Piezo-tolerant natural gas-producing microbes under accumulating p CO2

Abstract

Background: It is known that a part of natural gas is produced by biogenic degradation of organic matter, but the microbial pathways resulting in the formation of pressurized gas fields remain unknown. Autogeneration of biogas pressure of up to 20 bar has been shown to improve the quality of biogas to the level of biogenic natural gas as the fraction of CO₂ decreased. Still, the pCO₂ is higher compared to atmospheric digestion and this may affect the process in several ways. In this work, we investigated the effect of elevated pCO₂ of up to 0.5 MPa on Gibbs free energy, microbial community composition and substrate utilization kinetics in autogenerative high-pressure digestion.

Results: In this study, biogas pressure (up to 2.0 MPa) was batch-wise autogenerated for 268 days at 303 K in an 8-L bioreactor, resulting in a population dominated by archaeal Methanosaeta concilii, Methanobacterium formicicum and Mtb. beijingense and bacterial Kosmotoga-like (31% of total bacterial species), Propioniferax-like (25%) and Treponema-like (12%) species. Related microorganisms have also been detected in gas, oil and abandoned coal-bed reservoirs, where elevated pressure prevails. After 107 days autogeneration of biogas pressure up to 0.50 MPa of pCO₂, propionate accumulated whilst CH₄ formation declined. Alongside the Propioniferax-like organism, a putative propionate producer, increased in relative abundance in the period of propionate accumulation. Complementary experiments showed that specific propionate conversion rates decreased linearly from 30.3 mg g^-1 VS_added day^-1 by more than 90% to 2.2 mg g^-1 VS_added day^-1 after elevating pCO₂ from 0.10 to 0.50 MPa. Neither thermodynamic limitations, especially due to elevated pH₂, nor pH inhibition could sufficiently explain this phenomenon. The reduced propionate conversion could therefore be attributed to reversible CO₂-toxicity.

Conclusions: The results of this study suggest a generic role of the detected bacterial and archaeal species in biogenic methane formation at elevated pressure. The propionate conversion rate and subsequent methane production rate were inhibited by up to 90% by the accumulating pCO₂ up to 0.5 MPa in the pressure reactor, which opens opportunities for steering carboxylate production using reversible CO₂-toxicity in mixed-culture microbial electrosynthesis and fermentation.Graphical abstractThe role of pCO₂ in steering product formation in autogenerative high pressure digestion.

Keywords: Autogenerative high-pressure digestion; CO2-toxicity; Carboxylate platform; Gibbs free energy; Population dynamics; Propionate accumulation; Syntrophy.

Graphical abstract

The role of pCO₂ in steering product formation in autogenerative high pressure digestion

Fig. 1

Overview of experimental design

Fig. 2

Results of fed-batch reactor operation. a Pressure and pH, b measured pCH₄, measured pCO₂ and calculated pCO₂, c acetate and propionate; downward arrow indicates H₂ addition; P1–P6 indicate operational periods as described in Table 1

Fig. 3

FESEM micrographs from representative reactor samples. Rod (A), and filamentous (B) shaped (left) and coccus (C), spiral-shaped (D) organisms (middle). Smooth and tubular pore (E) cell surfaces are magnified on the right

Fig. 4

Archaeal and bacterial DGGE profiles and heat maps. Archaeal (a) and bacterial (c) DGGE profiles and heat maps of the relative intensities of major archaeal (b) and bacterial (d) DGGE bands. Numbered bands in a indicate the positions identical to the migration of clone samples closely related to (1–3) Methanosaeta concilii, (4) Methanobacterium formicicum, (5) Methanoregula boonei and/or Methanosarcina acetivorans, and (6) Methanoregula boonei and/or Methanobacterium formicicum. Numbered bands in b indicate the positions identical to the migration of clone samples closely related to (1) Brachymonas denitrificans and Tessaracoccus (2) Propionibacteriaceae, (3) Treponema, (4) Bacteroidales, (5) Bacteroidales and Victivallis, (6) Succiniclasticum, (7) Propioniferax, (8) Petrimonas, (9) Synergistaceae, Brachymonas denitrificans and Tessaracoccus, (10) Kosmotoga, (11) Clostridium quinii and Clostridia, and (12) Syntrophobacter fumaroxidans. Each band in c and d is labelled with the clone(s) with an identical migration pattern, followed in parentheses by the affiliation of the clone determined by Ribosomal Database Project classifier. Numbers indicate ratio (%) over the sum of band intensities of each sample (i.e., each lane in DGGE). P1–P6 and II, IV indicate operational periods and experiments described in Table 1

Fig. 5

Neighbour-joining tree illustrating the phylogenetic identities of archaeal communities in the pressure bioreactor. The archaeal 16S rRNA gene fragments were obtained from clone samples. Clone counts of each OTU are given in brackets; the first and the second numbers indicate the counts derived from samples A and L, respectively. Numbers at nodes are bootstrap values derived from 100 analyses. The scale bar represents an amount of nucleotide sequence change of 0.02

Fig. 6

Neighbour-joining tree illustrating the phylogenetic identities of bacterial communities in the pressure bioreactor. The bacterial 16S rRNA gene fragments were obtained from clone samples. Clone counts of each OTU are given in brackets; numbers in series indicate the counts derived from samples F, L and U, respectively. Numbers at nodes are bootstrap values derived from 100 analyses. The scale bar represents an amount of nucleotide sequence change of 0.03

Fig. 7

Results of the propionate degradation experiments (experiment III) under different pCO₂ conditions. a Propionate degradation profiles under different pCO₂ conditions. b Both acetate and propionate profiles of 0.50 MPa trial are shown for representation. Dashed lines represent curve fittings using modified Gompertz model