Enhanced understanding of nitrogen fixing bacteria through DNA extraction with polyvinylidene fluoride membrane

Abstract

Abstract The rhizobiota, particularly nitrogen-fixing bacteria, play a crucial role in plant functioning by providing essential nutrients and defense against pathogens. This study investigated the diversity of nitrogen-fixing bacteria in a relatively understudied habitat: technosoils developed from industrial soda production. To analyze the bacterial diversity in the rhizosphere soils of wheat (Triticum aestivum L.) and aster (Tripolium pannonicum Jacq.), regions of the nifH gene were amplified and sequenced from the resident bacterial communities. A polyvinylidene fluoride (PVDF) membrane was employed for metagenomic DNA extraction, enhancing the detection of nitrogen-fixing bacteria. Prior to standard DNA extraction, an enrichment step was conducted in nitrogen-free JMV medium at 26 °C for 24 h, with a modification that replaced soil with the PVDF membrane. This approach enabled a more comprehensive analysis of the rhizosphere bacterial community, revealing that unique amplicon sequence variants (ASVs) in aster and wheat membrane samples accounted for a notable proportion of all ASVs in the dataset (8.5% and 23%, respectively) that were not captured using the standard method. Additionally, our findings demonstrated higher alpha diversity of nitrogen-fixing bacteria in the wheat rhizosphere compared to the aster rhizosphere. In wheat, the dominant genus was Insolitispirillum (38.80%), followed by unclassified genera within Gammaproteobacteria (9.76%) and Rhodospirillaceae (4.74%). In contrast, the aster rhizosphere was predominantly occupied by Azotobacter (95.69%).