Elsevier

Bioresource Technology

Volume 172, November 2014, Pages 83-90
Bioresource Technology

Comparison of metabolomic profiles of microbial communities between stable and deteriorated methanogenic processes

https://doi.org/10.1016/j.biortech.2014.08.054Get rights and content

Highlights

  • Stable and deteriorated methanogenic processes were compared.

  • Intracellular metabolisms within complex microbial communities were shown.

  • Clear differences in the metabolites were observed in the processes.

  • Carbon flux in glycolysis and glutamate production were increased in stable process.

  • Pyruvate, acetyl-CoA, and succinate were accumulated in deteriorated process.

Abstract

Central metabolite profiles from glucose in microbial communities during methanogenic process were compared between a stable methanogenic reactor (MR) and a deteriorated reactor (DR). The concentrations of intracellular metabolites related to the Embden–Meyerhof and pentose phosphate pathways, with the exception of pyruvate, remained high in the MR, showing increased carbon flux in the glycolysis pathway during stable methanogenesis. Extracellular acetate temporarily accumulated in the MR, consistent with higher ATP level in the MR. Intracellular concentrations of the intermediates in the reductive branch of tricarboxylic acid cycle, malate, fumarate, and succinate were higher in the DR. Low NADH/NAD+ ratio both in the MR and DR would suggest NADH consumption during acetate and lactate/succinate production in the MR and DR, respectively. Intracellular glutamate levels were higher in the MR, correlating with lower NADPH/NADP+ ratio concentrations in the MR. These findings contribute to a better understanding of the metabolic state during stable methanogenesis.

Introduction

Anaerobic digestion, such as methane fermentation, produces renewable biogas from wastes (Risberg et al., 2013, Weiland, 2010). Methane fermentation involves a complex series of metabolic reactions that can be divided into at least four phases, hydrolysis, acidogenesis, acetogenesis/dehydrogenation, and methanogenesis by anaerobic microorganisms (Angelidaki et al., 2011, Weiland, 2010). During hydrolysis and acidogenesis, organic compounds are broken down into CO2 and organic acids, which are further degraded by acetogenic bacteria into acetate, CO2, and hydrogen. These substrates are then preferentially utilized by methanogens to produce methane (Mountfort and Asher, 1978, Stams and Plugge, 2009).

Under the deteriorated methanogenic process, intermediates, organic acids such as acetate, butyrate, and propionate accumulate (Angelidaki et al., 2003). Small subunit ribosomal DNA sequence-based methods have been used to examine changes in microbial community structure during deterioration of the methanogenic process (Hori et al., 2006, Sasaki et al., 2010). These analyses have revealed such deterioration is associated with a decrease in the proportion of methanogenic archaea among the total microbial population. This imbalance between different microbial groups results in the accumulation of organic acids, because their degradation is coupled to hydrogen consumption by hydrogenotrophic methanogens and acetate consumption by aceticlastic methanogens (Hassan and Nelson, 2012, Sasaki et al., 2011). The reduced activity of these methanogens results in decreased methane gas production, leading to further deterioration of methanogenic activity. However, changes in the intracellular metabolites produced by microorganisms during this deterioration have not yet been clarified.

Metabolomics studies are useful for elucidating the intracellular pathways that produce certain metabolites within a single microbial species and have the potential to bridge the gap between phenotype and genotype. The fermentation of monosaccharides by microorganisms such as Escherichia coli and Saccharomyces cerevisiae (Zhang et al., 2010) has been used to produce biofuels and chemicals (Toya and Shimizu, 2013). Although microbial communities found in methanogenic reactor are composed of diverse assemblages of microorganisms, most species, particularly bacteria that contribute to hydrolysis, acidogenesis, and acetogenesis/dehydrogenation degrade monosaccharides through the Embden–Meyerhof (EM) and/or pentose phosphate (PP) pathways. In anaerobic conditions, monosaccharides frequently enter an incomplete tricarboxylic acid (TCA) cycle after glycolysis (Feng et al., 2009, Trotter et al., 2011). Metabolomics can be applied to the analysis of microbial communities to clarify the central metabolic processes that are involved in the production of organic acids from monosaccharides during methane fermentation under stable and deteriorated conditions. Although nuclear magnetic resonance and gas chromatography-mass spectrometry (GC–MS)-based metabolomics approaches have been used to examine the extracellular metabolic dynamics during the anaerobic fermentation of glucose and cellulose-based substrates (Date et al., 2012, Yang et al., 2014), these approaches cannot sensitively identify intracellular metabolites.

In the present study, GC–quadrupole-MS (GC-Q-MS) and liquid chromatography triple-stage quadrupole MS (LC-QqQ-MS) were employed to examine the methane fermentation process by focusing on a wide range of metabolites involved in the EM and PP pathways, and TCA cycle. Methane fermentation was conducted at a pH of approximately 7.5 using glucose as the major carbon source until stable conditions were achieved, and deterioration was then induced by decreasing the pH to approximately 5.0. The reactor performance and intracellular metabolite profiles of microorganisms were compared under the stable and deteriorated methanogenic conditions.

Section snippets

Seed sludge and feed material

The supernatant from a 20% aqueous solution (wt/vol) of manure collected from a Japanese black cattle farm in Iwate Prefecture, Japan, was used as seed sludge. Synthetic medium containing (per 1 L) 10 g glucose (1%, wt/vol), 1.0 g yeast extract, 2.0 g NaHCO3, 1.0 g NH4Cl, 0.1 g KH2PO4, 0.2 g K2HPO4, 0.1 g MgCl2·6H2O, 0.1 g CaCl2·6H2O, 0.6 g NaCl, 10 mL Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany) medium 141 trace element solution, and 10 mL DSMZ medium 141 vitamin solution, was

MR and DR performances

The performances of the MR and DR were compared at HRT of 10 days (Fig. 1). The pH in the MR was maintained at approximately 7.5 to promote stable methanogenesis from glucose. As expected, stable gas production and only small amounts of acetate, butyrate, and propionate were detected in the MR (Fig. 1b, d, e, and f). Deterioration of the methanogenic process was induced in the DR after 10 days at an HRT of 10 days by decreasing the pH from 7.5 to approximately 5.0 (Fig. 1a). This increase in

Conclusions

The metabolite profiles for EM and PP pathways and TCA cycle in methanogenic microbial communities degrading glucose were compared between stable methane fermentation conditions at pH 7.5 and deteriorated fermentation conditions at pH 5.0. It was suggested the importance of activating EM and PP pathways to supply NADH and NADPH, extracellular acetate production for producing ATP and consuming NADH and NADPH, and intracellular glutamate production for NADPH consumption for stable methane

Acknowledgements

We are grateful to Yasuko Koura and Ayami Fujino for their analytical support with the experiments. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.

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