Application of a nifH microarray to assess the impact of environmental factors on free-living diazotrophs in a glacier forefield.

Glacier forefield environments are exposed to extreme and fluctuating climatic and nutritional conditions. The high diversity of free-living diazotrophic communities found in these environments indicates that nitrogen fixers are able to efficiently cope with such conditions. In this study, a nifH microarray was used to monitor changes in diazotrophic populations in the field over a season, in the presence or absence of plants and in 2 glacier forefields characterized by a different bedrock type (siliceous or calcareous), as well as at different temperatures (10 °C, 15 °C) and under different nitrogen fertilization regimes (0, 10, 40 kg N·ha(-1)·year(-1)) in laboratory systems. Population structures responded highly dynamically to environmental changes. Plant presence had the strongest impact, which decreased toward the end of the season and with high amounts of nitrogen fertilization. Temperature and nitrogen fertilization increases indirectly affected diazotrophic communities through their positive impact on plant growth. These results indicate strong carbon limitation in young glacier forefield soils. Phylotypes related to the genus Methylocystis strongly responded to environmental variations. These methanotrophic microorganisms, which are able to retrieve nitrogen and carbon from the atmospheric pool, are particularly adapted to the extreme nutritional conditions found in glacier forefields.

Development and experimental validation of a nifH oligonucleotide microarray to study diazotrophic communities in a glacier forefield.

Functional microarrays are powerful tools that allow the parallel detection of multiple strains at the species level and therefore to rapidly obtain information on microbial communities in the environment. However, the design of suitable probes is prone to uncertainties, as it is based so far on in silico predictions including weighted mismatch number and Gibbs free-energy values. This study describes the experimental selection of probes targeting subsequences of the nifH gene to study the community structure of diazotrophic populations present in Damma glacier (Swiss Central Alps) forefield soils. Using the Geniom One in situ synthesis technology (Febit, Germany), 2727 in silico designed candidate probes were tested. A total of 946 specific probes were selected and validated. This probe set covered a large diversity of the NifH phylotypes (35 out of the 45) found in the forefield. Hybridization predictors were tested statistically. Gibbs free-energy value for probe-target binding gave the best prediction for hybridization efficiency, while the weighted mismatch number was not significantly associated to probe specificity. In this study, we demonstrate that extensive experimental tests of probe-hybridization behaviour against sequences present in the studied environment remain a prerequisite for meaningful probe selection.

High diversity of diazotrophs in the forefield of a receding alpine glacier.

Forefields of receding glaciers are unique and sensitive environments representing natural chronosequences. In such habitats, microbial nitrogen fixation is of particular interest since the low concentration of bioavailable nitrogen is one of the key limitations for growth of plants and soil microorganisms. Asymbiotic nitrogen fixation in the Damma glacier (Swiss Central Alps) forefield soils was assessed using the acetylene reduction assay. Free-living diazotrophic diversity and population structure were resolved by assembling four NifH sequence libraries for bulk and rhizosphere soils at two soil age classes (8- and 70-year ice-free forefield). A total of 318 NifH sequences were analyzed and grouped into 45 unique phylotypes. Phylogenetic analyses revealed a higher diversity as well as a broader distribution of NifH sequences among phylogenetic clusters than formerly observed in other environments. This illustrates the importance of free-living diazotrophs and their potential contribution to the global nitrogen input in this nutrient-poor environment. NifH diversity in bulk soils was higher than in rhizosphere soils. Moreover, the four libraries displayed low similarity values. This indicated that both soil age and the presence of pioneer plants influence diversification and population structure of free-living diazotrophs.

Benzoate-driven dehalogenation of chlorinated ethenes in microbial cultures from a contaminated aquifer.

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene (cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to-cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate.