Physiological State of Microorganisms in Frozen Soil

No doubt, many microorganisms found in frozen soil should be in a dormant stage (endo- and exospores, cysts, non-spore anabiotic cells, etc.). This review does not touch dormancy, as we are looking only at metabolically active cells. They may be represented by normally growing organisms and those who maintain their viability and convert some substrates, but do not grow or multiply (so-called state of maintenance). The second state (maintenance) has been postulated for microorganisms active below freezing point (Bakermans and Nealson 2004; Price and Sowers 2004), but this hypothesis has not been tested until recently.

The state of maintenance is defined as zero growth rate with non-zero consumption rate of energy source (Panikov 1995); therefore yield at this physiological state should be zero. To measure the subzero growth yield of soil community, we incubated soil with 14C-ethanol and measured label partitioning between CO2 (oxidation of ethanol equivalent to respiration rate), cells (label incorporation equivalent to cell growth) and unused substrate + exometabolites (Panikov and Sizova 2007). It was found that the cooling of frozen soil from 0 to -16°C resulted in a dramatic decline of the respiration and incorporation rates, but their ratio remained almost constant; the growth yield remained practically constant (0.54 ± 0.09 g C-cell per g C-ethanol) and close to values reported for pure microbial cultures grown on unfrozen laboratory media.

In another experiment (Panikov and Sizova 2007), the eukaryotic consortium Leucosporidium-Geomyces was grown at -8°C and then subjected to a temperature shift-down by moving individual tubes with culture to various temperatures between -8 and -25°C. As shown in Fig. 9.9, the rates of respiration and DF after the temperature shift-down was biphasic: for the first 2-3 weeks the consortium remained

Fig. 9.9 Demonstration of subzero activity of microbial consortia isolated from permafrost during growth on microcrystalline cellulose with ethanol as a sole carbon and energy source (Panikov and Sizova 2007). The enrichment containing basidiomycetous fungi and leucosporidial yeasts was isolated from Fairbanks site on solid ethanol-mineral medium with cellulose powder at -8°C without antifreeze. At time zero, incubation temperature was shifted from -8°C to lower temperatures as indicated on legend. Main panel: CO2 evolution rate (microbial respiration) as dependent on incubation temperature Insert: plot of microbial respiration rate (•) and DF (o) vs temperature

Fig. 9.9 Demonstration of subzero activity of microbial consortia isolated from permafrost during growth on microcrystalline cellulose with ethanol as a sole carbon and energy source (Panikov and Sizova 2007). The enrichment containing basidiomycetous fungi and leucosporidial yeasts was isolated from Fairbanks site on solid ethanol-mineral medium with cellulose powder at -8°C without antifreeze. At time zero, incubation temperature was shifted from -8°C to lower temperatures as indicated on legend. Main panel: CO2 evolution rate (microbial respiration) as dependent on incubation temperature Insert: plot of microbial respiration rate (•) and DF (o) vs temperature active even at the lowest temperature of -25°C, then growth stopped in the temperature interval -25°C to -18°C, but continued at temperatures from -16°C to -8°C. The insert panel in Fig. 9.9 plots the respiration and DF vs temperature for the second growth phases. Again, we can clearly see that respiration (energy-generating process) and DF (anaplerotic process related to cellular biosynthesis and growth) do change synchronously with cooling. Both processes stopped between -16°C and -20°C, and we never observed even transiently that cessation of growth (zero DF) was associated with non-zero respiratory activity, as should be expected at the state of maintenance. Based on the described experiments, it can be concluded that the attractive hypothesis on maintenance state (Price and Sowers 2004) has not been confirmed experimentally, and can be safely rejected as inappropriate.

How can biphasic respiration dynamics be interpreted? Most probably, the first phase was endogenous respiration of reserves accumulated at -8°C. It could be specialized reserved compounds like poly-P-hydroxyalkanes or glycogen, or non-specific endogenous substrates, such as cellular proteins, nucleic acids and cell-wall components (Panikov 1995). In chemostat, an endogenous self-digestion is used as an energy source to drive cell entry into the stationary phase, to express gene rpoS and synthesize hundreds of new enzymes required for survival under starvation conditions (Reeve et al. 1984; Zgurskaya et al. 1997). In the case of temperature shift-down, endogenous respiration could play a similar role of cellular reconstruction to adjust the intracellular machinery to function under colder conditions.

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