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.

Laboratory Experiment and Application Evaluation of a Bio-Nano-depressurization and Injection-Increasing Composite System in Medium-Low Permeability Offshore Reservoirs.

Given the high injection pressure and insufficient injection volume in the offshore oilfield, Bohai Oilfield has developed a bio-nano-depressurization and injection-increasing composite system solution (bio-nano-injection-increasing solution) composed of bio-surfactants, hydrophobic nano-polysilicon particles, and dispersant additives. In response to the current problems, a new type of bio-nano-depressurization and injection enhancement technology has been studied, which has multiple functions such as nano-scale inhibition and wetting reversal. The new technology has the technical advantages of efficient decompression, long-term injection, and wide adaptation. However, there is still a lack of optimization schemes and application effect prediction methods, which hinder the further popularization and application of the bio-nano-composite system solution. To solve this problem, this paper takes Well A1 in the Bohai Sea as an example to optimize the injection volume, concentration, and speed of the bio-nano-augmentation fluid and evaluates the application effect by using the proposed well testing, water absorption index, and numerical simulation methods. The research results show that the bio-nano-injection fluid can effectively improve the reservoir permeability and reduce the injection pressure. The application effect evaluation method proposed is reliable and can provide some reference for similar depressurization and injection-increasing technologies.

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.

Optimization and characterization of biosurfactant produced by indigenous Brevibacillus borstelensis isolated from a low permeability reservoir for application in MEOR.

Biosurfactants are expected to be a key factor for microbial enhanced oil recovery (MEOR). In this study, we described the novel biosurfactant-producing strain Brevibacillus borstelensis YZ-2 isolated from a low permeability oil reservoir. We purified and characterized the biosurfactants produced by this YZ-2 strain via thin-layer chromatography and MALDI-TOF-MS, revealing them to be fengycins. We additionally used a Box-Behnken design approach to optimize fermentation conditions in order to maximize the biosurfactants production. Core flooding experiments showed that biosurfactants produced by YZ-2 can significantly enhance crude oil recovery. Micro-model tests showed that emulsification and IFT reduction was the main EOR mechanism of the YZ biosurfactant in the oil wet model. In summary, these findings highlight the potential of Brevibacillus borstelensis YZ-2 and its metabolites for MEOR.

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.

Genetic map construction and QTL analysis of leaf-related traits in soybean under monoculture and relay intercropping.

Soybean (Glycine max L.) is an important food and oil crop widely planted by intercropping in southwest China. The shade caused by intercropping changes plant growth traits, such as soybean leaf and dry mass, thereby reducing yields. To improve the yield and elucidate the genetic mechanism of the leaf-related traits in intercropped soybeans, we measured the F6:7-8 recombinant inbred lines (RILs) derived from the cross of 'Nandou 12' and 'Jiuyuehuang' for six leaf-related traits under monoculture and relay intercropping in 2015 and 2016. We found 6366 single-nucleotide polymorphisms (SNPs) markers that covered the whole genome of soybean distributed in 20 linkage groups, which spanned 2818.67 cM with an average interval of 0.44 cM between adjacent markers. Nineteen quantitative trait loci (QTLs) were detected in two environments in 2 years. Three candidate genes associated to leaf-related traits were found according to gene expression and GO enrichment analyses. These results revealed the susceptibility of leaf phenotype to shading and helped elucidate the mechanisms that control leaf-related traits.

Dynamics of a microbial community during an effective boost MEOR trial using high-throughput sequencing.

Using 454 pyrosequencing of 16S rRNA gene amplicons, microbial communities in samples of injection water and production water during a serial microbial enhanced oil recovery (MEOR) field trial in a water flooded high pour point oil reservoir were determined. There was a close microbial community compositional relationship between the injection water and the successful first round MEOR processed oil reservoir which was indicated by the result of 43 shared dominant operational taxonomic units detected in both the injection water and the production water. Alterations of microbial community after the injection of boost nutrients showed that microbes giving positive responses were mainly those belonging to the genera of Comamonas, Brevundimonas, Azospirillum, Achromobacter, Pseudomonas, and Hyphomonas, which were detected both in the injection water and in the production water and usually detected in oil reservoir environments or associated with hydrocarbon degradation. Additionally, microbes only dominant in the production waters were significantly inhibited with a sharp decline in their relative abundance. Based on these findings, a suggestion of re-optimization of the boost nutrients, targetting the microbes co-existing in the injection water and the oil reservoir and having survival ability in both surface and subsurface environments, rather than simple repeats for the subsequent in situ MEOR applications was proposed.

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.

Draft genome sequence of Paenibacillus sp. strain A2.

Paenibacillus sp. strain A2 is a Gram-negative rod-shaped bacterium isolated from a mixture of formation water and petroleum in Daqing oilfield, China. This facultative aerobic bacterium was found to have a broad capacity for metabolizing hydrocarbon and organosulfur compounds, which are the main reasons for the interest in sequencing its genome. Here we describe the features of Paenibacillus sp. strain A2, together with the genome sequence and its annotation. The 7,650,246 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 54.2 % and contains 7575 protein-coding and 49 RNA genes, including 3 rRNA genes. One putative alkane monooxygenase, one putative alkanesulfonate monooxygenase, one putative alkanesulfonate transporter and four putative sulfate transporters were found in the draft genome.

[Study on influence factors of seed germination and seeding growth of Lonicera macranthoides].

In order to improve reproductive efficiency and quality standard, the influence factors of seed germination and seeding growth of Lonicera macranthoides werew studied. The fruit and seed morphological characteristics of L. macranthoides were observed, the seed water absorbing capacity was determined, and different wet sand stratification time, temperature and germination bed treatment were set up. The effects of the parameters on seed germination and seedling growth were analysed. There was no obstacles of water absorption on L. macranthoides seed, quantity for 22 h water absorption was close to saturation. In the first 80 d, with the increase of the stratification time, seed initial germination time was shortened, germination rate and germination potential was improved. Stratification for 100 d, germination rate decreased. At 15 ℃, seed germination and seedling growth indicators were the best. The seedling cotyledon width in light was significantly higher than that in dark. Seeds on the top of paper and top of sand germination rate, germination potential, and germination index was significantly higher than that of other germination bed and mildew rate is low. The optimal conditions of seeds germination test was stratified in 4 ℃ wet sand for 80 d, 15 ℃ illuminate culture on the top of paper or top of sand. The first seeding counting time was the 4th day after beginning the test, the final time was the 23th day. The germination potential statistical time was the 13th day after beginning the test.

[Study on seed quality test and quality standard of Lonicera macranthoides].

Referring to the rules for agricultural seed testing (GB/T 3543-1995) issued by China, the test of sampling, purity, thousand seed weight, moisture, viability, relative conductivity and germination rate had been studied for seed quality test methods of Lonicera macranthoides. The seed quality from 38 different collection areas was measured to establish quality classification standard by K-means clustering. The results showed that at least 7.5 g seeds should be sampled, and passed 20-mesh sieve for purity analysis.The 500-seed method used to measure thousand seed weight. The moisture was determined by crushed seeds dried in high temperature (130±2) ℃ for 3 h.The viability determined by 25 ℃ 0.1% TTC stained 5h in dark. 1.0 g seeds soaked in 50 ml ultra pure water in 25 ℃ for 12 hours to determine the relative conductivity. The seed by 4 ℃stratification for 80 days were cultured on paper at 15 ℃. Quality of the seeds from different areas was divided into three grades. The primary seed quality classification standard was established.The I grade and II grade were recommend use in production.

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.

Permanent draft genome sequence of Geobacillus thermocatenulatus strain GS-1.

Geobacillus thermocatenulatus strain GS-1 is a thermophilic bacillus having a growth optimum at 60°C, capable of degrading alkanes. It was isolated from the formation water of a high-temperature deep oil reservoir in Qinghai oilfield, China. Here, we report the draft genome sequence with an estimated assembly size of 3.5Mb. A total of 3371 protein-coding sequences, including monooxygenase, alcohol dehydrogenase, aldehyde dehydrogenase, fatty acid-CoA ligase, acyl-CoA dehydrogenase, enoyl-CoA hydrogenase, hydroxyacyl-CoA dehydrogenase and thiolase, were detected in the genome, which are involved in the alkane degradation pathway. Our results may provide insights into the genetic basis of the adaptation of this strain to high-temperature oilfield ecosystems.