Abstract
Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically 13C-labeled glucose at three temperatures: +5 (control), -5, and -20°C. 13C was quantified in CO2, bulk soil, microbial biomass, and dissolved organic carbon (DOC) after 1, 3, and 10 days and also after 30 days for samples at -20°C. Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High 13C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13C recovery in microbial biomass at -5 than +5°C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at -20°C. Consequently, we attribute the fivefold higher extracellular 13C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms.
Highlights
Microbial processes in high-latitude ecosystems with permafrost or frozen soils during winter periods are important contributors to global carbon (C) and nitrogen (N) cycling
Attributes include the synthesis of cold-adapted enzymes that have high specific activities at low temperature (Herbert, 1989; Berry and Foegeding, 1997; Nakagawa et al, 2003), synthesis of antifreeze proteins (AFP) that bind to ice crystals, inhibiting their growth and recrystallization (Fletcher et al, 2001; Holt, 2003; Lorv et al, 2014), and adjustment of membrane composition by synthesis of unsaturated fatty acids to increase fluidity (Berry and Foegeding, 1997; Drotz et al, 2010)
SrCO3 precipitates were separated by centrifuging four times at 2000 × g for 10 min and washing in between with Millipore water until pH 7 was attained. δ13C values of the dried SrCO3 (1–2 g) were measured with a Flash 2000 elemental analyzer coupled by a ConFlo III interface to a Delta V Advantage isotope ratio mass spectrometer. 13C respired from the applied glucose was calculated according to a mixing model Eqs 1 and 2 (Gearing et al, 1991), where the C content of the background ([C]BG) in Eq 1 was determined by Eq 2
Summary
Microbial processes in high-latitude ecosystems with permafrost or frozen soils during winter periods are important contributors to global carbon (C) and nitrogen (N) cycling. The use of position- labeled glucose permits detailed reconstruction of microbial metabolic pathways and enables conclusions to be drawn on the microbial products formed under frozen conditions (Scandellari et al, 2009; Dijkstra et al, 2011a; Dippold and Kuzyakov, 2013; Apostel et al, 2015). This approach helps to identify the metabolic adaptations for overcoming the challenges under frozen conditions. This would result in higher 13C in the extracellular environment than in microbial biomass from the remaining glucose C positions due to the secreted EPS
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