Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation

Abstract

Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of ¹³C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.

Fig. 1

Subbituminous coal-seam, located in the western coalfields of NSW, field conditions for a long-term in situ methane stimulation trial. a Drilling of gas wells. b, cCoal cores after drilling. d Scanning electron microscopy of coal surface. e Schematic of the coal gas well

Fig. 2

(a) Methane formation, (b) Acetate concentration, (c) Cell numbers, and (d) sulfate concentrations in all four in situ treatments over an incubation time of 18 months. Addition of nutrients and acetate (red squares), nutrients and calcium peroxide (blue squares), nutrients (green squares) and no amendment (black squares)

Fig. 3

Cell numbers of archaea (white bars), methanogenic archaea (light grey bars), bacteria (dark grey bars) and sulfate reducing bacteria (grey bars) based on ribosomal RNA and the functional mcrA and dsrA genes determined by quantitative qPCR. Changes of absolute abundances of methanogens (coloured squares) and sulfate reducing bacteria (coloured circles) over an incubation time of 15–18 months

Fig. 4

a Changes in the bacterial and archaeal community composition in the coal formation water in response to different nutrient amendments over an incubation time of 15 months based on 16 S rRNA gene sequencing. From left to right: nutrients and acetate (positive control), nutrients and calcium peroxide, nutrients and no amendment (negative control). Colors indicate members of different phyla. Size of the bubble represents relative abundance of the genus or family in each sample. b Principal component analysis (PCA) comparing four different wells amended with nutrients and acetate (triangles), nutrients and calcium peroxide (circles), nutrients (squares) and no amendment (diamonds) over an incubation time of 15 months (white = 0 months to black = 15 months) and the showing the relationship to the different phyla (Bubbles are color coded as in a and methane production

Fig. 5

Changes in the bacterial and archaeal community composition on the coal surface in response to different nutrient amendments over an incubation time of 15 months based on 16 S rRNA gene sequencing. From left to right: nutrients and acetate (positive control), nutrients and calcium peroxide, nutrients and no amendment (negative control). Colours indicate members of different phyla. Size of the bubble represents relative abundance of the genus or family in each sample

Fig. 6

a, b Distribution and relative abundances of bacteria and archaea significantly associated with acetate utilisation in the ‘heavy’ and ‘light’ fractions of CsCl gradients derived from samples incubated in the presence of either ¹³C-labelled acetate or ¹²C-labelled acetate. c Acetate consumption and methane production in ¹³C- and ¹²C-labelled acetate amendments of coal formation water from the nutrient and acetate amended well (¹³C-acetate = black squares, ¹²C-acetate = black circles, ¹³C-methane = white squares, ¹²C-methane = white circles). DNA samples were taken from the ¹³C- and ¹²C-acetate enrichments during the time course of acetate consumption (red circles)

Fig. 7

Long-term microbial community succession in coal formation water biostimulated for enhanced methanogenesis was described. High sulfate concentrations and SRB did not prevent the stimulation of methanogenic archaea that are nutrient limited through the addition of a mineral nutrient amendment. Methanogenic archaea make up a major part of the coal associated biofilm communities carrying out acetoclastic methanogenesis in a gas-free coal seam. Acetate, and not H_2, is the central energy carrier in this coal reservoir. SRB as well as iron reducing bacteria (IRB) are energy (acetate) limited and compete with the methanogenic archaea for acetate. Only 28% of the amended acetate accounted for methane production, the majority was consumed by SRB and IRB