The ISME Journal (2011) 5, 122–130 & 2011 International Society for Microbial Ecology All rights reserved 1751-7362/11 www.nature.com/ismej ORIGINAL ARTICLE Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium This paper was corrected on 21st December 2010 to include contributing authors inadvertently ommitted from the original version of the paper Athanasios Lykidis1, Chia-Lung Chen2, Susannah G Tringe1, Alice C McHardy3, Alex Copeland1, Nikos C Kyrpides1, Philip Hugenholtz1, Herve´ Macarie4, Alejandro Olmos5, Oscar Monroy5 and Wen-Tso Liu2,6 1Joint Genome Institute, Lawrence National Berkeley Laboratory, Walnut Creek, CA, USA; 2Division of Environmental Science and Engineering, National University of Singapore, Singapore; 3Computational Genomics & Epidemiology, Max-Planck Institute for Informatics, Saarbru¨cken, Germany; 4IRD, UMR IMEP, PRAM, Le Lamentin, France; 5Departamento de Biotecnologı´a, Universidad Auto´noma Metropolitana- Iztapalapa, Mexico and 6Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, IL, USA Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium inside a hyper-mesophilic (that is, between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified b-oxidation to H2/CO2 and acetate. These intermediates are converted to CH4/CO2 by three novel hyper-mesophilic methano- gens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to CO2/H2 and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H2-producing syntroph–methanogen partnership that may serve to improve community stability. The ISME Journal (2011) 5, 122–130; doi:10.1038/ismej.2010.125; published online 5 August 2010 Subject Category: integrated genomics and post-genomics approaches in microbial ecology Keywords: metagenomics; methanogenesis; syntroph; microbial diversity; carbon cycling Introduction operated at hyper-mesophilic (46–50 1C) and ther- mophilic (B55 1C) temperatures may be preferable Terephthalate (TA) is used as the raw material for because of the ability to achieve higher loading the manufacture of numerous plastic products (for rate (van Lier et al., 1997; Chen et al., 2004), example, polyethylene TA bottles and textile fibers). which reduces the reactor volume. Moreover, TA During its production, TA-containing wastewater is 1 3 wastewater is usually generated at 54–60 C, and discharged in large volumes (as high as 300 million m does not require additional energy input for per year) and high concentration (up to 20 kg COD À3 maintaining reactor temperature (Chen et al., (chemical oxygen demand) m ) (Razo-Flores et al., 2004). The microbial biomass usually occurs in the 2006). This wastewater is generally treated by form of granules or biofilms attaching on the surface anaerobic biological processes under mesophilic of porous media. Under such environments, TA B 1 conditions ( 35 C). However, anaerobic processes degradation has been hypothesized (Kleerebezem et al., 1999) to be based on a syntrophic microbial Correspondence: W-T Liu, Department of Civil and Environmental relationship whereby fermentative H2-producing Engineering, University of Illinois at Urbana-Champaign, 3207 bacteria (syntrophs) convert TA through benzoate Newmark Civil Engineering Laboratory, MC-250, 205 North to acetate and H2/CO2, and acetoclastic and hydro- Mathews Avenue, Urbana, IL 61801, USA. E-mail: [email protected] genotrophic methanogens further convert the Received 7 April 2010; revised 14 June 2010; accepted 16 June intermediates to methane by physically positioning 2010; published online 5 August 2010 themselves close to the syntrophs to overcome the Metagenomics of a methanogenic consortium A Lykidis et al 123 thermodynamic barrier (Stams, 1994; Conrad, 1999; 2004) (see Supplementary Text). Biomass was Dolfing, 2001). collected from only porous packing filters on days In practice, the complexities of TA-degrading 221 and 280, and from both filters and sludge bed on communities are not as well known. The commu- days 346 and 430 for further analyses. These nities require a long maturation phase (4200–300 biomass samples were used for genomic DNA days), are difficult to maintain, and do not always extraction, library construction and sequencing result in a successful syntrophic interaction. If the according to standard protocols (http://www.jgi. syntrophic interaction is disturbed and the treatment doe.gov/sequencing/protocols/prots_production.html) rendered ineffective, the resulting high-concentration (see Supplementary Text). Detailed metagenome effluent must be treated with a more energy-intensive analysis methods are described in the Supplemen- down-stream aerobic biological process. These factors tary Text. Data can be accessed through the can significantly increase the operational cost of the Integrated Microbial Genome/Microbiome (http:// process and limit its application on a wider scale. img.jgi.doe.gov/m/) system. Several studies have investigated the microbial populations present in methanogenic TA degradation, often using laboratory-scale reactors operated at Results and discussion various temperatures (Kleerebezem et al., 1999; Wu et al., 2001; Chen et al., 2004). Using ribosomal RNA Bioreactor operation and performance (rRNA)-based molecular methods, these studies have An anaerobic hybrid reactor was successfully oper- found that TA-degrading consortia in bioreactors are ated with TA as the only carbon and energy source for dominated by two to three bacterial populations and 480 days. This reactor was constructed with an upper two types of methanogens (Wu et al., 2001; Chen et al., section filled with ring-shape porous filters to support 2004). The methanogens are relatively straightforward the growth of microbial biofilms, and a lower section to classify being mainly acetoclastic Methanosaeta- for the development of anaerobic granular sludge related species and a novel hydrogenotrophic metha- (Figure 1a). This reactor is unusual and novel, in that nogenic species in the family Methanomicrobiales. it was the first methanogenic reactor operated in the The syntrophic bacteria, however, are difficult to hyper-mesophilic temperature zone (46–50 1C), identify based on phylogenetic classification, and whereas previously published studies of TA-degrad- extremely difficult to obtain in pure culture. In the ing communities were at mesophilic (B35 1C) or last decade, only three bacterial species that can thermophilic (B55 1C) temperatures (Kleerebezem degrade TA and its isomers have been successfully et al., 1999; Wu et al., 2001; Chen et al., 2004). After co-cultured with methanogens under mesophilic achieving a good TA removal efficiency (Figure 1b), conditions (Qiu et al., 2006, 2008), and these isolates sludge samples were taken from the surface of the are different from those found under thermophilic filter media at days 221, 280, 346 and 430. Samples conditions (Chen et al., 2004). This greatly limits the were also taken from the sludge bed at days 346 and effort to understand the microbial interaction and 430. These samples taken were used for 16S rRNA function in the TA-degrading consortia. and metagenome analysis. A metagenome analysis was chosen for this study since it has been proven as an effective method for retrieving nearly complete microbial genomes of 16S rRNA-based community profiling dominant populations in relatively simple microbial The rarefaction curve analysis indicates that the ecosystems (Tyson et al., 2004; Martin et al., 2006). bacterial population diversity is much higher in terms In particular, this study aims to elucidate the of operational taxonomic unit number than the microbiology underpinning anaerobic TA-degrading archaeal population diversity in any given sample processes, including improved knowledge of the taken from the TA reactor (Supplementary Figure 1). diversity and physiology of participating syntrophs The phylogenetic distribution of bacterial 16S rRNA and methanogens, and the mechanism behind the clones (Figure 2) indicates that Peptococcaceaea establishment and maintenance of the partnership. (mostly Pelotomaculum), Thermotogae, Syntropha- This knowledge may lead to the generation of ceae, and candidate phyla OP5 and WWE1 were the principles, which could be applied to establish dominant bacterial lineages present. Between the different consortia for treating other chemicals biofilm samples (rings 1, 2, 3 and 5) and sludge bed discharged from industrial production lines, or to samples (rings 4 and 6) taken, differences in the treat contaminants in other environments. abundances of major phyla including Firmicutes, Thermotogae, Proteobacteria and OP5 were observed. These differences are likely attributed to the differ- Materials and methods ences in growth temperature (46 1Cvs501C) and form (biofilms vs granules). Archaeal representatives