Use of stable isotope probing to identify active free-living diazotrophs in soils from Malta

Nitrogen fixation by free-living diazotrophs can be a significant sour~e ofN in natural and agricultural ecosystems and in recent years molecular biology tools have advanced our knowledge of the diversity of free-living diazotrophs in soil. However, linking diversity to function remains one of the major challenges in microbial ecology. Stable isotope probing (SIP) using BC-labelled substrates was the first attempt to establish a link between microbial community structure and function using DNA. This technique depends upon the separation of labelled DNA from unlabelled DNA using CsCl density gradient centrifugation and identification of those microorganisms that were actively using the substrate. This project applies the principles of SIP using lsN-labelled dinitrogen gas to investigate diazotrophs that are actively fixing N in soil. Unlike BC-SIP, lSN_SIP is more technically challen~g due to the fact that the amount ofN in DNA is less than that of C. This limits the separation of lsN-labelled DNA from community unlabelled DNA to that DNA which has a high G+C content. To circumvent this problem, lSN_ SIP was used in conjunction with DGGE and changes in DNA buoyant density due to lSN incorporation were determined by comparing DGGE banding characteristics of the DNA in fractions from a CsCl gradient of DNA extracted from soil incubated under lSN2 with DGGE banding characteristics ofthe DNA in corresponding fractions .from a CsCl gradient ofDNA extracted from soil incubated under 14N2. Soils from Malta were characterised in terms of nitrogenase activity using the acetylene reduction assay and, after identifying a soil with high N fixing potential, nitrogenase activity in this soil was optimised by modifying the main limiting factors i during incubation. Microbial diversity in this soil was determined with molecular biology methods using the 16S rDNA and nijH genes, and the results compared with data obtained from traditional techniques using selective media and enrichment. This project has confirmed that 15N_SIP coupled with DGGE can identify diazotrophs in the natural environment that are actively fixing N. Using this approach, 15N_SIP has revealed that under the incubation conditions used in this project, diazotrophs related to Azotobacter and Methylobacterium spp. were actively fixing N in a Tal-Barrani soil.