Unraveling the functional instability of bacterial consortia in crude oil degradation via integrated co-occurrence networks.

Introduction: Soil ecosystems are threatened by crude oil contamination, requiring effective microbial remediation. However, our understanding of the key microbial taxa within the community, their interactions impacting crude oil degradation, and the stability of microbial functionality in oil degradation remain limited. Methods: To better understand these key points, we enriched a crude oil-degrading bacterial consortium generation 1 (G1) from contaminated soil and conducted three successive transfer passages (G2, G3, and G4). Integrated Co-occurrence Networks method was used to analyze microbial species correlation with crude oil components across G1-G4. Results and discussion: In this study, G1 achieved a total petroleum hydrocarbon (TPH) degradation rate of 32.29% within 10 days. Through three successive transfer passages, G2-G4 consortia were established, resulting in a gradual decrease in TPH degradation to 23.14% at the same time. Specifically, saturated hydrocarbon degradation rates ranged from 18.32% to 14.17% among G1-G4, and only G1 exhibited significant aromatic hydrocarbon degradation (15.59%). Functional annotation based on PICRUSt2 and FAPROTAX showed that functional potential of hydrocarbons degradation diminished across generations. These results demonstrated the functional instability of the bacterial consortium in crude oil degradation. The relative abundance of the Dietzia genus showed the highest positive correlation with the degradation efficiency of TPH and saturated hydrocarbons (19.48, 18.38, p < 0.05, respectively), Bacillus genus demonstrated the highest positive correlation (21.94, p < 0.05) with the efficiency of aromatic hydrocarbon degradation. The key scores of Dietzia genus decreased in successive generations. A significant positive correlation (16.56, p < 0.05) was observed between the Bacillus and Mycetocola genera exclusively in the G1 generation. The decline in crude oil degradation function during transfers was closely related to changes in the relative abundance of key genera such as Dietzia and Bacillus as well as their interactions with other genera including Mycetocola genus. Our study identified key bacterial genera involved in crude oil remediation microbiome construction, providing a theoretical basis for the next step in the construction of the oil pollution remediation microbiome.

Complications and long-term in-stent restenosis of endovascular treatment of severe symptomatic intracranial atherosclerotic stenosis and relevant risk factors.

To investigate the complications and in-stent restenosis of endovascular treatment of severe symptomatic intracranial atherosclerotic stenosis and relevant risk factors. Three hundred and fifty-four consecutive patients with intracranial atherosclerotic stenosis (70%-99%) were retrospectively enrolled. The clinical data, treatment outcomes, complications and in-stent restenosis at follow-up were analyzed. The endovascular treatment was composed of balloon dilatation only in 21 (5.93%) patients, and deployment of self-expandable stents in 232 (65.54%), balloon-expandable stents in 75 (21.19%), and both balloon- and self-expandable stents in 26 (7.34%), with a total of 359 stents being successfully deployed at the stenotic location. After treatment, the residual stenosis ranged 9.2%±1.5% (range 7%-19%), which was significantly (P < .05) smaller than that before treatment. Periprocedural complications occurred in 43 patients with a complication rate of 12.15% including arterial dissection in 4 (1.13%) patients, new cerebral infarction in 21 (5.93%), cerebral hemorrhage in 12 (3.3%), and subarachnoid hemorrhage in 6 (1.69%). Hyperlipidemia [odds ratio (OR) 10.35, 95% confidence interval (CI) 4.42-24.28, and P < .0001] and location at the middle cerebral artery (MCA) (OR 4.15, 95% CI 1.92-8.97, and P < .001) were significant (P < .05) risk factors for periprocedural complications, whereas hyperlipidemia (OR 11.28, 95% CI 4.65-30.60, and P < .0001), location at the MCA (or 5.26, 95% CI 2.03-15.08, and P < .001), and angulation (OR 1.02, 95% CI 1.00-1.04, and P = .02) were significant (P < .05) independent risk factors for periprocedural complications. Follow-up was performed in 287 (81.07%) patients at 6 to 36 (28 ± 6.7) months. In-stent restenosis was present in 36 (12.54%), and female sex (OR 2.53, and 95% CI 1.27-5.06) and periprocedural complications (OR 9.18, and 95% CI 3.52-23.96) were significant (P < .05) risk factors for in-stent restenosis, with periprocedural complication (OR 9.61, and 95% CI 3.48-27.23) as the only significant (P < .0001) independent risk factor for in-stent restenosis. A certain rate of periprocedural complications and in-stent stenosis may occur in endovascular treatment of severe intracranial stenosis, and the relevant risk factors may include hyperlipidemia, MCA location, angulation at the stenosis and female sex.

Circ VRK1/microRNA-17/PTEN axis modulates the angiogenesis of human brain microvascular endothelial cells to affect injury induced by oxygen-glucose deprivation/reperfusion.

BACKGROUND: Circular RNAs (circRNAs) can act as microRNA (miRNA) sponges, thus regulating gene expression. The role of circRNAs in the process of oxygen-glucose deprivation/reoxygenation (OGD/R) is unclear. Here, we explored the mechanism underlying Circ VRK1 in human brain microvascular endothelial cells (HBMVECs) injury induced by OGD/R. METHODS: The OGD/R cell model was established in HBMVECs. The microarray was applied to detect differentially expressed circRNAs, followed by subcellular fractionation assay. Colony formation assay, flow cytometry, ELISA, tube formation, Transwell and western blot assays were performed for loss-of-function assay. HE staining, TTC staining, immunohistochemistry and western blot were performed in an established mouse model. The relationships between Circ VRK1 and miR-17, and between miR-17 and PTEN were detected by bioinformatics and dual-luciferase assays. Rescue experiments were conducted in vitro and in vivo, and PI3K/AKT activity was detected by Western Blot. RESULTS: Circ VRK1, predominantly present in the cytoplasm of cells, was upregulated in the HBMVECs exposed to OGD/R. Circ VRK1 downregulation decreased proliferation, migration, tube formation, inflammatory factors and oxidative stress, while increased apoptosis in HBMVECs. Moreover, Circ VRK1 silencing reduced neurological damage, cerebral infarct size, CD34-positive cell counts and VEGF expression in mice. Circ VRK1 mediated PTEN expression and the PI3K/AKT pathway by targeting miR-17. Deletion of miR-17 inhibited the effects of Circ VRK1 siRNA, and silencing of PTEN suppressed the effects of miR-17 inhibitor. CONCLUSION: Circ VRK1 was upregulated during OGD/R. Circ VRK1 downregulation regulates PTEN expression by targeting miR-17, thereby promoting PI3K/AKT pathway activity to alleviate OGD/R injury.

Middle cerebral arterial bifurcation aneurysms are associated with bifurcation angle and high tortuosity.

PURPOSE: To investigate the association of middle cerebral artery (MCA) bifurcation aneurysms with bifurcation morphology. MATERIALS AND METHODS: 205 patients were enrolled, including 61 patients with MCA bifurcation aneurysms and 144 non-aneurysmal subjects. Aneurysmal cases were divided into types C (aneurysm neck on extension of the parent artery centerline) and D (deviating neck). The radius of the parent artery M1 (RP) and bilateral branches (RS and RL, respectively), smaller (φS) and larger (φL) lateral angles, bifurcation angle, and arterial tortuosity from parent vessel to bilateral branches (TS and TL, respectively) were analyzed. Logistic regression and receiver operator characteristic (ROC) curve analysis were performed to identify risk factors and predictive values for MCA aneurysm presence and types. RESULTS: In aneurysmal MCA bifurcations, bifurcating angle, TS, TL and RL were significantly larger (P<0.01), while φS was significantly smaller (P<0.001) than those in controls. The bifurcation angle, TS and LogitP were better morphological parameters for predicting MCA aneurysm presence with the AUC of 0.795, 0.932 and 0.951, respectively. Significant (P<0.05) differences were observed in the bifurcation angle, φL, RP, RL and TL between types C and D aneurysmal bifurcations. TL was an independent factor in discriminating types C from D aneurysms with an AUC of 0.802. CONCLUSIONS: Bifurcation angle and arterial tortuosity from the parent artery to the branch forming a smaller angle with the parent artery have a higher value in distinguishing MCA aneurysmal from non-aneurysmal ones, and the tortuosity from the parent artery to the contralateral branch is the best indicator for distinguishing types C from D aneurysmal bifurcations.

Viral Abundance and Diversity of Production Fluids in Oil Reservoirs.

Viruses are widely distributed in various ecosystems and have important impacts on microbial evolution, community structure and function and nutrient cycling in the environment. Viral abundance, diversity and distribution are important for a better understanding of ecosystem functioning and have often been investigated in marine, soil, and other environments. Though microbes have proven useful in oil recovery under extreme conditions, little is known about virus community dynamics in such systems. In this study, injection water and production fluids were sampled in two blocks of the Daqing oilfield limited company where water flooding and microbial flooding were continuously used to improve oil recovery. Virus-like particles (VLPs) and bacteria in these samples were extracted and enumerated with epifluorescence microscopy, and viromes of these samples were also sequenced with Illumina Hiseq PE150. The results showed that a large number of viruses existed in the oil reservoir, and VLPs abundance of production wells was 3.9 ± 0.7 × 108 mL-1 and virus to bacteria ratio (VBR) was 6.6 ± 1.1 during water flooding. Compared with water flooding, the production wells of microbial flooding had relative lower VLPs abundance (3.3 ± 0.3 × 108 mL-1) but higher VBR (7.9 ± 2.2). Assembled viral contigs were mapped to an in-house virus reference data separate from the GenBank non-redundant nucleotide (NT) database, and the sequences annotated as virus accounted for 35.34 and 55.04% of total sequences in samples of water flooding and microbial flooding, respectively. In water flooding, 7 and 6 viral families were identified in the injection and production wells, respectively. In microbial flooding, 6 viral families were identified in the injection and production wells. The total number of identified viral species in the injection well was higher than that in the production wells for both water flooding and microbial flooding. The Shannon diversity index was higher in the production well of water flooding than in the production well of microbial flooding. These results show that viruses are very abundant and diverse in the oil reservoir's ecosystem, and future efforts are needed to reveal the potential function of viral communities in this extreme environment.

Enhanced rhamnolipids production in Pseudomonas aeruginosa SG by selectively blocking metabolic bypasses of glycosyl and fatty acid precursors.

OBJECTIVE: To enhance rhamnolipids production in Pseudomonas aeruginosa, an optimization strategy based on selectively blocking the metabolic bypass that competed precursors with rhamnolipids biosynthesis pathway, containing exopolysaccharide (Psl and Pel) and polyhydroxyalkanoates (PHA) synthesis pathways. RESULTS: Blocking the synthesis of Psl and PHA by genes knockout, both mutants P. aeruginosa SG ∆pslAB and P. aeruginosa SG ∆phaC1DC2 can grow normally in fermentation medium and increase the production of rhamnolipids by 21% and 25.3%, respectively. While blocking the synthesis of Pel, the cell growth of the mutant strain P. aeruginosa SG ∆pelA was inhibited, thus its production yield of rhamnolipids was also decreased by 39.8%. In addition, simultaneously blocking the synthesis of Psl and PHA, a double mutant strain P. aeruginosa SG ∆pslAB ∆phaC1DC2 was constructed. Rhamnolipids production was significantly increased in strain SG ∆pslAB ∆phaC1DC2 by 69.7%. CONCLUSION: Through selectively blocking metabolic bypasses, increasing the amount of glycosyl and fatty acid precursors can significantly enhance rhamnolipids production in P. aeruginosa.

[Effect of N2O produced by indigenous denitrifiers in oil reservoir on the physical properties of crude oil]. / 油藏本源反硝化功能菌的产气作用(N2O)及对原油物性的影响.

The success of microbial enhanced oil recovery (MEOR) relies on complex microbial processes. Nevertheless, the contribution and mechanism of in-situ denitrification to microbial oil recovery remain unclear. In this study, eight denitrifying bacterial strains, designated T1, D1, D44, D46, D15, S1, S2 and S6, were isolated from the produced water of Xinjiang Oilfield, China, by a double layered plate method. The16S rDNA gene sequences of these denitrifying strains shared 100% similarity with Pseudomonas stutzeri (T1, D1, and D44), Pseudomonas putida (D46 and D15), and Pseudomonas aeruginosa (S1, S2, S6), respectively. The N2O production effects of these strains on the physical properties of crude oil were evaluated with batch experiment. Results showed that the highest total gas yield was observed with sucrose as carbon source, and the maximal concentration of N2O occurred with glycerol as carbon source. The denitrification process by these bacterial strains led to volume expansion and viscosity reduction of crude oil. Crude oil expansion rate was positively correlated with the concentration of N2O, with a correlation coefficient of 0.983, but not correlated with the volume of total gas production. Strain S1, S2, and S6 produced 530-730 mg·L-1 of surfactant using glycerol as ole carbon source, which could reduce surface tension and emulsify crude oil. However, these surfactant-producing strains produced less N2O, exhibited weaker effects on oil swelling and viscosity reduction, compared to the none-surfactant-producing denitrifying strains. Our results suggested that more attention should be paid to the ability of N2O production by denitrifying bacteria when exploiting microbial resources towards enhancing oil recovery.

Comparative studies on the structural composition, surface/interface activity and application potential of rhamnolipids produced by Pseudomonas aeruginosa using hydrophobic or hydrophilic substrates.

Pseudomonas aeruginosa SG produced 7.06 g/L and 10.32 g/L of rhamnolipids using glucose or soybean oil. Rhamnolipids produced from glucose contained 8 mono-rhamnolipids homologues and 6 di-rhamnolipids homologues with ratio of 61.46%:38.54%. Rhamnolipids produced from soybean oil contained 9 mono-rhamnolipids and 8 di-rhamnolipids homologues with ratio of 58.68%:41.32%. The main homologues produced from soybean oil possessed longer carbon chain of fatty acid. Rhamnolipids produced from glucose decreased surface tension to 26.3 mN/m with critical micelle concentration (CMC) of 50 mg/L; rhamnolipids produced from soybean oil reduced surface tension to 28.1 mN/m with CMC of 60 mg/L. Two rhamnolipids products were thermo-stable and halo-tolerant. Two rhamnolipids products efficiently cleaned oily sludge. Rhamnolipids produced from glucose possessed better surface activity and antimicrobial activity. Rhamnolipids produced from soybean oil exhibited greater emulsifying activity (EI24 = 76.1%) to better reduce oil viscosity. Different substrates make P. aeruginosa produce diverse rhamnolipids with distinct application potential.

Production of rhamnolipids with different proportions of mono-rhamnolipids using crude glycerol and a comparison of their application potential for oil recovery from oily sludge.

The use of efficient green cleaning agents, such as biosurfactants, is important in oil sludge treatment. Enhanced oil recovery from oily sludge by different rhamnolipids was comparatively evaluated. Using crude glycerol, the wild-type strain Pseudomonas aeruginosa SG and the recombinant strains P. aeruginosa PrhlAB and P. stutzeri Rhl produced 1.98 g L-1, 2.87 g L-1 and 0.87 g L-1 of rhamnolipids, respectively. The three bacterial strains produced different rhamnolipid mixtures under the same conditions. The proportions of mono-rhamnolipids in the three rhamnolipid products were 55.92%, 94.92% and 100%, respectively. These rhamnolipid products also possessed different bioactivities. Emulsifying activity became higher as the proportion of mono-rhamnolipids increased. The three rhamnolipid products were stable at temperatures lower than 121 °C, pH values from 5-11 and NaCl concentrations from 0-15%. All three rhamnolipid products could recover oil from oily sludge, but oil recovery efficiency was positively related to the proportion of mono-rhamnolipids. Mono-rhamnolipids produced by the recombinant strain Rhl exhibited the best oil recovery efficiency (53.81%). The results reveal that mono-rhamnolipids are the most promising for oil recovery from oily sludge.

[A Thermotolerant and Halotolerant Sulfate-reducing Bacterium in Produced Water from an Offshore High-temperature Oilfield in Bohai Bay, China: Isolation, Phenotypic Characterization, and Inhibition].

The growth and activity of sulfate-reducing prokaryotes (SRP) in oilfield environments could produce large amounts of H2S, leading to multifaceted problems, including oilfield souring and microbially-influenced corrosion, yet knowledge about the diversity and physiology of SRP therein was quite limited. To further understand the phenotypic characteristics of SRP residing in an offshore high-temperature oilfield at Bohai Bay, China, and to explore the potential methods for control of SRP-mediated problems, we isolated, using Hungate techniques, a thermotolerant, halotolerant SRP strain, designated BQ1, from the produced water of a high-temperature. We also presented the phenotypic features of BQ1, and investigated the efficacy of five biocides, or metabolic inhibitors, in suppressing the sulfidogenic activity of BQ1. Cells of BQ1 were motile, short rod-shaped, 1.2-2.5 µm in length and 0.5-0.8 µm in width. Although BQ1 shared 99% 16S rRNA gene sequence similarity with Desulfovibrio vulgaris Hildenborough, distinct phenotypic traits between them were observed. Isolated BQ1 could grow at 14-70℃(optimum at 30℃) and pH 6.0-9.0 (optimum pH 7.0), and in the presence of 0%-10% NaCl. Isolated BQ1 utilized a wide range of carbon substrates, including sodium formate, sodium lactate, and acetate. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors, but not nitrate or nitrite. Sodium hypochlorite (600 mg·L-1), Benzyltrimethylammonium chloride (300 mg·L-1), or nitrate (800 mg·L-1) failed to inhibit H2S production by BQ1. By contrast, glutaraldehyde (50 mg·L-1), bronopol (30 mg·L-1), chlorine dioxide (50 mg·L-1), and nitrite (70 mg·L-1) inhibited H2S production by BQ1 for at least 30 d, indicating that these compounds may be suitable for the mitigation of microbial souring in this specific, high-temperature, offshore oilfield at Bohai Bay, China.

Exopolysaccharide production by an indigenous isolate Pseudomonas stutzeri XP1 and its application potential in enhanced oil recovery.

In this study, a rapid and efficient method for screening biopolymer producers was established using 96-well plates. An indigenous biopolymer producer Pseudomonas stutzeri XP1 was isolated from Xinjiang oil reservoirs, China. Strain XP1 can grow and produce 16 g/l biopolymer using corn starch and nitrate. Produced biopolymer increased culture viscosity up to 2384 mPa s. Biopolymer showed rheological properties and pseudo-plastic behavior. The viscosity of 8 g/l biopolymer solution kept higher than 25 mPa s at 20-50 °C and pH values (5-9) and increased to 7600 mPa s with NaCl concentrations increasing to 2%. Gel permeation chromatography data showed that the biopolymer average molecular mass was 1.65 × 106 Da. Gas chromatography revealed that the monosaccharide composition in biopolymer was glucose. Core flooding experiments revealed that extra 13.56% of oil was recovered by in situ biopolymer production of strain XP1. Properties of strain XP1 and the biopolymer produced make them promising for enhanced oil recovery.

Oxygen effects on rhamnolipids production by Pseudomonas aeruginosa.

BACKGROUND: Rhamnolipids are the most extensively studied biosurfactants and has been successfully used in various areas from bioremediation to industrial fields. Rhamnolipids structural composition decide their physicochemical properties. Different physicochemical properties influence their application potential. Rhamnolipids can be produced at both aerobic conditions and anaerobic conditions by Pseudomonas aeruginosa. This study aims to evaluate the oxygen effects on the rhamnolipids yield, structural composition, physicochemical properties and the rhl-genes expression in P. aeruginosa SG. Results will guide researchers to regulate microbial cells to synthesize rhamnolipids with different activity according to diverse application requirements. RESULTS: Quantitative real-time PCR analysis revealed that rhlAB genes were down-regulated under anaerobic conditions. Therefore, strain P. aeruginosa SG anaerobically produced less rhamnolipids (0.68 g/L) than that (11.65 g/L) under aerobic conditions when grown in media containing glycerol and nitrate. HPLC-MS analysis showed that aerobically produced rhamnolipids mainly contained Rha-C8-C10, Rha-Rha-C10-C12:1 and Rha-Rha-C8-C10; anaerobically produced rhamnolipids mainly contained Rha-C10-C12 and Rha-C10-C10. Anaerobically produced rhamnolipids contained more mono-rhamnolipids (94.7%) than that (54.8%) in aerobically produced rhamnolipids. rhlC gene was also down-regulated under anaerobic conditions, catalyzing less mono-rhamnolipids to form di-rhamnolipids. Aerobically produced rhamnolipids decreased air-water surface tension (ST) from 72.2 to 27.9 mN/m with critical micelle concentration (CMC) of 60 mg/L; anaerobically produced rhamnolipids reduced ST to 33.1 mN/m with CMC of 80 mg/L. Anaerobically produced rhamnolipids emulsified crude oil with EI24 = 80.3%, and aerobically produced rhamnolipids emulsified crude oil with EI24 = 62.3%. Both two rhamnolipids products retained surface activity (ST < 35.0 mN/m) and emulsifying activity (EI24 > 60.0%) under temperatures (4-121 °C), pH values (4-10) and NaCl concentrations less than 90 g/L. CONCLUSIONS: Oxygen affected the rhl-genes expression in P. aeruginosa, thus altering the rhamnolipids yield, structural composition and physicochemical properties. Rhamnolipids produced at aerobic or anaerobic conditions was structurally distinct. Two rhamnolipids products had different application potential in diverse biotechnologies. Although both rhamnolipids products were thermo-stable and halo-tolerant, aerobically produced rhamnolipids possessed better surface activity, implying its well wetting activity and desorption property; anaerobically produced rhamnolipids exhibited better emulsifying activity, indicating its applicability for enhanced oil recovery and bioremediation of petroleum pollution.

Sphingomonas from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities

Abstract Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas -specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 105 CFUs g-1 soil. The number of yellow CFUs had a significant positive correlation (p < 0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas -specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples.

Medium factors on anaerobic production of rhamnolipids by Pseudomonas aeruginosa SG and a simplifying medium for in situ microbial enhanced oil recovery applications.

Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO4(2-) and Mg(2+) are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO3, 5.49 g/l KH2PO4, 6.9 g/l K2HPO4·3H2O and 0.40 g/l MgSO4 was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI24 = 82.5 %. The simplifying medium was promising for in situ MEOR applications.

Sphingomonas from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities.

Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas-specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 10(5)CFUsg(-1)soil. The number of yellow CFUs had a significant positive correlation (p<0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas-specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples.

Simultaneous inhibition of sulfate-reducing bacteria, removal of H2S and production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl: Applications for microbial enhanced oil recovery.

Sulfate-reducing bacteria (SRB) are widely existed in oil production system, and its H2S product inhibits rhamnolipid producing bacteria. In-situ production of rhamnolipid is promising for microbial enhanced oil recovery. Inhibition of SRB, removal of H2S and production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl were investigated. Strain Rhl can simultaneously remove S(2-) (>92%) and produce rhamnolipid (>136mg/l) under S(2-) stress below 33.3mg/l. Rhl reduced the SRB numbers from 10(9) to 10(5)cells/ml, and the production of H2S was delayed and decreased to below 2mg/l. Rhl also produced rhamnolipid and removed S(2-) under laboratory simulated oil reservoir conditions. High-throughput sequencing data demonstrated that addition of strain Rhl significantly changed the original microbial communities of oilfield production water and decreased the species and abundance of SRB. Bioaugmentation of strain Rhl in oilfield is promising for simultaneous control of SRB, removal of S(2-) and enhance oil recovery.

[Bacterial community structure and diversity in soils of different forest ages and types in Bao- tianman forest, Henan Province, China].

To compare the microbial compositions and diversities in soils of different forest ages and types in Baotianman forest, Henan Province, China, genomic DNA of forest soils was extracted for amplifying the 16S rRNA V4 hyper variable region by PCR and sequencing by Illumina MiSeq. The BIPES, UCHIME and QIIME were employed to analyze the soil bacterial community. It was shown that 60 phyla were identified, with Proteobacteria, Acidobacteria, and Verrucomicrobia representing the most dominant lineages and accounting for 29%, 18.5% and 10% of all sequences, respectively. At the genus level, 1209 genera were identified, the most abundant phylotypes were DA101 (6.3%), Acidobacteria-2 (5.9%), Candidatus Solibacter (2.9%) and Candidatus Nitrososphaera (2.6%). Different forest age and type soil samples had unique compositions and specific high and rare genus. Forest type and age both impacted the soil microbial community structure, and the influence of the former was stronger than the latter. The soil microbial diversity of the 80-year-old Quercus aliena forest was the lowest among all age and type forest soil samples. Soil pH, soil nitrogen and organic carbon contents were the most important factors affecting soil bacterial community structure.

[Construction and evaluation of an engineered bacterial strain for producing lipopeptide under anoxic conditions].

Biosurfactant-facilitated oil recovery is one of the most important aspects of microbial enhanced oil recovery (MEOR). However, the biosurfactant production by biosurfactant-producing microorganisms, most of which are aerobes, is severely suppressed due to the in-situ anoxic conditions within oil reservoirs. In this research, we successfully engineered a strain JD-3, which could grow rapidly and produce lipopeptide under anoxic conditions, by protoplast confusion using a Bacillus amyloliquefaciens strain BQ-2 which produces biosurfactant aerobically, and a facultative anaerobic Pseudomonas stutzeri strain DQ-1 as parent strains. The alignment of 16S rDNA sequence (99% similarity) and comparisons of cell colony morphology showed that fusant JD-3 was closer to the parental strain B. amyloliquefaciens BQ-2. The surface tension of culture broth of fusant JD-3, after 36-hour cultivation under anaerobic conditions, decreased from initially 63.0 to 32.5 mN · m(-1). The results of thin layer chromatography and infrared spectrum analysis demonstrated that the biosurfactant produced by JD-3 was lipopeptide. The surface-active lipopeptide had a low critical micelle concentration (CMC) of 90 mg · L(-1) and presented a good ability to emulsify various hydrocarbons such as crude oil, liquid paraffin, and kerosene. Strain JD-3 could utilize peptone as nitrogen source and sucrose, glucose, glycerin or other common organics as carbon sources for anaerobic lipopeptide synthesis. The subculture of fusant JD-3 showed a stable lipopeptide-producing ability even after ten serial passages. All these results indicated that fusant JD-3 holds a great potential to microbially enhance oil recovery under anoxic conditions.

Production of rhamnolipids by Pseudomonas aeruginosa is inhibited by H2S but resumes in a co-culture with P. stutzeri: applications for microbial enhanced oil recovery.

OBJECTIVES: Sulfate-reducing bacteria and H2S exist widely in oil production systems, and in situ production of rhamnolipids is promising for microbial enhanced oil recovery (MEOR). However, information of the effect of S(2-) on rhamnolipids production is scarce. RESULTS: Two facultative anaerobic rhamnolipids-producing bacterial strains, Pseudomonas aeruginosa SG and WJ-1, were used. Above 10 mg S(2-)/l, both cell growth and rhamnolipids production were inhibited. A large inoculum (9%, v/v) failed to completely relieve the inhibitory effect of 10 mg S(2-)/l. Below 30 mg S(2-)/l, both strains resumed rhamnolipid production through co-culturing with the denitrifying and sulphide-removing strain Pseudomonas stutzeri DQ1. CONCLUSIONS: H2S has a direct but reversible inhibitory effect on rhamnolipids production. Control of H2S in oilfields is indispensable to MEOR, and the co-culture method is effective in restoring rhamnolipid production in presence of S(2-).

[Inhibition of the activity of sulfate-reducing bacteria in produced water from oil reservoir by nitrate].

Growth and metabolic activity of sulfate-reducing bacteria (SRB) can result in souring of oil reservoirs, leading to various problems in aspects of environmental pollution and corrosion. Nitrate addition and management of nitrate-reducing bacteria (NRB) offer potential solutions to controlling souring in oil reservoirs. In this paper, a facultive chemolithotrophic NRB, designated as DNB-8, was isolated from the produced fluid of a water-flooded oil reservoir at Daqing oilfield. Then the efficacies and mechanisms of various concentrations of nitrate in combination with DNB-8 in the inhibition of the activity of SRB enriched culture were compared. Results showed that 1.0 mmol x L(-1) of nitrate or 0.45 mmol x L(-1) of nitrite inhibited the sulfate-reducing activity of SRB enrichments; the competitive reduction of nitrate by DNB-8 and the nitrite produced were responsible for the suppression. Besides, the SRB enrichment cultures showed a metabolic pathway of dissimilatory nitrate reduction to ammonium (DNRA) via nitrite. The SRB cultures could possibly alleviate the nitrite inhibition by DNRA when they were subjected to high-strength nitrate.