Page 82 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 4. Robustness of coal microbial community after nutrient amendment
temperature (20°C), and a 10 ml headspace was created in the vials by replacing 10 ml of liquid with 10 ml of nitrogen using a gastight glass syringe. Vials were inverted and equilibrated for 24 h. Methane from the headspace was analyzed using a Shimadzu GC-2010 gas chromatograph with flame ionization detection (GC-FID) fitted with a GASPRO PLOT column (60 m x 0.32 mm; Agilent Technologies, Mulgrave VIC, Australia). The carrier gas was helium (3 ml min-1), and inlet temperature was 250°C. Oven temperature program: isothermal 100°C (1 min) and then 25°C min-1 to 250°C and held for 1 min. Gas samples of 100 μl were withdrawn directly from the sampling flasks using a pressure lockable gastight glass syringe (SGE Analytical Science, Ringwood VIC, Australia) and injected into the GC. Compounds were quantified by comparison of the peak area of the unknown with an eight-point calibration curve with a lower detection limit of 0.2 μM. Calibration standards were made up in 60 ml serum vials.
For the analyses of acetate concentrations, water was filtered through a 0.2 μm syringe filter (Merck Millipore, Bayswater VIC, Australia) and subsequently 900 μl filtered water was acidified with 100 μl formic acid (10% v/v; Sigma-Aldrich, Castle Hill NSW, Australia) to a pH < 2. Acetate (1 μl) was analyzed by GC-FID (Shimadzu, Rydalmere NSW, Australia) using a DB-FFAP column (30 m x 0.32 mm; Agilent Technologies, Mulgrave, Australia) with helium (1 ml min-1) as carrier gas. Injection port was set at 250°C with split mode (1:30). Oven temperature was set to 60°C for 1 min and then 15°C min-1 to 250°C.
Cell counts
Coal formation water samples were immediately fixed by the addition of glutaric dialdehyde (0.2 μm filtered, 2% final concentration) and stored at 4°C in the dark. Prior to analysis, an aliquot was diluted 1000-fold in particle-free phosphate-buffered saline (0.9 g of NaCl, sodium phosphate buffer 15 mM, pH 7.4, 0.2 μm filtered), thoroughly shaken and transferred to a microscopic slide that was treated with a mounting medium (9.6% Mowiol 4-88, Sigma- Aldrich, Castle Hill NSW, Australia and 24% glycerol) prior to applications. Cells were stained using SybrGreen I (Sigma-Aldrich, Castle Hill NSW, Australia), and counting was performed using a BX51 epifluorescence microscopy (Olympus, Notting Hill VIC, Australia) as described by Lunau et al. (2005).
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