Methods: Sampling and Experimental set up

Samples from Movile Cave for these experiments were obtained by Dr Alexandra Hillebrand-Voicilescu, Mr Vlad Voicilescu, Prof Colin Murrell and Dr Rich Boden on the 13th April 2011. A sample of water and floating microbial mat was pooled from several regions of the air-water interface in air bell 2 of Movile Cave, and was subsequently aliqoted into 120 ml serum vials inside the cave environment just a few minutes after being sampled. The microcosms each contained 20 ml of the mat plus cave water. As far as was practical, each 20 ml of the mat sample contained

approximately the same amount of biomass.

The microcosms were spiked at the time of sampling with 2 ml of either 12C methane or 13C methane, resulting in a headspace concentration of 2.0 % (v/v) methane. There was no way of measuring the methane concentration in the microcosm until the samples had arrived back to the lab at Warwick University, 48 hours after sampling. Headspace methane concentration was measured by gas chromatograph (GC). On arrival into the lab, the microcosms were processed. 5 ml mat and water was removed from each microcosm 48 hours after sampling. The vial was resealed and methane was injected to 2.0 % (v/v). Subsequently, the methane concentration in the

headspace was monitored by GC (Figure 5.1). The microcosm was sampled again at 165 hours when another 5 ml of mat and water was removed. The serum vial stopper was replaced and the microcosm was spiked with methane, again to 2.0 % (v/v). The methane concentration in the serum vial headspace was monitored by GC until the microcosm was harvested at 261 hours. All samples were immediately stored at

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Figure 5.1: Consumption of methane in microcosms over time.

Plots indicate the consumption of methane over time from the DNA-SIP microcosms. Error bars represent 1 standard deviation from the mean of triplicate microcosms.

DNA was extracted from t=48, t=165 and t=261 samples and was quantified using the Nano-drop 1000. The DNA was then subject to isopycnic density gradient

ultracentrifugation, as outlined in the Neufeld et al., (2007) DNA SIP protocol paper. In brief, 3 µg of DNA was added to an ultracentrifuge tube along with gradient buffer and CsCl to a density of 1.725 g/ml-1. This was then inserted into a 5.1 ml

ultracentrifuge tube, the tube was heat sealed and tubes were spun in a Beckman Vti 65.2 rotor on a Beckman L-90K ultracentrifuge at 177,000 gav, at 20 ᴼC for 40 hours

under vacuum. Samples were then fractionated directly from the centrifuge tube by inserting a needle and tubing that was hooked up to a peristaltic pump. A second hole was made at the bottom of the centrifuge tube and the peristaltic pump turned on to

0 0.5 1 1.5 2 2.5 0 50 100 150 200 250 300 P er ce nta g e o f m et ha ne in hea ds pa ce

Incubation time in hours

12C 13C

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feed water into the top of the tube. This allowed the CsCl to drain at a controlled rate so as not to disturb the gradient, and fractions of ~425 µl were collected every minute. To ensure a gradient had formed, the CsCl density of each fraction was measured using a Reichart AR200 digital refractometer (Figure 5.2). Good gradients formed in both centrifuge tubes. The density of heavy DNA (1.725 g/ml-1) was found in fractions 6 and 7. The very low density of fraction 12 from both samples is due to a small amount of the displacement water mixing with the CsCl at the end of the fractionation process. DNA was precipitated from the fractionated samples using polyethylene glycol 6000 (Neufeld et al., 2007). Precipitated DNA was then stored at -20 ᴼC until processed.

Figure 5.2: Density gradients of CsCl measured from each fraction of the 12CH4 and 13

CH4 incubated samples after fractionation. 12C Blue trace, 13C Red trace.

In order to determine which microorganisms had assimilated the 13CH4, and also to

observe the shift in microbial community between heavy and light DNA, PCR 1.6500 1.6600 1.6700 1.6800 1.6900 1.7000 1.7100 1.7200 1.7300 1.7400 1.7500 0 1 2 3 4 5 6 7 8 9 10 11 12 D e n si ty g/ m l Fraction

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amplification of 16S rRNA genes of DNA from each of the fractions was carried out. The PCR primers were designed with GC clamps in order analyse the PCR products using DGGE. The PCR products were then run on a DGGE polyacrylamide gel with a denaturing gradient of 30-70 % urea and formamide (Figure 5.3). Bands from the DGGE profiles were excised using a razor blade after visualisation using a UV lamp table. Excised bands were placed into 20 µl of PCR grade water and left overnight at 4 ᴼC for the DNA to dissolve in the water. This solution was then used as template for 16S rRNA gene PCR amplification using standard 341F 907R primers without the GC clamp. The PCR reactions that yielded PCR products were sent for DNA

sequencing.

A clone library targeting the methane monooxygenase pmoA gene was constructed to analyse the methanotroph community labelled by the DNA-SIP process. The pmoA

gene PCR products were cloned using the Promega p-GEM-T Easy vector cloning kit. In brief, the pmoA gene PCR products were ligated into the p-GEM-T vector, which was then transformed into JM109 E.coli competent cells. The transformed cells were screened on LB agar with ampicillin, IPTG and X-Gal. White colonies were picked, re-streaked and used for whole cell colony PCR using the M13 primer pair (Messing, 1983).

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