بررسی تنوع میکروبی میدان نفتی سیلاب زنی نشده با دمای بالا در جنوب ایران

Introduction

The severe conditions within the oil reservoir make it possible for certain microorganisms to adapt to these conditions. These bacteria have been adapted to reservoirs conditions during many years. Various groups of microorganisms isolated from oil reservoirs with diverse physiological and metabolic characteristics and phylogenetic affiliations including sulfate reducing bacteria (SRB), fermentative bacteria, nitrate or manganese and iron reducers, sulphidogens, acetogens and methanogens. These microorganisms influence the quality of crude oil and reservoir conditions during metabolism activities (1-3). Therefore, in recent years, many studies have been carried out to identify varieties of microbial populations inhabiting oil reservoirs around the world (4). High-temperature oilfields are always considered more than other oil fields (5-8), for extending new technologies, microbial enhanced oil and energy recovery, control of souring, bioremediation and further understanding of biogeochemical procedures and life in extreme environments (9).

By molecular techniques and non-dependent culturing methods, a much larger range of different thermophilic microorganisms were detected from high temperature oilfields. Most of Archaea belong to CO₂-reducing methanogens including Methanobacteria, Methanococci and Thermococcales and Bacterial sequences affiliated with Firmicutes and Thermotoga in high-temperature oil reservoirs (2, 5-8).  Nevertheless, some moderate and non-thermophilic microorganisms were identified in various quantities in these oilfields. It has been hypothesized that the mesophilic microorganisms may originate from contaminant microorganisms that are capable to colonize the upper cooler parts of the oil well (9-11). Contamination of oilfields happens during drilling, sampling and enhanced oil recovery (EOR), especially during the water flooding processes (1, 9, 11, 12). It is difficult to distinguish indigenous microorganisms from allochthonous ones in these oil reservoirs.

In this study, for the first time, the microbial diversity of a non-water flooding oilfield with high temperature in southern Iran was evaluated using next generation sequencing of 16S rRNA gene analysis.

Materials andMethods

Reservoir Conditions:All inoculum sources originated from non-water flooding Zilaei (ZZ) oil reservoir which is located at the northeastern of Ahwaz, Iran, with a length of 3 km and a width of 8 km. According to internal report of National Iranian South Oil Company (NISOC, 2014), the study area was carbonate oil bed, having the salinity of 194000 ppm, with bottom-hole temperature of 85 - 90 °C, at a depth of approximately 3700 m above sea level. The reservoir contained light crude oil, holding an API gravity of 37 °C and pH of 8.0 (13).

Sampling: Five liters ZZ produced water sample were obtained from its oil-water processing site, during the period of July to December 2016. The bottles were filled completely and immediately taken to the laboratory for extraction of DNA (14).

DNA Extraction:All samples were filtered directly using 0.2-µm Startolab 150 V filter (Sartorius Biotech) and placed in 5 ml Homogenizer buffer (100 mM Tris–HCl (pH 8.2); 100 mM EDTA (pH 8); 1.5 M NaCl). The mixture was incubated under shaking conditions (150 rpm) for 12 h at room temperature for re-suspended microbial cells.The metagenomic DNA extraction was performed in a harsh manner which combined several lysis methods together. For achieving better result in DNA extraction, the treatment of the glass bead was used along with lysis buffer. The lysis buffer was formulated according to the method developed by Siddhapura et al. (15). The re-suspended microbial cells were applied to the enzymatic buffer (Tris-HCl pH 8; 20 mM, EDTA pH 8; 10 mM and Triton X-100 1.2%) with 20 mg/ml lysozyme and incubated overnight under shaking conditions. Before the addition of lysis buffer, the tubes were vigorously mixed with 1g glass beads by a vortex and subjected to intermittent freeze thaw in the liquid N₂ treatment. The next steps for the chemical lysis and purification were continued according to Siddhapura et al (15). The concentrations of double-stranded DNA in the extracts were determined using the Quant-iT dsDNA Assay Kit and the Qubit fluorometer (Invitrogen, USA).

16S rRNA Gene Amplicon Library and High-throughput Sequencing:The bacterial diversity was studied by pyrosequencing the amplified V1–V3 domain of the 16S rRNA gene amplifying a fragment of 520 bp by the 27F: 5'-AGAGTTTGATCCTGGCTCAG -3' and 534R: 5'- ATTACCGCGGCTGCTGG -3' primers (14). Ligated 454-adaptors were included in the forward primer, followed by a 10 bp Multiplex Identifier (MID). PCR amplification was done by 1X enzyme buffer, 0.2 mM dNTPs mixture  (Fermentas), 1 U High-Fidelity DNA Polymerase  (Fermentas), 0.5 μM of each primers (forward and reverse), 1.5 mM MgCl₂ and 2 μL of the DNA sample. The archaeal community analysis was performed by pyrosequencing the amplified 457 bp domain of the 16S rRNA gene amplifying by the 349F: 5'-GYGCASCAGKCGMGAAW -3' and 806R: 5'- GGACTACVSGGGTATCTAAT -3' primers with PCR reaction conditions as same as above (14, 16). The PCR incubation for bacterial and archaeal amplicon libraries products was performed according to Pournia and colleagues (14).  PCR products were purified and quantified by the Qubit flourometer (Invitorgen, USA). The amplicon pool was used for pyrosequencing on a GS Junior platform (454 Life Sciences, Roche, Macrogen) according to the manufacturer’s instructions using Titanium chemistry.

Bioinformatics Analysis:Raw reads were firstly filtered according to the 454 amplicon processing pipeline. Sequences were investigated by QIIME 1.6.0 software (17). Raw reads were demultiplexed and further filtered through the split library of QIIME. For 16S rRNA gene reads and the analysis were carried out as follows: sequences that passed the quality filter were denoised and singletons were excluded. OTUs defined by a 97% of similarity were picked using the uclust method and the representative sequences, selected as the most abundant in each cluster, were subjected to the RDPII classifier to get the taxonomy assignment and the relative abundance of each OTU using the Greengenes database (17).

Results

Rarefaction Curve:In this research, 6.88 μg archeal and 22.3 μg bacterial DNA per μL of the PCR product were obtained from the produced water of Zilaei oil field. During their sequencing, 6350 archeal and 14100 bacterial sequences were identified. The rarefaction curve is shown in Figure 1.

Fig. 1- Rarefaction Analysis of 16S rRNA Archaea and Bacteria Genes

Archaeal Community in Crude Oil: The 16S rRNA sequencing of archaeal genes showed that there are 7 different archeal species which belonged to the phylum of Euryarchaeota and one uncultured archaeon (Fig. 2).The family of Methanobacteriaceae, species of Methanothermobacter thermautotrophicus was observed with the frequency of 26.6% while Methanothermobacter thermophilus, Methanotorris formicicus, Methanococcus aeolicus, Halosimplex carlsbadense, Haloplanus natans and Picrophilus oshimae were characterized by less than 1% predominance (Table 1).

Table 1- Phylogenetic Affiliations and Relative Abundance of Archaeal Clone Library

Bacterial Community in Crude Oil: Phylogenetic association of bacterial 16S rRNA genes sequencesregain from ZZ produced water oilfield included members of Firmicutes; Bacilli (41.63%), Thermotogae (24.69%), Firmicutes; Clostridia (0.53%), Proteobacteria (0.43%), Actinobacteria (0.1%), Synergistetes (0.04%), Bacteroidetes (0.02%), and unclassified Bacteria, MPZ 101 (1.56%) as shown in Figure 2.

Sequence alignment studies showed that the major group of bacteria belonged to the family of Bacillaceae, genera of B. subtilis and B. licheniformis. While other main bacterial groups included the family of Thermotogaceae, genera of Petrotoga mobilis, Fervidobacterium pennivorans, Petrotoga miotherma and Thermotoga subterranea were observed. In addition, one unclassified genus has been identified with the frequency of 1.56%. The other recognized bacteria in this oil field were abundantly less than 0.5% (Table 2).

Fig. 2- Relative Abundance of Bacterial and Archaeal Groups within Clone Libraries

Discussion and Conclusion

Archaeal sequences with a close relationship to the family members of Methanobacteriaceae, genus of Methanothermobacter, had the greatest frequency in the samples taken from the ZZ reservoir (Table 1). The M. thermautotrophicus is a thermophilic, chemoautotroph, non-motive, obligatory anaerobic, which consumes H₂/CO₂ for methanogenesis (18). This methanogens and other species of Methanothermobacter have been reported many times from various oilfields at different temperatures by Orphan in South Elwood oil field, California (7), in Linch, Kalol and Nandasan oil wells, India (19), high temperature shengli oil reservoir, China (20-22), high temperature not water flooded Niburi oil field in Japan (23), high-temperature horizons of the Dagang and Kongdian oilfields, China (24-26), high temperature gas-water producing Niigata wells, Japan (27) and high temperature, water-flooded Huabei Petroleum (28).

Resource studies show that no comprehensive study has been carried out to identify methanogens in Iranian oilfields. However, Pournia et al. (14) by molecular methods identified several different methanogens from two non-water flooded oil reservoirs (LA and HK, located south of Iran) with an average temperature of 54 and 45 °C, respectively. The genus Methanofollis was observed with high presence, while genera of Methanothermobacter, Methanobrevibacter, Methanomethylovorans and Methanothermococcus were characterized with a lower abundance.

The major group of bacteria belonged to the division of Firmicutes (Bacilli) and Thermotogae (Fig. 2). Phylum of Firmicutes and Thermotogae are commonly more abundant taxa in non-water flooded oilfield, while in the flooded oil reservoirs, Proteobacteria is usually seen predominantly (7, 9, 23, 29). However, in this reservoir, only B. subtilis and B. licheniformis belonged to the phylum of Firmicutes which was present in large quantities, and the rest are much less abundant.

Table 2- Phylogenetic Affiliations and Relative Abundance of Bacterial Clone Library