceae ). The total abundance of theThermoanaerobacteriaceae is nearly 6 % of the pyrotags and1.7% of the library (Table S1). The Syntrophomonadaceae andSynergistaceae are both present at nearly 1 % of the pyrotags,but absent in the library.

Diversity and abundance of Archaea

Archaeal clone libraries constructed using primer sets 344F/915R and 109F/915R contained 38 and 81 clones, respectively.The Archaea were found to be comprised exclusively ofmethanogens, and two phylotypes were identified (Fig. 5).Both of the archaeal 16S rRNA libraries were dominated bysequences in the thermophilic subclade ofMethanobacteriales

(Wasserfallen et al. 2000). A BLAST search found that se-quences represented by clones L16 and Y13 (Fig. 5) are 99 %similar to Methanothermobacter thermautotrophicum . A phy-logenetic analysis of relationships within the thermophilicsubclade of methanogens demonstrates that the population inthis digester is clearly distinct from the other three species andlikely represents a new species (Fig. 5b). One clone (Y11) wasfound to be 99 % similar to several species of the genusMethanosarcina which are aceticlastic methanogens. Theabundance ofMethanothermobacter sequences in the archaeal16S rRNA library suggests that the hydrogen-utilizingmethanogens are dominant.

Discussion

This study profiles the microbial community found in a pilot-scale thermophilic CSTR digester stabilized on poultry litter,and begins to elucidate the structure/function relationships inthis methanogenic food web. The performance of this digesterin terms of methane production (57 %) and COD removal(54 %) is similar to digesters that treat other types of animalwastes (Sakar et al. 2009), and is efficient considering the lowC/N ratio of poultry litter (Khanal 2008). Previous research

Table 2 Comparison of theabundance and taxonomic affilia-tions of the 16S rRNA OTUsfrom the pyrotag data set calcu-lated at 97 % sequence similarity

Phylum Class OTU abundance

≥2 % <2 % to >0.5 % <0.5 %

Count (%) Count (%) Count (%)

Actinobacteria Actinobacteria 0 0 2 0.3 20 3.5

Bacteroidetes Bacteroidia 0 0 1 0.2 13 2.3

Chloroflexi Anaerolineae 0 0 0 0 2 0.3

Thermomicrobia 0 0 0 0 1 0.2

Total 0 0 0 0 3 0.5

Deinococcus-Thermus Deinococci 0 0 0 0 1 0.2

Firmicutes Bacilli 2 0.3 1 0.2 99 17.3

Clostridia 7 1.2 12 2.1 262 45.8

Erysipelotrichia 0 0 0 0 4 0.7

Negativicutes 0 0 0 0 9 1.6

Unclassified Firmicutes 0 0 3 0.5 53 9.3

Total 9 1.5 16 2.8 427 74.7

Proteobacteria Alphaproteobacteria 0 0 0 0 1 0.2

Betaproteobacteria 0 0 0 0 3 0.5

Gammaproteobacteria 0 0 0 0 5 0.9

Deltaproteobacteria 0 0 0 0 1 0.2

Epsilonproteobacteria 0 0 0 0 1 0.2

Total 0 0 0 0 11 2

Synergistetes Synergistia 0 0 1 0.2 3 0.5

Thermotogae Thermotogae 1 0.2 1 0.2 12 2.1

Unclassified Bacteria Unclassified Bacteria 2 0.3 4 0.7 45 7.9

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with this digester has also demonstrated its flexibility withregard to hydraulic retention time (Bombardiere et al. 2005)and co-digestion of poultry litter and thin stillage (Sharmaet al. 2013).

We used twomolecularmethods tomeasure biodiversity andreconstruct microbial community structure. The two methods(cloning and pyrosequencing) were in broad agreement, butsome significant differences were observed. The full pyrotagdata set did reveal greater taxonomic diversity at every level,but even a reduced pyrotag data set that matched the number ofclones in the library also showed higher taxonomic diversity.This was expected because the pyrosequencing primers hadbeen designed for greater phylogenetic coverage (Sul et al.2011). However, the same predominant bacterial phyla, classes,and orders were identified in both data sets. The pyrotags alsogreatly expanded our view of total microbial diversity. Using a97 % minimum rRNA similarity definition, the library

Fig. 4 Comparison of percentage relative abundances of bacterial taxonomic groups for the pyrotag and library data sets in terms of (a) phylum, (b)class, (c) order Clostridia, and (d) order Bacilli

Table 3 Comparison of the abundance of higher taxa in the pyrotag andclone data sets

No. of sequence Phylum Class Order Family

Pyrotag (total) 4,463 9 18 28 51

Pyrotag (partial) 412 8 12 18 32

Clone library 412 6 10 14 26

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Table4

Identificationandcomparisonof

themostabundant1

6SrRNAgene

OTUsat0.1and0.3divergence

from

thelibrary

andpyrotagdatasets

Phylotype

Relativeabundance(%

)Class

Order

Fam

ilyClosestaffiliatio

n(%

similarity)

Habitatfor

referencesequences

Clone

Pyrotag

0.01

0.03

0.01

0.03

OTU1

1212

46

Clostridia

Clostridiales

Unclassified

EF558949

(99.8%)a(98.2%)b

Therm

ophilic

anaerobicdigester

OTU2

1112

45

Clostridia

Clostridiales

Rum

inococcaceae

Clostridium

thermocellum

CP0

00568

(94.4%)a(92.4%)b

Therm

ophilic

anaerobic

OTU3

910

45

Clostridia

Clostridiales

Unclassified

AB114316

(100

%)a(99.1%)b

Therm

ophilic

anaerobicdigester

OTU4

56

11

Clostridia

Clostridiales

Unclassified

DQ887936

(99.5%)a

Therm

ophilic

anaerobicbioreactor

OTUA

0.9

1.5

24

Clostridia

Clostridiales

Unclassified

EF558949

(98.7%)b

Therm

ophilic

anaerobicdigester

OTU5

44

22

Bacilli

Lactobacillales

Carnobacteriaceae

Atopostipes

suicloacalis

AF4

45248

(99.8%)a(99.1%)b

Manurestoragepit

OTU6

44

23

Bacilli

Bacillales

Bacillaceae2

FJ380168(96.2%)a(96.4%)b

Ornith

ogenicsoil

OTU7

34

12

Clostridia

Clostridiales

Lachnospiraceae

AM947511

(100

%)a(99.1%)b

Anaerobically

digested

sludge

OTU8

34

12

Clostridia

Clostridiales

Rum

inococcaceae

HQ183790

(99.8%)a(100

%)b

Leachatesediment

OTU9

23

0.9

1Bacilli

Lactobacillales

Enterococcaceae

Enterococcussp.A

F445284(99.8%)a

Manurestoragepit

OTU10

22

22

Clostridia

Thermoanaerobacterales

Therm

oanaerobacteraceae

UnculturedSyntrophaceticus

JF417920

(98.7%)a(98.5%)b

Dry

anaerobicdigester

OTUB

0.7

1.5

12

Bacteroidia

Bacteroidales

Bacteroidaceae

UnclassifiedAcetomicrobium

GU455355

(98.7%)b

Therm

ophilic

anaerobicdigester

OTUC

00

22

Thermotogae

Thermotogales

Thermotogaceae

Defluviito

gatunisiensisFR

850164

(98.7%)b

Therm

ophilic

anaerobicdigester

OTU11

0.7

20.5

0.6

Clostridia

Clostridiales

Lachnospiraceae

Clostridium

phytofermentans

CP0

00885

(94.6%)a

Forestsoil

OTU12

0.9

20.5

0.5

Clostridia

Clostridiales

Rum

inococcaceae

FJ879103(97.4%)a

Ratfecalw

aste

aSequence

similarity

forOTUfrom

clonesequences

bSequence

similarity

forOTUfrom

pyrotagsequences

Appl Microbiol Biotechnol (2014) 98:2321–2334 2329

contained 94 bacterial OTUs while the pyrotags contained 577.The Chao 1 projections for total bacterial diversity were quitedifferent for the two data sets as well and higher than previousestimates for thermophilic digesters: 148 OTUs for the libraryand 949 OTUs for the pyrotags (Cheon et al. 2007; Weiss et al.2008; Goberna et al. 2009; Tang et al. 2011). Other studies havealso compared 16S rRNA pyrosequencing and traditional clon-ing methods, and found good qualitative agreement in theabundance of the major taxa as well as additional diversity inthe pyrosequences (Edwards et al. 2006; Kautz et al. 2013;Dunbar et al. 2012).

The predominant bacterial phylum in this thermophilicdigester was Firmicutes which represented 93% of the libraryand 76 % of the pyrotags. Of the Firmicutes , the largestgroups were classes Clostridia (52 % pyrotags) and Bacilli(13 % pyrotags). Other cloning-based studies of thermophilicdigesters have also found that the Firmicutes were dominantor co-dominant (e.g., Sasaki et al. 2011; Tang et al. 2004).Some thermophilic digesters also have a large percentage ofThermotogae , even reaching more than 60 % of clone librar-ies (Levén et al. 2007; Yabu et al. 2011), but this group waspresent at only about 2 % of the pyrotags. In addition, a largefraction of the bacterial diversity was not classifiable at orbelow the phylum level: >10% of the library and >26% of thepyrotags. Even within the most abundant class, Clostridia , asignificant percentage could not be classified at the level oforder. The large fraction of unclassified sequences may havebeen due to greater taxonomic ambiguity in the shorterpyrotags, or they may represent new taxa above the specieslevel. Other studies have also noted novel bacterial diversity indigesters (Chouari et al. 2005; Guermazi et al. 2008).

Two recent large-scale surveys of anaerobic digester mi-crobial diversity have been reported. In a meta-analysis,

Nelson et al. (2011) retrieved and classified more than16,000 bacterial 16S rRNA gene sequences from GenBankthat were derived from anaerobic digesters. Four phyla werefound to be dominant in terms of total number of OTUs:Proteobacteria were most abundant, Firmicutes second, andBacteroidetes and Chloroflexi about equally represented inthird place. Rivière et al. (2009) conducted a large cloning-based survey of 16S rRNA genes from seven full-scalemesophilic digesters in Europe in order to identify the “core”microbes in AD. They found that Proteobacteria andChloroflexi were much more abundant than Firmicutes . Incontrast, the bacterial taxonomic composition of this thermo-philic poultry litter digester was quite different.

The most abundant methanogen wasMethanothermobacterwhich is a hydrogenotrophic genus (Wasserfallen et al. 2000).The dominance of this metabolism and the low abundance ofacetate-utilizing methanogens imply that the majority of acetateis converted to methane through bacterial acetate oxidation.Sasaki et al. (2011) recently demonstrated that 80 % of acetatedecomposition in a thermophilic digester occurred through thenon-aceticlastic oxidative pathway. The dominance or co-dominance of Methanothermobacter has been found in otherthermophilic digesters as well (Goberna et al. 2010; Yabu et al.2011; Krakat et al. 2010).

The pathway for acetate breakdown can be influenced byseveral environmental factors. A shift in dominance fromaceticlastic Methanosarcina to Methanothermobacter hasbeen associated with an increase in temperature, hydrogenpartial pressure, and propionate concentration (Goberna et al.2010). In addition, high levels of ammonia may inhibit theaceticlastic pathway (Angelidaki and Ahring 1993; Koster andLettinga 1984), resulting in a shift to syntrophic acetate oxida-tion (Schnurer and Nordberg 2008; Schnurer et al. 1999).

Fig. 5 a Phylogenetic trees showing the relationships of Archaea in the digester. Four orders of methanogens are represented. The numbers inparentheses indicate the number of clones in that group. b The relationship of clones L16 and Y13 to members of the genus Methanothermobacter isshown in greater detail

2330 Appl Microbiol Biotechnol (2014) 98:2321–2334

Angenent et al. (2002) also observed a shift from aceticlastic tohydrogenotrophic methanogens in an anaerobic sequencingbatch reactor when ammonium levels rose. The average freeammonia concentration in our thermophilic digester was highat 2,000 mg/l. Therefore, environmental conditions furthersupport that acetate conversion to methane occurs primarilythrough bacterial acetate oxidation.

The structure/function relationships in ecological communi-ties are also reflected in the relative abundance of specificpopulations. The rank-abundance distribution of the OTUs inthis digester community indicates that the evenness is character-istic of other microbial environments which have few taxa ofhigh abundance and many rare taxa (Sogin et al. 2006). Weclassified the most abundant OTUs as those with 2 % or greaterabundance in either data set, and found that both data setsidentified most of the same fifteen dominant OTUs. Theserepresented 68 % of the library and 39 % of the pyrotags andare likely to be the dominant bacterial populations in the digest-er. Significantly, 14 of the top 15 most abundant OTUs werepresent in both datasets, and the top three most abundant OTUswere the same. The relative abundance ranking of the top 15OTUs was also similar except OTU 4was much more abundantin the library, and OTU C representing phylum Thermotogaewas absent in the library. In addition, each of these OTUs wasrepresented as a set of sequences sharing ≥99 % similarity inboth data sets. Because of this high level of sequence similarity,these OTUs are expected to represent ecologically differentiatedpopulations that are functionally distinct from the otherphylotypes within the deeper clades represented by 3 % 16SrRNA gene divergence (Zimmerman et al. 2013).

We also considered the composition of the uncommon andrare OTUs (<0.5 %). Roughly 75 % of the pyrotag sequencesunder 0.5 % abundance were Firmicutes . At the class level,46 % of the total rare pyrotags wereClostridia and 17% wereBacilli . While the pyrotag dataset did pick up greater taxo-nomic diversity, these groups represented a very small fractionof the total rare diversity. The highly skewed distribution ofboth abundant and rare populations toward the Firmicutesmay be due to clade-level environmental filtering within thedigester (Philippot et al. 2010). Rarefaction analysis of thepyrotags also showed that total bacterial diversity was stillrising at the 99 % rRNA similarity level while the 97 % levelwas reaching an asymptote. The Chao 1 estimate for totalbacterial diversity at 99 % rRNA similarity was projected tobe 3,045 OTUs. This indicates that a great deal of fine-scalegenetic diversity remains to be sampled and cautions againstidentifying the dominant populations in digesters simply interms of any phylotype within a 97 % similarity cluster whichis sometimes used to define species (Forney et al. 2004).

The roles of several of the top 15 OTUs can be inferred torepresent the first trophic level in the digester ecosystemwhere hydrolysis and fermentation of macromolecules occurs.Several of the major OTUs have similarity to groups known to

breakdown plant polymers. The family Ruminococcaceae isrepresented by three OTUs, one of which, OTU 2, is amongthe top three most abundant OTUs and is most similar toClostridium thermocellulum , a well-known thermophilic cel-lulose and hemicellulose degrader (Demain et al. 2005). TheRuminococcaceae are prominent members of animal guts,and some have an impressive arsenal of enzymes for plantbiomass depolymerization (Berg Miller et al. 2009). Anotherlikely candidate for plant biomass breakdown is OTU 11,which is most similar (94 % rRNA clone identity) toClostridium phytofermentans which can ferment both glucansand xylans (Jin et al. 2012). If OTUs 2 and 11 are indeedfunctionally similar to these two species, a possible metaboliclinkage is suggested. Some strains of C. thermocellulum donot utilize pentose sugars even though they producehemicellulases (Demain et al. 2005), implying that perhapsOTU 11 utilizes the pentoses liberated by OTU 2's depoly-merization. OTU 7 is an unidentified member of the familyLachnospiraceae which contains several genera found inanaerobic digesters (Goberna et al. 2009), wastewater treat-ment plants (McLellan et al. 2010), and as a major componentof some animal gastrointestinal tracts (Cotta and Forster2006).Members of this family can have diverse and specializedcapabilities for polysaccharide breakdown and proteolysis, andproduce a variety of fermentation products (Cotta and Forster2006). The abundance of Clostridium , Ruminococcaceae ,Lachnospiraceae , and Enterococcus (OTU 9) phylotypes cantherefore be associated with the breakdown of cellulosic bio-mass (Demain et al. 2005). This is consistent with the compo-sition of the poultry litter feedstock which not only containspoultry waste but a large component of sawdust and grain fromthe poultry bedding and feed. Furthermore, anaerobic cellulo-lytic species often specialize in their use of carbohydrates whichmay account for the diversity of Clostridiales (Brulc et al.2011; Lynd et al. 2002).

Because this digester has primarily hydrogenotrophicmethanogens, the sources and fate of acetate and hydrogenare particularly important. Another prominent phylotype,OTU 5, is nearly identical to Atopostipes suicloacale , whichis a nutritionally fastidious facultative anaerobe that is able todegrade cellobiose but not cellulose, and forms acetate fromglucose (Cotta et al. 2003, 2004). OTU B with abundance ofabout 1–2 % has high similarity to Acetomicrobium . The typespecies for this genus, Acetomicrobium flavidum , is a thermo-philic bacterium isolated from sewage sludge that is notablefor its fermentation of 1 mol glucose to 2 mol acetate andcarbon dioxide, and 4 mol hydrogen (Soutschek et al. 1984).OTU C is >98 % similar to a new genus, Defluviitoga , thatwas described from a mesophilic digester (Ben Hania et al.2011). The type species can ferment glucose, producing ace-tate, hydrogen, and CO2, and can grow on a variety of sugarsincluding pentoses. C. phytofermentans has also been foundto produce high concentrations of acetate during the

Appl Microbiol Biotechnol (2014) 98:2321–2334 2331

consolidated bioprocessing treatment of corn stover (Jin et al.2012). These four OTUs may be important fermenters thatsupply a significant fraction of the hydrogen and acetate fordownstream methanogenesis. The other dominant OTUs can-not be classified beyond order (Clostridiales ) or beyond fam-ily, limiting our ability to infer their functional roles.

The metabolic guild that occupies the trophic level betweenthe primary fermenters and methanogens is the fatty acid-oxidizing bacteria, including syntrophs (Hattori 2008; Schink2006). Three taxonomic groups that are known to containsyntrophs (Thermoanaerobacteriaceae , Syntrophomonad-aceae , and Synergistales) constitute about 8 % of the pyrotags.Fatty acid-oxidizing syntrophic bacteria have been previouslyidentified in thermophilic digesters (Hatamoto et al. 2007;Hattori 2008). The type species for Syntrophaceticus is anacetate-oxidizing syntroph collected from a mesophilic digesterwith high ammonium concentration (Westerholm et al. 2011).A more tenuous functional linkage is the low similarity (91–92%) betweenOTUs 1, 3, 4 andM. thermoacetica which is thebest characterized homoacetogen (Pierce et al. 2008). Otherhomoacetogens are known to oxidize acetate by reversing theWood–Ljungdahl pathway (Hattori 2008).

The roles of the low-abundance populations in anaerobicdigesters and other bioreactors remain an interesting question(Baiser et al. 2011). In the rumen, minor populations ofcellulolytic specialists have been shown to make a dispropor-tionately large contribution to plant biomass breakdown eventhough they may comprise less than 0.5 % of the total bacteria(Weimer et al. 1999). In this thermophilic digester, >93 % ofthe pyrotag OTUs (97 % similarity) were present as rarepopulations with an abundance under 0.5 %. We hypothesizethat the many rare Firmicutes and Clostridia populations inparticular are also making important specialized contributionsto the depolymerization of cellulosic biomass.

In conclusion, we have begun to characterize the microbialcommunity structure and function of a thermophilic anaerobicdigester stabilized on poultry litter. The overall diversity ofbacteria is distinct frommesophilic digesters and more similarto other thermophilic digesters. The predominance of hydro-genotrophic methanogens, as well as digester chemistry, im-plies that bacterial acetate oxidation controls this importantmetabolite. While projected total bacterial diversity surpasses570 phylotypes (OTUs) at 97 % rRNA similarity, the digesteris dominated by relatively few bacterial phylotypes, includingundescribed taxa above the species level. The stability of thedominant and rare members of this community needs to beexamined over time in order to discern whether specific pop-ulations represent core or flexible functions.

Acknowledgements This research was funded by USDA CSREESgrant 2004–02614.We also thank theGus R. Douglass LandGrant Institute(WVSU) for support, John Bombardiere and Jesus E. Chavarria Palma forassistance, and Nagamani Balagurusamy for helpful comments.

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