Soil Microcosm and Experimental Setup

Rice field soil was sampled at the CRA Agricultural Research Council, Rice Research Unit (Vercelli, Italy) in May 2010. Soil parameters and rice agricultural practices in the sampling field have been described previously (Krüger et al., 2001). Soil, sampled at 0–20 cm depth, was air-dried at room temperature (∼22°C), crushed, sieved (2 mm), and stored covered in plastic containers prior to experimental set up. Approximately 60 microcosms were setup. Each microcosm contained 10 g air-dried soil filled in a sterile petri dish and saturated with autoclaved de-ionized water (0.45 ml per g dry soil). The microcosm was incubated in a gas tight jar at 25°C under 10%_v/v methane in air in the dark. Headspace atmosphere in the jar was replenished every 2–3 days to ensure constant air and methane availability. The microcosm was pre-incubated for 14 days to acclimatize to the incubation condition. Desiccation was induced by placing the microcosm under the laminar flow cabinet (Clean Air ES/FB, Telstar Life Science Solutions, Utrecht, the Netherlands) overnight (16 h) at room temperature (∼25°C) which caused >94% gravimetric water loss. Desiccation was induced fortnightly and weekly, designated as moderate and severe disturbances, respectively. After desiccation, water loss in the microcosm was replaced by adding the corresponding amount of autoclaved de-ionized water, and methane uptake rate was determined in triplicate. Water loss (∼2–3% gravimetric water content) in the un-disturbed microcosm was also replenished. After methane uptake measurement, the three microcosms (un-disturbed and disturbed microcosms, each) representing independent replicates were destructively sampled. The remaining microcosms were returned to the jar, and incubation was resumed under 10%_v/v methane in air in the dark. Depending on the disturbance, methane uptake was measured again after 7 or 14 days to determine the recovery of activity. Microcosm not exposed to desiccation served as a reference. The soil was homogenized before sampling, and stored in aliquots in the -20°C freezer till further analysis.