metagenome from the oil reservoir
To obtain a sufficient amount of DNA, an enrichment culture method was used. Microorganisms were grown in M-medium consisting of 4 parts of Copro-medium, 4 parts of Def-medium, 1 part of Thermodesulfomedium, 1part of Alicy-medium and supplemented with 5 mM Lactate (Table 1) (Shlimon 2021). Target organisms for this medium include Deferribacteres, Alicycliphillus sp., Coprothermobacter sp., Thermodesulfobacteria. The components were dissolved in double-distilled water and sparged with N2 for 20 min before autoclaving. Stock solutions prepared and autoclaved separately. Cultivation was conducted in triplicate in 500 ml flasks filled with 400 ml of reaction medium and 2% (v/v) crude oil, then sealed under an N2/CO2 (70:30%) atmosphere. Microorganisms were incubated for 2 weeks at 28C in a GasPak Anaerobic System (BD Diagnostics, Franklin Lakes, New Jersey, USA).  Samples after incubation were separated into aqueous and oil phases in a separatory funnel and analyzed as described previously. MiSeq Illumina sequencing Preparation of the libraries was performed in accordance with the 16S Metagenomic Sequencing Library Preparation Protocol recommended for Illumina MiSeq. The first round of amplification of the V3-V4 region of the 16S rRNA gene was performed using the DNA Engine Tetrad 2 cycler (Bio-Rad, Munich, Germany) with specific primers  (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and B (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). PCR amplification was carried out according to the following protocol: initial denaturation for 3 min at 95C, 27 cycles of 30 s at 95C, 30 s at 55C, and 30 s at 72C; and a final extension for 5 min at 72C. The purification of amplicons was performed using the Agencourt AMPure XP purification kit (Beckman Coulter, USA). The second round of amplification was performed for double indexing of samples using the same cycle parameters.The library obtained was validated using the Bioanalyzer 2100 system and the Agilent DNA 1000 Kit (Agilent, USA) and was quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Quant-iT dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Purified amplicons were pooled at equal concentration. Further preparation of samples and sequencing were performed using MiSeq Reagent Kit v2 (300 cycles) and MiSeq-device (Illumina, USA) according to the manufacturers protocol.