Accumulation and Quantitative Assessment of Terrigenous Organic Matter in Upper Oligocene to Lower Miocene Marine Source Rocks of the Qiongdongnan Basin.

The intense collision between marine and terrestrial agents results in the dual-source (marine and terrigenous) characteristics of marine source rocks. Our research quantitatively assessed terrestrial organic matter and revealed the crucial role of terrestrial organic materials in the organic matter enrichment of lower Miocene to upper Oligocene marine source rocks in the Qiongdognnan Basin. The quantitative assessment was achieved using partial least-squares analysis with eight biomarker parameters associated with n-alkanes, isoprenoids, bicadinanes, taraxerane, tricyclic terpanes, and gammacerane. Differential unloading of terrestrial organic materials based on sedimentary facies of the delta-marginal sea system were observed through oleanane and bicadinane contents. It should be noted that the diagnostic ratio of oleanane was excluded from the quantitative analysis due to the dual influence from differential unloading and contact with seawater of the terrestrial organic materials. Calculation results show that the terrestrial organic matter was highest in the delta front at 70%, followed by prodelta at 59% and inner shallow marine at 57%. From the late Oligocene to the early Miocene, the proportion of terrestrial organic matter in marine source rocks continuously increased, with the highest average value observed in the second member of the Sanya Formation at 69% and the lowest occurring in the third member of the Lingshui Formation at 46%. Increasing terrestrial organic material inputs and preservation driven by the East Asian summer monsoon provided first-order control of the accumulation of organic carbon in the Qiongdongnan Basin during late Oligocene to early Miocene, rather than the bioproductivity of marine algae. The redox conditions of the water column determine the enrichment extent of organic matter.

Influence of HEM Drilling Fluid on Organic Geochemical Characteristics of Deep-Water Source Rocks in the Qiongdongnan Basin.

The influence of oil-based drilling fluid on the geochemical characteristics of source rocks has been widely reported in the northern South China Sea. However, contamination from water-based HEM drilling fluid has long been neglected in previous deep-water petroleum exploration. To further understand the impacts of HEM drilling fluid on deep-water source rocks, the organic matter abundance and type, kerogen maceral composition, and saturated biomarkers of the deep-water source rocks in the Qiongdongnan Basin were investigated. The influence of HEM drilling fluid on the organic geochemistry data of cuttings is significant but minor for sidecores. It is evident that the organic drilling additives in HEM drilling fluid can increase the organic matter abundance and optimize the organic matter types of shale cuttings. Specifically, the total organic carbon, S1, S2, and hydrogen index are increased by 28.5 ± 6.1, 90.8 ± 2.0, 34.2 ± 2.0, and 51.9 ± 4.0%, respectively. Furthermore, the organic drilling additives will greatly enhance the levels of C29 regular steranes, especially for C29-ααα-20R sterane, with the influence still persisting even when conducting GC-MS-MS analysis. The highly abundant 17α (H) -22, 29, 30-trisnorhopane, 17ß (H)-22, 29, 30-trisnorhopane, C29-3117α (H), 21ß (H) hopanes, and C29-31 17ß (H), 21α (H) hopanes may directly originate from the organic drilling additives. It is dangerous to directly use organic geochemical data from deep-water source rock cuttings contaminated by HEM drilling fluid as it may lead to conclusions that are entirely inconsistent with the basin's geological background.

Characterization of Crude Oil, Formation Water, and Fluid Inclusions of Hydrocarbon-Bearing Strata and Their Hydrocarbon Geological Significance in the Wuliyasitai Southern Sub-sag of the Erlian Basin, China.

The hydrocarbon-bearing inclusions, formation water, and crude oil contain a wealth of information regarding the process of accumulation of oil and gas. Their research has been one of the hotspots in the field of oil and gas geology. Chemical analysis data was systematically employed for the first time to identify the properties of liquid fluids in the oil-bearing system of the Wuliyasitai southern sag in the Erlian Basin. A biased lacustrine algal input was shown by the stable carbon isotopes (δ13C) of whole hydrocarbons, and the morphological characteristics of n-alkanes, isoprenoids, steranes, and terpenes were examined by gas chromatography-mass spectrometry. The δ13C values were determined by the salinity of the sedimentary water body and input of land-sourced materials. The signatures of the n-alkanes' molar fraction arrangement suggest that water washing, crystallization, and wax precipitation are regular occurrences in oil samples. The salinity of formation water and the proportion of characteristic biomarkers (gammacerane, ß-carotene) demonstrate that the salinity of the ancient water body ranges from fresh water to brackish water. The correlation between the stratigraphic water's characteristic coefficients and the total dissolved solids (TDS) is strong, particularly for the desulfurization coefficient, which illustrates a better hydrocarbon storage environment when TDS is >6 g/L. The metamorphic coefficients demonstrate that the existing reservoir preservation circumstances are not optimal, and the TDS and concentration degree of formation water are positively connected to the increasing depth, indicating poor longitudinal connectivity. The influence of atmospheric water infiltration is responsible for the fluid inclusions' temperatures of homogenization (60 °C). However, due to deep burial, crude oil has not undergone secondary significant biodegradation despite the percolation of atmospheric water. Besides, the distribution of reservoirs is highly correlated with formation water parameters such as the TDS, which can provide scientific guidance for further exploration of oil and gas in the study area or even in the Erlian Basin.

Shift in the Mode of Carbon Cycling Recorded by Biomarkers and Carbon Isotopic Compositions in the Yanchang Formation, Ordos Basin: Autotrophy vs Heterotrophy.

Organic-rich shales and mudstones have long been investigated regarding the control of source, environment, climate, etc. on the enrichment of organic carbon. However, little is documented about how autotrophy and heterotrophy influence organic carbon cycling/export. Here, we show molecular and carbon isotopic compositional changes of the shale or mudstone source rocks from the Chang 3 to 7 members of the Yanchang Formation. The Chang 7 member source rocks have higher quality (480-500 mg/g) and total organic carbon (TOC) (15.3% on average) than other member source rocks; the sterane/hopane ratio and the δ13C of organic carbon and kerogen decrease from the Chang 3 to 7 members, but Δδ ([average δ13C of n-C17 + n-C18] - [average δ13C of pristane + phytane]) increases, and no aryl isoprenoids and C40 aromatic carotenoids (e.g., isorenieratane) were observed. These low maturity biomarker features suggest that there were no water stratification, photic zone euxinia (PZE), and no obvious change in the organic matter source, and the water column is generally anoxic. A comparison of the δ13C of Pr and Ph with the δ13C of the n-C17 and n-C18 alkanes reveals a shift in the mode of carbon cycling/export (autotrophy versus heterotrophy) in the Yanchang Formation and that there was dominant heterotrophic bacterial activity or bacterial biomass in the Chang 7 member. The TOC spike in the Chang 7 member may result from boosted carbon cycling/export that improves organic carbon preservation than other members. Possible external forcings on the shift are abundant hydrothermal- or volcanic-derived metal salts as electron acceptors in the palaeowater, which is a reasonable explanation for enhanced heterotrophic bacterial activity. This finding improves our understanding of heterotrophic bacterial activity control on organic matter (OM) preservation and may be a significant supplement for understanding the ecological or environmental forcings in the Yanchang Formation, Ordos Basin.

Geochemical Characteristics of Three Oil Families and Their Possible Source Rocks in the Sub-Sag A of Weixinan Depression, Beibuwan Basin, Offshore South China Sea.

Three oil families from the sub-sag A of the Weixinan Depression are identified by integrated analysis of physical properties, stable carbon isotopes, and gas chromatography-mass spectrometry (GC-MS). Their similarities and differences in relative thermal maturities, depositional environments, and biological sources of organic matter (OM) are investigated. A possible oil-source correlation of this area is established. Group A1 oils, defined as low-maturity oils, are characterized by high density and high viscosity. They contain more terrigenous OM deposited in a freshwater environment with unstratified water columns reflected by a relatively high terrestrial/aquatic ratio and Pr/Ph values, low abundance of C30 4-methlysteranes, and low δ13C values. They are derived from the upper hydrocarbon supply combination. Group A2 oils are characterized by moderate density and viscosity and medium stable carbon isotope values. This group of oils has lower terrestrial/aquatic ratios and Pr/Ph values and a medium concentration of C30 4-methlysteranes and δ13C values, suggesting that the oils are derived from the shales which have more contribution from the algal input and are formed in a weakly oxidizing environment. They are a mixture generated from the source rocks in the middle and lower hydrocarbon supply combination. Groups A3 oils, defined as light oils, have low density and viscosity. The geochemical data of the A3 oils, including a less-negative stable carbon isotope, high abundance of C30 4-methylsteranes, low Pr/Ph values, and highest Ts/(Ts + Tm) ratios (Ts represents C27 18α(H)-22,29,30-trisnorneohopane and Tm represents C27 17α(H)-22,29,30-trisnorhopane), suggest that they are generated from the source rocks deposited in a subanoxic environment with the large input of dinoflagellates. The A3 oils are generated from the shales from the lower hydrocarbon supply combination. The oil-source correlation results can be further supported by the distribution of faults and structural ridge as the migration channel of petroleum developed around the sub-sag A.

Origin and Geochemical Implications of Hopanoids in Saline Lacustrine Crude Oils from Huanghekou East Sag and Laizhouwan Northeastern Sag, Bohai Bay Basin.

A suite of low-mature crude oils (five high-sulfur oils and six low-sulfur oils) from the Huanghekou and the Laizhouwan Sags, Bohai Bay Basin, are analyzed to investigate the fate of the hopanoids. Abundant hopanes, such as secohopanes, 25-norhopanes, benzohopanes, aromatized secohopanes, and sulfide hopanes, are identified, and their carbon isotope compositions are determined. Varying 13C isotope values of C31 hopane (-38.7-34.0‰) and C29-30 hopanes (-38.5-31.5‰) suggest different bacterial sources of these compounds. The presence of 25-norhopanes with enriched heavy carbon isotopes in severely biodegraded oils suggests that they are microbially mediated products. The detection of the isotopically depleted C29 and C30 D-ring-8,14-secohopanes (-45.6-41.2‰) indicates that secohopanes are from methane-oxidizing bacteria (methanotrophs). The presence of isorenieratane, lower aryl isoprenoid ratios, and a good correlation between the sulfur content and the gammacerane index indicate the presence of green sulfur bacteria (Chlorobiaceae) under photic zone euxinic conditions. Water column stratification results in good preservation of the organic matter, and it is in favor of diversity of aquatic microorganisms. The ratios of C35/C34 sulfide hopane, C35 sulfide hopane-2/C35 sulfide hopane-1, and C35/C34 benzohopane are influenced by the reducing environments in this region. In addition, the D-ring monoaromatized 8,14-secohopanoid/(D-ring monoaromatized 8,14-secohopanoid + benzohopanes) and C31-C35 secomoretanes/secohopanes are affected by the maturity. We hypothesize that the reducing environments and thermal effects are important markers for the hopanoid transformation, including the incorporation of inorganic sulfur in substituting functional groups, cyclizing, aromatizing, and opening ring C of the hopanoids.

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

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

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

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

Draft genome sequence of Paenibacillus sp. strain A2.

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

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

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

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

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

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

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

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

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

Genome sequence of Brevibacillus agri strain 5-2, isolated from the formation water of petroleum reservoir.

Brevibacillus agri strain 5-2 was isolated from the formation water of a deep oil reservoir in Changqing Oilfield, China. This bacterium was found to have a capacity for degrading tetradecane, hexadecane and alkanesulfonate. To gain insights into its efficient metabolic pathway for degrading hydrocarbon and organosulfur compounds, here, we report the high quality draft genome of this strain. Two putative alkane 1-monooxygenases, one putative alkanesulfonate monooxygenase, one putative alkanesulfonate transporter, one putative sulfate permease and five putative sulfate transporters were identified in the draft genome. The genomic data of strain 5-2 may provide insights into the mechanism of microorganisms adapt to the petroleum reservoir after chemical flooding.

Genomovar assignment of Pseudomonas stutzeri populations inhabiting produced oil reservoirs.

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

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

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

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

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

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

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