Identification of free-living nitrogen fixing bacteria isolated from EFB compost, molecular detection of nifH gene and measurement of the nitrogenase activity

A.K.R. Emmyrafedziawati and M. Stella

Abstract
This study aims to enhance the understanding of the free-living diazotroph isolated by culture-based method, determine the presence of nifH gene and measure the nitrogenase activity by means of using acetylene reduction assay (ARA). The free-living nitrogen-fixing bacteria (labelled as NC and NFC) were
isolated from the matured empty fruit bunch (EFB) compost. These pure colonies of N fixers are able to grow and multiply on Beijerinckia agar media (BJK). A total of 8 isolates were characterised and identified. 6 isolates were from bacilli group, which are comprised of Bacillus (5 isolates) and Lysinibacillus (1 isolate), and the other 2 isolates were from Klebsiella genus. Specific primer to amplify nitrogenase gene (nifH gene), namely Po1R and Po1F at about 400 bp length
was selected. Following the electhrophoresis, some isolates were not visible on the gel image, but perform the nitrogenase activity by acethylene reductase assay (ARA). It was found that NC4 isolates showed significantly higher in nitrogenase activity at 7.46 x 10^-3 μmol C₂H₄/mL/h, followed by NC10 at 6.20 x 10^-3 umol C₂H₄/mL/h after incubation for 24 hours in acetylene. Based on the results, it is evidenced that NC4 and NC10 showed good potential to be employed as inoculant in the development of biofertiliser. In conclusion, the presence of nifH gene indicated that those viable microorganisms on plates were nitrogen-fixing bacteria. Notably, the availability of nifH gene in diazotroph was not a factor to produce high amount of nitrogenase activity. Environmental factor, specifically exposure to oxygen was the main factor affecting the ethylene concentration of nitrogenase activity from the nitrogen-fixing bacteria metabolic reaction.

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