Global diversity and ecological functions of viruses inhabiting oil reservoirs.

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.

Appropriate characterization of reservoir properties and investigation of their effect on microbial enhanced oil recovery through simulated laboratory studies.

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.

Transitioning weathered oil fields towards new energy: A review on utilizing hydrogenotrophic methanogens for petroleum hydrocarbons remediation.

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 CO2 is a common remediation method, the complex structures and diverse types of petroleum hydrocarbons (PHs) involved can result in excessive CO2 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 H2 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.

Cultivation of novel Atribacterota from oil well provides new insight into their diversity, ecology, and evolution in anoxic, carbon-rich environments.

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.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, 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.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota.

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T 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 C16:0, iso-C15:0, and C18:0. 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 M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (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 M15T (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 M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Roseomonas oleicola sp. nov., isolated from an oil production mixture in Yumen Oilfield, and emended description of Roseomonas frigidaquae.

A pink, ovoid-shaped, Gram-stain-negative, strictly aerobic and motile bacterial strain, designated ROY-5-3T, 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-5-3T belongs to the genus Roseomonas and shared the highest pairwise similarities with Roseomonas frigidaquae CW67T (98.1%), Roseomonas selenitidurans BU-1T (97.8%), Roseomonas tokyonensis K-20T (97.7%) and Roseomonas stagni HS-69T (97.3%). The average nucleotide identity and digital DNA-DNA hybridization values between ROY-5-3T 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-5-3T 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 (C18 : 1 ω6c/C18 : 1 ω7c), summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c) and C16 : 0. Strain ROY-5-3T 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-5-3T represents a novel species of the genus Roseomonas for which the name Roseomonas oleicola sp. nov. is proposed. The type strain is ROY-5-3T (=CGMCC 1.13459T =KCTC 82484T).

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.

A review on anaerobic microorganisms isolated from oil reservoirs.

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 (H2S) 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.

Biodegradation of binary mixtures of octane with benzene, toluene, ethylbenzene or xylene (BTEX): insights on the potential of Burkholderia, Pseudomonas and Cupriavidus isolates.

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.

Long-Term Biocide Efficacy and Its Effect on a Souring Microbial Community.

Reservoir souring, which is the production of H2S 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 H2S 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.

Anaerobic biosynthesis of rhamnolipids by Pseudomonas aeruginosa: performance, mechanism and its application potential for enhanced oil recovery.

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 ß-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.

Prokaryotic community diversity during bioremediation of crude oil contaminated oilfield soil: effects of hydrocarbon concentration and salinity.

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 CO2-measuring respirometer. After the incubation, residual aliphatic hydrocarbons were quantified and were performed 16S rRNA gene sequencing. An increase in CO2 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, CO2 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.

Isolation and characterization of a halophilic Modicisalibacter sp. strain Wilcox from produced water.

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.

Characterization of microbiologically influenced corrosion by comprehensive metagenomic analysis of an inland oil field.

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.

Isolating, identifying and evaluating of oil degradation strains for the air-assisted microbial enhanced oil recovery process.

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.

Cupidesulfovibrio liaohensis gen. nov., sp. nov., a novel sulphate-reducing bacterium isolated from an oil reservoir and reclassification of Desulfovibrio oxamicus and Desulfovibrio termitidis as Cupidesulfovibrio oxamicus comb. nov. and Cupidesulfovibrio termitidis comb. nov.

A novel sulphate-reducing, Gram-stain-negative, anaerobic strain, isolate XJ01T, recovered from production fluid at the LiaoHe oilfield, PR China, was the subject of a polyphasic study. The isolate together with Desulfovibrio oxamicus NCIMB 9442T and Desulfovibrio termitidis DSM 5308T 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 XJ01T represents a novel genus and species for which the name Cupidesulfovibrio liaohensis gen. nov., sp. nov. is proposed with the type strain XJ01T (=CGMCC 1.5227T=DSM 107637T). It is also proposed that D. oxamicus and D. termitidis be reclassified as Cupidesulfovibrio oxamicus comb. nov. and Cupidesulfovibrio termitidis comb. nov., respectively.

Azospirillum oleiclasticum sp. nov, a nitrogen-fixing and heavy oil degrading bacterium isolated from an oil production mixture of Yumen Oilfield.

Two nitrogen-fixing and heavy oil degrading strains, designated RWY-5-1-1T 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-5-1-1T 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 (108 cells) and degrading heavy oil at a rate of 36.2 %. The major fatty acids and polar lipids were summed feature 8 (C18:1ω7c/C18:1ω6c), and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. Furthermore, a combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strains RWY-5-1-1T and ROY-1-1-2 represent a novel species, for which the name Azospirillum oleiclasticum sp. nov. is proposed. The type strain is RWY-5-1-1T (=CGMCC 1.13426T =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.

Koleobacter methoxysyntrophicus gen. nov., sp. nov., a novel anaerobic bacterium isolated from deep subsurface oil field and proposal of Koleobacteraceae fam. nov. and Koleobacterales ord. nov. within the class Clostridia of the phylum Firmicutes.

An anaerobic thermophilic, rod-shaped bacterium possessing a unique non-lipid sheathed-like structure enveloping a single-membraned cell, designated strain NRmbB1T 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 H2-scavenging partner, and growth on 3,4,5-trimethoxybenzoate was only observed under syntrophic condition. The predominant cellular fatty acids were C16:0, iso-C16:0, anteiso-C15:0, and iso-C14:0. Respiratory quinone was not detected. The genomic G+C content was 40.8mol%. Based on 16S rRNA gene phylogeny, strain NRmbB1T 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 2955T). In total, chemotaxonomic, phylogenetic and genomic characterization revealed that strain NRmbB1T (=KCTC 25035T, =JCM 39120T) 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.

Microbacterium algeriense sp. nov., a novel actinobacterium isolated from Algerian oil production waters.

A non-motile, straight-rod-shaped, Gram-stain-positive and facultative anaerobic bacterium (i.e., strain G1T) 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 G1T belonged to the genus Microbacterium. Strain G1 T was closely related to Microbacterium oxydans (DSM 20578T) and Microbacterium maritypicum (DSM 12512T) with 99.8 % sequence similarity and to Microbacterium saperdae (DSM 20169T) 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-C15:0, iso-C16:0 and anteiso-C17:0. The estimated DNA G+C content was 69.57 mol% based on its draft genome sequence. Genome annotation of strain G1T 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 G1T and M. oxydans (DSM 20578T) and M. maritypicum (DSM 12512T) 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 G1T was considered to represent a novel species for which the name Microbacterium algeriense is proposed with the type strain G1T (=DSM 109018T=LMG 31276T).

Composition of Xanthan gum produced by Xanthomonas campestris using produced water from a carbonated oil field through Raman spectroscopy.

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.