Page 87 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

of 450 L, a pH of 7.9, an oxidation reduction potential of -232 mV (chemical potential relative to the standard hydrogen electrode) and a temperature of 16°C. Groundwater flows were not assessed here but they likely cause exchange and mixing of the water body. After 25 months of operation, the pH was 8.2 and the redox potential -197 mV. The headspace gas volume is circa 1,000 L. The well was nutrient amended by adding a final concentration of 1.8 mM ammonium and 1.9 mM phosphate in addition to 20 mM acetate in two 10 mM additions at 0 and 4 months.
Nutrient and acetate concentrations were preassigned according to preliminary in vitro culture assays performed and modified according to Jones et al. (2010). Nutrient and acetate solutions were slowly released by drop tubing to 450 L coal formation water in contact with the coal seam with a feeding rate of about 0.5 L min-1 for a 2 L stock solution.
Amendment stimulated microbial growth and methane production
The amended well showed considerable methane production after 12 months, whereas in the unamended well, no methane production was observed over the course of the whole experiment (25 months, Fig. 1A). These observations are in line with previous studies on microbial coal conversion that showed that nutrient amendment stimulated methane production from non- producing coal systems (Laborda et al. 1997; Jones et al. 2010; Penner, Foght and Budwill 2010). Interestingly, methane emission was only observed after a prolonged lag phase of 12 months in conjunction with acetate degradation and stopped when acetate was depleted after about 18 months. This indicates that the microbial community at the start of the experiment was not yet able to or not abundant enough to convert acetate to methane. When we analyzed the cell numbers in the two coal wells we found that there was considerable growth in the amended well in the beginning of the incubation (3.0 x 104 cells ml-1 at 0 months; 9.9 x 107 cells ml-1 at 7 months, Fig. 1B) with a stabilization of the cell density after 7 months (2.4 x 108 cells ml-1 at 25 months) and absence of growth in the control well (1.5 x 104 cells ml-1 at 0 months; 1.6 x 104 cells ml-1 at 25 months, Fig. 1B). The increase in cell numbers was not correlated with a decrease in acetate concentration or concomitant methane production so it was presumably mainly due to the addition of nutrients that were also limiting in the unamended coal well. After about 11 months, acetate consumption and methane production were first observed, indicating a shift in microbial community functioning. From this, it can be concluded that acetate and nutrient amendment clearly stimulated microbial growth and enabled the microbial community
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