Trace elements affect methanogenic activity and diversity in enrichments from subsurface coal bed produced water

Abstract

Microbial methane from coal beds accounts for a significant and growing percentage of natural gas worldwide. Our knowledge of physical and geochemical factors regulating methanogenesis is still in its infancy. We hypothesized that in these closed systems, trace elements (as micronutrients) are a limiting factor for methanogenic growth and activity. Trace elements are essential components of enzymes or cofactors of metabolic pathways associated with methanogenesis. This study examined the effects of eight trace elements (iron, nickel, cobalt, molybdenum, zinc, manganese, boron, and copper) on methane production, on mcrA transcript levels, and on methanogenic community structure in enrichment cultures obtained from coal bed methane (CBM) well produced water samples from the Powder River Basin, Wyoming. Methane production was shown to be limited both by a lack of additional trace elements as well as by the addition of an overly concentrated trace element mixture. Addition of trace elements at concentrations optimized for standard media enhanced methane production by 37%. After 7 days of incubation, the levels of mcrA transcripts in enrichment cultures with trace element amendment were much higher than in cultures without amendment. Transcript levels of mcrA correlated positively with elevated rates of methane production in supplemented enrichments (R(2) = 0.95). Metabolically active methanogens, identified by clone sequences of mcrA mRNA retrieved from enrichment cultures, were closely related to Methanobacterium subterraneum and Methanobacterium formicicum. Enrichment cultures were dominated by M. subterraneum and had slightly higher predicted methanogenic richness, but less diversity than enrichment cultures without amendments. These results suggest that varying concentrations of trace elements in produced water from different subsurface coal wells may cause changing levels of CBM production and alter the composition of the active methanogenic community.

Keywords: coal bed methane; enrichments; mcrA transcript; methanogens; trace elements.

Figure 1

(A) Effect of increasing concentration of trace elements on cumulative methane production over time. No-TES is the enrichment without addition of any trace elements, 1×-TES, 2.5×-TES, and 5×-TES are the enrichments amended with increasing concentrations of trace element mix solutions. 1×-TES is detailed in the Section “Materials and Methods.” Error bars represent standard deviations (n = 3). Probability values were calculated for cumulative methane production at the end of 6 weeks. P < 0.001 for 1×-TES and 2.5×-TES; and P = 0.822 for 5×-TES. (B) Changes in pH value and total cell number (lines; DAPI total direct counts), and cumulative methane production (bars) over time in 1× -TES-amended and unamended cultures (No-TES) (n = 3).

Figure 2

Levels of mcrA transcripts in enrichment cultures with (trace element amendment) and without (no trace element) addition of trace elements over time.

Figure 3

Methane production rate correlated with mcrA transcription in enrichment cultures with addition of trace elements (regression coefficient R² = 0.95) and without addition of trace elements (regression coefficient R² = 0.69). Black circle: Enrichment cultures amended with trace element mix solution, white circle: enrichment cultures without addition of any trace elements.

Figure 4

Methanogenic community structure in trace element-amended and unamended enrichments. Startup enrichment culture (T0), trace element amended (T1:TES), and unamended (T1:No TES) enrichments. Blue bars: Methanobacterium subterraneum-like sequences; red bars: Methanobacterium formicicum-like sequences.

Figure 5

Rarefaction curves indicating mcrA richness within clone libraries derived from the startup enrichment culture (T0), trace element amended (T1:TES), and trace element unamended (T1:No TES) enrichment cultures. Sequences were grouped into phylotypes based on 97% sequence similarity.

Figure 6

Phylogenetic tree of 15 selected mcrA mRNA sequences retrieved from methanogenic enrichment cultures in this study and their relationship to reference sequences of cultivated methanogens and to other environmental clone sequences. Numbers in parentheses reflect the number of sequences in each OTU. Red square, trace element amended enrichment culture (TES); blue circle, non-amended enrichment culture (No); green triangle, startup enrichment culture (T0). Evolutionary history was inferred using the Neighbor-Joining method. Bootstrap values are shown for nodes in an analysis of 1000 replicates. Evolutionary distances were computed based on amino acid sequences, the scale bar represents 0.05 substitutions per amino acid position. Methanopyrus kandleri, the deepest branching methanogen, was chosen as out-group (Burggraf et al., 1991).