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Microbial Diversity of Long-Duration Gas Injection Oil Reservoir Based on Next Generation Sequencing in South of Iran

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Microbial Diversity of Long-Duration Gas Injection Oil Reservoir Based on Next Generation Sequencing in South of Iran Nature Environment and Pollution Technology p-ISSN: 0972-6268 Vol. 17 No. 2 pp. 413-420 2018 An International Quarterly Scientific Journal e-ISSN: 2395-3454 Original Research Paper Open Access Microbial Diversity of Long-Duration Gas Injection Oil Reservoir Based on Next Generation Sequencing in South of Iran Mohsen Pournia*(**), Nima Bahador**† and Ghasem Hosseini Salekdeh*** *Department of Microbiology, Science and Research Branch, Islamic Azad University, Fars, Iran **Department of Microbiology, College of Science, Agriculture and Modern Technology, Shiraz Branch, Islamic Azad University, Shiraz, Iran ***Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran †Corresponding author: Nima Bahador ABSTRACT Nat. Env. & Poll. Tech. Website: www.neptjournal.com Recent studies have shown that oilfield harbours diverse microbial communities. Water-flooding is Received: 12-07-2017 believed to be the main contaminating factor of oil reservoirs, however, less attempts have been made Accepted: 20-09-2017 to study the effect of natural gas injection on microbial community of oilfields. Molecular methods were used to evaluate the microbial diversity of Haftkel (HK) and Lali (LA) petroleum reservoirs in the south Key Words: of Iran. The HK oilfield has been injected with natural gas for long duration, but LA oilfield had no Iranian oil reservoir enhanced oil recovery (EOR) process. Next generation sequencing (NGS) of 16S rRNA archaeal Natural gas injection genes indicated that the genus Methanofollis was observed with high presence in 89.7% and 92.6% Microbial community frequency in the HK and LA oilfields, respectively. Most abundant phyla in HK oilfield were Synergistes 16S rRNA (58.1%), Firmicutes (17%), Proteobacteria (Betaproteobacteria) 12.8% and unclassified bacteria (5.2%), while Thermotogae (78%), Firmicutes (10.8%), Synergistes (4%) and unclassified bacteria (3.8%) were observed in LA oilfield. The comparison of the results indicated that the injection of natural gas could increase bacterial diversity and most probable cause to increase frequency of bacterial genus Anaerobaculum belonging to the Synergistes. INTRODUCTION and methanogenesis (Youssef et al. 2009, Ollivierand Magot 2005, Magot et al. 2000, Fan Zhang et al. 2010). There are large number of the petroleum reserves in world, which show the existence of a specific range of indigenous Culture dependent approaches are considered to reveal bacteria, with diverse physiological and metabolic functions limited efficiency to describe the microbial communities of and phylogenetic relationships. These bacteria have adapted ecosystems completely (Martin & Karsten 2014), while cul- to reservoir’s conditions during several years (Youssef et al. ture-independent 16S rRNA gene based investigations and 2009, Ollivierand Magot 2005, Magot et al., 2000). Con- metagenomic study are extremely valuable in providing an tamination of oil reservoirs occurs during sampling and en- overall view of the community composition in a specific hanced oil recovery (EOR) processes which affects the native ecosystem (Martin & Karsten 2014). microbial community (Youssef et al. 2009, Ollivierand Magot Hence, this is the first molecular study conducted to 2005, Fan Zhang et al. 2010). The water-flooding is the main determine the microbial communities of crude oil samples contaminating factor of oil reservoirs; however, there is a obtained from a petroleum reservoir, which has been in- challenge to understand the microbial community during gas jected by natural gas. The results were then compared with injection (Youssef et al. 2009, Ollivierand Magot 2005, another similar and close oilfield (LA) with no natural gas Magot et al. 2000, Fan Zhang et al. 2010). injection in south Iran and Middle East oilfields. Normally, oil reservoirs provide an appropriate envi- MATERIALS AND METHODS ronment for anaerobic and facultative anaerobic microor- ganisms (Ollivierand Magot 2005, Magot et al. 2000). Ma- Reservoir conditions: All samples originated from the jor electron donors are hydrogen, volatile fatty acids, petro- Haftkel (HK) and Lali (LA) oilfields, located at the south of leum hydrocarbons and inorganic electrons, while sulphate Iran. The distance between HK and LA oilfield is about 70 and carbonate minerals play important role as electron ac- km. The production of the crude oil from HK oil field was ceptors in many oil reservoirs. Therefore, the main metabolic started in 1927; this reservoir has a small gas cap and a processes of indigenous microbes in petroleum reservoirs are fracture reservoir using the miscible gas injection, which sulphate reduction, iron reduction, fermentation, acetogenesis could increase the oil recovery factor. Hydrocarbon con- 414 Mohsen Pournia et al. tents of both oilfields were almost similar, but no EOR (60s) with final extension 72°C (5 min) and 94°C for 3 min, processing has been performed in LA oil reservoir. followed by 35 cycles of 94°C (45s), 50°C (60s), 72°C (90s) Sampling: Five litres of HK crude oil samples were col- with a final extension of 72°C for 10 min. After agarose gel lected from two distinct operating oil wells (HK-1 and HK- electrophoresis, PCR products were purified and then quan- 2) and five litres LA produced water obtained from its oil- tified using the Qubit fluorometer. An equimolar pool was water processing site (Haft-Shahid), during the period of obtained prior to further processing. The amplicon pool was June to October 2015. The bottles were filled completely used for pyrosequencing on a GS junior platform (454 Life and sealed to maintain anoxic conditions and immediately Sciences, Roche, Macrogen) according to the manufactur- taken to the laboratory for extraction of DNA (Korenblum er’s instructions using titanium chemistry. et al. 2012, Irene et al. 2008). Bioinformatics analysis: Raw reads were firstly filtered DNA extraction: Since the amount of water accompanied according to the 454 amplicon processing pipeline. Se- by the HK crude oil was low, 100 mL of each sample was quences were then analysed by QIIME 1.6.0 software mixed with the same volume of the sterile Winogradsky’s (Caporaso et al. 2010). Raw reads were demultiplexed and buffer (Korenblum et al. 2012, Irene et al. 2008). The aque- further filtered through the split_library.py script of QIIME. ous phase was separated from crude oil by decantation at For 16S rRNA, gene reads and the analysis was carried out room temperature. This procedure was repeated five times, as follows: Sequences that passed the quality filter were until all of the crude oil was mixed and about five litres of denoised and singletons were excluded. OTUs defined by a aqueous phase were obtained. All aquatic phases and five 97% of similarity were picked using the uclust method and litres of LA produced water were filtered directly using 0.2µm the representative sequences, chosen as the most abundant Startolab 150 V filter (Sartorius Biotech). All filters were in each cluster, were subjected to the RDPII classifier to placed in 5 mL homogenizer buffer (100 mM Tris-HCl (pH obtain the taxonomy assignment and the relative abundance 8.2); 100 mM EDTA (pH 8); 1.5 M NaCl) and incubated of each OTU using the Greengenes 16S rRNA gene data- under shaking conditions overnight for re-suspended mi- base. Alpha and beta diversity were evaluated through crobial cells (Grabowski et al. 2005, Siddhapura et al. 2010). QIIME as recently described. Connections were drawn be- The genomic DNA extraction was performed in a harsh man- tween samples and OTUs, with edge weights defined as the ner which combined several lysis methods together accord- number of sequences from each OTU that occurred in each ing to Siddhapura et al. (2010). The concentrations of DNA sample. Network was visualized using Cytoscape 2.5.2 in the extracts were determined using the Quant-iT dsDNA (Caporaso et al. 2010). assay kit and the qubit fluorometer (Invitrogen, USA). RESULTS AND DISCUSSION 16S rRNA gene amplicon library and high-throughput Physicochemical characteristics of HK and LA oil fields: sequencing: The bacterial diversity was studied by The characteristics of HK and LA oil reservoirs with typical pyrosequencing the amplified V1-V3 region of the 16S rRNA hydrocarbon contents and properties of the natural gas cap gene amplifying a fragment of 520 bp using the 27F: 5'- according to internal report of National Iranian South Oil AGAGTTTGATCCTGGCTCAG-3' and 534R: 5'- Company (NISOC 2014) are given in Table 1. ATTACCGCGGCTGCTGG -3' primers (Tasharrofi et al. 2011). 454-adaptors were included in the forward primer, The injection of the natural gas into HK oil field studied followed by a 10 bp sample-specific Multiplex Identifier has been made since 1976, typical with following character- istics per (mole %): 82% CH , 10% C H , 3.5% C H , 1.7% (MID). PCR amplification was done using 1X enzyme buffer, 4 2 6 3 8 C H , 0.6% C H , 0.3% C H , 0.7% C H , 1.2% nitrogen, 0.2 mM dNTPs mixture (Fermentas), 1U High-Fidelity DNA 4 10 5 12 6 14 7 16 Polymerase (Fermentas), 0.5 µM forward and reverse prim- 0.2% carbon dioxide, hydrogen sulfide etc. in trace range. ers, 1.5 mM MgCl2 and 2 µL of the diluted DNA sample. Phylogenetic association of archaeal 16S rRNA gene se- The archaeal community analysis was carried out by quences: The 16S rRNA sequencing of archaeal genes re- pyrosequencing the amplified 457 bp region of the 16S trieved from HK samples indicated the abundance of 5 dif- rRNA gene amplifying using the 349F: 5'-GYGCASCAGK ferent archeal genera with one unclassified genus, which CGMGAAW-3' and 806R: 5'-GGACTACVSGGG belonged to the phylum of Euryarchaeota. The genus TATCTAAT-3' primers with PCR reaction conditions as same Methanofollis was observed with the frequency of 89.65%, as above (Takai et al. 2000). PCR incubation for bacterial while genera of Methanothermobacter and Methanobrevi and archaeal 16S amplicon libraries construction were per- bacter were characterized with 3.25% and 1.2% respectively.
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