Effect of organic loading on the microbiota in a

Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6738-3

ENVIRONMENTAL BIOTECHNOLOGY

Effect of organic loading on the microbiota in a temperature-phased anaerobic digestion (TPAD) system co-digesting dairy manure and waste whey Yueh-Fen Li 1 & Christopher Abraham 2 & Michael C. Nelson 4 & Po-Hsu Chen 3 & Joerg Graf 4 & Zhongtang Yu 1,2

Received: 30 January 2015 / Revised: 27 May 2015 / Accepted: 29 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Temperature-phased anaerobic digestion (TPAD) has gained increasing attention because it provides the flexibility to operate digesters under conditions that enhance overall digester performance. However, research on impact of organic overloading rate (OLR) to microbiota of TPAD systems was limited. In this study, we investigated the composition and successions of the microbiota in both the thermophilic and the mesophilic digesters of a laboratory-scale TPAD system codigesting dairy manure and waste whey before and during organic overloading. The thermophilic and the mesophilic digesters were operated at 50 and 35 °C, respectively, with a hydraulic retention time (HRT) of 10 days for each digester. High OLR (dairy manure with 5 % total solid and waste whey of ≥60.4 g chemical oxygen demand (COD)/l/day) resulted in decrease in pH and in biogas production and accumulation of volatile fatty acids (VFAs) in the thermophilic digester, while the mesophilic digester remained unchanged except a transient increase in biogas production. Both denaturant gradient gel electrophoresis (DGGE) and Illumina sequencing of 16S

ribosomal RNA (rRNA) gene amplicons showed dramatic change in microbiota composition and profound successions of both bacterial and methanogenic communities. During the overloading, Thermotogae was replaced by Proteobacteria, while Methanobrevibacter and archaeon classified as WCHD3-02 grew in predominance at the expense of Methanoculleus in the thermophilic digester, whereas Methanosarcina dominated the methanogenic community, while Methanobacterium and Methanobrevibacter became less predominant in the mesophilic digester. Canonical correspondence analysis (CCA) revealed that digester temperature and pH were the most influential environmental factors that explained much of the variations of the microbiota in this TPAD system when it was overloaded. Keywords DGGE . Illumina sequencing . Microbiota . Organic overloading . TPAD

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6738-3) contains supplementary material, which is available to authorized users. * Zhongtang Yu [email protected] 1

Environmental Science Graduate Program, The Ohio State University, Columbus, OH, USA

2

Department of Animal Sciences, The Ohio State University, 2029 Fyffe Court, Columbus, OH 43210, USA

3

Department of Statistics, The Ohio State University, Columbus, OH, USA

4

Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA

Anaerobic digestion (AD) has been a biotechnology widely used to treat wastewater and municipal sludge for many decades. In recent years, AD attracted additional attention because the biogas produced thereby can be used as renewable energy. The microbiological process of AD can be conceptually divided into four major steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Different guilds, or functional groups, of microorganisms mediate each of these steps. An efficient and stable operation of an AD process requires balanced activities among the different guilds of microorganisms, especially between acidogens and methanogens, the latter of which is particularly fastidious and susceptible to changes in different environmental factors, such as pH, concentrations of ammonia, and volatile fatty acids (Chen et al. 2008).

Appl Microbiol Biotechnol

Failure and upset of AD systems due to un-balanced microbial growth and metabolism, especially acidogenesis and methanogenesis, has been repeatedly reported (Banks et al. 2008; Batstone et al. 2002; Ghanimeh et al. 2013; Yan et al. 1993). Thus, great efforts have been put into the design and operation of AD systems to stabilize AD process by balancing acidogenesis and methanogenesis. Temperature-phased anaerobic digestion (TPAD) is a relatively new design to the AD field with the advantages of improved solid removal, methane production, and pathogen control (Kim et al. 2004; Riau et al. 2010; Santha et al. 2006). It is an AD process that involves both a thermophilic phase and a mesophilic phase in sequence (Lv et al. 2010). Conceptually, in a TPAD system, hydrolysis and the acidogenesis primarily occur in a thermophilic digester, while acetogenesis and methanogenesis occur in a mesophilic digester. Such a separation enables optimizing the environmental conditions to enhance the metabolism of different guilds of microorganisms in two separate digesters. However, if the thermophilic phase is maintained at near neutral pH (referred to as NTTPAD), all the four steps of the AD process happen in both the thermophilic and the mesophilic digesters (Lv et al. 2013b). In either case, the mesophilic digester has more permissive conditions than the thermophilic digester, and it is protected from fluctuations in operation. As such, compared to the thermophilic digester, the mesophilic digester of a TPAD system is less likely affected, or affected to a smaller extent, by operational variations, such as organic loading rate (OLR). OLR is one of the most important operational factors that profoundly affect the stability of AD systems. In the pursuit of increased volumetric biogas yield (volumes of biogas produced per unit of digester per day) of a digester, operation of AD systems tends to be aggressive by feeding as much feedstock as possible. Depending on the digestibility and composition of the feedstocks, high OLR can have a dramatic impact on the microbiota in the digester and the stability of the AD process. Several studies have examined changes in microbiota structure of anaerobic digesters in response to organic overloading (Chen et al. 2012; Lerm et al. 2012; McMahon et al. 2004; Tale et al. 2011). Although these studies covered a number of designs of digesters, ranging from laboratory scale to full scale, all of them focused on single-stage digesters, and no study has been reported that investigated how the microbiota in a TPAD system responds to organic overloading. In the present longitudinal study, we monitored the performance of a TPAD system co-digesting dairy manure and waste whey and investigated the effect of OLR on the composition and succession of the microbiota in both the thermophilic and the mesophilic digesters of the TPAD system. Furthermore, canonical correspondence analysis (CCA) was also performed to identify plausible correlation between microbial populations and the environmental variables/digester

performance in each of the two digesters. The knowledge learned may help understand and improve stability of TPAD systems by understanding the effects of organic loading on different groups of bacteria and methanogens.

Materials and methods TPAD setup, feedstock, operation, and sample collection The TPAD system consisted of two BioFlo 3000 fermentation units (New Brunswick Scientific Co. Inc., NJ) connected in sequence via tubing and a Masterflex I/P pump (Cole Parmer Instrument Co, Vernon Hills, IL). Each unit has a continuously stirred tank reactor (CSTR) with a working volume of 5 l. The temperature for the first thermophilic digester was maintained at 50 °C, while that of the second mesophilic digester was set at 35 °C. The mixing propeller of each digester was set at 50 rpm. The thermophilic and the mesophilic digesters were respectively seeded with the digestate of the thermophilic and the mesophilic digesters of a TPAD system that had been operated in batch mode (Lv et al. 2013a). Dairy manure was collected weekly from the Krause Dairy Farm of the Ohio Agricultural Research and Development Center (located in Wooster, Ohio) and stored at 4 °C, while waste whey was collected from a large maker of dairy products located near Wooster and refrigerated. In the first 2 weeks of the acclimation period, the thermophilic digester was fed once daily with 100 ml of waste whey (20 %, w/v) and 100 ml dairy manure slurry with a total solid (TS) content of 5 %. The daily feeding resulted in an OLR of 16.16 g chemical oxygen demand (COD) from the waste whey and 5 g manure TS per liter per day. The TPAD system was operated in batch mode. Before feeding the above mixture to the thermophilic digester, 200 ml of the mesophilic digester content was discharged, and 200 ml was pumped directly from the thermophilic to the mesophilic reactors. Samples were collected from the effluent of each digester for chemical and microbial analysis (see below). This resulted in a hydraulic retention time (HRT) (also solid retention time (SRT)) of 25 days in each reactor. The biogas production from each digester was measured continuously using a gas flow meter. After the initial 2 weeks of the system startup, the volume of the daily feeding was increased to 500 ml consisting of 250 ml dairy manure slurry (5 % TS) and 250 ml waste whey, which resulted in a HRT of 10 days in each reactor. While the OLR of dairy manure slurry was unchanged, the OLR of the whey was increased in a stepwise manner: from days 15 to 39, 32.3 g COD/l/day; from days 40 to 69, 48.5 g COD/l/day; and from day 70 to the end of the study (day 167), 60.4 g COD/l/day (Fig. 1a). The feeding and sampling were done as described above. Samples were collected periodically from both digesters throughout the whole experimental period and kept at −80 °C before further


e)

b)

f)

c)

g)

Inﬂuent COD (g/l)

a)

Biogas (l/d/l)

d)

h) 10

10

8

8

6

6

4

4

2

2

0

0

Eﬄuent COD (g/l)

i)

Fig. 1 Summary of organic loading rate (OLR) and digester performance of the thermophilic (a–e) and the mesophilic digester (e–i). a, e OLR (note, the effluent of the thermophilic digester was the influent of the mesophilic digester). b, f VFA concentration. c, g Digester pH. d, h

Volumetric biogas yield. e, i, Effluent COD of the thermophilic digester and the mesophilic digester. The arrows on the top with corresponding sample ID indicated the samples that were selected for microbial analysis

chemical analysis. Samples taken at days 30, 44, 58, 72, 79, 93, 98, 121, 135, 154, and 169 were subjected to microbial analysis. The samples from the thermophilic digesters were designated as t1 to t11, while the samples from the mesophilic digester were designated as m1 to m11, in a timely order corresponding to the aforementioned sampling dates (Fig. 1).

conductivity detector, while concentrations of individual volatile fatty acids (VFAs) in the samples were also analyzed using a gas chromatograph (HP 5890 series, Agilent Technologies) fitted with a flame ionization detector and a Chromosorb W AW packed glass column (Supelco, USA) as described previously (Zhou et al. 2011). TS content and total volatile solid (VS) content were determined following the standard methods (American Public Health American Public Health Association, American Water Works Association, Water Environment Federation 2005). COD was determined using a DR2500 spectrophotometer (Hach, Loveland, CO) following Method 8000 in the Water Analysis Handbook (Hach, Loveland, CO).

Chemical analysis The pH values and daily gas production were continuously monitored and recorded. The methane content of the biogas was determined using gas chromatograph (HP 5890 Series, Agilent Technologies, USA) equipped with a HP-PLOT Q capillary column and a thermal


DNA extraction, PCR-DGGE, and Illumina sequencing Total metagenomic DNA was extracted from 1.5 to 2.1 g of each of the digester samples collected on selected days (as indicated in Fig. 1) using the repeated bead beating plus column purification (RBB + C) method (Yu and Morrison 2004b). The integrity of the extracted DNA was evaluated using agarose gel (0.8 %) electrophoresis, while the concentrations were quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). The bacterial community and the archaeal community were profiled using PCR-DGGE using domain-specific primers as described previously (Yu and Morrison 2004a; Yu et al. 2008). Briefly, the V3 hypervariable region of the 16S ribosomal RNA (rRNA) gene was amplified from 50 ng of each metagenomic DNA sample using the primer set GC-357f/ 519r for bacteria and 344f/GC-519r for archaea. Bovine serum albumin (BSA) was included (670 ng/μl) in all the PCR to attenuate potential inhibition to the PCR reaction. A touchdown thermal program (61 °C with 0.5 °C/cycle decrement for 10 cycles followed by 25 cycles at 56 °C for primer annealing) was used to maximize specificity. The PCR was ended with a final extension step at 72 °C for 30 min to eliminate artifactual double DGGE bands created from possible heteroduplex (Janse et al. 2004). Size and quality of the PCR products were verified using agarose gel (1.0 %) electrophoresis before running DGGE using a PhorU system (Ingeny, Leiden, NL). The gel contained 8 % of acrylamide with a 40 to 60 % denaturant gradient, and the electrophoresis was carried out at 80 V for an hour followed by 160 V for 15 h. The gels were stained with SYBR Green I (Invitrogen, Carlsbad, CA) and visualized using a Kodak Gel Logic 200 imaging system (Eastman Kodak Company, Rochester, NY). Preparation of amplicon libraries of 16S rRNA genes and subsequent Illumina sequencing were conducted as described previously (Caporaso et al. 2012). Briefly, the V4 hypervariable region of prokaryote 16S gene was amplified using primers 515F and 806R, a set of prokaryotic universal primers that was well-validated and used as the standard protocol for the Earth Microbiome Project (http://www.earthmicrobiome. org/), with a unique identifier sequence for each sample. The amplicon libraries were sequenced using a 2×250-bp pairedend protocol on an Illumina MiSeq system at the University of Connecticut. Illumina sequencing data processing and analysis The sequencing data were first processed for demultiplexing, trimming of primer sequences, and assembly of pair-ended reads into single sequences using the software MiSeq Reporter v 2.0 (Illumina Inc., San Diego, CA), FastX Trimmer (http://hannonlab.cshl.edu/fastx_toolkit/) and SeqPrep

(https://github.com/jstjohn/SeqPrep) prior to further analysis using the shell scripts developed recently (Nelson et al. 2014) for the use in QIIME v 1.5 (Caporaso et al. 2010). Briefly, sequences were clustered at 97 % similarity against the V4-V5 region of the Greengenes reference operational taxonomic unit (OTU) alignment using ulust (Edgar 2010) as recommended by Werner et al. (2012). For those sequences that failed to cluster with the reference sequences, a de novo OTU clustering was performed. Chimeric check was performed on the representative sequences picked from each OTU using ChimeraSlayer (Haas et al. 2011). The nonchimeric sequences were combined, and then, taxonomic assignment was made using the Ribosomal Database Project (RDP) Classifier (Wang et al. 2007). An approximately maximum likelihood phylogenetic tree was generated using FastTree (Price et al. 2010). OTUs that were each represented by less than 0.005 % of the total bacterial sequences or by less than 0.25 % (because a much smaller number of archaeal sequences were obtained) of the total archaeal sequences for archaea were filtered out and discarded before performing further diversity analysis. To compare the diversity of the microbiota in the samples, alpha diversity and beta diversity were calculated using the software tools included in QIIME. Rarefaction was performed (rarefied to 89,778 sequences) before the diversity analysis. Shannon and Simpson diversity indices along with the phylogenetic distance were calculated as the indicators for alpha diversity. For beta diversity analysis, a distance matrix of the samples was generated using the OTU-based Bray-Curtis and binary Ochiai methods and the phylogenetic tree-based UniFrac (both weighted and unweighted) method. Ordination was then performed using principal coordinate analysis (PCoA). CCA was performed using the Vegan Community Ecology package for R language (http://cran.r-project.org/web/packages/vegan/) to elucidate the correlation between microbial populations and operational parameters/performance of the TPAD digesters, including temperature; pH; biogas production; TS content; effluent COD; and the concentrations of acetic acid, propionic acid, and butyric acid. The CCA was performed with the phyla identified, the OTUs that were each represented by more than 0.1 % of total sequences, and the OTUs that were assigned to known genera. The distributions of the OTUs obtained were visualized using the heatmap and clustering method implemented in the software GAP (http://gap.stat.sinica.edu.tw/Software/GAP/). The abundance (number of sequences) of these OTUs was first log transformed for normalization before generation of the heatmap. Pearson’s correlation coefficients were calculated to examine the community similarity among the samples. Hierarchical clustering trees were generated using the rank-two ellipse seriation method (Chen 2002; Wu et al. 2010) to sort the samples.


Data availability The demultiplexed sequence data set obtained in this study are available in the NCBI Sequence Read Archive (SRA) database under the BioProject ID PRJNA260489.

Results Digester performance Increase in OLR was achieved by increasing the amount of waste whey fed at days 15, 40, and 69 while the amount of dairy manure slurry fed remained unchanged (Fig. 1a). The performance-related data including pH, TS content, individual VFA concentrations, and biogas production are summarized in Fig. 1. The first and the second increase in OLR at days 15 and 40, respectively, did not significantly affect the pH of either digester (Fig. 1c, g). The total VFA concentration of the thermophilic digester stayed ≤8 mM until after the second OLR increase when it increased to 20 mM (±2) between days 51 and 63, during which propionic acid reached a higher concentration than any other VFA (Fig. 1b). In the mesophilic digester, total VFA concentration remained below 6 mM even after the OLR was increased twice, and acetate and propionate were the major VFAs (Fig. 1f). The COD values in the effluent of the thermophilic digester and the mesophilic digester remained unaffected, at 10,000 and 3000 mg/L, respectively (Fig. 1e, i). During this period, the thermophilic digester produced more biogas than the mesophilic digester (Fig. 1d, h). These results indicated that the TPAD system tolerated an OLR of 5 % manure solid plus 48 g COD/l/day as waste whey during codigestion. The third OLR increase, from 48.5 to 60.4 g COD/l/day, from day 69 onward dramatically affected the performance of the thermophilic digester (Fig. 1). The pH decreased by about 2.5 units over about the first 10 days and then slowly decreased to about 4.3 by the end of the operation (Fig. 1c), and all the VFAs increased in concentration (Fig. 1b). Acetate concentration increased to 54 mM and remained at that level to the end of the operation, while concentrations of the other VFAs increased but then gradually decreased. The third OLR increase caused substantial decrease in biogas production in the thermophilic digester within about a week of the third OLR increase, and then, biogas production became barely measurable (Fig. 1d). The COD concentration of the thermophilic digester effluent showed an opposite trend as that of the biogas production (Fig. 1e). The organic overloading only had limited, and delayed, impacts on the mesophilic digester. The concentration of the

three major VFAs increased about 1 week after the overloading started, but that of propionate and butyrate then decreased to levels comparable to the pre-overloading concentration, except a sharp increase in propionate concentration toward the end of the operation (Fig. 1f). However, the concentration of acetate remained much higher than that seen before the overloading. The pH fluctuated but remained above pH 7.0 (Fig. 1g). The COD value of the mesophilic digester effluent increased only by a small margin (Fig. 1i), suggesting that much of the increased COD fed from the thermophilic digester was removed in the mesophilic digester. Biogas production in the mesophilic digester increased after the overloading started and peaked around day 97, reaching a volumetric biogas yield of about 4, but then started to decrease afterward (Fig. 1h). Concurrent with the decrease in biogas production, the pH dropped about 0.5 unit and foaming started about 1 month after the beginning of the overloading. A 50 % decrease in OLR to the mesophilic digester and concurrent increase of HRT to 20 days (by discarding 50 % of the effluent from the thermophilic digester) for another 64 days did not remove the foaming (data not shown) or resume the biogas production. Profiling of bacterial and archaeal communities by PCR-DGGE The DGGE profiles clearly showed that the thermophilic and the mesophilic digesters each had distinct populations of archaea and bacteria, and the organic overloading induced successions of several archaeal and bacterial populations (Supplementary material Fig. S1). For the thermophilic digester, two major archaeal bands became weaker and disappeared eventually during the overloading, while a few bands at different migration distances intensified. Similar trends were also observed for the mesophilic samples. The DGGE profiles of the mesophilic digester samples could be roughly divided into three distinct stages, corresponding to before the overloading, during the overloading when biogas production increased, and, thereafter, when biogas production fell to preoverloading level, and different groups of bacteria and archaea dominated at different stages Summary of the Illumina sequencing data After quality checking and removal of likely artificial sequences, 1.74 million sequences were obtained and about 98 % of them were assigned to known phyla of domain Bacteria. Twenty bacteria phyla were represented, with the Firmicutes (36.3 % of total bacterial sequences) and Bacteroidetes (30.7 %) being the most predominant followed by Thermotogae (19.5 %), Proteobacteria (5.8 %), Synergistetes (3.0 %), and Spirochaetes (1.3 %). Other phyla was each represented by ≥1 % of total bacterial sequences and


included Actinobacteria; Armatimonadetes; Chloroflexi; Fibrobacteres; Fusobacteria; Lentisphaerae; Planctomycetes; Tenericutes; Verrucomicrobia; and candidate phyla NKB19, OP9, and WPS-2. The archaeal sequences accounted for about 1 % of the total sequences, and all of them were assigned to the phylum Euryarchaeota. In total, 955 species-level OTUs of bacteria and archaea (clustered at 97 % sequence similarity) were obtained. Unweighted UniFrac (weighted UniFrac produced similar results, and only the results of unweighted UniFrac analysis were used) and PCoA showed profound difference in the microbiotas between the two digesters and microbiota successions at different stages of the operation (Fig. 2). PC1, which explained 46.8 % of total variation, separated the samples from the two digesters except the thermophilic digester samples collected before the overloading. On the other hand, PC2, which explained 33 % of total variation, separated the samples collected during the overloading from those collected before the overloading for both digesters, with the largest separation being seen for the thermophilic digester. The samples clustered into four groups based on digester and OLR. A close examination of the grouping showed that the microbiota in each digester was relatively stable before the overloading but underwent considerable successions during the overloading. The microbiota successions were greater

PCoA2 (33.1 % of total variance explained)

0.3

0.2

t8 0.1

m11 m9 m7

t10

t9

t11

m8 m10

m6

t7 t6

m5

0

m2 m4

m1 m3

-0.1 t5

-0.2

-0.3

-0.4 -0.6

t4

-0.4

-0.2

t2 t3 0

t1

0.2

0.4

PCoA1 (46.8 % of total variance explained)

Fig. 2 Principal coordinates analysis (PCoA) of the microbiotas in the thermophilic (diamond) and mesophilic (circle) digester of the TPAD system before (open) and after (filled) the start of the organic overloading. PCoA analysis shown was done using the unweighted UniFrac method. t1 to t11 and m1 to m11 correspond to the samples collected from the thermophilic and the mesophilic digesters over the course of the study (please see Fig. 1 for the times of sample collection). The arrows indicate the transition following the organic overloading

and lasted longer in the thermophilic digester than in the mesophilic digester during the organic overloading. The microbiota successions were delayed in the mesophilic digester. In addition, noticeable increases in microbial diversity and evenness were observed about 24 days into the overloading in the thermophilic digester, but not in the mesophilic digester (Table 1). Overall, the microbiota in the mesophilic digester was much less impacted than that in the thermophilic digester by the overloading. Bacterial community composition and structure at phylum level Ten bacterial phyla were each represented by more than 1 % of total bacterial sequences in at least one sample, and they together accounted for 99.3 % of the bacterial sequences (Fig. 3). From system start (t1 in Fig. 3) to 10 days into the overloading (t5), the bacterial community in the thermophilic digester was quite stable at phylum level, with Thermotogae being the most predominant followed by Firmicutes, Bacteroidetes, and Synergistetes. At days 93 and 98 (t6 and t7, 24 and 30 days into the overloading, respectively), Firmicutes increased in relative abundance at the expense of that of Thermotogae. Concomitantly, Bacteroidetes maintained a rather stable population, but Proteobacteria and Actinobacteria started to appear and reached a sizeable population by day 98. Tenericutes and Spirochaetes also increased their relative abundance, whereas Synergistetes population decreased to almost undetected. From day 120 (t8) to the end, the relative abundance of Firmicutes continued to increase, while Thermotogae b e c a m e a m i n o r p h y l u m . P ro t e o b a c t e r i a a n d Bacteroidetes together represented 35–55 % of the bacterial population after day 120. The relative abundance of these two phyla seemed inversely correlated. A c t i n ob a c t e r i a , Ten e r ic u te s, S p i ro c ha e t e s , a n d Fusobacteria were also found at low relative abundance (0.4–2 %) when the thermophilic was stressed by the overloading. In the mesophilic digester, the bacterial phyla maintained their relatively stable relative abundance before the overloading (Fig. 3). Before and during the first 10 days of the overloading, Bacteroidetes, Firmicutes, and Thermotogae were the three most abundant phyla. During this period, Synergistetes (1.4–4 %), Spirochaetes (1.7– 4.3 %), and Verrucomicrobia (1–2.5 %) were also detected at greater proportion in the mesophilic digester than in the thermophilic digester. Transitions of bacterial phyla were observed after the overloading started, with Bacteroidetes, Firmicutes, and Tenericutes slightly increasing, while other phyla declining, especially Thermotogae, in relative abundance. A new stable phylum distribution was established

Appl Microbiol Biotechnol Table 1

Alpha diversity analysis on the TPAD samples

Digester

Overloading Sampling day

First-stage thermophilic Before

During

Second-stage mesophilic

Before

During

Observed OTUs

Shannon (H)

Simpson (D)

Phylogenetic distance

Shannon’s equitability (EH)

30 44 58 72

357 385 348 398

3.46 3.53 3.06 3.39

0.74 0.74 0.69 0.74

43.13 46.99 42.04 49.05

0.59 0.59 0.52 0.57

79 93 98 121 135 154 169 30 44 58 72 79 93 98 121 135 154 169

549 697 679 502 406 668 456 549 589 585 583 602 519 497 448 473 471 472

3.28 5.37 5.37 5.13 4.67 5.61 4.88 5.85 6.09 5.62 4.92 5.77 5.84 5.57 5.06 5.50 5.48 5.58

0.72 0.87 0.89 0.90 0.88 0.92 0.92 0.94 0.95 0.92 0.87 0.93 0.96 0.94 0.91 0.93 0.93 0.94

62.17 74.17 72.23 58.12 46.98 68.96 50.73 57.52 61.65 61.84 62.12 63.31 57.50 55.54 50.72 53.84 53.29 52.42

0.52 0.82 0.82 0.82 0.78 0.86 0.80 0.93 0.95 0.88 0.77 0.90 0.93 0.90 0.83 0.89 0.89 0.91

other phyla with relative abundance >1 % (Spirochaetes and Synergistetes) were observed also.

Percentage

later (at day 120) and maintained, with Bacteroidetes and Firmicutes becoming the two predominant phyla, though

Thermophilic

Mesophilic

Fig. 3 Major bacterial phyla (each represented by >1 % of total sequences in at least one sample) found in the TPAD system. Samples were arranged in a timely order. The arrows on the top indicated the start of the organic overloading


Bacterial community composition and structure at OTU level The relative abundance of OTUs was visualized using heatmap (Fig. 4), and the taxonomic lineage and occurrence of these OTUs were supplied as Supplementary material Table S1. Consistent with the PCoA analysis, the grouping based on Pearson’s coefficients (Fig. 4b) showed that clustering of microbiota was influenced by operation temperature and changes in other chemical factors induced by the overloading. The successions of the bacterial communities in both digesters in response to the overloading could be revealed by the hierarchical clustering of the samples (Fig. 4d). Based on the distribution pattern, OTUs were clustered into six groups. It was obvious that some OTUs were more temperaturesensitive, and they appeared only in either the thermophilic (groups I and II) or the mesophilic digester (groups IV, V, and VI). However, some OTUs were more related to other factors that might be caused by the overloading, and these OTUs (e.g., group III) were more ubiquitous at both operation temperatures. Group III contained 216 OTUs, and most of them were detected in both digesters prior to the overloading (before day 79). Some of these OTUs were predominant (collectively accounting for one-third of total bacterial sequences) and assigned to the genus Petrotoga (12 OTUs), the family Porphyromonadaceae (11 OTUs), the candidate order M B A 0 8 (i n Clostridia , 20 O T U s), or th e gen us Anaerobaculum (7 OTUs). Some of the OTUs were assigned to genera containing known homoacetogens, such as Te p i d i m i c r o b i u m , Te p i d a n a e r o b a c t e r , a n d Thermacetogenium. In addition, some OTUs were assigned to Clostridium and Ruminococcus, both of which contain many fibrolytic bacterial species, and Syntrophomonas, which hosts known butyrate-degrading bacteria. Apparent changes in community structure were observed starting from day 93, which was 25 days into the overloading. The 315 OTUs clustered in group II were mostly observed in the thermophilic digester when it was overloaded. These OTUs were assigned to Bifidobacterium, Lactobacillus, Acetobacter, Bacteroides, the order Bacteroidales, and the family Porphyromonadaceae. Erysipelothrix and Treponema, both of which contain known homoacetogens, were also well presented by this group of OTUs. In addition to the group II OTUs, several group I OTUs classified to Proteobacteria were found at day 135 in the thermophilic digester. Group I OTUs were almost exclusively detected in this sample, including one OTU each assigned to Providencia, Achromobacter, Alcaligenaceae, and Acetobacteraceae. The OTU assigned to Acetobacteraceae was most predominant at day 169. In addition to the group III OTUs, OTUs in groups IV and V were also components of the bacterial community in the

mesophilic digester before the overloading, but group IV was found only before the overloading, while group V was found both before and during the overloading. Fifty-six OTUs were clustered in group IV, with the largest OTU classified to Bacteroidales (1.2 % of the total bacterial sequences). OTUs classified to Acholeplasmataceae (in the phylum Tenericutes), phylum Spirochaetes, and Clostridia were found relatively abundant in this group. Group V contained 191 OTUs, and 72 of them (representing 14 % of the total bacterial sequences) were classified to Bacteroidales, with some of them being further classified to Bacteroidaceae, Porphyromonadaceae, and Marinilabiaceae. Eighty percent of the OTUs in group V classified to Firmicutes were assigned to Clostridiales (represented 9 % of the total bacterial sequences), and many of these OTUs were further classified to Sedimentibacter (12 OTUs), Clostridium (27 OTUs), Pelotomaculum (2 OTUs), and Syntrophomonas (5 OTUs). The former two genera contain fermenting bacteria and bacteria that degrade amino acid and peptides, while the latter two genera contain known syntrophic propionate- and butyrate-oxidizing bacteria. Five OTUs assigned to the phylum Verrucomicrobia and one OTU assigned to the phylum Spirochaetes showed higher relative abundance before the overloading, while two OTUs assigned to the genus Aminobacterium were more predominant 25 days into the overloading in the mesophilic digester. Similar as the group V OTUs, most of the OTUs clustered in group VI were also components of the mesophilic digester bacterial community during the overloading. The group VI OTUs were assigned to the order Bacteroidales (3.4 % of the total bacterial sequences); to genera Trichococcus, Enterococcus, and Streptococcus in the order Lactobacillales; to families Clostridiaceae and Ruminococcaceae; to genera Clostridium, Tepidanaerobacter, Thermacetogenium, and Sphaerochaeta; and to the class Mollicutes (in the phylum Tenericutes). Archaeal community Eighteen archaeal OTUs were obtained from the Illumina sequencing data, and all these OTUs were assigned to six genera and one family in the phylum Euryarchaeota (Table 2). Methanoculleus was the most predominant genus, and it was represented by two OTUs, followed by Methanobrevibacter (represented by four OTUs) and Methanosarcina (represented by three OTUs). Other genera were each represented by a few small OTUs. The relative abundance of archaeal genera differed between the two digesters and was affected by the overloading (Table 2). In the thermophilic digester, Methanosarcina and Methanoculleus, followed by Methanobrevibacter, were the major genera together representing 95 % of the total archaeal community. However, Methanoculleus expanded its relative abundance to 86.2 % as the operation


b) Sample-sample Correlaon Matrix -1 1 T9 T10 T11 T8 T7 T6 T5 T4 T3 T2 T1 M4 M3 M2 M1 M5 M6 M7 M10 M9 M8 M11

Group I

II

III

IV

V VI 0

4.61 a) Log abundance of OTUs

-1

1

c) OTU-OTU correlaon matrix

d) Hierarchical clustering tree of OTUs

Fig. 4 Generalized association plots for the OTUs from the 22 data sets in the TPAD system. a The log abundance heatmap of the OTUs. b The sample-sample correlation map among the 22 data sets. c The OTU-OTU correlation map among the OTUs. d The hierarchical clustering tree for

sorting the OTU-OTU correlation map in c. The 22 data sets and the OTUs were sorted based on corresponding OTU-OTU correlation and sample-sample correlation, respectively

proceeded until day 72 (t4 in Table 2), while the relative abundance of Methanosarcina decreased to only 8.8 %. Ten days into the overloading (t5 in Table 2), however, Methanoculleus shrank its relative abundance to 40.2 %, though still the most predominant genus in the thermophilic digester. Concurrently, the Methanobrevibacter population doubled to 27.5 % and it became the second most predominant genus, and Methanobacterium became the third largest genus, accounting for 18.8 % of total archaea. In the later period of the operation into the overloading (t6 and onward), Methanobrevibacter became the most dominant genus, followed by uncultured archaeon WCHD3-02 classified in the genus Thermogymnomonas, whereas Methanosarcina went almost undetected. In the mesophilic digester, Methanobacterium and Methanobrevibacter were more predominant than they

were in the thermophilic digester and represented 8 to 25 % of the archaeal sequences before the organic overloading (during the first 72 days of operation). Two OTUs classified to Methanosarcina together expanded their relative abundance from 54.1 to 67.3 % of the archaeal community. From day 79 to day 134 of the operation (m5 to m9 in Table 2), one of the Methanosarcina OTUs decreased, while the other continued to increase. Together, they represented 49–68 % of the archaeal population. In this period, Methanobacterium and Methanobrevibacter became the second and the third largest genera, respectively. After day 153, although Methanoculleus remained abundant (22.2 % of total archaea), Methanosarcina became the most predominant genus throughout the rest of the operation, reaching a relative abundance of 82.9 % of total archaea by day 169.

Methanosarcina Methanosarcina WCHD3-02 WCHD3-02 WCHD3-02 WCHD3-02

592689 N63383 843408 136107 570725 282634 0.7

59.5 5.7 2.0

30.2 1.0 3.7

1.4

0.2 0.5

0.3 1.4

19.9

2.0

4.4

47.1 0.3

6.5 3.0

0.3 2.4 7.5

2.0

10.9 0.3

79.6 1.3 0.9

0.7

0.1 1.7 2.6

1.4

8.6

84.6 1.6 0.2

0.1 0.1

0.7

1.0 1.7

t4

1.6 0.8

7.8

0.8 1.6 34.9 6.3

10.6 0.4

18.8 16.5

t5

0.2 0.3 0.3 1.1 2.0

0.3 0.5 1.1 2.5

0.3 12.2 0.9

0.4 2.8 36.1 0.1 38.9 0.8 1.4 2.2

t7

0.4

1.0 5.2 37.0 0.3 38.1 0.5 0.4 0.6 0.5 0.6 10.6 0.4

t6

1.2

0.8 8.3

6.2

39.0 0.4 1.2 1.7 7.1

34.0

t8

4.5 17.9

47.8 4.5

25.4

t9

3.3 29.6 0.3 4.4

0.3

1.8 0.3 45.0 0.9

0.3 6.8 0.6 6.5

t10

1.7

3.4 22.7

0.8

5.9

0.8 2.5

34.5 6.7 21.0

t11

0.4

34.9 0.4 0.4

14.2 14.6 1.5 8.8 1.1 1.9 1.1 0.4 0.8 0.4 19.2

0.7 1.4

33.8 0.7

12.8

1.0

2.4 1.7

7.6

1.7 19.3 16.9

m2

0.6 0.6

46.7

4.8 0.9 19.8

1.2 0.3

1.8

2.1 15.0 6.3

m3

0.7

52.0 0.5

4.2 1.2 15.3

11.1 10.2 0.5 3.2 0.2 0.5 0.5

m4

m1

t3

t1

t2

Mesophilic second stagea

Thermophilic first stagea

The samples collected after the start of the organic overloading are italicized

Methanobacterium Methanobacterium Methanobrevibacter Methanobrevibacter Methanobrevibacter Methanobrevibacter Methanosphaera Methanosphaera Methanocorpusculum Methanoculleus Methanoculleus Methanosarcina

551498 N101286 842598 167356 153168 470690 589886 150477 212362 108784 840393 4027690

a

Taxonomic assignment

Relative abundance of archaeal (percentage of the total archaeal sequences in respective sample) found in the TPAD system

OTD ID

Table 2

33.7 1.1

0.4 1.9 20.3

2.3

4.6

2.7 27.2 5.7

m5

1.2 7.1

20.2 1.2

4.8 46.4

1.2

2.4

10.7 4.8

m6

1.3

15.0 1.3

1.3 50.0

5.0 22.5 3.8

m7

2.9

67.6

14.7 8.8 5.9

m8

7.8 1.1 8.9

2.2 4.4

22.2 46.7

1.1

1.1

2.2 2.2

m9

0.6

1.2

1.2 1.7

5.2 81.5

0.6

6.9 1.2

m10

1.0

1.0

1.0

3.1 81.9

3.1 3.1 2.6 3.1

m11



Correlation between microbiota and digester performance and conditions To investigate the correlation between the digester performance/conditions and the microbial taxa in the TPAD system, canonical correspondence analysis (CCA) was performed. Because operation temperature, pH, concentrations of VFA (acetic, propionic, and butyric acids), TS content, effluent COD, and biogas yield were the most influential factors, they were included in the CCA. Separate CCA was performed at phylum level using the phyla detected, at OTU level using the major OTUs that each was represented by ≥0.1 % of total sequences, and at genus level using the known genera that were represented by the major OTUs (Fig. 5). At any of these three taxonomic levels, the environmental variables included in the CCA analysis (i.e., temperature, pH, biogas yield, VFA concentrations, effluent COD, and TS) explained 57–79 % of the variations of relative abundance, suggesting that these environmental variables primarily affected or reflected the distribution of the taxa. Furthermore, CCA1 and CCA2 together explained 73–94 % of the constrained inertia at any of the three taxonomic levels, which indicates that these two coordinates are adequate to represent the CCA results. The clustering of samples on the CCA plots was consistent with the clustering on the PCoA plot and further attributed the variation of the microbial community to the environmental factors that we measured. Temperature and pH were the two most influential factors that corresponded to much of the variations in the microbial communities. Samples from the thermophilic and the mesophilic digesters were separated along the temperature gradient. Content of TS, effluent COD, and concentrations of acetic acid and propionic acid were highly correlated with each other. These factors were highly correlated positively with the relative abundance of Actinobacteria, Fusobacteria, and Proteobacteria (phyla marked 3, 9, and 14, respectively, in Fig. 5a). The relative abundances of phyla Synergistetes and Thermotogae were highly correlated positively with the biogas production (phyla marked 16 and 18). The major OTUs were grouped into three groups on the CCA plot. One group was positively correlated with biogas production, and it contained five OTUs classified to the genera Anaerobaculum, Petrotoga, Bacteroidales, candidate MBA08 (in the class Clostridia), and Porphyromonadaceae. Another group of OTUs was highly correlated positively with acetic acid concentration, TS content, and effluent COD, and it included OTUs classified to a number of genera including Lactobacillus, Acetobacter, Bifidobacterium, and Erysipelothrix. The third group was found positively correlated with pH but inversely correlated with temperature and VFA (top left cluster on Fig. 5b, c).

Discussion Co-digestion of livestock manure, which is rich in nutrients (nitrogen and phosphorus) but contains little readily digestible carbohydrates and food wastes or food-processing wastes that have opposite compositional features, is an attractive strategy to boost energy yield from anaerobic digesters. Co-digestion of dairy manure and waste whey generated from makers of dairy products is an attractive option because dairy farms and makers of dairy products are typically located in the same area and because dairy manure can buffer the low pH of waste whey and its rapid acidogenesis from readily fermentable sugars. In co-digestion of these types of feedstocks, organic overloading with increased proportion of the food/foodprocessing waste can cause digester failure. Such failure has been documented and attributed to inhibition of methanogens by low pH and accumulation of VFA, particularly propionate (Chen et al. 2008). The advancement of metagenomics empowered by NGS provided opportunities to examine how organic overloading affects the entire microbial community in detail. In this study, we investigated both the archaeal and the bacterial communities in a TPAD system co-digesting dairy manure and waste whey and their successions in response to organic overloading using both DGGE profiling and Illumina sequencing. Both methods consistently showed that the thermophilic and the mesophilic digesters had different microbiota with respect to composition and structure. The microbiota in the thermophilic digester was impacted by the organic overloading to a much greater extent than that in the mesophilic digester. Some of the identified microbial taxa appeared to be correlated with performance data or environmental factors, helping understand the functions of different microbial guilds involving in the AD process and improve stability and performance of AD system. The thermophilic digester performance, such as biogas production and COD removal, deteriorated rapidly soon after the organic overloading started and was unable to recover as the overloading continued. These results also implied acidogens continued fermentation, but both acetoclastic methanogens and hydrogenotrophic methanogens were inhibited. The similar COD values in the effluent and the influent of the thermophilic digester and the lack of biogas production suggest that little H2 or CO2 was generated from acidogenesis therein. On the other hand, the mesophilic digester increased biogas production considerably for more than 6 weeks after the overloading started. Such increased performance by the mesophilic digester was apparently attributed to the increased COD input from the thermophilic digester before the mesophilic digester was also stressed by the overloading. Our observation was in agreement with the finding that biogas was produced mainly in the thermophilic digester of TPAD systems during normal feeding, but the mesophilic digester became the major producer of biogas during organic


Fig. 5

Canonical correspondence analysis at levels of phylum (a), predominant OTUs (b), and OTUs that were classified to a genus (c). Each dot represents one sample. Triangles represent individual taxa. Arrows represent the direction of increase of the environmental variables or digester performance data. Temp operation temperature, biogas daily biogas yield, TS TS content, Eff_COD effluent COD. The length of each arrow is proportional to the degree of correlation with the ordination axes. Corresponding phyla in a: 1 Euryarchaeota, 2 unclassified bacteria, 3 Actinobacteria, 4 Armatimonadetes, 5 Bacteroidetes, 6 Chloroflexi, 7 Fibrobacteres, 8 Firmicutes, 9 Fusobacteria, 10 Lentisphaerae, 11 candidate division NKB19, 12 candidate division OP9, 13 Planctomycetes, 14 Proteobacteria, 15 Spirochaetes, 16 Synergistetes, 17 Tenericutes, 18 Thermotogae, 19 Verrucomicrobia, 20 candidate division WPS-2

overloading (Zuo et al. 2013). These results also corroborated protection of the mesophilic digester from shocking loading by the first-stage thermophilic digester. Therefore, TPAD system is more tolerant to overloading than single-staged digesters. Although ecologically, a microbiota with greater diversity is more stable and resilience, it is not known to what extent the greater diversity seen in the mesophilic digester contributed to its tolerance to the shocking of organic overloading. Our results showed that the thermophilic and the mesophilic digesters in the TPAD system harbored different m i c r o b i a l p o p u l a t i o n s . I n p a r t i c u l a r, t h e p h y l a Verrucomicrobia and Spirochaetes were barely detected in the thermophilic digester but were observed in the mesophilic digester before the overloading. Most of the members of Verrucomicrobia are mesophilic (Sangwan et al. 2004) and are present ubiquitously in soil (Bergmann et al. 2011). This phylum has also been found in a mesophilic full-scale CSTR treating corn straw (Qiao et al. 2013); however, its ecological relevance or role in the environment or digesters remains to be determined. Compared to Verrucomicrobia, Spirochaetes was often observed in AD systems fed with different substrates (Chouari et al. 2005; Delbes et al. 2000; Lee et al. 2013). Because its relative abundance increased with increased glucose loading, this phylum has been suggested to be sugar fermenters in digesters (Delbes et al. 2000). However, a recent study on the Spirochaetes population in seven full-scale digesters suggested that Spirochaetes might also be involved in syntrophic acetate oxidation (Lee et al. 2013). Because the sugars fed to the thermophilic digester were probably converted to VFA therein, the Spirochaetes found in the mesophilic digester in the present study might be primarily involved in syntrophic acetate oxidation. The occurrence of this phylum corresponded with the accumulation of VFA in the thermophilic digester during the organic overloading, supporting the above premise. As the effluent of the thermophilic digester was fed to the mesophilic digester, bacteria and methanogens of the thermophilic digester were transferred to the mesophilic digester during feeding. Some of these carried-over bacteria, such as the


OTUs assigned to the families Thermotogaceae and Porphyromonadaceae and the class Clostridia (group III in Fig. 4), might have survived in the mesophilic digester. This finding supported the result from a previous study where microorganisms from the thermophilic digester survived and were probably involved in the metabolism in the mesophilic digester in a TPAD system (Vandenburgh and Ellis 2002). Consistent with previous findings (Levén et al. 2007; Pycke et al. 2011; Sundberg et al. 2013), we also observed that the microbiota in the mesophilic digester had a greater diversity than that in the thermophilic digester. Bacteroidetes and Firmicutes were predominant in both digesters both before and during the overloading, while Thermotogae was predominant in the thermophilic digester before the overloading, but Thermotogae was replaced by Proteobacteria and diminished to being almost undetected during the overloading. The above predominance trends of these three phyla were consistent with those observed in both a mesophilic and a thermophilic CSTR digesters treating organic household waste (Levén et al. 2007). The predominance of these three phyla may be explained by the ability of many of their members to utilize a broad range of substrates in the anaerobic digesters. With respect to Thermotogae, its ability to thrive at high temperature (Huber and Hanning 2005) and to adapt to mesophilic temperature, at least members of Thermotogales (Nesbø et al. 2006), might be another reason contributing to its predominance in both digesters in the TPAD system observed in the present study. It should also be noted that all the Thermotogae sequences obtained in the present study were classified to the genus Petrotoga, which contains thermophilic xylanolytic species (Miranda-Tello et al. 2004) and has been repeatedly documented as DNA sequences in anaerobic digesters (Weiss et al. 2008; Jabbour et al. 2013). This group of bacteria may have important role in anaerobic digesters, especially in thermophilic digesters. Considerable succession of bacterial community in the thermophilic digester was observed during the organic overloading to the TPAD system. Before the overloading, Petrotoga and Anaerobaculum, both of which contain peptide-fermenting species (Maune and Tanner 2012a, b; Menes and Muxí 2002), predominated. These two genera were likely important in the TPAD system at normal OLR. The appearance of lactic acid producer (e.g., Lactobacillus and Bifidobacterium) and acetic acid producer (e.g., Acetobacter) during the overloading might be owing to their ability to live in acidic environment and to utilize the lactose present in the influent. The cooccurrence of Thermoacetogenium, members of which are syntrophic acetate oxidizers (Hattori et al. 2000), and hydrogenotrophic methanogens in the thermophilic digester before the overloading might imply the importance of methane production through syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis.

Both the DGGE data and the Illumina sequencing data consistently showed profound successions of the archaeal community in both digesters during the overloading, corroborating the sensitivity of methanogens to OLR. During the stable operation (days 1–72), both hydrogenotrophic Methanoculleus and acetoclastic Methanosarcina were abundant in the thermophilic digester, with the former increasing and the later decreasing as the OLR increased over time. Although the thermophilic digester had not suffered from organic overloading before day 72, the stepwise increase in OLR might have resulted in substrate availability and/or chemical conditions that favored Methanoculleus. This observation suggests that hydrogenotrophic methanogenesis contributed more to the biogas production in the thermophilic digester than the acetoclastic pathway during the stable operation period. This premise is consistent with the results from several previous studies that showed hydrogenotrophic methanogenesis as the primary pathway for methane production in thermophilic digesters (Hori et al. 2006; Karakashev et al. 2005; Sasaki et al. 2011; Sundberg et al. 2013). During the overloading, Methanobrevibacter, a hydrogenotrophic genus, and another group closely related to an uncultured clone (WCHD3-02) in the genus Thermogymnomonas (based on RDP classification) became the predominant methanogens, accounting for 62–75 and 18–30 % of the archaeal population, respectively, in the thermophilic digester. Methanobrevibacter is the most predominant genus of methanogens in the rumen and hind gut of mammalian animals (Kim et al. 2011), but it has not been widely reported in anaerobic digester. Most species of this genus are acid tolerant, and only one species of Methanobrevibacter has been isolated from a sour acidogenic anaerobic digester (Savant et al. 2002). However, the minimal biogas production from the thermophilic digester during the overloading suggests that the methanogens might not actively producing methane. The increased relative abundance of Methanobrevibacter could be simply due to decrease in other methanogens. Quantitative analysis using qPCR is needed to understand the population dynamics of this genus (and also other genera). Although thermophilic and acid-tolerant, the genus Thermogymnomonas has only one cultured representative, and no member of the class Thermoplasmata was found to be methanogenic except the recently reported isolates of Methanomassiliicoccus luminyensis (Dridi et al. 2012) and Candidatus Methanogranum caenicola (Iino et al. 2013), which were isolated from human faces and anaerobic digester sludge, respectively. Thus, although its predominance can be explained by the acidic and thermophilic conditions in the thermophilic digester during the overloading, the function and importance of this WCHD3-02 archaeal group in the thermophilic digester remain unclear. It will be intriguing to


understand and investigate the metabolic capacity of this group and how similar it is taxonomically compared to other methanogens in the class Thermoplasmata. The lack of active biogas production is consistent with previous researches that low pH induced by organic overloading inhibits methanogenic activity (Alkaya and Demirer 2011; Zuo et al. 2013). In the mesophilic digester, Methanosarcina increased while Methanobacterium and Methanobrevibacter decreased in relative abundance shortly after the organic overloading started. Such a shift in these genera can be explained by the ability of Methanosarcina and the inability of Methanobacterium to utilize acetate and the increase in acetic acid influx from the thermophilic digester caused by the overloading. Such an explanation is consistent with previous reports that high acetate in anaerobic digesters favored Methanosarcina (Blume et al. 2010; Hori et al. 2006; McMahon et al. 2004; Steinberg and Regan 2011). However, Methanosaeta, an obligate acetoclastic methanogens, was hardly detected in our study, though it was the most predominant methanogens in the mesophilic digester of a TPAD system fed diary manure only (Lv et al. 2013a). The large population of Methanosarcina may be attributed to its high μmax and high Ks (Conklin et al. 2006). It should be noted that although Methanosarcina expanded its population during the overloading with concurrent increase in biogas production and decrease in acetate concentration in the mesophilic digester, the biogas production peaked and then decreased gradually. The persistent overloading might have changed the conditions under which Methanosarcina gradually decreased metabolic activity. Therefore, although abundance information provides some insight into the function, confirmation of their actual activity will need further investigation using functional analysis such as the transcriptomics or methanogenic activity assay. Multivariate analysis has been used widely in microbial ecology to help understand potential relationship between environmental factors and specific microbial populations (review in Ramette 2007). In this study, the correlations between the relative abundance of microbial taxa and the important factors of digester operation and performance were investigated using CCA. As expected, temperature and pH were the two most important operation parameters that affected the composition of the microbiota in this TPAD system. The relative abundance of some taxa including Anaerobaculum, Petrotoga, Bacteroidales, candidate MBA08 (in the class Clostridia), and Porphyromonadaceae was found highly positively correlated to biogas production, while that of some other taxa including Lactobacillus, Acetobacter, Bifidobacterium, and Erysipelothrix was found positively correlated to VFAs and the degradation of feedstocks. These genera may play important roles in AD, at least co-digestion of

dairy manure and waste whey. However, as discussed above, the causality between the environmental factors or performance measurements observed and the population dynamics of these taxa remains to be determined. Future researches using functionality-based analysis, such as transcriptomics, proteomics, and stable isotope probing (SIP), can help determine the causality. Nevertheless, the aforementioned taxa may serve as indicator for AD performance and can guide future research on efficiency and stability of AD systems. Further, those directly contribute to digester performance and stability may be used as bioaugmentation organisms to restore stressed or failed digesters. Taken together, the information on the microbiota in the thermophilic and the mesophilic digesters and the dynamic successions of the microbiota induced by the organic overloading provided new insight into the composition and stability of microbiota in the digesters in response to changed OLR. The correlation revealed between some of the microbial populations and performance data/environmental factors may help future studies to improve efficiency and stability of anaerobic digesters. Acknowledgments This study was partially supported by a Department of Energy (DOE) award (DE-FG36-05GO85010) and a US Department of Agriculture/National Institute of Food and Agriculture (USDA/NIFA) award (2012-10008-20302). Conflict of interest The authors declare that they have no conflict of interest. Compliance with ethical standard This article does not contain any studies with human participants or animals performed by any of the authors.

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