Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase
Nitrogen inputs for sustainable crop production for a growing population require the enhancement of biological nitrogen fixation. Efforts to increase biological nitrogen fixation include bioprospecting for more effective nitrogen-fixing bacteria. As bacterial nitrogenases are extremely sensitive to...
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2025-01-01
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| author | Amrit Koirala Nabilah Ali Alshibli Bikram K. Das Volker S. Brözel |
| author_facet | Amrit Koirala Nabilah Ali Alshibli Bikram K. Das Volker S. Brözel |
| author_sort | Amrit Koirala |
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| description | Nitrogen inputs for sustainable crop production for a growing population require the enhancement of biological nitrogen fixation. Efforts to increase biological nitrogen fixation include bioprospecting for more effective nitrogen-fixing bacteria. As bacterial nitrogenases are extremely sensitive to oxygen, most primary isolation methods rely on the use of semisolid agar or broth to limit oxygen exposure. Without physical separation, only the most competitive strains are obtained. The distance between strains provided by plating on solid media in reduced oxygen environments has been found to increase the diversity of culturable potential diazotrophic bacteria. To obtain diverse nitrogen-fixing isolates from natural grasslands, we plated soil suspensions from 27 samples onto solid nitrogen-free agar and incubated them under atmospheric and oxygen-reducing conditions. Putative nitrogen fixers were confirmed by subculturing in liquid nitrogen-free media and PCR amplification of the <i>nifH</i> genes. Streaking of the 432 isolates on nitrogen-rich R2A revealed many cocultures. In most cases, only one community member then grew on NFA, indicating the coexistence of nonfixers in coculture with fixers when growing under nitrogen-limited conditions. To exclude isolates able to scavenge residual nitrogen, such as that from vitamins, we used a stringent nitrogen-free medium containing only 6.42 μmol/L total nitrogen and recultured them in a nitrogen-depleted atmosphere. Surprisingly, PCR amplification of <i>nifH</i> using various primer pairs yielded amplicons from only 17% of the 442 isolates. The majority of the <i>nifH</i> PCR-negative isolates were <i>Bacillus</i> and <i>Streptomyces</i>. It is unclear whether these isolates have highly effective uptake systems or nitrogen reduction systems that are not closely aligned with known nitrogenase families. We advise caution in determining the nitrogen fixation ability of plants from growth on nitrogen-free media, even where the total nitrogen is very limited. |
| format | Article |
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| language | English |
| publishDate | 2025-01-01 |
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| series | Microorganisms |
| spelling | doaj-art-aa96364c225c482d93750a373b8675032025-01-24T13:42:38ZengMDPI AGMicroorganisms2076-26072025-01-011319610.3390/microorganisms13010096Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without NitrogenaseAmrit Koirala0Nabilah Ali Alshibli1Bikram K. Das2Volker S. Brözel3Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USADepartment of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USADepartment of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USADepartment of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USANitrogen inputs for sustainable crop production for a growing population require the enhancement of biological nitrogen fixation. Efforts to increase biological nitrogen fixation include bioprospecting for more effective nitrogen-fixing bacteria. As bacterial nitrogenases are extremely sensitive to oxygen, most primary isolation methods rely on the use of semisolid agar or broth to limit oxygen exposure. Without physical separation, only the most competitive strains are obtained. The distance between strains provided by plating on solid media in reduced oxygen environments has been found to increase the diversity of culturable potential diazotrophic bacteria. To obtain diverse nitrogen-fixing isolates from natural grasslands, we plated soil suspensions from 27 samples onto solid nitrogen-free agar and incubated them under atmospheric and oxygen-reducing conditions. Putative nitrogen fixers were confirmed by subculturing in liquid nitrogen-free media and PCR amplification of the <i>nifH</i> genes. Streaking of the 432 isolates on nitrogen-rich R2A revealed many cocultures. In most cases, only one community member then grew on NFA, indicating the coexistence of nonfixers in coculture with fixers when growing under nitrogen-limited conditions. To exclude isolates able to scavenge residual nitrogen, such as that from vitamins, we used a stringent nitrogen-free medium containing only 6.42 μmol/L total nitrogen and recultured them in a nitrogen-depleted atmosphere. Surprisingly, PCR amplification of <i>nifH</i> using various primer pairs yielded amplicons from only 17% of the 442 isolates. The majority of the <i>nifH</i> PCR-negative isolates were <i>Bacillus</i> and <i>Streptomyces</i>. It is unclear whether these isolates have highly effective uptake systems or nitrogen reduction systems that are not closely aligned with known nitrogenase families. We advise caution in determining the nitrogen fixation ability of plants from growth on nitrogen-free media, even where the total nitrogen is very limited.https://www.mdpi.com/2076-2607/13/1/96culturablediazotrophnitrogenasenitrogen-fixingprairiesoil |
| spellingShingle | Amrit Koirala Nabilah Ali Alshibli Bikram K. Das Volker S. Brözel Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase Microorganisms culturable diazotroph nitrogenase nitrogen-fixing prairie soil |
| title | Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase |
| title_full | Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase |
| title_fullStr | Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase |
| title_full_unstemmed | Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase |
| title_short | Bacterial Isolation from Natural Grassland on Nitrogen-Free Agar Yields Many Strains Without Nitrogenase |
| title_sort | bacterial isolation from natural grassland on nitrogen free agar yields many strains without nitrogenase |
| topic | culturable diazotroph nitrogenase nitrogen-fixing prairie soil |
| url | https://www.mdpi.com/2076-2607/13/1/96 |
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