Bacterial community shift in nutrient-treated oil-bearing sandstones from the subsurface strata of an onshore oil reservoir and its potential use in Microbial Enhanced Oil Recovery

Microbial Enhanced Oil Recovery (MEOR) is a promising strategy to improve recovery of residual oil in reservoirs, which can be performed by promoting specific indigenous microorganisms. In this study, bacterial communities and the effects of elemental nutrient treatment of oil-bearing sandstone cores originated from six oil wells of an onshore reservoir was determined by tagged 16S rRNA gene amplicon sequencing, using Ion Torrent Metagenomic Sequencing Analysis. A total number of sequences were taxonomically classified into 43 phyla, 320 families, and 584 genera, with the dominant bacterial populations being related to Deinococcus-Thermus, and Betaproteobacteria. The nutrient treatment resulted in markedly increase in the relative abundance of Gammaproteobacteria. Thermus, Acinetobacter, and Pseudomonas were the most abundant genera. To our knowledge, this is the first report on the effect of elemental nutrients on alteration of bacteria communities attached to the oil-bearing rock. It provides comprehensive data on bacterial, physical, and chemical structures within a reservoir and demonstrates how these parameters can be co-analyzed to serve as a basis for designing a MEOR process. It also provides a model of how a bacterial community in reservoirs’ strata can be altered by nutrient treatment to enhance the efficiency of MEOR applications.

120 core analysis (performed by GENLabs, Thailand). The lithology of core rock was analysed using 121 an X-Ray fluorescence (XRF) spectrometer.

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Produced water from the production wells was analysed for pH and salinity using a 123 standard pH meter (Ohaus starter3100, USA) and an electrochemical analyser (Consort C933, 124 Belgium), respectively. Concentrations of sulfate and nitrate in the produced water were 125 determined using ion chromatography (Dionex ICS-3000, USA). Iron content was analysed 126 using inductively coupled plasma optical emission spectrometry (ICP-OES).  The enriched core samples were separated from the fluids by centrifugation at 4,766 g for 143 5 min. The genomic DNA was extracted from 10 grams of the samples using 10 ml of lysis 144 buffer (100 mM Tris-HCl, 100 mM Na-EDTA, 100 mM NaPO 4 , pH 8.0, 1.5M NaCl and 1% 145 (v/v) CTAB) and 100 µl of proteinase K (Zhou et al. 1996 Table 1. It can be seen that the lithological nature of the cores, their 196 porosity and grain density were slightly different, but the degrees of permeability, which reflect the 197 mobility of petroleum liquid, had a greater variation among the samples.

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199 Table 1 Characteristics of core samples and the wells from which core samples originated.    Produced water from the oil wells was analysed for chemical properties, including pH 219 and salinity. In addition, the concentrations of sulfate, nitrate, and iron, which can be the source 220 of nutrition for microorganisms, were also determined. We found that the produced water from 221 most wells (excluding well 3, which is presently abandoned) does not vary significantly in 222 salinity, which is in the normal range of the salinity of onshore reservoirs. The pH of the 223 produced water from the 6 wells tested showed that the wells were slightly alkaline. As for the 224 elemental compounds, sulfate was not detected in the produced water from most wells, while 225 nitrate was present in every well, and iron in 4 out of 5 wells (Table 4). An analysis of a  243 than the control (Fig 2A). The highest bacterial richness was observed in sample C5N, which 244 was in accordance with the diversity estimator, Chao 1. According to Shannon's diversity 245 indices, greater bacterial diversities were observed in the controls than in the nutrient-treated 246 samples, with the exception of sample C4, in which bacterial diversity increased after being 247 treated with elemental nutrient (Fig 2B). The diversity indices are summarised in Table 5.  Bacterial communities were analysed in the oil-bearing part of the core samples 261 submerged in the produced water and additional elemental nutrients. According to the shared 262 OTUs chart (Fig 2C), bacterial groups in the sandstone core were altered after being treated with 263 elemental nutrients. Smaller numbers of shared OTUs between the two treatments were observed 264 compared to unique OTUs found in either produced water-treated or nutrient-treated samples.
265 More than one thousand unique OTUs were found in produced water-treated samples C1P -C4P 266 and nutrient-treated samples C3N -C5N. Principle Coordinated Analysis (PCoA) was performed 267 to review the relationship of bacterial communities in the cores subjected to two treatments.
268 PCoA indicated that the bacterial community profiles were associated with the treatment 269 conditions used (Fig 2D). Comparison of the bacterial community under the different treatments Bacterial community profiles were studied between the two treatments at the phylum 276 level (Fig 3). More diverse bacterial groups were observed among the control samples than in the 277 nutrient-treated samples. In the controls, bacteria in phyla Deinococcus-Thermus,

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We also determined the effects of different treatment conditions on bacterial diversity at 303 the genus level. The abundances of bacterial genera were different between the samples 304 submerged in produced water and those treated with elemental nutrient (Fig 4) 379 We have studied a microbial community in the Mae Soon oil-bearing sand reservoir. The 380 oil-bearing sand from cores retrieved from six wells was placed under conditions resembling the 381 reservoir's conditions, flooded with the produced water in an anoxic condition at a raised 382 temperature. We also investigated the microbial communities of the sand core samples from the 383 reservoir after the addition of elemental nutrients. We did this to evaluate the impact of a change 384 in extrinsic conditions on the bacterial communities, which will assist in designing a microbial 385 enhanced oil recovery process.

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The relative abundance of predominant taxa found in the oil-bearing sand core samples of

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In the genus level, sequencing of 16S rRNA gene amplicons from the reservoir samples 400 revealed several potential bacteria for microbial recovery process including Thermus,