[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.

A Rapid Detection Method for On-site Screening of Estazolam in Beverages with Au@Ag Core-shell Nanoparticles Paper-based SERS Substrate.

Estazolam (EST) is a common sedative-hypnotic drug with a risk of abuse. Therefore, rapid on-site detection of EST is necessary to control the abuse of EST. In this paper, a fast, simple, and sensitive method is demonstrated for the detection of EST in both water and beverages, using surface-enhanced Raman spectroscopy (SERS) techniques. Au@Ag core-shell nanoparticles (NPs) assembled on the filter paper as a SERS substrate exhibit good applicability and practicality. At the same time, density functional theory (DFT) is used to assign the vibration mode of the EST molecules, which can be used as a guide for subsequent experiments. The lowest detectable concentration of EST in aqueous solution can be as low as 5 mg/L, and signal uniformity is excellent (RSD687 = 5.56%, RSD1000 = 4.35%). In addition, EST components artificially added to orange juice and pomegranate juice can be effectively detected by simple pretreatment with a minimum detection concentration as low as 10 mg/L. Therefore, this study found that the use of Au@Ag core-shell nanoparticles paper-based SERS substrate provides a quick and easy method for the detection of illegally added drugs in beverages.

Rapid and Sensitive Surface-enhanced Raman Spectroscopy Method for Determination of Ketamine in Urine.

Surface-enhanced Raman spectroscopy (SERS) has gradually proved to be a powerful tool with wide applications in various fields. Here, a simple and rapid SERS method was developed for the determination of ketamine in urine based on silver aggregates as a SERS substrate. Ketamine in urine were demonstrated by the SERS technique with silver sol aggregated by a 0.5 M NaBr solution. The limit of detection for ketamine in urine could be obtained as low as 7.5 ppm, and a linear relationship for ketamine in urine between the Raman intensity and the concentrations was achieved in the range from 7.5 to 150 ppm (R2 = 0.977). Additionally, the recovery of this method ranged from 95.7 to 104.9%, which laid a favorable foundation for the rapid and reliable quantitative detection of ketamine in urine. Therefore, this SERS approach with high sensitivity and simplicity has a great prospect for the real-world application of ketamine in urine.

[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.

Detection and Quantification of Bucinnazine Hydrochloride Injection Based on SERS Technology.

In this investigation, surface-enhanced Raman spectroscopy (SERS) technology was performed to detect bucinnazine hydrochloride (BH) injection in water and urine. The theoretical Raman spectrum of BH with characteristic peaks was calculated and identified by density functional theory (DFT). Employing an improved silver sol as a SERS active substrate, the SERS spectra of a BH solution with different concentrations were acquired with a 0.5 mol/L KI solution as an aggregation agent. It was determined that the limit of detection (LOD) was low, to 0.01 µg/mL. A good linear relationship of BH between the Raman intensity and the concentrations was obtained in water at a concentration range from 0.5 to 6 µg/mL (R2 = 0.9914), which laid a favorable foundation for quantitative analysis. In addition, the recovery rate of spiked samples from 95.13 to 120.54% were calculated. Finally, the detection of BH injection in artificial urine was completed and the detection limit could reach 0.5 µg/mL, which met the requirements of a rapid on-site detection of drugs in urine. As a result, it indicates that the inspection of BH by the SERS method is with simplicity and high sensitivity, having a great potential for real-time detection.

Detection of Scopolamine Hydrobromide via Surface-enhanced Raman Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) was used to measure scopolamine hydrobromide. First, the Raman characteristic peaks of scopolamine hydrobromide were assigned, and the characteristic peaks were determined. The optimal aggregation agent was potassium iodide based on a comparative experimental study. Finally, the SERS spectrum of scopolamine hydrobromide was detected in aqueous solution, and the semi-quantitative analysis and the recovery rate were determined via a linear fitting. The detection limit of scopolamine hydrobromide in aqueous solution was 0.5 µg/mL. From 0 - 10 µg/mL, the curve of the intensity of the Raman characteristic peak of scopolamine hydrobromide at 1002 cm-1 is y = 4017.76 + 642.47x. The correlation coefficient was R2 = 0.983, the recovery was 98.5 - 109.7%, and the relative standard deviation (RSD) was about 5.5%. This method is fast, accurate, non-destructive and simple for the detection of scopolamine hydrobromide.

Rapid Detection of Diazepam Injection Based on Surface Enhanced Raman Spectroscopy.

Diazepam injection was detected based on a droplet surface enhanced Raman spectroscopy (SERS) platform, which overcomes the disadvantages of the poor uniformity and time-consuming sample treatment process of the conventional "drop and dry" detection strategy. The Raman peak positions of diazepam injection were determined and identified, they are mainly located in the 689, 1002, 1170 and 1598 cm-1, etc. Different concentrations of diazepam injection were detected. It was found that the intensity of 1002 cm-1 increases linearly with concentration in the range from 0.05 to 10 µg/mL and the linear correlation coefficient is 0.988. The limit of detection can reach 0.05 µg/mL. The SERS method is easy, fast and efficient. The results are accurate and reliable. It has favorable application potential in the detection of diazepam injection.

[Characterization of microbial community in produced water from a petroleum reservoir subjected to alkali-surfactant-polymer ASP flooding]. / ä¸‰å ƒ(碱-表面活性剂-聚合物)复合驱油藏采出水微生物群落特征.

Injection of alkali, surfactant and polymer (ASP) into oil reservoir can substantially increase oil recovery compared with water-flooding strategy. However, the effects of these agents on the microbial diversity and community structure, which is important for water management and corrosion control in oil industry, are hitherto poorly understood. Here, we disclosed the microbial diversity and community structure in the produced water collected from four producing wells of an ASP-flooded oilfield at Daqing, China, using high-throughput sequencing technique. Results showed that the average pH in produced water was as high as 9.65. The microbial diversity varied from well to well, and the Shannon diversity index was between 2.00 to 3.56. The Proteobacteria (85.5%-98.3%), γ-proteobacteria (83.7%-97.8%), and alkaliphilic Nitrincola (51.8%-82.5%) were the most dominant phylogenetic taxa at the phylum, class, and genus levels, respectively. A total of 12 potentially sulfide-producing genera were detected, and the most abundant taxon was Sulfurospirillum (0.4%-7.4%). The microbial community of ASP-flooded petroleum reservoir was distinct, showing an alkaliphilic or alkalitolerant potential; a reduced diversity and more simple structure were observed compared with those of the water-flooded petroleum reservoirs that were previously reported.

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.

[Effects of the frequency and intensity of nitrogen addition on soil pH, the contents of carbon, nitrogen and phosphorus in temperate steppe in Inner Mongolia, China.] / æ–½æ°®é¢‘çŽ‡å’Œå¼ºåº¦å¯¹å† è’™å¤æ¸©å¸¦è‰åŽŸåœŸå£¤pH及碳、氮、磷含量的影响.

A four-year simulated nitrogen (N) deposition experiment involving nine N gradients and two N deposition frequencies (N was added either twice yearly or monthly) was conducted in Inner Mongolian grassland, to examine the effects of frequency and intensity of N addition on pH and the contents of carbon, nitrogen and phosphorus in soil. The results indicated that the soil pH and total phosphorus content, regardless of the N addition frequency, gradually decreased with the increase of N addition intensity. By contrast, the contents of soil available nitrogen and available phosphorus showed an increasing trend, while no significant variation in dissolved organic carbon (DOC) content was observed, and the contents of soil total carbon and total nitrogen had no change. Compared with the monthly N addition, the twice-a-year N addition substantially overestimated the effects of N deposition on decreasing the soil pH and increasing the available phosphorus content, but underestimated the effects of N deposition on increasing the soil available nitrogen content, and the significant difference was found in 0-5 cm soil layer.

[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.

Fabrication of transparent SERS platform via interface self-assembly of gold nanorods and gel trapping technique for on-site real time detection.

A transparent SERS platform was fabricated via the gel-trapping method coupled with a liquid/liquid interface self-assembly technique. We employed gold nanorods as the building blocks for interface self-assembly because of their strong localized surface plasmons upon excitation by infrared radiation. Based on a "top cover" configuration, this transparent SERS platform endows high signal reproducibility for directly detecting liquid samples by confining the sample droplet into a regular shape. The Au NR PDMS platform was able to directly detect crystal violet in aqueous solutions down to 10 ppb level with high enhancement factor (0.87 × 10(5)) and signal uniformity (RSD = 3.9%). Furthermore, SERS-based anti-fungal agent detection on a fish scale was demonstrated by simply covering the fish scale with a tailored GNRs PDMS film. The experimental results clearly show that the Au NR PDMS SERS platform has great potential for on-site real time detection of contaminants in water as well as on curved surfaces.

[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.

[CaCl2-heat shock preparation of competent cells of three Pseudomonas strains and related transformation conditions].

Pseudomonas, due to its diversity in habitat and metabolic type, makes it have broad prospects applying in bioremediation, bioconversion, and biocontrol, while the introduction of exogenous gene is the key link to genetically modified Pseudomonas. The preparation and transformation of competent cells are the important methodological basis of the introduction of exogenous gene. In this paper, three Pseudomonas strains (P. putida TS11, P. stutzeri DNB, and P. mendocina JJ12) isolated from a petroleum-contaminated soil were taken as the recipient strains, and a three-factor and four-level orthogonal experiment was conducted to investigate the effects of CaCl2 concentration, heat shock duration, and recovery duration on the preparation and transformation efficiency of the strains competent cells. The results showed that CaCl2 concentration was the most important factor affecting the transformation efficiency (P<0.05), and the transformation efficiency was improved markedly when the Pseudomonas cells were repeatedly washed with sterile distilled water before the preparation of competent cells. When the P. putida TS11 cells were treated with 100 mmol L-1 of CaC12, heat-shocked for 3 minutes at 42 degrees, and incubated for 1.5 hours at 30 degrees C, the P. stutzeri DNB cells were treated with 50 mmol . L-1 of CaCl2, heat-shocked for 6 minutes, and incubated for 1.5 hours, and the P. mendocina JJ12 cells were treated with 75 mmol . L-1 of CaCl2, heat-shocked for 4. 5 minutes, and incubated for 0. 5 hours, the transformation efficiency of exogenous plasmids in the three strains all achieved 10(5) cells . microg-1 DNA.

[Temporal variations of soil microbial biomass and enzyme activities during the secondary succession of primary broadleaved-Pinus koraiensis forests in Changbai Mountains of Northeast].

By the method of space-for-time Substitution, and taking the matured (>200 years old) and over-matured (>200 years old) primary broadleaved-Pinus koraiensis forests and, their secondary forests at different succession stages (20-, 30-, 50-, 80-, and 100 years old Betula platphylla forests) in Changbai Mountains of Northeast China as test objects, this paper studied the temporal variations of soil organic carbon, soil microbial biomass, and soil enzyme activities during the secondary succession of primary broadleaved-Pinus koraiensis forests in the Mountains. Under the 20- and 80 years old B. platphylla forests, the soil organic carbon content in humus layer was the highest (154.8 and 154.3 g.kg-1, respectively); while under the matured and over-matured primary broad-leaved-Pinus koraiensis forests, this organic carbon content was relatively low, being 141. 8 and 133. 4 g.kg , respectively. The soil microbial biomass carbon and microbial quotient and the activities of soil cellulase, peroxidase, acid phosphatase, and cellobiase under the 50- and 80 years old B. platphylla forests were the highest, but the activity of soil polyphenol oxidase was the lowest, which revealed that under middle-aged and matured B. platphylla forests, soil organic carbon had a faster turnover rate, and was probably in a stronger accumulation phase. Statistical analysis showed that the soil microbial biomass carbon had significant positive correlations with the soil organic carbon, total nitrogen, and available phosphorus (r = 0.943, 0. 963, and 0.953, respectively;

[Effects of elevated CO2 on forest soil CH4 consumption in Changbai Mountains].

Elevated atmospheric CO2 concentration may affect the oxidation rate of methane (CH4 ) in forest soil. In this study, the effects of a 6-year exposure to elevated CO2 concentration (500 micromol x mol(-1)) on the soil microbial process of CH4 oxidation under Quercus mongolica seedlings were investigated with open top chamber (OTC), and specific 16S rRNA and pmoA gene fragment primers were adopted to analyze the diversity and abundance of soil methanotrophs. Comparing with that under ambient CO2 and open-air, the soil methane consumption under elevated atmospheric CO2 during growth season was reduced by 4% and 22%, respectively. The specific 16S rRNA PCR-DGGE analysis showed that under elevated CO2, the community structure of methane-oxidizing bacteria (MOB) changed, and the diversity index decreased. Elevated CO2 concentration had no distinct effects on the abundance of Type I MOB, but decreased the amount of Type II MOB significantly. The pmoA gene copy number under elevated CO2 concentration decreased by 15% and 46%, respectively, as compared with that under ambient CO2 and open-air. Our results suggested that elevated atmospheric CO2 decreased the abundance and activity of soil methanotrophs, and the main cause could be the increase of soil moisture content.