Bioresource Technology 101 (2010) 63366344

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Microbial community dynamics associated with biomass granulation in low-temperature (15 C) anaerobic wastewater treatment bioreactors
Joe OReilly a, Changsoo Lee a,1, Fabio Chinalia b, Gavin Collins c,d, Thrse Mahony a, Vincent OFlaherty a,d,*
Microbial Ecology Laboratory, Microbiology School of Natural Sciences and Environmental Change Institute, National University of Ireland, Galway, University Road, Galway, Ireland b Centre for Resource Management and Efciency, School of Applied Science, Craneld University, College Road, Craneld, Bedfordshire MK43 0AL, UK c Microbial Ecophysiology Research Group, Microbiology School of Natural Sciences and Environmental Change Institute, National University of Ireland, Galway, University Road, Galway, Ireland d Bioenergy Research Group, Energy Research Centre, Environmental Change Institute, National University of Ireland, Galway, University Road, Galway, Ireland
a

a r t i c l e

i n f o

a b s t r a c t
Granular biolms underpin the operation of several categories of anaerobic wastewater treatment bioreactors. Recent studies have demonstrated the feasibility of treating both industrial and domestic wastewaters at their discharge temperatures (usually <18 C), thereby avoiding the heating expenses of mesophilic (2045 C) or thermophilic (4565 C) treatments. Previous low-temperature trials used mesophilic inocula and little information is available on the viability of low-temperature anaerobic granulation. Six laboratory-scale, expanded granular sludge bed bioreactors (R16) were operated at 15 C (R12 and R45) and 37 C (R3 and R6). R13 were fed glucose-based wastewater and R46 were fed volatile fatty acid-based wastewater. Quantitative real-time PCR and qualitative denaturing gradient gel electrophoresis of 16S rRNA genes identied the dominance of Methanomicrobiales (mainly Methanocorpusculum-like organisms) during low-temperature granulation. Granulation only occurred in glucose-fed bioreactors. The results suggest that (i) granulation is feasible in low-temperature bioreactors; (ii) carbohydrate decomposition likely favoured granulation, (iii) Methanocorpusculum-like organisms play a critical role in low-temperature granulation. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 11 November 2009 Received in revised form 5 March 2010 Accepted 10 March 2010 Available online 7 April 2010 Keywords: Anaerobic granules DGGE Low-temperature anaerobic digestion Methanogens Quantitative PCR

1. Introduction Anaerobic digestion (AD) is an increasingly popular treatment option for both domestic and industrial wastewaters. AD confers several advantages over traditional treatments, including the (i) elimination of costly aeration processes and (ii) production of energy in the form of methane (Liu et al., 2003; McCarty, 2001). Wastewater carbon is converted to biogas through a series of interlinked processes involving phylogenetically- and functionally-distinct microbial groups (Batstone et al., 2004). Anaerobic granular biolms form by the self-immobilisation of those diverse groups in response to bioreactor operating conditions, such as liquid upow velocity and associated shear forces. The high biomass concentration and optimal organisation of microbial trophic groups
* Corresponding author at: Microbial Ecology Laboratory, Microbiology School of Natural Sciences and Environmental Change Institute, National University of Ireland, Galway, University Road, Galway, Ireland. Tel.: +353 91 493734; fax: +353 91 494598. E-mail address: vincent.oaherty@nuigalway.ie (V. OFlaherty). 1 Present address: Division of Environmental and Water Resources Engineering, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore. 0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.03.049

in anaerobic sludge granules, facilitates applied organic loading rates in excess of 50 kg chemical oxygen demand (COD) m3 d1 and underpinned the development of several categories of highrate anaerobic wastewater treatment systems (Hulshoff Pol et al., 2004). Full-scale AD plants are mainly operated within the mesophilic (2445 C) or thermophilic (4565 C) temperature ranges (Lettinga et al., 2001). The majority of domestic and industrial wastewaters, however, are discharged 618 C, thus incurring a considerable additional heating cost for mesophilic or thermophilic systems (Connaughton et al., 2006). The economic attractiveness of AD could be enhanced by treating wastewaters at their discharge temperature. Sub-ambient or low-temperature treatments were previously not considered feasible due to lower microbial activity, increased gas solubility and liquid viscosity (Lettinga et al., 1999; Rebac et al., 1999). Recently, several trials reported the successful treatment of both real and synthetic wastewaters at temperatures as low as 4 C, and demonstrated comparable performances to mesophilic systems (McKeown et al., 2009). However, most previous low-temperature anaerobic digestion (LTAD) studies used pre-granulated, mesophilic sludge to seed anaerobic bioreactors. Gradually, the microbial communities adapted to the

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Phase 3 day 255300

2.1. Bioreactor operation Anaerobic granular sludge (, 0.83.5 mm) was obtained from a full-scale internal circulation bioreactor (Carbery Milk Products Ltd., Balineen, Ireland) treating industrial alcohol production wastewater at 37 C. The granular sludge was nely crushed and sieved (, <0.2 mm), and 88 g of volatile solids (VS) were used to inoculate each of six identical EGSB bioreactors (R16), each with a 3.5-l working volume. The rst three bioreactors, R13, were fed with a synthetic, glucose-based wastewater, while R46 were fed a synthetic, volatile fatty acid (VFA)-based wastewater consisting of acetate, propionate, butyrate and ethanol in the chemical oxygen demand (COD) ratio of 1:1:1:1. Of these bioreactors, R1, R2, R4 and R5 were operated psychrophically (15 1 C), and R3 and R6 were operated mesophilically (37 1 C). The inuent wastewaters were buffered with NaHCO3 to pH 7.1 0.2 and fortied with macro- (10 ml l1) and micro- (1 ml l1) nutrients, as previously described by Shelton and Tiedje (1984). The trial was divided into three operational phases (Table 1), which were characterised by changes in the hydraulic retention time (HRT) or applied organic loading rate (OLR). Each bioreactor was operated at a 36-h HRT during Phase 1 and at a reduced HRT of 12 h during Phases 2 and 3. An OLR of 2 kg COD m3 d1 was applied through out Phases 1 and 2. Phase 3 was characterised by the doubling of the OLR to 4 kg COD m3 d1. The COD concentration and pH, and CH4 content of efuent and biogas, respectively, were determined every 23 days. 2.2. Granulation rate determination The granulation rate was dened as the proportion of granules >1.0 mm in diameter and was determined for each bioreactor at the end of each operational phase (Table 1). Typically, most lowtemperature granular anaerobic bioreactors are operated at higher up-ow velocities to counter the effects of increased liquid viscosity (McKeown et al., 2009). Under these operating conditions, increased biomass washout of smaller less dense aggregates is likely and, in the authors experience, a diameter of 1 mm represents a suitable indicator size of retention/granulation for these systems. Approximately 30 ml of sludge from each bioreactor was graded in a step-wise manner, through a series of sieves with pore sizes of 0.21.4 mm. Each size fraction was dried at 110 C for 24 h, and the granulation rate was calculated based on
Table 1 Bioreactor operating parameters and performance data. V 15 2 12 69 11 1.25 63 NAj 1.4 NAj Feed Temperature (C) OLRb HRTc CREd V ue M%f BGRg SHARh GHARi
a

R5

Phase 2 day 120255

Phase 1 day 0120

G 15 2 36 80 18 1.25 59 NAj 3.1 NAj

R1

G 15 2 36 78 18 1.25 61 NAj 3.1 NAj

R2

G 37 2 36 82 17 1.25 66 NAj 1.2 NAj

R3

V 15 2 36 80 13 1.25 64 NAj 3.1 NAj

R4

V 15 2 36 77 15 1.25 61 NAj 3.1 NAj

R5

V 37 2 36 84 17 1.25 72 NAj 1.2 NAj

R6

G 15 2 12 64 10 1.25 62 14 1.5 2.4

R1

G 15 2 12 68 9 1.25 61 16 1.6 1.9

R2

G 37 2 12 73 8 1.25 67 36 1.2 1

R3

V 15 2 12 67 12 1.25 67 NAj 1.3 NAj

R4

V 37 2 12 82 8 1.25 74 10 1.2 0.9

2. Methods
R6

G, synthetic glucose wastewater; V, synthetic VFA wastewater. Organic loading rate (kg COD m3 d1). Hydraulic retention time (h). Chemical oxygen demand removal efciencies, determined every 2 or 3 days (standard deviation). Up-ow velocity (m h1). Methane content of biogas produced, determined every 2 or 3 days. Biomass granulation rate, determined on the nal day of each phase, measured as proportion (%) of biomass >1.0 mm. Ratio of hydrogenotrophic to aceticlastic SMA values in suspended biomass, determined for seed inoculum (Phase 1) and thereafter at the end of each operational phase. Ratio of hydrogenotrophic to aceticlastic SMA values in granular biomass, was determined at the end of each operational phase. Not applicable.

G 15 4 12 54 12 1.25 64 32 2 1.9

R1

lower temperatures, leading to the emergence of distinct, psychrotolerent communities (Connaughton et al., 2006; OReilly et al., 2009a). Psychrophilically-cultivated biomass would likely facilitate shorter start-up times, as well as the application of higher organic loads, improving the stability and cost-effectiveness of LTAD (Akila and Chandra, 2007; McKeown et al., 2009). Thus, it is clear that a greater insight into the populations involved in the anaerobic granulation process, particularly under low-temperature conditions, is required. Currently, little information is available on the feasibility of low-temperature granulation, and its regulation by ecological and process factors is not yet understood. In light of this, we compared the microbial community development in six expanded granular sludge bed (EGSB) bioreactors (R1 6), which were inoculated with non-granular biomass and were used to treat synthetic glucose-based wastewater or volatile fatty acid (VFA)-based wastewater at psychrophilic or mesophilic temperatures. Temporal population dynamics were monitored in the suspended and granulating biomass using a combination of qualitative and quantitative molecular tools.

G 15 4 12 54 14 1.25 65 38 1.9 2.1

R2

G 37 4 12 76 9 1.25 72 61 1.3 1.1

R3

V 15 4 12 52 16 1.25 61 NAj 1.4 NAj

R4

V 15 4 12 61 13 1.25 60 NAj 1.6 NAj

R5

V 37 4 12 80 11 1.25 71 20 1.3 1.2

R6

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the proportion of biomass, measured as dry weight, in each fraction. Biomass fractions with diameters of <0.2 mm and >1.0 mm were dened as suspended and granular biomass, respectively. 2.3. Specic methanogenic activity (SMA) SMA was determined for seed sludge (day 0) and bioreactor biomass on days 255 and 300, using the pressure transducer technique (Colleran et al., 1992). From day 255 onwards the SMA of both suspended and granular biomass was determined (where applicable). Acetate (30 mM) and H2/CO2 (80:20, v/v) were used as direct methanogenic substrates (Coates et al., 1996; Colleran et al., 1992). SMA assays were conducted in triplicate with 3 g VS l1 per assay. The SMA assays were conducted at the operating temperature of each bioreactor. 2.4. DNA extraction Total DNA was extracted from the seed biomass and from each bioreactor biomass on days 120, 255 and 300 using an automated nucleic acid extractor (Magtration 12 GC, PSS Co., Chiba, Japan). For each bioreactor (where applicable), the extraction was performed using granular ( > 1.0 mm) as well as suspended ( < 0.2 mm) biomass. Prior to extraction, samples of suspended biomass were concentrated by centrifuging at 10,000g for 3.5 min. Each granular or suspended biomass sample (0.5 g wet weight) was nely crushed using a mortar and pestle, and then re-suspended in 50 ml of sterile double distilled water. A 100-ll aliquot of the biomass suspension was loaded per extraction. In parallel, the VS concentration of each re-suspension was measured to estimate the amount of biomass used for DNA extraction. Each extraction was performed in duplicate and the extracted DNA was eluted in TrisHCl buffer (pH 8.0) and stored at 20 C. 2.5. DGGE and phylogenetic analyses Archaeal 16S rRNA gene fragments were amplied with the primer set 787F-1059R (Takai and Horikoshi, 2000). Bacterial 16S rRNA genes were targeted with the primer set 338F-805R (Raskin et al., 1995). A 40-bp GC-clamp was attached at the 50 -end of each forward primer to stabilise the melting behaviour of the PCR products (Muyzer et al., 1993). The PCR amplication was performed as follows: initial denaturation at 95 C for 3 min; a touch-down thermal cycling of denaturation at 95 C for 1.5 min (archaeal) or 45 s (bacterial), annealing at 6555 C for 1.5 min (reducing 1 C per cycle), and elongation at 72 C for 1.5 min (archaeal) or 1 min (bacterial); an additional 20 cycles of (for archaeal or bacterial, respectively): 95 C for 1.5 min or 45 s, 55 C for 1.5 min or 45 s, and 72 C for 1.5 min or 1 min; a nal extension at 72 C for 4 min. A 20-ll aliquot of each PCR product was loaded onto a 10% (w/v) polyacrylamide gel containing a denaturing gradient of 3060% (archaeal) or 3070% (bacterial) (100% denaturant contained 7 M urea and 40% (v/v) formamide). Electrophoresis was run in a D-Code system (BioRad, Hercules, CA). The DGGE gel was ethidium bromide-stained and photographed under UV transillumination. A binary matrix was generated by scoring the presence (1) or absence (0) of each band, and then statistically analyzed with non-metric multidimensional scaling (NMS) using Sorensen (Bray-Curtis) distance measurement, in PC-ORD software version 5.0 (Grandin, 2006). For further sequencing and phylogenetic analyses, bands of interest were excised from the gel using a sterile scalpel blade. For accurate correlation of band homology between different DGGE gels, a selection of bands were excised and sequenced from multiple gels. The excised bands were eluted in 25 ll of sterile water, and then re-amplied with the same primer sets (without

GC-clamp). The PCR products were gel-puried and cloned into the TOPO TA 2.1 vector (Invitrogen, Carlsbad, CA). PCR fragments were sequenced using M13 primers and compared against the GenBank and RDP10 databases. A neighbour-joining tree was constructed using MEGA 4 software (Tamura et al., 2007). The nucleotide sequences were deposited in the GenBank database under accession numbers GQ304250-GQ304275. 2.6. Real-time PCR analysis Quantitative real-time PCR was performed using a LightCycler 480 (Roche, Mannheim, Germany) with ve methanogenic primer and probe sets, specic to three orders (Methanomicrobiales, Methanobacteriales, and Methanococcales) and two families (Methanosaetaceae and Methanosarcinaceae) covering most methanogens present in anaerobic digesters (Lee et al., 2009; Yu et al., 2005). All DNA samples were analyzed with each primer and probe set in duplicate. Each reaction mixture was prepared using the LightCycler TaqMan Master kit (Roche): 8 ll of PCR-grade water, 1 ll of the probe (nal concentration 200 nM), 1 ll of each primer (nal concentration 500 nM), 4 ll of 5 reaction solution, and 5 ll of DNA template. Amplication was carried out using a two-step thermal cycling protocol consisting of predenaturation for 10 min at 94 C followed by 40 cycles of 10 s at 94 C and 30 s at 60 C (exception: 63 C for Methanobacteriales-set as out lined by Yu et al. (2005). Quantitative standard curves were constructed using the standard plasmids containing the full-length 16S rRNA gene sequences from the representative strains of the target methanogenic groups as previously described (Lee et al., 2009; Yu et al., 2005). For each primer and probe set, an equimolar mixture of its corresponding standard plasmids was used as the template solution for constructing the standard curve. The mass concentration of each plasmid was measured in duplicate using a Qubit system (Invitrogen) and converted into its copy concentration as previously described (Lee et al., 2009). A 10-fold serial dilution series (101109 copies ll1 was generated for each standard solution and analyzed by real-time PCR in triplicate with its corresponding primer and probe set. The threshold cycle (CT) values determined were plotted against the logarithm of their input copy concentrations. The 16S rRNA gene copy concentrations of target groups were then estimated against the corresponding standard curves within the linear range (r2 > 0.995). The volume-based concentration (copies ll1) were converted into the biomass-based concentration (copies g [VS]1) using the VS concentration of each sludge sample used for the DNA extraction. In this study, the real-time PCR detection limit for each primer and probe set was <107 copies g VS1 level. 2.7. Analytical methods Solids and COD were analyzed following the procedure in Standard Methods (APHA-AWWA-WEF, 2000). Methane content was determined using a Philips PYE Unicam 304 gas chromatograph (Cambridge, UK) equipped with a Propak Q packed capillary column (100120 mesh). Nitrogen was used as a carrier gas at a ow rate of 60 ml min1. All samples were analyzed in duplicate. 3. Results and discussion 3.1. Bioreactor performance Table 1 summarises bioreactor R16 operating parameters and performance. During Phase 1, no granular biomass ( > 1.0 mm) developed in any of the bioreactors, regardless of the operating

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temperature. Upon commencement of Phase 2, the HRT was decreased from 36 to 12 h, and by the end of Phase 2, the granulation rates had reached 1416% in the glucose-fed low-temperature bioreactors R1 and 2% and 36% in the glucose-fed mesophilic R3 (Table 1). Notably, Phase 2 was characterised by reduced COD removal efciencies in R13 of 916%. However, the standard deviations decreased by almost half, indicating less variable COD removal (Table 1). The low-temperature VFA-fed R4 and 5 displayed a similar COD removal efciency to R12, though no granulation was observed during this period (Table 1). The applied OLR was increased to 4 kg COD m3 d1 on day 255 (Phase 3), and the granulation rates in the glucose-fed bioreactors almost doubled to 32 38% in R12; 61% in R3. Interestingly, throughout the trial, the most efcient COD removal of 8084% along with the highest methane content of 7174%, was observed in the mesophilically operated, VFA-fed, R6 (Table 1). In contrast to the glucose wastewater-fed bioreactors (R13), the biomass granulation rate was signicantly lower (10% on day 255; 20% on day 300). 3.2. Methanogenic activity of biomass The hydrogenotrophic to aceticlastic SMA ratio was consistently higher in the low-temperature bioreactors and this phenomenon was more pronounced in the bioreactors treating glucose wastewater and, in particular the granular biomass (Table 1). Interestingly, the highest SMA ratio of 3.1 was observed in the nely crushed seed inoculum ( < 0.2 mm) tested at 15 C (Table 1). This was likely due to the increased solubility and accessibility of H2/ CO2 for methanogenic organisms normally present at the core of spherical mesophilic granules (Kotsyurbenko 2005; Satoh et al., 2007).

3.3. DGGE analyses of archaeal and bacterial communities DGGE proles and the NMS analysis of their binary matrices revealed a replicated archaeal community structure between the glucose-fed low-temperature bioreactors R1 and 2. A similar trend was also observed in the low-temperature VFA-fed R4 and 5 through out the trial (Figs. 1a and 2a). Notably, post the development of granular biomass in R1 and 2 (day 255) no changes in community structure in either suspended or granular biomass was observed for the trial duration (Table 1). The R3 (mesophilic glucose fed) community structure changed most between days 120 and 255, while the proles from day 255 and 300, both granular and suspended biomass grouped closely together. Through out the trial the R6 (mesophilic VFA-fed), proles clustered closely together. Additionally, the granular and suspended communities from day 300 were identical to each other, and interestingly, also to the seed sludge prole. Signicantly, no differences between the granular and suspended communities were observed in any bioreactors at any time points assayed, except the R6 day 255 prole (Fig. 2a). A total of ve bands (DA15) were retrieved from the archaeal DGGE gel and several ribotypes were identied (Fig. 1a and Table 2). DA1, dominant in R1 and 2 from day 255 onwards, was closely related to three Methanocorpusculum species (99.6% sequence similarity), M. parvum, M. bavaricum and M. labreanum. DA2 was only detected in R3 on day 300 and showed 98.2% similarity to Methanoculleus receptaculi. DA3, which appears in all lanes, and DA4 were assigned to Methanosaeta concilii with 100% and 99.6% similarities, respectively (Fig. 1a). DA5, detected in all samples except the day 255 and 300 in R1 and 2, exhibited 100% similarities to Methanobacterium foricium and Methanobacterium palustre (Table 2). Conse-

Fig. 1. Archaeal (A) and bacterial (B) denaturing gradient gel electrophoresis image: S, suspended biomass; G, granular biomass.

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Fig. 2. Archaeal (A) and bacterial (B) non-metric multidimensional scaling: S, suspended biomass; G, granular biomass.

Table 2 Phylogenetic afliations of the archaeal DGGE band sequences. Band DA 1 Nearest species or taxon Methanocorpusculum parvum Methanocorpusculum labreanum Methanocorpusculum bavaricum Methanoculleus receptaculi Methanosaeta concilii Methanosaeta concilii Methanobacterium formicicum Methanobacterium palustre Accession No. AY260435 AF095267 AY196676 DQ787475 X16932 X16932 AF169245 AF093061 % Similarity 99.6 99.6 99.6 98.2 100 99.6 100 100 Phylogenetic group (order/family) Methanomicrobiales/Methanocorpusculaceae

DA DA DA DA

2 3 4 5

Methanomicrobiales/Methanomicrobiaceae Methanosarcinales/Methanosaetaceae Methanosarcinales/Methanosaetaceae Methanobacteriales/Methanobacteriaceae

quently, three out of the ve deduced sequences were afliated with hydrogenotrophs (DA1, 2 and 5) and the remaining two with aceticlastic methanogens (DA3 and 4). The bacterial DGGE gels contained more complex banding patterns with greater variations compared to the archaeal prole (Fig. 1b). The NMS statistical analysis highlighted that, the bacterial communities from the glucose wastewater- and VFA wastewaterfed bioreactors mostly grouped separately, similar to the archaeal analysis (Fig. 2b). Unlike the archaeal results, in most samples considerable community variations were observed between the replicate bioreactors, and also between the granular and suspended biomass proles. This indicates that bacterial community structure was signicantly more complicated and changeable than archaeal community structure in all bioreactors. It has previously been suggested in several studies that the methanogenic community remains stable over time, in terms of population diversity, while the bacterial community is highly variable (Akarsubasi et al., 2005; Zumstein et al., 2000). A total of 21 bacterial DGGE bands (DB121) were further sequenced for phylogenetic afliation (Table 3). Fourteen of them were assigned to three phyla Bacteriodetes (7), Proteobacteria (5), and Firmicutes (2), which are prominent in anaerobic treatment

systems. Only seven of the deduced sequences were closely related (>97% sequence similarity) to previously characterised organisms, which could be useful to infer functional characteristics. The majority were closely related to yet uncharacterised environmental clones. Additionally, ve of the remaining sequences (DB10, 12, 13, 16 and 20) displayed <97% similarities to any database sequence, indicating the likely presence of novel populations in our bioreactors. 3.4. Quantitative community dynamics of methanogens The real-time PCR results demonstrated signicant quantitative changes in the methanogenic community composition throughout the trial (Fig. 3). In terms of 16S rRNA gene concentration, the aceticlastic family Methanosaetaceae was dominant accounting for >98% of the total measured methanogenic 16S rRNA gene concentration (TMC; as detected with the primer and probe sets used) in the seed inoculum. This nding is in agreement with previous reports on the high abundance of Methanosaetaceae populations in stable anaerobic granular systems (Diaz et al., 2006; Satoh et al., 2007). In the glucose wastewater-fed bioreactors (R13), the suspended Methanosaetaceae populations decreased by up to 33-fold

J. OReilly et al. / Bioresource Technology 101 (2010) 63366344 Table 3 Phylogenetic afliations of the bacterial DGGE band sequences. Band DB 1 DB 2 DB 3 DB 4 DB 5 BD 6 DB 7 DB 8 DB DB DB DB DB 9 10 11 12 13 Nearest taxon Paludibacter propionicigenes WB4 Uncultured bacterium PeM47 Uncultured bacterium 2E12_cons Uncultured bacterium 99 Uncultured bacterium PL-7B5 Uncultured bacterium A4 Uncultured bacterium A_4 Bacteriodes sp. XDT-1 Uncultured bacterium 29d03 Uncultured bacterium TSBZ10 Uncultured bacterium LS4227 Uncultured bacterium M79 Uncultured bacterium R1B-4 Geobacter sp. TMJ1 Uncultured bacterium i9-104 Uncultured bacterium R4b16 Smithella propionica LYP Pelobacter propionicus OttBd1 Geobacter chapelleii Desulfovibrio carbinoliphilus Desulfovibrio sp. zt10e Uncultured bacterium C144 Clostridium botulinum Eklund 202F Clostridium puniceum DSM 2619 Syntrophomonas zehnderi OL-4 Syntrophomonas sp. TB-6 Uncultured bacterium N09 Salana multivorans Uncultured bacterium 6F6_cons Uncultured bacterium SR_EBR_L1 Accession no. AB078842 AJ576410 EF688167 FJ535039 AY570638 AY540495 DQ080179 AB363973 EF515505 AB186818 AB234247 AY692052 FJ167441 EU711072 DQ383313 AF482441 AF126282 X70954 U41561 DQ186200 AF109469 FJ466245 X68171 X71857 DQ898277 AB098336 AB195906 AJ400672 EF688256 AY340834 % Similarity 98.2 97.5 100 100 99.1 99.1 99.1 99.8 100 98.2 98.2 99.5 92.5 99.3 95.0 95.2 95.0 100 99.3 98.2 98.2 88.2 99.1 98.6 97.9 96.8 100 95.9 96.1 100

6341

Phylogenetic group (phylum/order) Bacteroidetes/Bacteroidales Candidate division TM7 Bacteroidetes/Bacteroidales Bacteroidetes/unclassied Bacteroidetes/unclassied Bacteroidetes/Bacteroidales Bacteroidetes/Bacteroidales Bacteroidetes/unclassied Unclassied bacterium Unclassied bacterium Proteobacteria (d)/Desulfuromonadales Proteobacteria (d)/Syntrophobacterales Proteobacteria (d)/Syntrophobacterales Proteobacteria (d)/Desulfuromonadales Proteobacteria (d)/Desulfovibrionales Unclassied bacterium Firmicutes/Clostridiales Firmicutes/Clostridiales Actinobacteridae/Actinomycetales Actinobacteridae/Actinomycetales Nitrospirae/Nitrospirales

DB 14 DB 15 DB 16 DB 17 DB 18 DB 19 DB 20 DB 21

Fig. 3. Quantitative changes in the 16S rRNA gene concentrations of methanogens.

in R13 after the initial operational period. At the trial completion the concentrations of Methanosaetaceae (suspended biomass) in R1

and 2 had increased to approximately 1.13.6 1010 copies g VS1. Granular biomass was detected in R13 on day 255. Its Methano-

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saetaceae concentration varied over time in R1 and 2 while it was maintained at around 1 1011 copies g VS1 for the trial duration in R3. On the other hand, in R4 and 5, the suspended Methanosaetaceae population gradually declined to 1.32.2 1010 copies g VS1 with no signicant uctuations. In R6, it decreased 5fold by day 120 and remained at approximately 1 1011 copies g VS1 thereafter. A similar concentration was observed in the R6 granular biomass from day 255 onwards (Fig. 3). A second aceticlastic family, Methanosarcinaceae, was detected only in the suspended biomass samples from R5 and 6, at relatively low concentrations of 2.76.7 108 copies g VS1, at the end of Phase 3 (day 300). Notably, this phase was characterised by the doubling of the OLR to 4 kg COD m3 d1 (Table 1), which likely resulted in an increased residual acetate concentration favoured by Methanosarcinaceae populations (Boone et al., 2001). The populations of Methanobacteriales in the suspended biomass increased signicantly in all bioreactors by 1.57.5-fold during the rst 120 days. After which, it was roughly maintained within 1091010 copies g VS1 in all bioreactors. Notably where granular biomass was detected, a higher concentration of Methanobacteriales was observed in the granular relative to the suspended biomass (Fig. 3). It remained fairly constant, except for a decrease on day 300 in R1 and 2 to 1.0 1010 copies g VS1 and a rise on day 300 in R6 to 1.3 1011 copies g VS1. The hydrogen utilising methanogenic order Methanomicrobiales, was present in the seed biomass at a concentration of 5.8 109 copies g VS1. Its population in the suspended biomass remained at or under the initial concentration throughout the trial in R36, except at the trial conclusion where concentrations of 2.0 1010 and 4.0 1010 copies g VS1, were observed in R3 and 6, respectively (Fig. 3). By contrast, the increase and dominance of Methanomicrobiales, in the low-temperature, glucose wastewater fed, R1 and 2 coincided with the detection of granular biomass. In both bioreactors, its 16S rRNA gene concentration in the suspended biomass increased up to 3.65.2 1011 copies g VS1 on day 255, which is >1700-fold higher than its minimum concentration on day 120 (Fig. 3). The high abundance of Methanomicrobiales in R1 and 2 was also observed in the granular proles, with concentrations of 3.0 1010 to 4.0 1011 copies g VS1, from day 255 onwards. Notably, it was one of the least abundant methanogenic groups (<9.6 109 copies g VS1) detected in the granular biomass of the mesophilically operated R3 and 6. These ndings, together with the granulation assays (Table 1), suggest that Methanomicrobiales populations may have been signicantly involved in the mechanism of psychrophilic biomass granulation in R1 and 2 (Fig. 3). Among the ve target methanogenic groups, the order Methanococcales was not detected in our experimental trial. This is likely due to the requirement of high salt concentration for growth (0.39.4% (w/v) NaCl) (Boone et al., 2001).

4. Discussion The replicate low-temperature bioreactors, glucose-fed R12 and VFA-fed R45, displayed similar performance levels (Table 1), and the methanogenic community composition was qualitatively and quantitatively comparable between the replicates. These results imply that the low-temperature bioreactors were reproducible in terms of methanogenic community dynamics as well as system performance. High COD removal efciency of 7784% was observed in all bioreactors during Phase 1, with no meaningful difference according to the substrate treated or the operational temperature. In the VFA wastewater trial, biomass granulation was observed only in the mesophilic R6. In contrast, in the glucose trial, granulation was observed in all three bioreactors during Phase 2

and, notably, the mesophilic R3 had a 1.62.6-fold higher granulation rate than the low-temperature R1 and R2. Phase 2 was characterised by the 3-fold decrease in HRT to 12 h, which resulted in a considerable drop in the COD removal efciency of all bioreactors. Interestingly, the COD standard deviations decreased by almost half indicating less variable COD removal (Table 1). The combination of reducing the HRT while maintaining the same OLR and up-ow velocity, appeared to be fundamental in selecting for a psychrophilic granular community. It likely favoured species with higher growth rates. An exception was in R6, where it is likely the simple VFA constituents were easily degraded under mesophilic conditions. The data indicates that both wastewater and bioreactor operating temperature/parameters had a major effect on biomass granulation. Previously reports have indicated that exocellular polysaccharides (ECP) are important for granulation. It is proposed they aid initial microbial adhesion and maintain the architectural integrity of granules by hydrating the granule surface, protecting against excessive shear forces (Diaz et al., 2006; Schmidt and Ahring, 1996; Tay et al., 2000). Higher levels of ECP are typically generated through acidogenesis of carbohydrates, rather than from acetogenesis and methanogenesis of VFAs (Liu et al., 2003). Therefore, the poor granulation in R46 may be due to the absence of carbohydrates in the VFA wastewater. Additionally, the composition of the synthetic VFA wastewater (acetate:propionate:butyrate:ethanol = 1:1:1:1 in COD equivalent) may be unfavourable for granulation compared to the composition of the acidogenic intermediates produced from the decomposition of a glucose-based wastewater. The NMS analysis of the DGGE proles demonstrated that the archaeal community was, in terms of diversity, much simpler and less varied than the bacterial community. Notably, after the development of granular biomass (day 255), the archaeal diversity in granular and suspended biomass from R12 remained identical throughout the trial duration (Figs. 1a and 2a). This indicates that the archaeal community in R1 and R2 was highly stable after the formation of granules. Furthermore, in the R13 DGGE proles the emergence of DA1 coincided with the observation of granular biomass (Fig. 1a). DA1 is afliated with Methanocorpusculum-like organisms and was the only species in R1 and R2 belonging to the order Methanomicrobiales. This corresponded well to the quantitative assays, which revealed the numerical dominance of Methanomicrobiales during the corresponding period in R1 and R2 (Fig. 3). However, due to the limitations of DGGE, which is prone to overlook numerically minor populations in mixed microbial communities (Sanz and Kochling 2007; Talbot et al., 2008), no Methanomicrobiales-related bands were detected from R46, despite the detection of this order by qPCR throughout the trial (Fig. 3). Similarly, the Methanobacteriales order were detected by qPCR at each sampling point, but a corresponding ribotype was not detected in R13 DGGE proles on days 255 and 300. The family Methanosaetaceae varied greatly according to both operating temperature and wastewater composition (Fig. 3). Methanosaetaceae is known as a dominant and vital methanogenic group in mesophilic anaerobic granulation (Diaz et al., 2006; Hulshoff Pol et al., 2004). In this study, we also observed that this group composed a signicant part of both granular and suspended methanogenic communities (rst or second most abundant group) during the period of biomass granulation. R3 and 6 had identical operating conditions, except for wastewater composition (Table 1). However, despite similar concentrations of Methanosaetaceae in both granular and suspended biomass, the granulation rate was in excess of three-times greater in R3 at the trial completion. It appears wastewater composition and resultant bacterial community dynamics have a profound effect on Methanosaetaceae developing into granular biolms, even at a mesophilic temperature of 37 C (Fig. 3).

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In contrast to Methanosaetaceae, the aceticlastic family Methanosarcinaceae was not detected in any granular/suspended biomass sample, except from R5 and 6 at the end of Phase 3 (Fig. 3). This operational period was characterised by the doubling of the OLR to 4 kg COD m3 d1 (Table 1). The members of this family are typically favoured in substrate rich environments high in organic acids, particularly acetate >300 mg l1 (Boone et al., 2001). Several previous studies have linked Methanosarcinaceae-related populations to high residual acetate concentrations often associated with poor COD removal (Hulshoff Pol et al., 2004; OReilly et al., 2009b). The limited detection of this family, in R5 and 6 was likely due to the increased OLR in Phase 3, directly increasing inuent acetate concentrations. Although the low-temperature bioreactors were operated at 15 C for 300 days, all DGGE sequences were assigned and related mainly to organisms, known to grow in and/or retrieved from mesophilic environments (Tables 2 and 3). A number of authors have also highlighted a similar trend, indicating that the emergence of psychrophilic organisms does not appear to be necessary for successful low-temperature anaerobic biotreatment. The majority of mesophilic granular sludges appear to have pre-existing populations that are highly psychrotolerant (McKeown et al., 2009; OReilly et al., 2009a). However, several studies have reported the emergence and/or increased abundance of species within the mesophilic consortia. Most notably is the apparent increase in Methanocorpusculum species as mesophilic granular sludge adapt to lower temperatures (Collins et al., 2003; OReilly et al., 2009b). This study also highlighted a similar trend. Both the DGGE and real-time PCR analyses demonstrated that the order Methanomicrobiales varied considerably as granulation occurred in R1 and 2 (Fig. 3). In terms of the 16S rRNA gene concentration, this hydrogenotrophic order was the dominant group in both suspended and granular methanogenic communities (38.696.6% of TMC) during the period of granulation from day 255 onwards in R1 and R2. Interestingly, it was the least abundant methanogenic group measured as granulation occurred in R3. Our results suggest that Methanomicrobiales may be a key microbial group in the formation of granular biolms in LTAD. Although DGGE is not robustly quantitative, the emergence and high band intensity of DA1 (Methanocorpusculum-like species), the only Methanomicrobiales-related ribotype, in R1 and R2 (Fig. 1a) is in agreement with the quantitative results and supports the potential importance of Methanomicrobiales in psychrophilic biomass granulation. Physiological analysis of the biomass highlighted that the hydrogenotrophic to aceticlastic SMA ratio (HAR) was, as expected, signicantly higher in R1 and R2 than in R3, and was more pronounced in the granular biomass (Table 1). On the other hand, the VFA-fed bioreactors with poor granulation (R46) showed no considerable differences in HAR between different temperatures or between the suspended and granular biomass. This further suggests the importance of hydrogenotrophic methanogens in aspects of both performance and granulation in LTAD. It has previously been reported that hydrogen is metabolically and thermodynamically more favourable for methanogenesis than acetate at lowtemperatures. Further, higher levels of hydrogen are retained due to increased gas solubility at low-temperatures (Kotsyurbenko, 2005; Lettinga et al., 2001).

conditions. In this study, Methanomicrobiales and, in particular, Methanocorpusculum species appeared to play a key role in the psychrophilic granulation process. Future work will focus on the operational requirements to facilitate enhanced psychrophilic granulation, with a view towards more systematic operational control of ambient or low-temperature anaerobic digestion. Acknowledgements This publication emanates from research conducted with the nancial support of Science Foundation Ireland. References
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