Degradation and enhanced oil recovery potential of Alcanivorax borkumensis through production of bio-enzyme and bio-surfactant.

Microbial enhanced oil recovery (EOR) has become the focus of oilfield research due to its low cost, environmental friendliness and sustainability. The degradation and EOR capacity of A. borkumensis through the production of bio-enzyme and bio-surfactant were first investigated in this study. The total protein concentration, acetylcholinesterase, esterase, lipase, alkane hydroxylase activity, surface tension, and emulsification index (EI) were determined at different culture times. The bio-surfactant was identified as glycolipid compound, and the yield was 2.6 ± 0.2 g/L. The nC12 and nC13 of crude oil were completely degraded, and more than 40.0 % of nC14-nC24 was degraded by by A. borkumensis. The results of the microscopic etching model displacement and core flooding experiments showed that emulsification was the main mechanism of EOR. A. borkumensis enhanced the recovery rate by 20.2 %. This study offers novel insights for the development of environmentally friendly and efficient oil fields.

Cultivation and biogeochemical analyses reveal insights into biomineralization caused by piezotolerant iron-reducing bacteria from petroleum reservoirs and their application in MEOR.

Interactions between minerals and iron-reducing bacteria under in-situ pressure and temperature conditions play important roles in oil extraction, residual oil methanation, and CO2 storage in petroleum reservoirs. However, the impacts of pressure on dissimilatory iron-reducing bacteria (DIRB) are poorly understood. Herein, the interactions between clay minerals and microbes under elevated hydrostatic pressure conditions were elucidated through enrichment experiments. Bioreduction experiments were performed under hydrostatic pressures of 0.1-40 MPa. Microbial diversity analysis revealed that high pressures significantly increased microbial diversity in petroleum reservoirs, which is helpful for restoring underground ecosystems in situ. The key piezotolerant iron-reducing bacteria in the samples were Shewanella and Flaviflexus. These two genera were isolated for the first time from petroleum reservoirs and identified as piezophiles. The SEM results clearly showed mineral surface dissolution. Moreover, nanoscale secondary minerals were produced during biomineralization. XRD analysis revealed that illite, albite, and clinoptilolite were present after bioreduction. The isolates showed the capacity to inhibit hydro-swelling and prevent plugging-related damage in reservoirs.

Microbial dynamics and biogenic methane production responses to the addition of glycine betaine in shales.

Biogenic methane production depends on microbial community compositions in shale gas reservoirs, and glycine betaine plays an important role in methanogenic metabolic pathways. Previous studies have mainly focused on the microbial community dynamics in the water produced by shale hydraulic fracturing. Here, we used fresh shale as a sample and obtained the methane (CH4) and carbon dioxide (CO2) concentrations, microbial communities, and methanogenic functional gene numbers of solid and liquid groups in anaerobic bottles through gas chromatography, 16S rDNA sequencing (60 samples) and quantitative real-time PCR analysis in all culture stages. With glycine betaine addition, the total CH4 concentrations of the S1, S2 and Sw samples were 1.56, 1.05 and 4.48 times, while CO2 increased by 2.54-, 4.80- and 0.43-fold compared with samples without glycine betaine after 28 days of incubation, respectively. The alpha diversity was reduced when glycine betaine was added. The significant differences in bacterial community abundance at the genus level in samples with glycine betaine were Bacillus, Oceanobacillus, Acinetobacter, and Legionella. The bacterial and archaeal community changes implied that the addition of glycine betaine may promote CH4 production mainly by first forming CO2 and then generating CH4. The results of mrtA, mcrA, and pmoA gene numbers showed that the shale had great potential for producing methane. The addition of glycine betaine to shale changed the original microbial networks and increased the nodes and taxon connectedness of the Spearman association network. Our analyses indicate that the addition of glycine betaine enhances CH4 concentrations, causing the microbial network to be more complex and sustainable which supports the survival and adaptation of microbes in shale formations.

Synthesis and Enhanced Oil Recovery Potential of the Bio-Nano-Oil Displacement System.

Nanoparticles (NPs) have attracted great attention in the tertiary oil recovery process due to their unique properties. As an economical and efficient green synthesis method, biosynthesized nanoparticles have the advantages of low toxicity, fast preparation, and high yield. In this study, with the theme of biotechnology, for the first time, the bio-nanoparticles reduced by iron-reducing bacteria were compounded with the biosurfactant produced by Bacillus to form a stable bio-nano flooding system, revealing the oil flooding mechanism and enhanced oil recovery (EOR) potential of the bio-nano flooding system. The interfacial properties of the bio-nano-oil displacement system were studied by interfacial tension and wettability change experiments. The enhanced oil recovery potential of the bio-nano-oil displacement agent was measured by microscopic oil displacement experiments and core flooding experiments. The bio-nano-oil displacement system with different nanoparticle concentrations can form a stable dispersion system. The oil-water interfacial tension and contact angle decreased with the increase in concentration of the bio-nano flooding system, which also has a high salt tolerance. Microscopic oil displacement experiments proved the efficient oil displacement of the bio-nano-oil displacement system and revealed its main oil displacement mechanism. The effects of concentration and temperature on the recovery of the nano-biological flooding system were investigated by core displacement experiments. The results showed that the recovery rate increased from 4.53 to 15.26% with the increase of the concentration of the system. The optimum experimental temperature was 60 °C, and the maximum recovery rate was 15.63%.

Research on Characterization Technology and Field Test of Biological Nano-oil Displacement in Offshore Medium- and Low-Permeability Reservoirs.

At present, the water displacement recovery in some medium- and low-permeability reservoirs that cannot be injected and produced in offshore oil fields because of small pores and complex structures is less than 18%. This amount is far lower than 25-40%, which is obtained after water displacement and chemical displacement in medium- and high-permeability reservoirs. Given the current situation of water injection in offshore medium- and low-permeability reservoirs, a new green and environmentally friendly nano-oil displacement technology must be urgently developed to improve the sweep coefficient and oil displacement efficiency of injected water. In this study, the experimental laboratory investigation of a biological nano-oil displacement system suitable for medium- and low-permeability reservoirs is performed. The oil displacement effects, such as changing interfacial tension, viscosity reduction, and oil flushing ability, are also evaluated. The partial differential mathematical model of multicomponent isothermal multiphase seepage is deduced, the mechanism of biological nano-oil displacement technology is finely characterized, and a set of numerical simulation optimization charts of the biological nano-oil displacement process parameters is established. Results show that the biological nano-oil displacement system has adsorption characteristics in porous media, effective miscibility with crude oil, and a minimum contact angle reaching 14.3°. Its interfacial tension can be reduced to the 10-3 level, the viscosity reduction efficiency can reach more than 90%, and the oil washing efficiency can reach more than 70%. Compared with the conventional water and chemical displacement systems, the displacement system in this study has a good oil rock flushing effect and improves oil recovery by 15%. When the injection-production ratio is comprehensively considered, the recommended injection cycle is 6000 ppm. The field test of the biological nano-oil displacement system has been completed, with a validity period of 1 year and a cumulative oil increase of 1.2 × 104 m3, which is still effective. This study provides environmentally friendly solutions for the new chemical displacement of offshore medium- and low-permeability reservoirs. The established process parameter optimization chart has important guiding relevance for the optimization of technical schemes and improvement of the oil increase effect in chemical displacement.

Culture-dependent and culture-independent methods reveal microbe-clay mineral interactions by dissimilatory iron-reducing bacteria in an integral oilfield.

Fe(III) may be reasonably considered as one of the most important electron acceptors in petroleum reservoir ecosystems. The microbial mineralization of clay minerals, especially montmorillonite, is also of great significance to the exploration of petroleum and gas reservoirs. The bioreduction mechanisms of iron-poor minerals in petroleum reservoirs have been poorly investigated. This study investigated the bioreduction of montmorillonite by dissimilatory iron-reducing bacteria (DIRB) in petroleum reservoirs based on culture-independent and culture-dependent methods. Microbial diversity analysis revealed that Halolactibacillus, Bacillus, Alkaliphilus, Shewanella, Clostridium, and Pseudomonas were the key genera involved in the bioreduction of Fe(III). Through the traditional culture-dependent method, most of the key genera were isolated from the samples collected from petroleum reservoirs. Traditional culture-dependent methods can be used to reveal the metabolic characteristics of microorganisms (such as iron-reduction efficiency) to further elucidate the roles of different species (B. subtilis and B. alkalitelluris) in the environment. Moreover, many species with high iron-reduction efficiencies and relatively low abundances in the samples, such as Tessaracoccus and Flaviflexus, were isolated from petroleum reservoirs for the first time. The combination of culture-dependent and culture-independent methods can be used to further the understanding of the microbial communities and the metabolic characteristics of DIRB in petroleum reservoirs. Structural alterations that occurred during the interactions of microorganisms and montmorillonite were revealed through scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (XRD). The physical and chemical analysis results demonstrated that microorganisms from petroleum reservoirs can dissolve iron-poor montmorillonite and promote the release of interlayer water. The secondary minerals illite and clinoptilolite were observed in bioreduced smectite. The formation of secondary minerals was closely related to the dissolution degrees of minerals based on iron reduction.

Elucidate microbial characteristics in a full-scale treatment plant for offshore oil produced wastewater.

Oil-produced wastewater treatment plants, especially those involving biological treatment processes, harbor rich and diverse microbes. However, knowledge of microbial ecology and microbial interactions determining the efficiency of plants for oil-produced wastewater is limited. Here, we performed 16S rDNA amplicon sequencing to elucidate the microbial composition and potential microbial functions in a full-scale well-worked offshore oil-produced wastewater treatment plant. Results showed that microbes that inhabited the plant were diverse and originated from oil and marine associated environments. The upstream physical and chemical treatments resulted in low microbial diversity. Organic pollutants were digested in the anaerobic baffled reactor (ABR) dominantly through fermentation combined with sulfur compounds respiration. Three aerobic parallel reactors (APRs) harbored different microbial groups that performed similar potential functions, such as hydrocarbon degradation, acidogenesis, photosynthetic assimilation, and nitrogen removal. Microbial characteristics were important to the performance of oil-produced wastewater treatment plants with biological processes.

Tracking alterations of alkyl side chains of N1 species in heavy crude oil after anaerobic biodegradation with negative-ion electrospray ionization coupled with high-field Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: Heteroatomic compounds are relatively abundant and believed to be bio-resistant in heavy crude oils. However, few studies have focused on the biodegradation of these heteroatomic compounds. METHODS: Heteroatoms, especially N1 species, in a blank crude oil and in three treated oils co-incubated with anaerobic sulfate-reducing bacteria, nitrate-reducing bacteria and fermentative consortia cultures were detected using negative-ion electrospray ionization coupled with high-field Fourier transform ion cyclotron resonance mass spectrometry. RESULTS: The relative abundance of N1 species in the three treated oils decreased, while the relative abundance of O2 species increased. Remarkably, the relative abundances of N1 species with low carbon number increased and those with higher carbon number decreased. CONCLUSIONS: These results revealed that the anaerobic biodegradations of heavy crude oil occurred. With direct evidences, the degradations of alkyl side chains of N1 species by the anaerobic microbes could be deduced.

Rhamnolipids Produced by Indigenous Acinetobacter junii from Petroleum Reservoir and its Potential in Enhanced Oil Recovery.

Biosurfactant producers are crucial for incremental oil production in microbial enhanced oil recovery (MEOR) processes. The isolation of biosurfactant-producing bacteria from oil reservoirs is important because they are considered suitable for the extreme conditions of the reservoir. In this work, a novel biosurfactant-producing strain Acinetobacter junii BD was isolated from a reservoir to reduce surface tension and emulsify crude oil. The biosurfactants produced by the strain were purified and then identified via electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR-MS). The biosurfactants generated by the strain were concluded to be rhamnolipids, the dominant rhamnolipids were C26H48O9, C28H52O9, and C32H58O13. The optimal carbon source and nitrogen source for biomass and biosurfactant production were NaNO3 and soybean oil. The results showed that the content of acid components increased with the progress of crude oil biodegradation. A glass micromodel test demonstrated that the strain significantly increased oil recovery through interfacial tension reduction, wettability alteration and the mobility of microorganisms. In summary, the findings of this study indicate that the newly developed BD strain and its metabolites have great potential in MEOR.

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains.

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301(T)) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.

High quality genome sequence and description of Enterobacter mori strain 5-4, isolated from a mixture of formation water and crude-oil.

Enterobacter mori strain 5-4 is a Gram-negative, motile, rod shaped, and facultatively anaerobic bacterium, which was isolated from a mixture of formation water (also known as oil-reservior water) and crude-oil in Karamay oilfield, China. To date, there is only one E. mori genome has been sequenced and very little knowledge about the mechanism of E. mori adapted to the petroleum reservoir. Here, we report the second E. mori genome sequence and annotation, together with the description of features for this organism. The 4,621,281 bp assembly genome exhibits a G + C content of 56.24% and contains 4,317 protein-coding and 65 RNA genes, including 5 rRNA genes.

Permanent draft genome sequence of Bacillus flexus strain T6186-2, a multidrug-resistant bacterium isolated from a deep-subsurface oil reservoir.

Previous studies suggest that antibiotic resistance genes have an ancient origin, which is not always linked to the use of antibiotics but can be enhanced by human activities. Bacillus flexus strain T6186-2 was isolated from the formation water sample of a deep-subsurface oil reservoir. Interestingly, antimicrobial susceptibility testing showed that this strain is susceptible to kanamycin, however, resistant to ampicillin, erythromycin, gentamicin, vancomycin, fosfomycin, fosmidomycin, tetracycline and teicoplanin. To explore our knowledge about the origins of antibiotic resistance genes (ARGs) in the relatively pristine environment, we sequenced the genome of B. flexus strain T6186-2 as a permanent draft. It represents the evidence for the existence of a reservoir of ARGs in nature among microbial populations from deep-subsurface oil reservoirs.

Genomovar assignment of Pseudomonas stutzeri populations inhabiting produced oil reservoirs.

Oil reservoirs are specific habitats for the survival and growth of microorganisms in general. Pseudomonas stutzeri which is believed to be an exogenous organism inoculated into oil reservoirs during the process of oil production was detected frequently in samples from oil reservoirs. Very little is known, however, about the distribution and genetic structure of P. stutzeri in the special environment of oil reservoirs. In this study, we collected 59 P. stutzeri 16S rRNA gene sequences that were identified in 42 samples from 25 different oil reservoirs and we isolated 11 cultured strains from two representative oil reservoirs aiming to analyze the diversity and genomovar assignment of the species in oil reservoirs. High diversity of P. stutzeri was observed, which was exemplified in the detection of sequences assigned to four known genomovars 1, 2, 3, 20 and eight unknown genomic groups of P. stutzeri. The frequent detection and predominance of strains belonging to genomovar 1 in most of the oil reservoirs under study indicated an association of genomovars of P. stutzeri with the oil field environments.

Microbial diversity in long-term water-flooded oil reservoirs with different in situ temperatures in China.

Water-flooded oil reservoirs have specific ecological environments due to continual water injection and oil production and water recycling. Using 16S rRNA gene clone library analysis, the microbial communities present in injected waters and produced waters from four typical water-flooded oil reservoirs with different in situ temperatures of 25 °C, 40 °C, 55 °C and 70 °C were examined. The results obtained showed that the higher the in situ temperatures of the oil reservoirs is, the less the effects of microorganisms in the injected waters on microbial community compositions in the produced waters is. In addition, microbes inhabiting in the produced waters of the four water-flooded oil reservoirs were varied but all dominated by Proteobacteria. Moreover, most of the detected microbes were not identified as indigenous. The objective of this study was to expand the pictures of the microbial ecosystem of water-flooded oil reservoirs.

Impact of an indigenous microbial enhanced oil recovery field trial on microbial community structure in a high pour-point oil reservoir.

Based on preliminary investigation of microbial populations in a high pour-point oil reservoir, an indigenous microbial enhanced oil recovery (MEOR) field trial was carried out. The purpose of the study is to reveal the impact of the indigenous MEOR process on microbial community structure in the oil reservoir using 16Sr DNA clone library technique. The detailed monitoring results showed significant response of microbial communities during the field trial and large discrepancies of stimulated microorganisms in the laboratory and in the natural oil reservoir. More specifically, after nutrients injection, the original dominant populations of Petrobacter and Alishewanella in the production wells almost disappeared. The expected desirable population of Pseudomonas aeruginosa, determined by enrichment experiments in laboratory, was stimulated successfully in two wells of the five monitored wells. Unexpectedly, another potential population of Pseudomonas pseudoalcaligenes which were not detected in the enrichment culture in laboratory was stimulated in the other three monitored production wells. In this study, monitoring of microbial community displayed a comprehensive alteration of microbial populations during the field trial to remedy the deficiency of culture-dependent monitoring methods. The results would help to develop and apply more MEOR processes.

Investigation of biosurfactant-producing indigenous microorganisms that enhance residue oil recovery in an oil reservoir after polymer flooding.

Three biosurfactant-producing indigenous microorganisms (XDS1, XDS2, XDS3) were isolated from a petroleum reservoir in the Daqing Oilfield (China) after polymer flooding. Their metabolic, biochemical, and oil-degradation characteristics, as well as their oil displacement in the core were studied. These indigenous microorganisms were identified as short rod bacillus bacteria with white color, round shape, a protruding structure, and a rough surface. Strains have peritrichous flagella, are able to produce endospores, are sporangia, and are clearly swollen and terminal. Bacterial cultures show that the oil-spreading values of the fermentation fluid containing all three strains are more than 4.5 cm (diameter) with an approximate 25 mN/m surface tension. The hydrocarbon degradation rates of each of the three strains exceeded 50%, with the highest achieving 84%. Several oil recovery agents were produced following degradation. At the same time, the heavy components of crude oil were degraded into light components, and their flow characteristics were also improved. The surface tension and viscosity of the crude oil decreased after being treated by the three strains of microorganisms. The core-flooding tests showed that the incremental oil recoveries were 4.89-6.96%. Thus, XDS123 treatment may represent a viable method for microbial-enhanced oil recovery.

Molecular biologic techniques applied to the microbial prospecting of oil and gas in the Ban 876 gas and oil field in China.

Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.