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1406 © IWA Publishing 2011 Water Science & Technology | 64.7 | 2011

Comparative 16S rRNA gene surveys of granular sludge from three upflow anaerobic bioreactors treating purified terephthalic acid (PTA) wastewater S. D. Perkins, N. B. Scalfone and L. T. Angenent

ABSTRACT

The microbial communities from three upflow anaerobic bioreactors treating purified terephthalic S. D. Perkins Department of Energy, acid (PTA) wastewater were characterized with 16S ribosomal RNA gene sequencing surveys. Environment and Chemical Engineering, Washington University in St. Louis, Universal bacterial and archaeal primers were used to compare the bioreactor communities to each St. Louis, MO 63130, USA other. A total of 1,733 bacterial sequences and 383 archaeal sequences were characterized. The high Syntrophus Pelotomaculum fi N. B. Scalfone number of spp. and spp. found within these reactors indicates ef cient L. T. Angenent (corresponding author) removal of benzoate and terephthalate. Under anaerobic conditions benzoate can be degraded Biological and Environmental Engineering, Cornell University, through syntrophic associations between these bacteria and hydrogen-scavenging microbes, such as 214 Riley-Robb Hall, Ithaca, NY 14853, Desulfovibrio spp. and hydrogenotrophic methanogens, which remove H2 to force the USA E-mail: [email protected] thermodynamically unfavourable reactions to take place. The authors did not observe a relatively high percentage of hydrogenotrophic methanogens with the archaeal gene survey because of a high acetate flux (acetate is a main component in PTA wastewater and is the main degradation product of terephthalate/benzoate fermentation), and because of the presence of Desulfovibrio spp. (a sulfate reducer that scavenges hydrogen). The high acetate flux also explains the high percentage of acetoclastic methanogens from the genus Methanosaeta among the archaeal sequences. A group of uncultured bacteria (OD1) may be involved in the degradation of p-toluate (4-methyl benzoate), which is a component of PTA wastewater. Key words | anaerobic digestion, methanogens, p-toluate, PTA wastewater, 16S rRNA gene survey, terephthalate

INTRODUCTION

Purified terephthalic (p-phthalic or 1,4-benzenedicar- which can be accomplished in upflow bioreactors, such as boxylic) acid (PTA) wastewater has been successfully upflow anaerobic sludge blanket (UASB) reactors and its off- treated in anaerobic digesters at chemical companies that shoots, with granular biomass to sustain long residence produce polyethylene terephthalate to manufacture, for times (Kleerebezem et al. ). But even after a sustainable example, plastic bottles for the beverage industry (Qiu period of terephthalate degradation, high concentrations of et al. ; Kleerebezem et al. b, ). This wastewater acetate and benzoate (itself the product of terephthalate contains terephthalate, acetate, benzoate, and p-toluate degradation) can disrupt this (Kleerebezem et al. b). (4-methyl-benzoate) among other chemicals (Zhang et al. These authors also discussed that the same bacterium may ). Even though these chemicals are not inhibiting the be degrading both terephthalate and benzoate. p-Toluate methanogenic biomass at the concentrations observed in can be removed, albeit at even lower rates than terephthal- PTA wastewater, terephthalate and p-toluate are not ate in methanogenic environments, which is why some always degraded in anaerobic upflow bioreactors, while have suggested the use of aerobic post-treatment to remove the biodegradation of acetate and benzoate is relatively p-toluate (Zhang et al. ). In high-rate anaerobic bio- easy (Kleerebezem et al. ). Terephthalate under anaero- reactors, such as UASB reactors or expanded sludge bed bic conditions is degraded at low rates after long lag periods, reactors (EGSBs), the high concentration of granular doi: 10.2166/wst.2011.552 1407 S. D. Perkins et al. | Comparative 16s rRNA gene surveys of granular sludge from anaerobic bioreactors Water Science & Technology | 64.7 | 2011

biomass and long cell residence times may overcome the consists of a tank with an internal diameter of 13 m and slow removal rates of p-toluate. Here, we surveyed three aheightof15m(∼2,000 m3 wet volume). This reactor granular samples from upflow anaerobic bioreactors treating was started ∼4 years prior to sampling with a sCOD PTA wastewater to characterize their microbial commu- removal efficiency of ∼60%. This relatively low sCOD nities with nonculturing methods. We used Sanger removal efficiency indicates that the most difficult to be sequencing to sequence near full-length sequences of 16S removed chemicals, such as p-toluate, were not degraded ribosomal RNA (rRNA) genes that were amplified with bac- in the anaerobic system. terial or archaeal specific PCR primers. Our goal was to compare the communities of the upflow bioreactors with DNA extraction and PCR amplification different operating conditions and performance, especially in regard to the removal of p-toluate. Received samples were immediately stored at –80 W C until further processing. Granules were crushed using a sterile spa- tula (Corning 3003, Corning, NY) before 0.5 ml of sample METHODS was submitted to a bead-beating and phenol:chloroform extraction protocol. The 16S rRNA genes from bacteria Granular samples were amplified with a 30-cycle PCR (initial 2 min denaturing step at 94 W C, followed by 30 cycles at 92 W C for 30 s 45 W C We surveyed granular samples from two lab-scale UASB for 90 s, 72 W C for 90 s, and a final 72 W C extension for reactors (samples U1 and U2, received 17 June 2008); 15 min). The 50-μl solution contained 1.25 units of GoTaq and from a full-scale expanded granular sludge bed (Promega Corp, Madison, WI), 0.4 pmol/μl forward and (EGSB)-Biobed reactor (E1, received 5 August 2008). The reverse primers (8F-50 AGAGTTTGATCCTGGCTCAG-30; UASB reactors had an internal diameter of 0.10 m with a 1391R-50 GACGGGCGGTGWGTRCA-30), 0.5 mmol/L height of 1.2 m (∼9 L wet volume). U1 was operated with MgCl2, 0.2 mmol/L dNTPs, 0.8 mg/mL BSA, and 2-μlof an upflow velocity of 1.9 m/h, a temperature of 38 W C, template. Archaeal 16S rRNA genes were amplified with a and an average pH of 7.2 during a three-month period 30-cycle PCR (initial 2 min denaturing step at 94 W C, followed before sampling. This reactor had been operating for a by 30 cycles at 94 W C for 60 s, 55 W C for 60 s, 72 W C for 60 s, and period of 15 months after inoculation with granular a final 72 W C extension for 10 min). A similar PCR solution sludge from upflow anaerobic bioreactors located at a brew- was used with different primers: 21F-50 AAGGAGGTGATC ery (70% v/v) and a papermill (8%), and from U2 (22%). A CAGCC-30; 1492R-50 GGTTACCTTGTTACGACTT-30. Con- synthetic PTA wastewater was fed to U1 at a loading rate of trol positive and negative reactions were included with 42.6 g COD/L/day, a hydraulic retention time (HRT) of 14 each reaction set. h, and a soluble COD (sCOD) removal efficiency of 98.7%. U2 was operated with an upflow velocity of 1.7 m/h, a Cloning and sequence analysis temperature of 38 W C, and an average pH of 7.2 during a three-month period. This reactor had been operating for PCR products were cleaned and concentrated (Montage a period of 21 months after inoculation with granular PCR cleaning kit, UFC7PCR50, Millipore, Billerica, MA) sludge from upflow anaerobic bioreactors at two breweries and sent to the Genome Sequencing Center, Washington and a petrochemical company. A synthetic PTA wastewater University School of Medicine in St. Louis, for cloning, puri- was fed to U2 at a loading rate of 27.4 g COD/L/day, a fication, and Sanger sequencing on an ABI 3730 capillary HRT of 12 h, and a sCOD removal efficiency of 99.0%. sequencer (Applied Biosystems, Foster City, CA). Three The difference between U1 and U2 was the composition independent PCR amplicon materials were sequenced per of the PTA wastewater, which mimicked two different pro- sample for bacteria and one for Archaea. 16S rRNA gene cessing plants. The high sCOD removal efficiencies show sequences were edited and assembled into consensus that almost all chemicals (including p-toluate) in this type sequences using PHRED and PHRAP aided by XplorSeq of wastewater were completely removed under methano- (Frank ). Bases with a PHRAP quality score <20 were genic conditions and converted to biogas, and that the trimmed from the dataset before being aligned using the intermediate volatile fatty acids were maintained at very NAST online tool (DeSantis et al. b). Chimeras were low concentrations in the effluent. The full-scale E1 detected and removed using Bellerophon (Huber et al. system is located at a petrochemical industrial site and ). Nonchimeric sequences were compared to the 1408 S. D. Perkins et al. | Comparative 16s rRNA gene surveys of granular sludge from anaerobic bioreactors Water Science & Technology | 64.7 | 2011

greengenes public database and imported into ARB Nucleotide sequence accession numbers (Ludwig et al. ). A pair-wise identity of 97% or greater was used to bin sequences into ‘species’-level operational Near full-length bacterial 16S rRNA gene sequences were taxonomic units (OTUs). Distance matrices constructed in deposited in GenBank under accession numbers GQ181242- DOTUR were used to assess the number of OTUs and clus- GQ182974. Near full-length archaeal 16S rRNA gene ter them by pair-wise identity (percent ID) with a furthest- sequences were deposited in GenBank under accession num- neighbour algorithm and a precision of 0.01 (Schloss & bers GQ201538-GQ201920. Handelsman ). Assignment of the majority of bacterial sequences to phyla was based on their position after parsi- mony insertion to the ARB dendrogram in the greengenes RESULTS AND DISCUSSION database (DeSantis et al. a). Bacteria phyla classifi- cations were double-checked and a percent ID was Bacterial and archaeal sequences showed a diverse calculated for nonchimeric sequences with the Ribosomal granular community Database Project II (RDP) (Cole et al. ). However, for archaeal sequences only RDP was used to classify to the We obtained 1,733 near full-length nonchimeric bacterial family level. A phylogenetic tree model was found using 16S rRNA gene sequences for the three granular samples. jModeltest 0.1 and an Adaike Information Criterion to The phylum Proteobacteria was the dominant bacterial rank 88 candidate models with an objective scoring taxonomic phyla with the class of Deltaproteobacteria dom- method (Posada ). The best-fit model was used to inating the sequences from all three reactors (Figure 1(a)). In create a maximum likelihood phylogenetic tree with addition, the phyla Firmicutes and Chloroflexi were abun- PAUP (Wilgenbusch & Swofford ). Finally, UniFrac dant in all three reactors. Further analyses were performed was used to conduct a jackknife replication weighted and to bin sequences, to assess diversity, to compare commu- normalized test, replicated 100 times to compare the three nities, and to classify 16S rRNA gene sequences at the reactor environments by clustering them based upon phylo- species level. We found 538 unique OTUs for the 1,733 bac- genetic similarity between sample communities (Lozupone terial sequences (Figure 1(a)). Next, rarefaction curves et al. ). showed that the community from E1 was more diverse

Figure 1 | Comparison of bacterial 16S rRNA gene sequences for each upflow anaerobic bioreactor (U1, U2, and E1): (a) Phylum or class distribution (proteobacteria was divided into the classes alpha-, beta-, gamma-, and deltaproteobacteria); (b) Rarefaction curves, which represent diversity based on the number of OTUs; and (c) Cluster analysis (weighted and normalized UniFrac). Percentages indicate bootstrap from 100 jack-knifed subsamples. 1409 S. D. Perkins et al. | Comparative 16s rRNA gene surveys of granular sludge from anaerobic bioreactors Water Science & Technology | 64.7 | 2011

(steeper curve) than from U1 and U2 (Figure 1(b)), which accepted that low acetate concentrations allow for slow- makes sense because of the more complex nature of real growing Methanosaeta spp. to have a competitive advan- PTA wastewater (E1) compared to synthetic wastewater tage over Methanosarcina spp. (Jetten et al. ). Thus, (U1 and U2). All three curves also show that the granular the abundance of Methanosaetaceae found within these biomass samples were not fully characterized (no asymp- anaerobic digesters is indicative of a low acetate concen- totes; Figure 1(b)), or, in other words, more sequencing trationintheeffluent. would have resulted in additional unique OTUs. We used the UniFrac distance method to compare the three commu- Syntrophy between Syntrophus spp. and nities to each other by quantifying the taxonomical hydrogenotrophic methanogens or Desulfovibrio differences between samples, and found that U1 and U2 spp. explains benzoate degradation were more similar to each other than E1 (Figure 1(c)). This may have identified an effect of differences in reactor Almost 46% (804/1733) of the bacterial sequences in this operating conditions (upflow velocity), in wastewater con- study were from Syntrophus spp. This genus (in the class ditions (real versus synthetic PTA wastewater), and in of Deltaproteobacteria) is known to degrade fatty acids reactor performance (complete versus incomplete removal or aromatic acids with, for example, a hydrogenotrophic of chemicals) between the UASB and EGSB reactors on methanogen or Desulfovibrio spp. (a sulfate reducer that the community structure. In almost all bacterial phyla, clo- scavenges hydrogen) in a syntrophic partnership. Wu sely related OTUs were represented from all three et al.() had found 78% of 106 bacterial sequences reactors, which clearly shows a similar functioning commu- in granular biomass degrading terephthalate to be of this nity (data not shown). class, including several Syntrophus spp.-related sequences. We obtained 383 near full-length nonchimeric archaeal We performed maximum likelihood analysis within the 16S rRNA gene sequences. In all three reactors, more than phyla of Proteobacteria, and found that the majority of 90% of the archaeal sequences were from the family Metha- Syntrophus spp. sequences for U1 and U2 matched nosaetaceae, while the rest of the archaeal sequences were the pure culture strain S. buswellii, while for E1 the only from three other methanogenic families (Table 1). Syntrophus spp. sequences matched environmental Wu et al.() had found more than 80% of 72 archaeal sequences. Mountfort et al.() isolated S. buswellii sequences in granular biomass degrading terephthalate to from anaerobic digester sludge and aquatic be closely affiliated with Methanosaeta spp.,which,there- sediments; their work characterized S. buswellii as a fi fore, veri es the relatively large percentage of this family benzoate catabolizer (C7H5O2 þ 7H2O ↔ 3H2 þ HCO3 þ þ W 0 in a similar type of reactor. In our study, maximum likeli- 3CH3COO þ 3H ; ΔG ¼þ59.6 kJ/mol (Kleerebezem hood analysis (data not shown) showed that the majority et al. a)). Anaerobic benzoate degradation is not of the members of the Methanosaetaceae family were clo- thermodynamically favourable unless syntrophic hydroge- sely related to a sequence from a cultured Methanosaeta notrophic microbes within the community scavenge the concilii, which is an acetoclastic methanogen (Boone hydrogen to keep the hydrogen partial pressure below et al. ). Acetate is an important constituent of PTA 10 3–10 5 bar (Auburger & Winter ). These authors wastewater and is also a product from benzoate degradation further characterized the syntrophic relationship between (3 mol of acetate per mol of benzoate), and this explains the S. buswellii and the hydrogenotrophs Methanospirillum abundance of these acetoclastic methanogens. It is well hungatei and Desulfovibrio spp. using benzoate. Our

Table 1 | RDP classification of archaeal sequences from reactors U1, U2, and E1

U1 U2 E1 Family

Methanobacteriaceae 4 2.8% 0 0.0% 1 0.5% Methanomicrobiaceae 0 0.0% 3 7.9% 0 0.0% Methanosaetaceae 134 93.1% 35 92.1% 195 97.0% Methanosarcinaceae 6 4.2% 0 0.0% 5 2.5% Total 144 38 201

A total of 383 sequences were classified to the family level. For each sample the amount of sequences is given and its relative presence. 1410 S. D. Perkins et al. | Comparative 16s rRNA gene surveys of granular sludge from anaerobic bioreactors Water Science & Technology | 64.7 | 2011

maximum likelihood distribution analyses matched Syntrophy between Pelotomaculum spp. and Desulfovibrio spp. to cultured sequences for all three hydrogenotrophic methanogens explains terephthalate reactors (Figure 2), and M. hungatei was identified in reac- degradation tor U2 (data not shown). Therefore, both Desulfovibrio spp. and the hydrogen-utilizing methanogens of the A study conducted by Qiu et al.() enriched and isolated families Methanobacteriaceae, Methanomicrobiaceae, terephthalate-degrading microbes from anaerobic sludge or Methanosarcinaceae were the likely synergistic partner from digesters treating PTA wastewater. One of the isolates of the Deltaproteobacteria in our study to explain benzoate (Pelotomaculum spp. in the Desulfotomaculum lineage I) removal. was able to degrade terephthalate (and also benzoate) in

Figure 2 | Maximum likelihood phylogenetic distribution of select 16S rRNA gene sequences from our study and closely related pure-culture sequences from the phylum Proteobacteria. Reference sequences from the NCBI database are included with their accession numbers. Individual sequences that represent one OTU are labelled with U1, U2, and E1 identifiers followed by the sample label and the unique sequence identity. The number of sequences in the OTU found in this study are in parenthesis. The scale bar represents base changes per site. 1411 S. D. Perkins et al. | Comparative 16s rRNA gene surveys of granular sludge from anaerobic bioreactors Water Science & Technology | 64.7 | 2011

Figure 3 | Maximum likelihood phylogenetic distribution of select 16S rRNA gene sequences from our study and closely related pure-culture sequences from the phyla Firmicutes. Reference sequences from the NCBI database are included with their accession numbers. Individual sequences that represent one OTU are labelled with U1, U2, and E1 identifiers followed by the sample label and the unique sequence identity. The number of sequences in the OTU found in this study are in parenthesis. The scale bar represents base changes per site. co-culture with the hydrogenotroph M. hungatei. Here, an uncultured candidate division recently split from the Pelotomaculum spp. were present in all reactor samples in uncultured candidate division OP11. Its members are relatively high numbers (Figure 3). Similarly to benzoate often found within marine and aquifer sediments (Harris degradation, terephthalate removal (C8H4O4 þ 8H2O ↔ et al. ), but their function is unknown. The important þ W 0 3H2 þ 2HCO3 þ 3CH3COO þ 3H ; ΔG ¼þ38.9 kJ/mol difference between U1/U2 and E1 was that the former reac- (Kleerebezem et al. a)) also requires a syntrophic part- tors were able to convert p-toluate, while the latter reactor ner to remove thermodynamic constraints. The presence missed this important characteristic. Therefore, members of hydrogenotrophic methanogens (including M. hungatei of OD1 may have been involved in p-toluate biodegradation. in U2) – the relatively low percentage of archaeal sequences However, further time-resolved studies with highly parallel (Table 1) does not exclude these methanogens as important sequencing efforts would be necessary to prove this hypoth- partners – explains the syntrophic removal of terephthalate esis. The enrichment or isolation of this group of bacteria from all our reactors. may be very difficult under laboratory conditions.

Other organisms found within the reactors play key roles in the overall community dynamics CONCLUSIONS

The phylum Chloroflexi was the third most abundant The community structure of three upflow anaerobic bio- phylum in the reactors (after Firmicutes and Proteobac- reactors with granular biomass explains their teria), and has been recognized as an important bioconversion of PTA wastewater into methane. A very component of mesophilic and thermophilic granular high level of syntrophic partnerships between benzoate- sludge. The filamentous web-like structures formed by and terephthalic-degrading bacteria and hydrogenotrophic these bacteria play an important role in maintaining the methanogens or Desulfovibrio spp. was found. The genera structural integrity of sludge granules (Yamada et al. ). Syntrophus and Pelotomaculum were the likely degraders It may, therefore, have an important function in these of benzoate and terephthalate. We have identified a possible upflow bioreactors. As mentioned earlier, many OTUs in group of uncultured bacteria that could be responsible for our study overlapped between the three reactors (Figures 2 p-toluate degradation, but further work is necessary to and 3), and almost all phyla included OTUs from all three explore this hypothesis. Finally, acetate was degraded by reactors. One distinct discrepancy to this was the phyla the acetoclastic methanogens in the genus Methanosaeta. OD1 found in reactors U1 and U2, but not in E1. OD1 is The relatively high percentage of these methanogens 1412 S. D. Perkins et al. | Comparative 16s rRNA gene surveys of granular sludge from anaerobic bioreactors Water Science & Technology | 64.7 | 2011

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First received 3 February 2011; accepted in revised form 10 February 2011