Techniques to Assess Microbial Activity in Frozen Ground 921 Permafrost Sampling and Sample Processing

It is usual practice to ensure that permafrost cores remain uncontaminated with chemicals or alien microorganisms (Shi et al. 1997; Rivkina et al. 2004). To achieve this goal, all mechanical parts of cutting equipment (auger, chisel, disk saw) are cleaned (e.g., with ethanol or other antiseptics) before collecting the next sample. This condition is relaxed when we analyze the surface frozen soil, which is normally subjected to intensive colonization by allochtonous forms (atmospheric deposition, run-off, borrowing animals, addition of manure, etc.) The extracted cores are immediately sealed in plastic bags and kept frozen during transportation and storage; at each step the temperature is monitored by microloggers to exclude accidental warming of samples. Microbiological analysis such as plating or DNA extraction is preceded by surface shaving of the cores with a sterile scalpel.

Several additional requirements come forward when the task is to measure subzero microbial activity:

(a) How to split frozen cores into small-size aggregates to be filled into test tubes for subsequent activity detection

(b) How to add soluble (glucose, succinate, etc.), or insoluble substrates (starch, cellulose) with minimal disturbance of frozen soil and its community

(c) How to avoid oxygen stress on strict anaerobes

(d) How to prevent sample desiccation during long-term incubations, etc.

The developed procedure (Fig. 9.2) satisfies the majority of required conditions. The homogenization is achieved by splitting the original 30-50 cm core into 2-3 cm sections following crushing into 3-8 mm aggregates inside a polypropylene sleeve under continuous cooling and N2 flow. The easiest way to amend substrate is to use gases and volatile compounds (CO2, ethanol, methane and other hydrocarbons, volatile fatty acids, etc.) and add them to the headspace over crushed soil. Soluble substrates require preliminary short-term permafrost melting. We tried to add soluble and insoluble substrates (cellulose) as frozen powder, but never detected their transformation below -10°C. Horizontal chest freezers are advantageus over vertical freezers, and a circulating thermostat with appropriate bath fluid (ethanol, poly-dimethylsiloxane) is obviously to be preferred over a dry freezer due to higher temperature stability (±0.1°C) even under frequent access. Alcohol bath (at constant temperature) and aluminum block (temperature gradient) provide a unique opportunity for headspace gas sampling: the vial with incubated permafrost is kept at a given

Fig. 9.2 Sampling and processing of permafrost samples for the measurement of metabolic activity of indigenous permafrost microorganisms including strictly anaerobes

below-zero temperature while the rubber septum stays outside the cooling range, remaining soft and resilient to multiple needle piercing and accessible to analysis.

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