Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase

Area of Science:

• Microbiology
• Agricultural Science
• Biotechnology

Background:

• Sustainable crop production necessitates enhanced biological nitrogen fixation (BNF) through bioprospecting for effective nitrogen-fixing bacteria.
• Traditional isolation methods using low-oxygen conditions often yield only the most competitive strains, limiting microbial diversity.
• Plating on solid media in reduced oxygen environments can increase the diversity of culturable diazotrophic bacteria.

Purpose of the Study:

• To isolate diverse nitrogen-fixing bacteria from natural grassland soil samples.
• To confirm nitrogen fixation ability using stringent nitrogen-free media and PCR amplification of the nitrogenase gene (nifH).
• To evaluate the reliability of growth on nitrogen-free media for identifying nitrogen-fixing bacteria.

Main Methods:

• Soil suspensions from 27 grassland samples were plated on solid nitrogen-free agar.
• Incubation was performed under atmospheric and oxygen-reducing conditions.
• Putative nitrogen fixers were confirmed by subculturing in liquid nitrogen-free media and PCR amplification of nifH genes; isolates were further tested on stringent nitrogen-free media and in nitrogen-depleted atmospheres.

Main Results:

• Of 442 isolates, only 17% yielded amplicons for nifH genes, indicating a low success rate in confirming nitrogen fixation.
• Many isolates initially appearing to fix nitrogen on nitrogen-free media were found to be cocultures, with only one member growing under nitrogen limitation.
• Common soil bacteria like Bacillus and Streptomyces, which were nifH PCR-negative, were frequently isolated, suggesting potential alternative nitrogen acquisition or novel nitrogen reduction systems.

Conclusions:

• Standard methods for isolating and confirming nitrogen-fixing bacteria may overestimate their prevalence and diversity.
• Caution is advised when assessing nitrogen fixation based solely on growth in nitrogen-free media, even under stringent conditions.
• Further research is needed to understand the nitrogen metabolism of nifH-negative isolates, particularly those from the Bacillus and Streptomyces genera.