Community and Proteomic Analysis of Methanogenic Consortia Degrading Terephthalate

Abstract

c Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122-130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H 2 /CO 2 , and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.T erephthalate (TA) is produced in large quantities in plastic, textile, and petroleum-related industries, and wastewater generated by the TA manufacturing process is often treated with anaerobic methanogenic processes (1). In these processes, TA is converted to CH 4 and CO 2 through the microbial syntrophy established among the enriched microbial populations. It is hypothesized that the hydrogen-producing syntrophic bacteria degrade TA to acetate and H 2 /CO 2 , which are immediately used by the methanogenic archaea adjacent to final gaseous products (2, 3). To validate the syntrophic interaction, a full-cycle 16S rRNA approach was used to successfully identify the identities of the syntrophic bacteria and methanogenic archaea inside the granules and biofilms of different laboratory-scale TA-degrading methanogenic reactors (3, 4). A recent study further used a metagenomic approach to reveal the diversity and physiological traits of the syntrophs and methanogens involved in the TA degradation under moderately thermophilic conditions (46 to 50°C) (5). All of these studies have provided strong evidence to elucidate the degradation of TA based on the microbial populations and the metabolic pathways involved.Furthermore, to directly observe the global expression of microbial functions within the TA-degrading consortium, community-level proteomics can be used (6). To do so, total proteins recovered from an environmental sample are first separated and enzymatically fragmented. The resulting peptide mixture is analyzed using advanced liquid chromatography-mass spectrometry (LC-MS) technology, and information related to the key proteins is identified using in silico analysis of the protein sequence database. ...

Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor

“…These Pelotomaculum indeed encode and express genes for syntrophic energy conservation (Hdr-Ifo and ECHyd) and a previously observed pathway for TA degradation to acetate, CO 2 and H 2 (Figure 2 and Supplementary Table S4) (McInerney et al, 2007;Lykidis et al, 2011). In addition, we newly identify expression of a clostridial electron-bifurcating butyryl-CoA dehydrogenase in TAPelo3 that may facilitate the previously hypothesized Sporotomaculum-like energy-conserving butyrate generation from aromatic compound degradation (Qiu et al, 2003;Buckel and Thauer, 2013;Wu et al, 2013;Nobu et al, 2014), albeit refuting the involvement of previously identified non-energy-conserving acylCoA dehydrogenase (Lykidis et al, 2011;Wu et al, 2013). Although butyrate-fermenting TA degradation is thermodynamically more favorable, it sacrifices precious adenosine triphosphate from substrate-level phosphorylation.…”

“…Our recent metagenomic and proteomic studies proposed that Pelotomaculum may produce acetate, H 2 and butyrate as by-products and that uncultivated taxa (Caldiserica and 'Ca. Cloacimonetes', formerly OP5 and WWE1) may further metabolize H 2 and butyrate (Lykidis et al, 2011;Wu et al, 2013). This provided evidence that non-methanogen community members may serve as syntrophic partners (secondary degraders) to metabolize TA degradation by-products alongside aceticlastic and hydrogenotrophic methanogens.…”

“…Syntrophic TA degradation by Pelotomaculum and Syntrophorhabdus with methanogens Organisms of the genus Pelotomaculum and family Syntrophorhabdaceae are thought to be responsible for syntrophic TA degradation in reactor systems (Lykidis et al, 2011;Wu et al, 2013). We identify diverse Pelotomaculum (bins TAPelo1-TAPelo4) predominate the community, accounting for 16.8% and 31.7% of the bacterial community and metratranscriptome.…”

Application of Metagenomics in Environmental Anaerobic Technology

“…Later, the rapidly decreasing sequencing cost and the prevalence of NGS technologies other than 454 pyrosequencing allowed for more extensive applications of HTS-based metagenomics approaches, including 16S rRNA gene amplicons sequencing and metagenome for novel microbial insights into the anaerobic digestion processes operated for bioenergy production (e.g., methane, biohydrogen, bioethanol, fatty acids) and/or pollution control (e.g., refractory compounds, bacterial pathogens, antibiotics resistance genes). A few pioneering studies even applied metatranscriptome (Xia et al 2014;Zakrzewski et al 2012) or metaproteome (Lü et al 2013;Kohrs et al 2013;Hanreich et al 2013;Hanreich et al 2012;Abram et al 2011;Heyer et al 2013;Wu et al 2013) to gain some preliminary insights into the gene expression and active enzymes in anaerobic digestion processes.…”

Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

“…As shown in Table 1, granule samples 1 and 2 were collected from full-scale up-flow anaerobic sludge bed (UASB) reactors treating purified terephthalic acid (PTA) wastewater, in which acetate, benzoate, terephthalate, and p-toluate account for the major components in the wastewater stream (23). The terephthalate-degrading biofilm sample (sample 3) was taken from a thermophilic (50°C) fixed film reactor (24), and the p-toluate-degrading granule sample (sample 4) was obtained from a mesophilic (35°C) fed-batch reactor in the laboratory. Both reactors inoculated with sludge sample 2 were operated for at least 6 months.…”