Indirect Evidence for Subzero Microbial Activity

Although reported in occasional publications starting in the 1960s (see below), subzero activity remains a matter of serious doubt, and is not unconditionally accepted as a significant factor in ecosystem dynamics of boreal and polar regions. The majority of texts assume that subzero temperatures reduce the intensity of biological processes to a negligible level. The definition of psychrophiles is based on their upper temperature limit of 20°C (Morita 1975; Helmke and Weyland 2004), while the low-temperature boundary is left undefined or is assumed to be around zero. Skepticism with regard to subzero activity by the majority of biologists is based on the deeply rooted postulate that life functions are to be supported by the running of the key metabolic processes above a certain threshold level; if cooling slows metabolic reactions below this level, then cells die. Another important restriction factor is claimed to be a lack or severe deficiency in the amount of liquid water in frozen habitats. Without liquid water, the majority of cellular bio-catalysts, such as DNA, RNA, enzymes, semi-fluidic membranes etc., remain functionally disabled (Kushner 1981).

In spite of these persuasive a priori arguments, there are several areas of indirect evidence which support the existence of subzero metabolic activity:

(1) Most biological processes are chemical reactions, so chemical kinetics at different temperatures, including ultra-low temperatures, may be instructive for explaining subzero metabolic activity. Generally, rates of abiotic chemical reactions decrease with cooling, but do not stop completely below the freezing point, having a global minimum in the vicinity of 0 K. It is remarkable that some chemical reactions, e.g., neutral free radicals reactions of O(3P) with hydrocarbon (Sabbah et al. 2007), have been shown to remain rapid down to temperatures as low as 20 K, and the rate coefficients increase as the temperature is lowered (Fig. 9.1). These data clearly demonstrate that temperature per se could not be the only restrictive factor; some chemical and maybe biochemical reactions may be accelerated below the freezing point (0°C, 273 K).

(2) It was shown many years ago (Michener and Elliott 1964; Gill and Lowry 1982; Geiges 1996) that frozen food is slowly degraded by bacteria and fungi. Although temperatures in industrial freezers are not maintained perfectly constant, it was concluded that frozen meat can support subzero growth down to -12°C. A psy-chrophilic community developing on a slaughtered cow in a freezer is probably initiated by opportunistic pathogens or by accidental saprotrophic contamination of freezers. Time for adaptation is measured at no longer than several years, which is nothing, compared with, say, Yedoma subsoils developed under permanent freezing during 30,000 years (Zimov et al. 2006). Therefore, we may safely assume that the lowest permissible temperature for a microbial community in frozen soils or subsoils may be essentially lower than -12°C.

(3) Similar observations were made recently on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen (Bielanski et al. 2003). After such multiyear storage, plating and DNA retrieval permitted the identification of 32 bacterial and 1 fungal species which represented commensal or environmental microorganisms. Stenotrophomonas maltophilia was the most common organism. No doubt, significant parts of detected microbes were just survival forms, but some of them could preserve activity and slowly multiply. Indirect evidence comes from the fact that the spectrum of detected species in cryopreserved material was not identical before and after long-term storage.

Fig. 9.1 Experimental data on the rates of reactions between O(3P) atoms and various alkenes at different subzero temperatures. The dashed lines show the results of calculations based on the modified microcanonical transition state theory (Sabbah et al. 2007). Note that cooling just below the water freezing point (273 K) decreases reaction rates but further cooling leads to acceleration of these radical reactions with approaching the maximum close to 10 K (with permission of Science magazine)

Fig. 9.1 Experimental data on the rates of reactions between O(3P) atoms and various alkenes at different subzero temperatures. The dashed lines show the results of calculations based on the modified microcanonical transition state theory (Sabbah et al. 2007). Note that cooling just below the water freezing point (273 K) decreases reaction rates but further cooling leads to acceleration of these radical reactions with approaching the maximum close to 10 K (with permission of Science magazine)

(4) Finally, the most impressive indirect evidence of subzero activity was recently demonstrated by the fact that winter tundra and boreal soils emit various gases: CO2, CH4, N2O. The cumulative cold-season C-fluxes can account for 2-20% of the annual methane emission and up to 60% of the net CO2 efflux from soil to atmosphere (Whalen and Reeburgh 1988; Dise 1992; Zimov et al. 1993; Melloh and Crill 1996; Brooks et al. 1997; Oechel et al. 1997; Fahnestock et al. 1998, 1999; Grogan and Chapin 1999; Panikov and Dedysh 2000). The mechanism behind the winter emission was a matter of hot discussion. Coyne and Kelley (1971) interpreted it as physical gas ejection from the soil by progressing freezing front; Zimov et al. (1993) hypothesized that soil microorganisms warm themselves up by biogenic heat production; Oechel et al. (1997) and Panikov and Dedysh (2000) suggested that cold-season C-emission was due to instant winter activity of yet unknown organisms.

All these factors are really indirect. Paradoxically, winter-season gas fluxes turned out not to be valid evidence for instant subzero activity of soil microorganisms. To explain this, the next section will focus on critical analysis of available techniques.

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