Influence of Freeze-Thaw Stress on the Structure and Function of Microbial Communities and Denitrifying Populations in Soil

Abstract

Microbial N 2 O release during the course of thawing of soil was investigated in model experiment focusing on denitrification, since freeze-thaw has been shown to cause significant physical and biological changes in soil, including a surge of N 2 O and CO 2 . The origin of these is still controversially discussed. The increase in denitrification after thawing may be attributed to the diffusion of organic substrates newly available to denitrifiers from disrupted soil aggregates, leading to an increase in microbial activity. Laboratory experiments with upper soil layer of a grassland were conducted in microcosms for real-time gas measurements during the entire phase of freeze and thaw. Shifts in microbial communities were evident on resolution of 16S and 18S rRNA genes and transcripts by denaturing gradient gel electrophoresis (DGGE). Microbial expression profiles were compared by RNA-arbitrarily primed PCR technique and subsequent resolution of amplified products on acrylamide gels. Differences in expression levels of periplasmic nitrate reductase gene (napA) and cytochrome cd 1 nitrite reductase (nirS) were observed by most-probable-number-reverse transcription-PCR, with higher levels of expression occurring just after thawing began, followed by a decrease. napA and nirS DGGE profiles showed no change in banding patterns with fingerprints derived from DNA, whereas those derived from cDNA showed a clear succession of denitrifying bacteria, with the most complex pattern being observed at the end of the N 2 O surge. This study provides insight into the structural community changes and expression dynamics of denitrifiers as a result of freeze-thaw stress. Also, the results presented here support the belief that the gas fluxes observed during thawing is a result of freezing initiated high microbial activity.The main process that forms the trace gas nitrous oxide (N 2 O) in agricultural soils is denitrification. It contributes significantly to the greenhouse effect. The concentration of N 2 O in the troposphere has increased from 270 ppb in 1750 to concentration of 316 ppb in the year 2000 and continues to rise (15). Moreover, it is involved in the destruction of stratospheric ozone. Therefore, identifying sources of N 2 O has been a subject of research over the last two decades. Several field studies in the temperate regions have indicated that N 2 O emissions in winter and spring, due to freezing and thawing of agricultural soils, can reach between 20 and 70% of the annual budget (28,35).

Wavelet analysis of wintertime and spring thaw CO2 and N2O fluxes from agricultural fields

“…These features were also present in WCO analysis, showing phase-locked behaviour with N 2 O fluxes leading CO 2 by 1/6l-1/4l in 2001 and 2003. This would support the argument that N 2 O and CO 2 fluxes produced at thaw events are due to microbial respiration (Azam et al, 2002;Azam and Mü ller, 2003;Garcia-Montiel et al, 2003;Sharma et al, 2006). The production of CO 2 and N 2 O confirms that microbial respiration can occur at temperatures near freezing (Dö rsch et al, 2004;Sharma et al, 2006).…”

“…This would support the argument that N 2 O and CO 2 fluxes produced at thaw events are due to microbial respiration (Azam et al, 2002;Azam and Mü ller, 2003;Garcia-Montiel et al, 2003;Sharma et al, 2006). The production of CO 2 and N 2 O confirms that microbial respiration can occur at temperatures near freezing (Dö rsch et al, 2004;Sharma et al, 2006). Denitrification at thaw events may be high due to the high soil water content (Nyborg et al, 1997) and available substrate (Christensen and Christensen, 1991).…”

“…Denitrification reactions are temperature dependent, and it has been observed that this process is active at temperature as low as À2 8C (Dorland and Beauchamp, 1991). Denitrification has been shown to occur at temperatures expected during winter and thaw as seen in field (Dö rsch et al, 2004) and laboratory studies (Sharma et al, 2006). Accumulation of N 2 O at low temperatures has been attributed to suppression of nitrous oxide reductase, the enzyme needed for conversion of N 2 O to N 2 (Melin and Nommik, 1983).…”

“…Recently, molecular analysis of microbial communities associated with freeze/thaw stress indicated very low or no expression for nosZ (gene encoding for nitrous oxide reductase), even though this gene was present in the gene pool (Sharma et al, 2006).…”

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

“…Nitrate concentrations approximate 2 mM in the pore water of such unvegetated cryoturbated peat soil, and are B1000 Â higher than in adjacent vegetated unturbated peat areas where N 2 O emissions are negligible (Repo et al, 2009). Repeated freezing and thawing of the cryoturbated soil leads to breakdown of soil aggregates, renders decomposable organic carbon more easily accessible to microbes and may thereby activate the microbial community including N 2 O producers (Mørkved et al, 2006;Sharma et al, 2006). Thus, cryoturbated peat circles represent acidic 'hotspots' of microbial N 2 O emission in the tundra (Repo et al, 2009;Marushchak et al, 2011).…”