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The weathering process can cause the volatilization of light components in crude oil, leading to the accumulation of total petroleum hydrocarbons (TPH) in weathered oil field soils. These TPH compounds are relatively resistant to biodegradation, posing a significant environmental hazard by contributing to soil degradation. TPH represents a complex mixture of petroleum-based hydrocarbons classified as persistent organic pollutants in soil and groundwater. The release of TPH pollutants into the environment poses serious threats to ecosystems and human health. Currently, various methods are available for TPH-contaminated soil remediation, with bioremediation technology recognized as an environmentally friendly and cost-effective approach. While converting TPH to CO 2 is a common remediation method, the complex structures and diverse types of petroleum hydrocarbons (PHs) involved can result in excessive CO 2 generation, potentially exacerbating the greenhouse effect. Alternatively, transforming TPH into energy forms like methane through bioremediation, followed by collection and reuse, can reduce greenhouse gas emissions and energy consumption. This process relies on the synergistic interaction between Methanogens archaea and syntrophic bacteria, forming a consortium known as the oil-degrading bacterial consortium. Methanogens produce methane through anaerobic digestion (AD), with hydrogenotrophic methanogens (HTMs) utilizing H 2 as an electron donor, playing a crucial role in biomethane production. Candidatus Methanoliparia (Ca. Methanoliparia) was found in the petroleum archaeal community of weathered Oil field in northeast China. Ca. Methanoliparia has demonstrated its independent ability to decompose and produce new energy (biomethane) without symbiosis, contribute to transitioning weathered oil fields towards new energy. Therefore, this review focuses on the principles, mechanisms, and developmental pathways of HTMs during new energy production in the degradation of PHs. It also discusses strategies to enhance TPH degradation and recovery methods., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Oil reservoirs, being one of the significant subsurface repositories of energy and carbon, host diverse microbial communities affecting energy production and carbon emissions. Viruses play crucial roles in the ecology of microbiomes, however, their distribution and ecological significance in oil reservoirs remain undetermined. Here, we assemble a catalogue encompassing viral and prokaryotic genomes sourced from oil reservoirs. The catalogue comprises 7229 prokaryotic genomes and 3,886 viral Operational Taxonomic Units (vOTUs) from 182 oil reservoir metagenomes. The results show that viruses are widely distributed in oil reservoirs, and 85% vOTUs in oil reservoir are detected in less than 10% of the samples, highlighting the heterogeneous nature of viral communities within oil reservoirs. Through combined microcosm enrichment experiments and bioinformatics analysis, we validate the ecological roles of viruses in regulating the community structure of sulfate reducing microorganisms, primarily through a virulent lifestyle. Taken together, this study uncovers a rich diversity of viruses and their ecological functions within oil reservoirs, offering a comprehensive understanding of the role of viral communities in the biogeochemical cycles of the deep biosphere., (© 2024. The Author(s).)

The genus Fictibacillus contains twelve species significant in the synthesis of cellulose-degrading enzymes and phenylalanine dehydrogenase, isolated mainly from marine sedimentary environments. Here, we report a new biosurfactant-producing strain, Fictibacillus nanhaiensis ME46, isolated from Daqing oil field in China. The biosurfactant extracted from Strain ME46 was determined as surfactin, one of the representative families of lipopeptide biosurfactants. The yield of the surfactin produced by strain ME46 was 0.62 g·L -1 as determined by high-performance liquid chromatography, and the critical micelle concentration (CMC) of the surfactin was estimated to be about 68 mg·L -1 and the surface tension at CMC was 35.1 mN·m -1 . This study extended our knowledge about the role of the species Fictibacillus nanhaiensis in the ecosystem of natural environments such as the oil field.

Background: The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood., Results: We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34 TS , both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11 T and Phoenicimicrobium oleiphilum HX-OS.bin.34 TS ferment sugars and n-alkanes, respectively, producing H 2 , CO 2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments., Conclusion: Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory. Video Abstract., (© 2024. The Author(s).)

Appropriate characterization of reservoir properties and investigation of the effect of these properties on microbial metabolism and oil recovery under simulated reservoir conditions can aid in development of a sustainable microbial enhanced oil recovery (MEOR) process. Our present study has unveiled the promising potential of the hyperthermophilic archaeon, identified as Thermococcus petroboostus sp. nov. 101C5, to positively influence the microenvironment within simulated oil reservoirs, by producing significant amounts of metabolites, such as biosurfactants, biopolymers, biomass, acids, solvents, gases. These MEOR desired metabolites were found to cause a series of desirable changes in the physicochemical properties of crude oil and reservoir rocks, thereby enhancing oil recovery. Furthermore, our study demonstrated that the microbial activity of 101C5 led to the mobilization of crude oil, consequently resulting in enhanced production rates and increased efficiency in simulated sand pack trials. 101C5 exhibited considerable potential as a versatile microorganism for MEOR applications across diverse reservoir conditions, mediating significant light as well as heavy oil recovery from Berea/carbonaceous nature of rock bearing intergranular/vugular/fracture porosity at extreme reservoir conditions characterized by high temperature (80-101 °C) and high pressure (700-1300 psi). Core flood study, which truly mimicked the reservoir conditions demonstrated 29.5% incremental oil recovery by 101C5 action from Berea sandstone at 900 psi and 96 °C, underscoring the potential of strain 101C5 for application in the depleted high temperature oil wells., (© 2024. The Author(s).)

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15 T , was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15 T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45-75 °C (optimum 70-75 °C) and pH 6.5-8.5 (optimum pH 7.5-8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C 16:0 , iso-C 15:0 , and C 18:0 . The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15 T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761 T , suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15 T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761 T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15 T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15 T (=JCM39389 T , =KCTC25731 T ) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Hydrocarbon-degrading consortia (HDC) play an important role in petroleum exploitation. However, the real composition and metabolic mechanism of HDC in the microbial enhanced oil recovery (MEOR) process remain unclear. By combining 13 C-DNA stable isotope probing microcosms with metagenomics, some newly reported phyla, including Chloroflexi, Synergistetes, Thermotogae, and Planctomycetes, dominated the HDC in the oil reservoirs. In the field trials, the HDC in the aerobic-facultative-anaerobic stage of oilfields jointly promoted the MEOR process, with monthly oil increments of up to 189 tons. Pseudomonas can improve oil recovery by producing rhamnolipid in the facultative condition. Roseovarius was the novel taxa potentially oxidizing alkane and producing acetate to improve oil porosity and permeability in the aerobic condition. Ca. Bacteroidia were the new members potentially degrading hydrocarbons by fumarate addition in the anaerobic environment. Comprehensive identification of the active HDC in oil reservoirs provides a novel theoretical basis for oilfield regulatory scheme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In subsurface biodegraded oil reservoirs, methanogenic biodegradation of crude oil is a common process. This process was previously assigned to the syntrophy of hydrocarbon-degrading bacteria and methanogenic archaea. Recent studies showed that archaea of the Candidatus Methanoliparum named as alkylotrophic methanogens couple hydrocarbon degradation and methane production in a single archaeon. To assess the geochemical role of Ca. Methanoliparum, we analyzed the chemical and microbial composition and metabolites of 209 samples from 15 subsurface oil reservoirs across China. Gas chromatography-mass spectrometry analysis revealed that 92% of the tested samples were substantially degraded. Molecular analysis showed that 85% of the tested samples contained Ca. Methanoliparum, and 52% of the tested samples harbored multiple alkyl-coenzyme M derivatives, the intercellular metabolites of alkylotrophic archaea. According to metagenomic and metatranscriptomic analyses, Ca. Methanoliparum dominates hydrocarbon degradation in biodegraded samples from the Changqing, Jiangsu, and Shengli (SL) oilfields, and it is persistently present as shown in a 15-year-long sampling effort at the Shengli oilfield. Together, these findings demonstrate that Ca. Methanoliparum is a widely distributed oil degrader in reservoirs of China, suggesting that alkylotrophic methanogenesis by archaea plays a key role in the alteration of oil reservoirs, thereby expanding our understanding of biogeochemical process in the deep biosphere., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

A pink, ovoid-shaped, Gram-stain-negative, strictly aerobic and motile bacterial strain, designated ROY-5-3 T , was isolated from an oil production mixture from Yumen Oilfield in PR China. The strain grew at 4-42 °C (optimum, 30 °C), at pH 5-10 (optimum, 7) and with 0-5 % (w/v) NaCl (optimum, 0%). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that ROY-5-3 T belongs to the genus Roseomonas and shared the highest pairwise similarities with Roseomonas frigidaquae CW67 T (98.1%), Roseomonas selenitidurans BU-1 T (97.8%), Roseomonas tokyonensis K-20 T (97.7%) and Roseomonas stagni HS-69 T (97.3%). The average nucleotide identity and digital DNA-DNA hybridization values between ROY-5-3 T and other related type strains of Roseomonas species were less than 84.08 and 28.60 %, respectively, both below the species delineation threshold. Pan-genomic analysis showed that the novel isolate ROY-5-3 T shared 3265 core gene families with the four closely related type strains in Roseomonas , and the number of strain-specific gene families was 513. The major fatty acids were identified as summed feature 8 (C 18 : 1 ω 6 c /C 18 : 1 ω 7 c ), summed feature 3 (C 16 : 1 ω 6 c /C 16 : 1 ω 7 c ) and C 16 : 0 . Strain ROY-5-3 T contained Q-10 as the main ubiquinone and the genomic DNA G+C content was 69.8 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. Based on the phylogenetic, morphological, physiological, chemotaxonomic and genome analyses, strain ROY-5-3 T represents a novel species of the genus Roseomonas for which the name Roseomonas oleicola sp. nov. is proposed. The type strain is ROY-5-3 T (=CGMCC 1.13459 T =KCTC 82484 T ).

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., Competing Interests: The authors have read the journal’s policy and have the following competing interests: WL is an employee of Sinopec Shengli Oilfield, and LW is an employee of CNOOC Energy Development Co. Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Reservoir souring, which is the production of H 2 S mainly by sulfate-reducing microorganisms (SRM) in oil reservoirs, has been a long-standing issue for the oil industry. While biocides have been frequently applied to control biogenic souring, the effects of biocide treatment are usually temporary, and biocides eventually fail. The reasons for biocide failure and the long-term response of the microbial community remain poorly understood. In this study, one-time biocide treatments with glutaraldehyde (GA) and an aldehyde-releasing biocide (ARB) at low (100 ppm) and high (750 ppm) doses were individually applied to a complex SRM community, followed by 1 year of monitoring of the chemical responses and the microbial community succession. The chemical results showed that souring control failed after 7 days at a dose of 100 ppm regardless of the biocide type and lasting souring control for the entire 1-year period was achieved only with ARB at 750 ppm. Microbial community analyses suggested that the high-dose biocide treatments resulted in 1 order of magnitude lower average total microbial abundance and average SRM abundance, compared to the low-dose treatments. The recurrence of souring was associated with reduction of alpha diversity and with long-term microbial community structure changes; therefore, monitoring changes in microbial community metrics may provide early warnings of the failure of a biocide-based souring control program in the field. Furthermore, spore-forming sulfate reducers ( Desulfotomaculum and Desulfurispora ) were enriched and became dominant in both GA-treated groups, which could cause challenges for the design of long-lasting remedial souring control strategies. IMPORTANCE Reservoir souring is a problem for the oil and gas industry, because H 2 S corrodes the steel infrastructure, downgrades oil quality, and poses substantial risks to field personnel and the environment. Biocides have been widely applied to remedy souring, but the long-term performance of biocide treatments is hard to predict or to optimize due to limited understanding of the microbial ecology affected by biocide treatment. This study investigates the long-term biocide performance and associated changes in the abundance, diversity, and structure of the souring microbial community, thus advancing the knowledge toward a deeper understanding of the microbial ecology of biocide-treated systems and contributing to the improvement of current biocide-based souring control practices. The study showcases the potential application of incorporating microbial community analyses to forecast souring, and it highlights the long-term consequences of biocide treatment in the microbial communities, with relevance to both operators and regulators.

The contamination of the environment by crude oil and its by-products, mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Cupriavidus sp. B1, Pseudomonas sp. T1, and another Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

The Role of microorganisms in the petroleum industry is wide-ranging. To understand the role of microorganisms in hydrocarbon transformation, identification of such microorganisms is vital, especially the ones capable of in situ degradation. Microorganisms play a pivotal role in the degradation of hydrocarbons and remediation of heavy metals. Anaerobic microorganisms such as Sulphate Reducing Bacteria (SRB), responsible for the production of hydrogen sulphide (H 2 S) within the reservoir, reduces the oil quality by causing reservoir souring and reduction in oil viscosity. This paper reviews the diversity of SRB, methanogens, Nitrogen Reducing Bacteria (NRB), and fermentative bacteria present in oil reservoirs. It also reviews the extensive diversity of these microorganisms, their applications in petroleum industries, characteristics and adaptability to survive in different conditions, the potential to alter the petroleum hydrocarbons properties, the propensity to petroleum hydrocarbon degradation, and remediation of metals.

Crude oil extracted from oilfield reservoirs brings together hypersaline produced water. Failure in pipelines transporting this mixture causes contamination of the soil with oil and hypersaline water. Soil salinization is harmful to biological populations, impairing the biodegradation of contaminants. We simulated the contamination of a soil from an oilfield with produced water containing different concentrations of NaCl and crude oil, in order to evaluate the effect of salinity and hydrocarbon concentration on prokaryote community structure and biodegradation activity. Microcosms were incubated in CO 2 -measuring respirometer. After the incubation, residual aliphatic hydrocarbons were quantified and were performed 16S rRNA gene sequencing. An increase in CO 2 emission and hydrocarbon biodegradation was observed with increasing oil concentration up to 100 g kg -1 . Alpha diversity decreased in oil-contaminated soils with an increase in the relative abundance of Actinobacteria and reduction of Bacteroidetes with increasing oil concentration. In the NaCl-contaminated soils, alpha diversity, CO 2 emission, and hydrocarbon biodegradation decreased with increasing NaCl concentration. There was an increase in the relative abundance of Firmicutes and Proteobacteria and a reduction of Actinobacteria with increasing salt concentration. Our results highlight the need to adopt specific bioremediation strategies in soils impacted by mixtures of crude oil and hypersaline produced water.

Background: Pseudomonas aeruginosa, the rhamnolipids-producer, is one of dominant bacteria in oil reservoirs. Although P. aeruginosa strains are facultative bacteria, the anaerobic biosynthesis mechanism of rhamnolipids is unclear. Considering the oxygen scarcity within oil reservoirs, revealing the anaerobic biosynthesis mechanism of rhamnolipids are significant for improving the in-situ production of rhamnolipids in oil reservoirs to enhance oil recovery., Results: Pseudomonas aeruginosa SG anaerobically produced rhamnolipids using glycerol rather than glucose as carbon sources. Two possible hypotheses on anaerobic biosynthesis of rhamnolipids were proposed, the new anaerobic biosynthetic pathway (hypothesis 1) and the highly anaerobic expression of key genes (hypothesis 2). Knockout strain SGΔrmlB failed to anaerobically produce rhamnolipids using glycerol. Comparative transcriptomics analysis results revealed that glucose inhibited the anaerobic expression of genes rmlBDAC, fabABG, rhlABRI, rhlC and lasI. Using glycerol as carbon source, the anaerobic expression of key genes in P. aeruginosa SG was significantly up-regulated. The anaerobic biosynthetic pathway of rhamnolipids in P. aeruginosa SG were confirmed, involving the gluconeogenesis from glycerol, the biosynthesis of dTDP-L-rhamnose and β-hydroxy fatty acids, and the rhamnosyl transfer process. The engineered strain P. aeruginosa PrhlAB constructed in previous work enhanced 9.67% of oil recovery higher than the wild-type strain P. aeruginosa SG enhancing 8.33% of oil recovery., Conclusion: The highly anaerobic expression of key genes enables P. aeruginosa SG to anaerobically biosynthesize rhamnolipids. The genes, rmlBDAC, fabABG, rhlABRI, rhlC and lasI, are key genes for anaerobic biosynthesis of rhamnolipid by P. aeruginosa. Improving the anaerobic production of rhamnolipids better enhanced oil recovery in core flooding test. This study fills the gaps in the anaerobic biosynthesis mechanism of rhamnolipids. Results are significant for the metabolic engineering of P. aeruginosa to enhance anaerobic production of rhamnolipids.

Corrosion in pipelines and reservoir tanks in oil plants is a serious problem in the global energy industry because it causes substantial economic losses associated with frequent part replacement and can lead to potential damage to entire crude oil fields. Previous studies revealed that corrosion is mainly caused by microbial activities in a process currently termed microbiologically influenced corrosion or biocorrosion. Identifying the bacteria responsible for biocorrosion is crucial for its suppression. In this study, we analyzed the microbial communities present at corrosion sites in oil plant pipelines using comparative metagenomic analysis along with bioinformatics and statistics. We analyzed the microbial communities in pipelines in an oil field in which groundwater is used as injection water. We collected samples from four different facilities in the oil field. Metagenomic analysis revealed that the microbial community structures greatly differed even among samples from the same facility. Treatments such as biocide administration and demineralization at each location in the pipeline may have independently affected the microbial community structure. The results indicated that microbial inspection throughout the pipeline network is essential to prevent biocorrosion at industrial plants. By identifying the bacterial species responsible for biocorrosion, this study provides bacterial indicators to detect and classify biocorrosion. Furthermore, these species may serve as biomarkers to detect biocorrosion at an early stage. Then, appropriate management such as treatment with suitable biocides can be performed immediately and appropriately. Thus, our study will serve as a platform for obtaining microbial information related to biocorrosion to enable the development of a practical approach to prevent its occurrence., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

We report the isolation a halophilic bacterium that degrades both aromatic and aliphatic hydrocarbons as the sole sources of carbon at high salinity from produced water. Phylogenetic analysis of 16S rRNA-gene sequences shows the isolate is a close relative of Modicisalibacter tunisiensis isolated from an oil-field water in Tunisia. We designate our isolate as Modicisalibacter sp. strain Wilcox. Genome analysis of strain Wilcox revealed the presence of a repertoire of genes involved in the metabolism of aliphatic and aromatic hydrocarbons. Laboratory culture studies corroborated the predicted hydrocarbon degradation potential. The strain degraded benzene, toluene, ethylbenzene, and xylenes at salinities ranging from 0.016 to 4.0 M NaCl, with optimal degradation at 1 M NaCl. Also, the strain degraded phenol, benzoate, biphenyl and phenylacetate as the sole sources of carbon at 2.5 M NaCl. Among aliphatic compounds, the strain degraded n-decane and n-hexadecane as the sole sources of carbon at 2.5 M NaCl. Genome analysis also predicted the presence of many heavy metal resistance genes including genes for metal efflux pumps, transport proteins, and enzymatic detoxification. Overall, due to its ability to degrade many hydrocarbons and withstand high salt and heavy metals, strain Wilcox may prove useful for remediation of produced waters.

A novel sulphate-reducing, Gram-stain-negative, anaerobic strain, isolate XJ01 T , recovered from production fluid at the LiaoHe oilfield, PR China, was the subject of a polyphasic study. The isolate together with Desulfovibrio oxamicus NCIMB 9442 T and Desulfovibrio termitidis DSM 5308 T formed a distinct, well-supported clade in the Desulfovibrionaceae 16S rRNA gene tree. The taxonomic status of the clade was underscored by complementary phenotypic data. The three isolates comprising the clade formed distinct phyletic branches and were distinguished using a combination of physiological features and by low average nucleotide identity and digital DNA-DNA hybridization values. Consequently, it is proposed that isolate XJ01 T represents a novel genus and species for which the name Cupidesulfovibrio liaohensis gen. nov., sp. nov. is proposed with the type strain XJ01 T (=CGMCC 1.5227 T =DSM 107637 T ). It is also proposed that D. oxamicus and D. termitidis be reclassified as Cupidesulfovibrio oxamicus comb. nov. and Cupidesulfovibrio termitidis comb. nov., respectively.

Due to the inefficient reproduction of microorganisms in oxygen-deprived environments of the reservoir, the applications of microbial enhanced oil recovery (MEOR) are restricted. To overcome this problem, a new type of air-assisted MEOR process was investigated. Three compounding oil degradation strains were screened using biochemical experiments. Their performances in bacterial suspensions with different amounts of dissolved oxygen were evaluated. Water flooding, microbial flooding and air-assisted microbial flooding core flow experiments were carried out. Carbon distribution curve of biodegraded oil with different oxygen concentration was determined by chromatographic analysis. The long-chain alkanes are degraded by microorganisms. A simulation model was established to take into account the change in oxygen concentration in the reservoir. The results showed that the optimal dissolved oxygen concentration for microbial growth was 4.5~5.5mg/L. The main oxygen consumption in the reservoir happened in the stationary and declining phases of the microbial growth systems. In order to reduce the oxygen concentration to a safe level, the minimum radius of oxygen consumption was found to be about 145m. These results demonstrate that the air-assisted MEOR process can overcome the shortcomings of traditional microbial flooding techniques. The findings of this study can help for better understanding of microbial enhanced oil recovery and improving the efficiency of microbial oil displacement., Competing Interests: The authors have declared that no competing interests exist.

An anaerobic thermophilic, rod-shaped bacterium possessing a unique non-lipid sheathed-like structure enveloping a single-membraned cell, designated strain NRmbB1 T was isolated from at the deep subsurface oil field located in Yamagata Prefecture, Japan. Growth occurred with 40-60°C (optimum, 55°C), 0-2% (2%), NaCl and pH 6.0-8.5 (8.0). Fermentative growth with various sugars was observed. Glucose-grown cells generated acetate, hydrogen, pyruvate and lactate as the main end products. Syntrophic growth occurred with glucose, pyruvate and 3,4,5-trimethoxybenzoate in the presence of an H 2 -scavenging partner, and growth on 3,4,5-trimethoxybenzoate was only observed under syntrophic condition. The predominant cellular fatty acids were C 16:0, iso-C 16:0 , anteiso-C 15:0 , and iso-C 14:0 . Respiratory quinone was not detected. The genomic G+C content was 40.8mol%. Based on 16S rRNA gene phylogeny, strain NRmbB1 T belongs to a distinct order-level clade in the class Clostridia of the phylum Firmicutes, sharing low similarity with other isolated organisms (i.e., 87.5% for top hit Moorella thermoacetica DSM 2955 T ). In total, chemotaxonomic, phylogenetic and genomic characterization revealed that strain NRmbB1 T (=KCTC 25035 T , =JCM 39120 T ) represents a novel species of a new genus. In addition, we also propose the associated family and order as Koleobacteraceae fam. nov and Koleobacterales ord. nov., respectively., (Copyright © 2020. Published by Elsevier GmbH.)

Two nitrogen-fixing and heavy oil degrading strains, designated RWY-5-1-1 T and ROY-1-1-2, were isolated from an oil production mixture from Yumen Oilfield in China. The 16S rRNA gene sequence showed they belong to Azospirillum and have less than 96.1 % pairwise similarity with each species in this genus. The average nucleotide identity and digital DNA-DNA hybridization values between them and other type strains of Azospirillum species were less than 75.69 % and 22.0 %, respectively, both below the species delineation threshold. Pan-genomic analysis showed that the novel isolate RWY-5-1-1 T shared 2145 core gene families with other type strains in Azospirillum, and the number of strain-specific gene families was 1623, almost two times more than the number known from other species. Furthermore, genes related to nitrogenase, hydrocarbon degradation and biosurfactant production were found in the isolates' genomes. Also, this strain was capable of reducing acetylene to ethylene at a rate of 22nmol ethylene h -1 (10 8 cells) and degrading heavy oil at a rate of 36.2 %. The major fatty acids and polar lipids were summed feature 8 (C 18:1 ω7c/C 18:1 ω6c), and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. Furthermore, a combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strains RWY-5-1-1 T and ROY-1-1-2 represent a novel species, for which the name Azospirillum oleiclasticum sp. nov. is proposed. The type strain is RWY-5-1-1 T (=CGMCC 1.13426 T =KCTC 72259 T ). Azospirillum novel strains with the ability of heavy oil degradation associated with the promotion of plant growth has never been reported to date., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Produced water (PW) is a by-product generated throughout oil exploration. Geological formation and geographical location of the reservoir influence its physical, chemical and biological characteristics. Xanthan gum (XG), an exopolysaccharide (EPS) produced by Xanthomonas campestris, has been widely used in enhanced oil recovery (EOR) technology because of its high viscosity, pseudoplastic behavior, stability in function of salinity, temperature and alkaline conditions. The production of XG may be affected by the composition of the PW, where the acetyl and pyruvyl radicals may be present in the mannoses. The aim of this study was to evaluate the composition of XG produced by X. campestris, particularly the amount of Xanthan, acetyl and pyruvyl groups, in culture mediums containing distilled (DW) or produced (PW) water in different concentrations, by means of dispersive Raman spectroscopy (1064 nm). The spectra of XG showed peaks referred to the main constituents of the Xanthan (glucose, mannose and glucuronic acid). Spectral features assigned to pyruvyl were seen in all samples mainly at ~1010 cm -1 , with higher intensity when using DW and 25% PW. PCA loadings showed that the peaks assigned to pyruvyl are consistent to presence of sodium pyruvate (~1040/~1050 and ~ 1432 cm -1 ) and were higher in the samples obtained in 25% PW. ANOVA GLM applied to Raman peaks of interest (~1010 and ~ 1090 cm -1 ) and to PCA scores (Score 1 to Score 3) showed that both were influenced by the type of water used in the culture medium, where the XG were strongly reduced in the groups PW compared to DW while the pyruvyl content increased proportionally with the concentration of PW. The results suggest that the composition of the water used in the bacteria's culture medium influenced the composition of XG, including the amount of Xanthan and particularly the pyruvyl content, and therefore needs to be considered when using this approach of injecting XG in oil fields as pyruvyl content affects viscosity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A non-motile, straight-rod-shaped, Gram-stain-positive and facultative anaerobic bacterium (i . e., strain G1 T ) was isolated from production waters from an Algerian oilfield. Growth was observed in the presence of 0.3-3.5 % (w/v) NaCl, at 20-50 °C and at pH 6.0-9.0. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that strain G1 T belonged to the genus Microbacterium . Strain G1 T was closely related to Microbacterium oxydans (DSM 20578 T ) and Microbacterium maritypicum (DSM 12512 T ) with 99.8 % sequence similarity and to Microbacterium saperdae (DSM 20169 T ) with 99.6 % sequence similarity. Strain G1 T contained MK9, MK10, MK11, MK12 and MK13 as respiratory quinones, and phosphatidylglycerol, diphosphatidylglycerol and glycolipid as the major polar lipids. The major cellular fatty acids were anteiso-C 15:0 , iso-C 16:0 and anteiso-C 17:0 . The estimated DNA G+C content was 69.57 mol% based on its draft genome sequence. Genome annotation of strain G1 T predicted the presence of 3511 genes, of which 3483 were protein-coding and 47 were tRNA genes. The DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) values between strain G1 T and M. oxydans (DSM 20578 T ) and M. maritypicum (DSM 12512 T ) were in both cases far below the respective species boundary thresholds (27.5 and 28.0 % for DDH; and 84.40 and 84.82% for ANI, respectively). Based on the data presented above, strain G1 T was considered to represent a novel species for which the name Microbacterium algeriense is proposed with the type strain G1 T (=DSM 109018 T =LMG 31276 T ).

Synthetic biology and adaptive laboratory evolution are key tools for developing biotechnology platforms for the remediation of oil sands tailings. However, field deployment and subsequent regulation of engineered and/or evolved strains is rife with uncertainties and risks. Here, we detail an innovation strategy to derisk and deploy engineered bioremediation platforms., (Copyright © 2020. Published by Elsevier Ltd.)

Temperature is supposed to be one of the primary drivers for the bacterial diversification as well as hydrocarbon formation process of oil reservoirs. However, the bacterial community compositions are not systematically elucidated in oil reservoirs with different temperatures. Herein, the diversity of indigenous bacteria and the functional species in the water samples from oil reservoirs with different in situ temperatures was investigated by high-throughput sequencing technology. The results showed that samples in the high (65 °C) and super high (80 °C) temperature oil reservoir had significantly high bacterial richness, even more than twice as much as moderate temperature (36 °C) ones, which showed relatively high bacterial diversity. Meanwhile, the bacterial compositions were almost similar in the high temperature oil reservoirs but there were different relative abundances of the bacterial communities. Phylogenetic analysis revealed that indigenous bacteria fell into 20 phylotypes in which Proteobacteria were the principal phylum in all of samples. At the genus level, 10 out of 22 major genera displayed statistically significant differences. Among of them, Pseudomonas was extremely dominant in all of samples, while Halomonas, Caldicoprobacter, Arcobacter, and Marinobacter tended to be enriched in the high temperature oil reservoirs. Moreover, the abundance of bacterial populations exhibited important distinction in oil reservoir such as hydrocarbon-oxidizing, fermentative, nitrate-reducing, sulfate-reducing, and methanogenic bacteria. Those bacteria were strongly correlated to in situ temperature variation.

Anaerobic, fermentative, halotolerant bacteria, strains 4-11 T and 585, were isolated from production water of two low-temperature petroleum reservoirs (Russia) and were characterized by using a polyphasic approach. Cells of the strains were spherical, non-motile and 0.30-2.5 µm in diameter. Strain 4-11 T grew optimally at 35 °C, pH 6.0 and 1.0-2.0% (w/v) NaCl. Both strains grew chemoorganotrophically with mono-, di- and trisaccharides. The major cellular fatty acids of both strains were C 14:0 , C 16:0 , C 16:1 ω9 and C 18:0 3-OH. Major polar lipids were glycolipids and phospholipids. The 16S rRNA gene sequences of the strains 4-11 T and 585 had 99.9% similarity and were most closely related to the sequence of Sphaerochaeta associata GLS2 T (96.9, and 97.0% similarity, respectively). The G+C content of the genomic DNA of strains 4-11 T and 585 were 46.8 and 46.9%, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 4-11 T and S. associata GLS2 T were 73.0 and 16.9%, respectively. Results of phylogenomic metrics analysis of the genomes and 120 core proteins of strains 4-11 T and 585 and their physiological and biochemical characteristics confirmed that the strains represented a novel species of the genus Sphaerochaeta , for which the name Sphaerochaeta halotolerans sp. nov. is proposed, with the type strain 4-11 T (=VKM B-3269 T =KCTC 15833 T ). Based on the results of phylogenetic analysis, Sphaerochaeta coccoides was reclassified as member of a new genus Parasphaerochaeta gen. nov., Parasphaerochaeta coccoides comb. nov. The genera Sphaerochaeta and Parasphaerochaeta form a separate clade, for which a novel family, Sphaerochaetaceae fam. nov., is proposed.

A Gram-negative, aerobic, motile, non-spore-forming and rod-shaped bacterium, designated strain B18-69  T , was isolated from oil-well production liquid in Baolige oilfield, China. The strain was able to grow at pH 6-9.5 (optimum at pH 7), in 0-4% (w/v) NaCl (optimum at 0.5-1%, w/v) and at 35-60 °C (optimum at 55 °C). Major cellular fatty acids were C 16:0 , C 19:0 cyclo ω8c, C 17:0 cyclo and C 18:1 ω7c. The predominant respiratory quinone was ubiquinone 8. Major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B18-69  T was most closely related to Tepidiphilus margaritifer DSM 15129  T (98.8% similarity). The draft genome of strain B18-69  T was composed of 2,250,419 bp, and the G+C content was 64.6 mol%. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain B18-69  T and T. margaritifer DSM 15129  T were 90.9% and 68.9%, respectively. Genotypic and phenotypic features indicate that strain B18-69  T represents a novel species of the genus Tepidiphilus, for which the name Tepidiphilus baoligensis sp. nov. is proposed. The type strain is B18-69  T (= CGMCC 1.13573  T  = KCTC 62782  T ).

Strain 16W4-4-3  T was isolated from the oil-well production water in Qinghai Oilfield, China. Cells were Gram-stain-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile by single polar flagellum. The 16S rRNA gene sequences of strain 16W4-4-3  T showed the highest similarities with Pseudomonas profundi M5 T (98.8%), P. pelagia CL-AP6 T (98.0%), P. salina XCD-X85 T (97.7%), and P. sabulinigri J64 T (97.5%). The phylogenetic trees based on multilocus sequence analyses with concatenating 16S rRNA, gyrB, rpoD and rpoB genes suggested that this strain should be affiliated to the genus Pseudomonas but remotely related from other species. In addition, whole genome analyses revealed that the digital DNA-DNA hybridization values and average nucleotide identities of strain 16W4-4-3  T against its close relatives were all below 28.8% and 86.5%, respectively. Furthermore, the isolate had totally different whole cell protein profile as compared to those of other species. Major fatty acids were summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c), C 16:0 , summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) and C 17:0 cyclo. Major isoprenoid quinone was ubiquinone (Q-9), and major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The DNA G + C content was 58.5 mol%. Therefore, phenotypic, phylogenetic, genomic, chemotaxonomic, and proteomic traits showed that the isolate represented a novel species of the genus Pseudomonas, the name Pseudomonas saliphila sp. nov. is proposed. Type strain is 16W4-4-3  T (= CGMCC 1.13350  T  = KCTC 72619  T ).

A novel, moderately thermophilic, Gram-stain-negative bacterium, designated strain J18 T , was isolated from a water-flooded oil reservoir. Cells were aerobic, oxidase- and catalase-positive, with a polar flagellum. Growth occurred at 35-60 °C and at pH 6-8.5. The respiratory quinones were ubiquinone 8 and ubiquinone 9. The dominant cellular fatty acids were C 16 : 0 , C 17 : 0 cyclo, C 19 : 0 cyclo ω 8 c and summed feature 8 (C 18 : 1  ω 7 c /C 18 : 1  ω 6 c ). The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, an unidentified phospholipid and an unidentified aminophospholipid. The strain showed the highest 16S rRNA gene sequence similarities to Tepidiphilus margaritifer DSM 15129 T (98.6 %), Tepidiphilus succinatimandens DSM 15512 T (98.4 %) and Tepidiphilus thermophilus DSM 27220 T (98.1 %), respectively, and the similarity to other species was lower than 93 %. In the phylogenetic trees, it constituted a unique sub-cluster within the genus Tepidiphilus . The DNA G+C content of strain J18 T was 64.44 mol%. As compared with the type strains, the genome-to-genome distances of strain J18 T were 34.7-40 %. These results confirmed the separate species status of J18 T with its close relatives. On the basis of physiological, chemotaxonomic and phylogenetic analyses along with the low levels of identity at the whole-genome level, it can be concluded that strain J18 T represents a new species of the genus Tepidiphilus , for which the name Tepidiphilus olei sp. nov. is proposed. The type strain of T. olei is J18 T (=CGMCC 1.16800 T =LMG 31400 T ).

A Gram-stain-negative, rod-shaped bacterium, designated XJ4 T , was isolated from oil-contaminated water, collected from Xinjiang Province, north-west PR China (45° 1' 27″ N, 85° 6' 14″ E). Growth occurred at 20-45 °C (optimum, 30 °C) and pH 6.0-10.0 (optimum, pH 6.0-7.0). Strain XJ4 T could tolerate up to 7 % (w/v) NaCl and grow optimally in the absence of NaCl. Phylogenetic analysis based on comparative sequence analysis of 16S rRNA gene sequences indicated that strain XJ4 T belonged to the genus Frigidibacter , and that was closely related to Frigidibacter mobilis cai42 T (97.2 %), Frigidibacter albus SP32 T (97.0 %) and Rhodobacter aestuarii JA296 T (97.0 %). The average nucleotide identity values between XJ4 T and three type strains were 77.9, 77.6 and 71.9 %, respectively. The DNA G+C content of strain XJ4 T was 69.5 mol%. The sole respiratory quinone was Q-10. The major cellular fatty acid was summed feature 8 (C 18 : 1   ω7 c and/or C 18 : 1   ω6 c ), C 18 : 0 and 11-methyl C 18 : 1   ω7 c . The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, an unidentified aminolipid and unidentified lipids. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain XJ4 T represents a novel species of the genus Frigidibacter , for which the name Frigidibacter oleivorans sp. nov. is proposed. The type strain is XJ4 T (=CGMCC 1.13778 T =LMG 30952 T ).

Microbial communities ultimately control the fate of petroleum hydrocarbons (PHCs) that enter the natural environment, but the interactions of microbes with PHCs and the environment are highly complex and poorly understood. Genome-resolved metagenomics can help unravel these complex interactions. However, the lack of a comprehensive database that integrates existing genomic/metagenomic data from oil environments with physicochemical parameters known to regulate the fate of PHCs currently limits data analysis and interpretations. Here, we curated a comprehensive, searchable database that documents microbial populations in natural oil ecosystems and oil spills, along with available underlying physicochemical data, geocoded via geographic information system to reveal their geographic distribution patterns. Analysis of the ~2000 metagenome-assembled genomes (MAGs) available in the database revealed strong ecological niche specialization within habitats. Over 95% of the recovered MAGs represented novel taxa underscoring the limited representation of cultured organisms from oil-contaminated and oil reservoir ecosystems. The majority of MAGs linked to oil-contaminated ecosystems were detectable in non-oiled samples from the Gulf of Mexico but not in comparable samples from elsewhere, indicating that the Gulf is primed for oil biodegradation. The repository should facilitate future work toward a predictive understanding of the microbial taxa and their activities that control the fate of oil spills., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A Gram-stain-negative, facultative aerobic, non-spore-forming, non-motile, non-flagellated, rod-shaped bacterium, designated strain NAU-18 T was isolated from an oil-contaminated soil in China. Strain NAU-18 T could grow at 10-42 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, 7.0) and in the presence of 0-2.0% (w/v) NaCl (optimum, 0.5% NaCl in R2A). The predominant fatty acids were C 18:1 ω7c (71.2%) and Summed feature 2 (5.1%), representing 76.3% of the total fatty acids. The major respiratory quinones were Q9 and Q10. The DNA G + C content of strain NAU-18 T was 61.4 mol% based on its draft genome sequence. Genome annotation of strain NAU-18 T predicted the presence of 6668 genes, of which 6588 are coding proteins and 80 are RNA genes. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NAU-18 T was a member of the genus Rhizobium and showed 96.93% (with 93.2% coverage) and 96.81% (with 100% coverage) identities with those of Neorhizobium alkalisoli CCBAU 01393 T and Rhizobium oryzicola ZYY136 T , respectively. In the phylogenetic analysis, strain NAU-18 T and R. oryzicola ZYY136 T are consistently placed in the same branch. Strain NAU-18 T represents a novel species within the genus Rhizobium, for which the name Rhizobium terrae sp. nov. is proposed, with the type strain NAU-18 T (=KCTC 62418 T  = CCTCC AB 2018075 T ).

A novel Bacillus licheniformis strain (DM-1) was isolated from a mature reservoir in Dagang oilfield of China. DM-1 showed unique properties to utilize petroleum hydrocarbons and agroindustrial by-product (molasses) for exopolysaccharide (EPS) production under oil recovery conditions. The DM-1 EPS was proven to be a proteoglycan with a molecular weight of 568 kDa. The EPS showed shear thinning properties and had high viscosities at dilute concentrations (<1%, w/v), high salinities, and elevated temperatures. Strain DM-1 could degrade long-chain n-alkanes up to C36. Viscosity reduction test have shown that the viscosity of the crude oil was reduced by 40% compared with that before DM-1 treatment. Sand pack flooding test results under simulated reservoir conditions have shown that the enhanced oil recovery efficiency was 19.2% after 7 days of in-situ bioaugmentation with B. licheniformis DM-1. The obtained results indicate that strain DM-1 is a promising candidate for in situ microbial enhanced oil recovery (MEOR).

Most of the microbial degradation in oil reservoirs is believed to take place at the oil-water transition zone (OWTZ). However, a recent study indicates that there is microbial life enclosed in microliter-sized water droplets dispersed in heavy oil of Pitch Lake in Trinidad and Tobago. This life in oil suggests that microbial degradation of oil also takes place in water pockets in the oil-bearing rock of an oil leg independent of the OWTZ. However, it is unknown whether microbial life in water droplets dispersed in oil is a generic property of oil reservoirs rather than an exotic exception. Hence, we took samples from three heavy-oil seeps, Pitch Lake (Trinidad and Tobago), the La Brea Tar Pits (California, USA), and an oil seep on the McKittrick oil field (California, USA). All three tested oil seeps contained dispersed water droplets. Larger droplets between 1 and 10 μl revealed high cell densities of up to 10 9 cells ml -1 Testing for ATP content and LIVE/DEAD staining showed that these populations consist of active and viable microbial cells with an average of 60% membrane-intact cells and ATP concentrations comparable to those of other subsurface ecosystems. Microbial community analyses based on 16S rRNA gene amplicon sequencing revealed the presence of known anaerobic oil-degrading microorganisms. Surprisingly, the community analyses showed similarities between all three oil seeps, revealing common OTUs, although the sampling sites were thousands of kilometers apart. Our results indicate that small water inclusions are densely populated microhabitats in heavy oil and possibly a generic trait of degraded-oil reservoirs. IMPORTANCE Our results confirmed that small water droplets in oil are densely populated microhabitats containing active microbial communities. Since these microhabitats occurred in three tested oil seeps which are located thousands of kilometers away from each other, such populated water droplets might be a generic trait of biodegraded oil reservoirs and might be involved in the overall oil degradation process. Microbial degradation might thus also take place in water pockets in the oil-bearing oil legs of the reservoir rock rather than only at the oil-water transition zone., (Copyright © 2020 Pannekens et al.)

Oil reservoirs have been shown to house numerous microbial lineages that differ based on the in-situ pH, salinity and temperature of the subsurface environment. Lineages of Firmicutes, including Clostridiales, have been frequently detected in oil reservoirs, but are typically not considered impactful or relevant due to their spore-forming nature. Here we show, using metagenomics, a high temperature oil reservoir of marine salinity contains a microbial population that is predominantly from within the Order Clostridiales. These organisms form an oil-reservoir specific clade based on the phylogenies of both 16S rRNA genes and ribosomal proteins, which we propose to name U Petromonas tenebris, meaning they are single-celled organisms from dark rocks. Metagenome-assembled genomes (MAGs) of these Petromonas sp. were obtained and used to determine that these populations, while capable of spore-formation, were also likely replicating in situ in the reservoir. We compared these MAGs to closely related genomes and show that these subsurface Clostridiales differ, from the surface derived genomes, showing signatures of the ability to degrade plant-related compounds, whereas subsurface genomes only show the ability to process simple sugars. The estimation of in-situ replication from genomic data suggest that U Petromonas tenebris lineages are functional in-situ and may be specifically adapted to inhabit oil reservoirs.

The purpose of the study involves the development of an anaerobic, thermophilic microbial consortium TERIK from the high temperature reservoir of Gujarat for enhance oil recovery. To isolate indigenous microbial consortia, anaerobic baltch media were prepared and inoculated with the formation water; incubated at 65°C for 10 days. Further, the microbial metabolites were analyzed by gas chromatography, FTIR and surface tension. The efficiency of isolated consortia towards enhancing oil recovery was analyzed through core flood assay. The novelty of studied consortia was that, it produces biomass (600 mg/l), bio-surfactant (325 mg/l), and volatile fatty acids (250 mg/l) at 65°C in the span of 10 days, that are adequate to alter the surface tension (70 to 34 mNm -1) and sweep efficiency of zones facilitating the displacement of oil. TERIK was identified as Clostridium sp. The FTIR spectra of biosurfactant indicate the presence of N-H stretch, amides and polysaccharide. A core flooding assay was designed to explore the potential of TERIK towards enhancing oil recovery. The results showed an effective reduction in permeability at residual oil saturation from 2.14 ± 0.1 to 1.39 ± 0.05 mD and 19% incremental oil recovery., Competing Interests: We hereby confirm that the Mr Vipin Kukreti is an employ of IRS-ONGC. There is no patent, product in development or marketed products associated with this research study. This will not alter our adherence to PLOS ONE policies on sharing data and materials.

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S 0 ) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S 0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY 1 Z 1 AB and soxCDY 2 Z 2 H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY 2 Z 2 H cluster and not soxXY 1 Z 1 AB. Despite the absence of the soxXY 1 Z 1 AB cluster, strain CVO oxidized S 0 to thiosulfate and sulfate, demonstrating that soxCDY 2 Z 2 H genes alone are sufficient for S 0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY 2 Z 2 H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A novel obligately anaerobic, thermophilic and formate-utilizing bacterium K32 T was isolated from Shengli oilfield of China. Cells were straight rods (0.4-0.8 µm × 2.5-8.0 µm), Gram-stain-positive, non-spore-forming and slightly motile. Optimum growth occurred with pH of 7 and 0.5 g l -1 NaCl under temperature of 55-60 °C. Nitrate could be reduced into nitrite, syntrophic formate oxidation to methane and carbon dioxide occurred when co-culturing strain K32 T and Methanothermobacter thermautotrophicus ΔH. The main cellular fatty acids were iso-C 15 : 0 (24.0 %), anteiso-C 15 : 0 (21.7 %), C 16 : 0 (12.7 %) and C 14 : 0 (10.8 %), and the main polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 46.3 mol%. The 16S rRNA gene sequence of K32 T shared ≤90.4 % of sequence similarity to closest type strains of Desulfitibacter alkalitolerans , Calderihabitans maritimus and members of the genus Moorella . Based on the phenotypic, biochemical and genotypic characterization, Zhaonella formicivorans gen. nov., sp. nov. is proposed with K32 T (=CCAM 584 T =DSM 107278 T =CGMCC1.5297 T ) as the type strain, which is the first representative of Zhaonellaceae fam. nov. In addition, the order Thermoanaerobacterales a nd family Peptococcaceae were reclassified, and three novel families in the novel order of Moorellales ord. nov. were also proposed.

A novel Gram-staining-negative, spiral-shaped bacterium, designated strain 64-1 T , was isolated from oil reservoir water collected from Liaohe oilfield, north-eastern China. Growth occurred at 15-55 °C and pH 6.0-10.0. The sole respiratory quinone was Q-10. The predominant cellular fatty acids were summed feature 8 (C 18 : 1  ω 7 c /C 18 : 1  ω 6 c ), C 16 : 0 and C 19 : 0 cyclo ω 8 c . The polar lipids consisted of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), an unidentified aminophospholipid (UAPL), an unidentified aminolipid (UAL) and two unidentified polar lipids (UPL). The genomic DNA G+C content of strain 64-1 T was 64.5 mol%. Strain 64-1 T shared the highest 16S rRNA gene sequence similarities with Phaeospirillum chandramohanii JA145 T (92.0 %) and Telmatospirillum siberiense 26-4b1 T (91.8 %). In the phylogenetic trees, the strain constituted a sub-cluster within the family Rhodospirillaceae . Based on the results of morphological, physiological, biochemical and phylogenetic analysis, strain 64-1 T represents a new species of a novel genus within the family Rhodospirillaceae , for which the name Oleiliquidispirillum nitrogeniifigens gen. nov., sp. nov. is proposed. The type strain is 64-1 T (=CGMCC 1.16798 T =LMG 31399 T ).

An aerobic methane oxidizing bacterium, designated XLMV4 T , was isolated from the oxic surface layer of an oil sands tailings pond in Alberta, Canada. Strain XLMV4 T is capable of growth on methane and methanol as energy sources. NH 4 Cl and sodium nitrate are nitrogen sources. Cells are Gram-negative, beige to yellow-pigmented, motile (via a single polar flagellum), short rods 2.0-3.3 µm in length and 1.0-1.6 µm in width. A thick capsule is produced. Surface glycoprotein or cup shape proteins typical of the genera Methylococcus, Methylothermus and Methylomicrobium were not observed. Major isoprenoid quinones are Q-8 and Q-7 at an approximate molar ratio of 71 : 22. Major polar lipids are phosphoglycerol and ornithine lipids. Major fatty acids are C 16 : 1  ω8+C 16 : 1  ω7 (34 %), C 16 : 1  ω5 (16 %), and C 18 : 1  ω7 (11 %). Optimum growth is observed at pH 8.0 and 25 °C. The DNA G+C content based on a draft genome sequence is 46.7 mol%. Phylogenetic analysis of 16S rRNA genes and a larger set of conserved genes place strain XLMV4 T within the class Gammaproteobacteria and family Methylococcaceae , most closely related to members of the genera Methylomicrobium and Methylobacter (95.0-97.1 % 16S rRNA gene sequence identity). In silico genomic predictions of DNA-DNA hybridization values of strain XLMV4 T to the nearest phylogenetic neighbours were all below 26 %. On the basis of the data presented, strain XLMV4 T is considered to represent a new genus and species for which the name Methylicorpusculum oleiharenae is proposed. Strain XLMV4 T (=DSMZ DSM 27269=ATCC TSD-186) is the type strain.

A Gram-stain-negative, rod-shaped bacterial strain JS15-10A1 T was isolated from oil production water. Its optimum growth was observed at 37 °C, pH 7.0, and 3% (w/v) NaCl. The 16S rRNA gene sequence of strain JS15-10A1 T showed the highest similarities with Pseudomonas parafulva CB-1 T (97.6%) and P. fulva IAM 1529 T (97.5%). In addition, phylogenetic analyses based on multilocus sequence analyses with concatenating 16S rRNA, gyrB, rpoD, and rpoB genes indicated that strain JS15-10A1 T was a member of genus Pseudomonas but discriminated from other species. Furthermore, whole-genome analyses revealed that average nucleotide identities and in silico DNA-DNA hybridization values of strain JS15-10A1 T against its closest relatives were all below 76.7% and 21.1%, respectively. The major cellular fatty acids of strain JS15-10A1 T were summed feature 8 (C 18:1 ω7c/C 18:1 ω6c), C 16:0 , C 12:0 , C 17:0 cyclo, summed feature 3 (C 16:1 ω7c/C 16:1 ω6c), C 12:0 3-OH, C 10:0 3-OH, and C 19:0 cyclo ω8c. The predominant quinone was ubiquinone Q-9. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown amino-lipid, and two unidentified lipids. The genome DNA G + C content was 60.0 mol%. On the basis of phylogenetic, phenotypic, physiological, and chemotaxonomic analyses, it can be concluded that strain JS15-10A1 T represents a novel species in genus Pseudomonas, for which the name Pseudomonas jilinensis sp. nov. is proposed. The type strain is JS15-10A1 T (= CGMCC 1.16072 T  = LMG 30036 T ).

A Gram-stain-negative, non-motile and ovoid bacterial strain, designated 4-2 T , was isolated from oil-contaminated water which was collected from Xinjiang Province, north-west PR China. The 16S rRNA gene sequence analysis showed that strain 4-2 T belonged to the genus Paracoccus . The species with highest similarity to strain 4-2 T was Paracoccus saliphilus YIM 90738 T (97.83 %), followed by ' Paracoccus siganidrum ' M26 (97.83 %) and Paracoccus endophyticus SYSUP0003 T (97.25 %). The average nucleotide identity values between 4-2 T and three type strains were 84.69, 77.88 and 74.07 %, respectively. The genomic DNA G+C content of strain 4-2 T was 61.4 mol%. Chemotaxonomical characteristic results showed that the respiratory quinone was ubiquinone Q-10 and the major fatty acids were summed feature 8 (C 18 : 1 ω 7 c or C 18 : 1 ω 6 c ) and C 19 : 0 cyclo ω 8 c . The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, unidentified phospholipids, an unidentified aminolipid and an unidentified polar lipid. The predominant polyamines were putrescine, cadaverine and spermidine. On the basis of phenotypic, chemotaxonomic and phylogenetic inferences, strain 4-2 T represents a novel species of the genus Paracoccus , for which the name Paracoccus alkanivorans sp. nov. is proposed. The type strain is 4-2 T (=CGMCC 1.13669 T =LMG 30882 T ).

We report on the characterization of a novel genomic assembly (ARYD3) recovered from formation water (17.6% salinity) and crude oil enrichment amended by isolated soy proteins (0.2%), and incubated for 100 days under anaerobic conditions at 50°C. Phylogenetic and phylogenomic analysis demonstrated that the ARYD3 is unaffiliated with all currently described bacterial phyla and candidate phyla, as evident by the low AAI (34.7%), shared gene content (19.4%), and 78.9% 16S rRNA gene sequence similarity to Halothiobacillus neapolitanus, its closest cultured relative. Genomic characterization predicts a slow-growing, non-spore forming, and non-motile Gram-negative rod. Adaptation to high salinity is potentially mediated by the production of the compatible solutes cyclic 2,3-diphosphoglycerate (cDPG), α-glucosylglycerate, as well as the uptake of glycine betaine. Metabolically, the genome encodes primarily aminolytic capabilities for a wide range of amino acids and peptides. Interestingly, evidence of propionate degradation to succinate via methyl-malonyl CoA was identified, suggesting possible capability for syntrophic propionate degradation. Analysis of ARYD3 global distribution patterns identified its occurrence in a very small fraction of Earth Microbiome Project datasets examined (318/27,068), where it consistently represented an extremely rare fraction (maximum 0.28%, average 0.004%) of the overall community. We propose the Candidatus name Mcinerneyibacterium aminivorans gen. nov, sp. nov. for ARYD3 T , with the genome serving as the type material for the novel family Mcinerneyibacteriaceae fam. nov., order Mcinerneyibacteriales ord. nov., class Mcinerneyibacteria class nov., and phylum Mcinerneyibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number VSIX00000000., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

A novel Gram-stain-positive, rod shaped and anaerobic bacterium, designated as W6 T , was isolated from Shengli oilfield in China. Strain W6 T was observed to grow from 20 to 45 °C with pH 6.5-9.0 (optimally at 40 °C and pH of 7.5) and without addition of NaCl. The major cellular fatty acids were iso-C 15 : 0 (29.1%), C 14 : 0 (27.0%) and C 16 : 0 (12.2%), and the main polar lipids were lipids (L) and aminolipids (AL). The DNA G+C content is 42.9 mol%. Based on 16S rRNA gene sequence analysis, strain W6 T showed highest similarities to Tissierella creatinini DSM 9508 T (94.9%) and Soehngenia saccharolytica DSM 12858 T (94.1%). The morphological, physiological, biochemical, phylogenetic and phylogenomic analyses demonstrated strain W6 T (CCAM 534 T =DSM 28124 T =CGMCC 1.5291 T ) represents a novel species in a new genus , for which the name Gudongella oleilytica gen. nov. sp. nov. is proposed. The family Tissierellaceae is proposed as a new family containing the genera Anaerosalibacter , Gudongella , Keratinibaculum , Soehngenia , Sporanaerobacter , Tepidimicrobium , Tissierella , Urmitella and species Clostridium ultunense based on the phylogenetic and phylogenomic analyses.

Polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are toxic contaminants of environmental concern found in process water and mature fine tailings, or tailings, from the oil sands industry. BioTiger™, a patented microbial consortium of twelve natural environmental isolates, was found to cometabolically biodegrade the NA hexanoic acid and the PAH phenanthrene in the presence of tailings. Hexanoamide was found to be produced and consumed during cometabolism of hexanoic acid. Mechanistic analysis demonstrated three of the BioTiger™ strains generated biosurfactants with the bacterial adhesion to hydrocarbons assay, seven with the methylene blue active substances assay, and nine with a hemolysis assay. Serial transfers of the BioTiger™ consortium demonstrated the stability of hexanoic acid degradation over several generations. The results demonstrate that BioTiger™ cometabolically biodegrades combinations of phenanthrene and hexanoic acid in tailings. This work reveals the potential for in situ bioremediation of tailings with this natural microbial consortium.

Aims: We aimed to explore a new Methylobacterium isolate to produce polyhydroxybutyrate (PHB) by using methanol as a sole carbon resource and improve PHB production., Methods and Results: A new PHB-producing isolate (Methylobacterium sp. 1805) was obtained from oil fields by using methanol as a sole carbon source. The fermentation situation of PHB production was further optimized by using Box-Behnken response surface methodology (RSM). Before optimization, the cell biomass was 0·6 g l -1 after 3-day culture and 0·3 g l -1 PHB was produced after 5-day methanol-inducing stage. The RSM growth medium was optimized as 15 g l -1 glycerol, 10 g l -1 beef extract and 0·65 g l -1 MgSO 4 ·7H 2 O. The RSM methanol-inducing medium was optimized as 0·65 g l -1 MgSO 4 (metal ions), 20 mmol l -1 PBS pH 6·5 and final 2% methanol (v/v). The biomass and PHB production reached 1·0 and 0·55 g l -1 after 3-day culture, respectively. The PHB yield increased by about 80% when compared with before optimization., Conclusions: The optimization of a two-stage fermentation process improved PHB production from methanol by using Methylobacterium sp. 1805., Significance and Impact of the Study: A new Methylobacterium isolate was isolated and produced high-level PHB by using methanol as a sole carbon resource. The bacteria will provide a potential tool for C1 resource in producing PHB., (© 2019 The Society for Applied Microbiology.)

The expansion of oil sands has made remediation of oil sands process-affected water (OSPW) critical. As naphthenic acids (NAs) are the primary contributors to toxicity, remediation is required. Bioremediation by native microorganisms is potentially effective, however, toxicity of NAs towards native microorganisms is poorly understood. The aim of this study was to isolate microorganisms from OSPW, assess tolerance to stressors, including naturally sourced NAs and examine exposure effect of NAs on cell membranes. Microorganisms were isolated from OSPW, including the first reported isolation of a fungus (Trichoderma harzianum) and yeast (Rhodotorula mucilaginosa). Isolates tolerated alkaline pH, high salinity, and NA concentrations far exceeding those typical of OSPW indicating toxic effects of OSPW are likely the result of interactions between OSPW components. Comparisons of toxicity determined that OSPW exhibited higher cytotoxicity than NAs. The fungal isolate was able to grow using commercial NAs as its sole carbon source, indicating high resistance to NAs' cytotoxic effects. Future studies will focus on the organisms' ability to degrade NAs, and subsequent effects on toxicity. Characterization of OSPW constituents should be investigated with focus on the synergistic toxic effects of dissolved compounds. A better understanding of OSPW toxicity would enable more effective and targeted bioremediation schemes by native microorganisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A Gram-stain-negative, rod-shaped, chemoorganotrophic and anaerobic bacterium, strain SK-G1 T , was isolated from oily sludge sampled at the Shengli oilfield in PR China. Growth occurred with 0-30 g l -1 NaCl, at 40-65 °C and at pH 6.0-8.5. The predominant fatty acids were C 14 : 0 and C 13 : 0 , and the major cellular polar lipids were phosphatidylglycerol and phosphatidylethanolamine. No respiratory quinone was detected. The genomic G+C content was 43.9 mol%. The strain had highest 16S rRNA gene sequence similarity (93.2 % identity) to Tepidanaerobacter syntrophicus DSM 15584 T . The phylogenetic, phenotypic and chemotaxonomic data showed that strain SK-G1 T (=CCAM 530 T =KCTC 15783 T =JCM 33158 T ) represents a novel species of a new genus Biomaibacteracetigenes gen. nov., sp. nov. The results of phylogenetic and phylogenomic analyses indicated that the genera Biomaibacter , Caldanaerovirga , Fervidicola , Tepidanaerobacter , Thermosediminibacter , Thermovenabulum and Thermovorax formed a clade with high bootstrap support distinguishing to other taxon within the order Thermoanaerobacterales. This clade is proposed as Thermosediminibacterales ord. nov. and includes Tepidanaerobacteraceae fam. nov. and Thermosediminibacteraceae fam. nov. Emended descriptions of the order Thermoanaerobacterales and family Syntrophomonadaceae are also provided.

In the last decade, extensive application of hydraulic fracturing technologies to unconventional low-permeability hydrocarbon-rich formations has significantly increased natural-gas production in the United States and abroad. The injection of surface-sourced fluids to generate fractures in the deep subsurface introduces microbial cells and substrates to low-permeability rock. A subset of injected organic additives has been investigated for their ability to support biological growth in shale microbial community members; however, to date, little is known on how complex xenobiotic organic compounds undergo biotransformations in this deep rock ecosystem. Here, high-resolution chemical, metagenomic, and proteomic analyses reveal that widely-used surfactants are degraded by the shale-associated taxa Halanaerobium, both in situ and under laboratory conditions. These halotolerant bacteria exhibit surfactant substrate specificities, preferring polymeric propoxylated glycols (PPGs) and longer alkyl polyethoxylates (AEOs) over polyethylene glycols (PEGs) and shorter AEOs. Enzymatic transformation occurs through repeated terminal-end polyglycol chain shortening during co-metabolic growth through the methylglyoxal bypass. This work provides the first evidence that shale microorganisms can transform xenobiotic surfactants in fracture fluid formulations, potentially affecting the efficiency of hydrocarbon recovery, and demonstrating an important association between injected substrates and microbial growth in an engineered subsurface ecosystem.

Naphthenic acids (NAs) originate from bitumen and are considered a major contributor to acute toxicity in oil sands process-affected water (OSPW) produced from bitumen extraction processes. To reclaim oil sands tailings and remediate OSPW, in-pit fluid fine tailings can be water-capped as end pit lakes (EPL). Addressing NAs present in OSPW, either through removal, dilution or degradation, is an objective for oil sands reclamation. EPLs can remediate NAs through degradation or dilution or both. To assess and understand degradation potential, Chlorella kessleri and Botryococcus braunii were tested for their tolerance to, and ability to biodegrade, three model NAs (cyclohexanecarboxylic acid, cyclohexaneacetic acid, and cyclohexanebutyric acid). Water sourced from the industry's first EPL, the Base Mine Lake (BML), was used alone as an inoculum or co-cultured with C. kessleri to biodegrade cyclohexanecarboxylic acid and cyclohexanebutyric acid. All cultures metabolized the model compounds via β-oxidation. Biodegradation by the co-culture of C. kessleri and BML inoculum was most effective and rapid: the cyclohexaneacetic acid generated from cyclohexanebutyric acid could be further degraded by the co-culture, while the cyclohexaneacetic acid generated could not be consumed by pure algal cultures or BML inoculum alone. Adding C. kessleri greatly increased the diversity of the microbial community in the BML inoculum; many known hydrocarbon and NA degraders were identified from the 16S rRNA gene sequencing from this co-culture. This more diverse microbial community could have potential for EPL remediation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)