Metagenomic Analysis of N-FIX Consortia

For community analysis (amplicon metabarcoding and Denaturing Gradient Gel Electrophoresis, DGGE), total DNA from both the original consortia and the consortia obtained after specific time points following serial transfers in N-FIX medium was analyzed using an FastDNA™ SPIN Kit for Soil (MP Biomedicals) per the standard protocol. For metagenomic analysis, a consortium sample obtained after 10 transfers via the N-FIX medium was used for DNA extraction. To improve cell recovery, 1 mL of 10% (volume/mass of approximately 0.03 M) sodium dodecyl sulfate solution was added to each flask containing culture medium, with a final concentration of approximately 0.25% (0.0008 M). The contents of three glass bottles (120 mL) containing the full-grown consortia were filtered through sterile 0.22-µm Millipore membranes; the membranes were then directly subjected to DNA extraction, and instead of the single lysis step in the FastPrep™ system (Bio 101, Inc., La Jolla, CA, USA), we performed the lysis step again. We used the FastPrep™ system at 6.0 rpm for 40 s. Nanodrop™ and Qubit™ (Thermo Fisher Scientific) were used to assess the quality (260/230 and 260/280 ratios) and amount of the extracted DNA, respectively. Electrophoresis was performed using 1.0% agarose gel for 40 min at 90 V, after which the gels were stained with SYBR^™ Safe (Thermo Fisher Scientific) and observed under an ultraviolet (UV) transilluminator to assess DNA integrity.