[Effects of Different Land Use Typess on the Molecular Ecological Network of Soil Bacteria].

The bacterial community composition in four land-use types was determined and the visualized bacterial network was constructed by 16S rDNA Illumina MiSeq high-throughput sequencing technology and a molecular ecological network method. The results show that Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Planctomycetes, Verrucomicrobia, Cyanobacteria, Gemmatimonadetes, Firmicutes, Nitrospirae, and Chlorobi are the main bacteria in this area. The number of nodes of urban green land, paddy field, and dry field bacteria networks is higher, and that of natural forest land is lower. The number of connections and average connectivity of dry fields are the highest; following are those of urban green land and paddy field, and those of natural forest land are the lowest. The four bacterial networks are dominated by positive correlation, and the ratio of competition relationship is TL > LD > HT > ST. The average network path and modularity of the soil bacteria networks of paddy field and dry land are small, while the average connectivity and clustering coefficient are higher. Some flora of Acidobacteria, Firmicutes, and Proteobacteria play an important role in the soil bacterial network in this area. The classification of operational taxonomic units is different among the key nodes of different bacterial molecular ecological networks, and there is almost no overlap. The relative abundance of bacteria of some key nodes in the four bacterial networks is low (<1%), and these are not the main bacteria in this area. The soil microflora in dry land are mainly affected by TP (P<0.05), the soil microflora in paddy field were mainly affected by clay, silt, and water content (P<0.05), and that in natural forest land and urban green land were mainly affected by C/N (P<0.05). The above results show that different land-use patterns lead to changes in soil physical and chemical properties and the interaction between soil bacteria species. The bacterial network of dry land soil is larger and the relationship between species is more complex. The bacteria in different land-use types are mainly cooperative, and the competition is weak. Compared with other land-use types, there is stronger competition between the bacteria in natural forest soil. The soil bacteria in paddy field and dry land are the most sensitive to the external environment, respond more quickly, and the community structure is easier to change. The response of soil bacteria in natural forest land and urban green land is slower, and the disturbance of environmental factors does not affect the whole bacterial ecological network in a short time, and thus the community structure is more stable. Some bacteria have the phenomenon of species role transformation between networks. The abundance and community distribution of microorganisms cannot indicate the strength of their connectivity between network nodes; low-abundance bacteria in soil play an important role in the construction of bacterial networks.

[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.

[Soil bacterial community structure and predicted functions in the larch forest during succession at the Greater Khingan Mountains of Northeast China]. / å¤§å ´å®‰å²­è½å¶æ¾æž—ä¸åŒæ¼”æ›¿é˜¶æ®µåœŸå£¤ç»†èŒç¾¤è½ç»“æž„ä¸ŽåŠŸèƒ½æ½œåŠ¿.

To reveal soil bacterial community structure and potential functions in larch forest during succession at Greater Khingan Mountains (Hanma National Nature Reserve), 16S rDNA was sequencing by Illumina Miseq. The results showed that the Proteobacteria, Acidobacteria, Verrucomicrobia, Bacteroidetes, Actinobacteria, Planctomycetes and Chloroflexi were the most dominant phyla in soils of larch forests at various successional stages. Along forest succession, Acidobacteria increased, while Chloroflexi decreased. Relative abundance of dominant phyla was different at various successional stages. The α diversity, Chao1, Shannon index and Simpson index of soil bacterial community had no significant difference among five succession stages, while significant differences in soil bacterial community structure were observed between young and medium larch, between young and over mature larch, and between near mature and mature larch. Bacterial community structure was mainly influenced by redox potential, pH and available phosphorus. The redox potential was the most important factor influencing soil bacterial community structure. Along the succession of larch forest, N-fixation, denitrification, ammonia oxidation and lignin breakdown decreased, dissimilatory sulfate reduction had down-up trend, carbon fixation had up-down trend, and alkaline phosphatase had no apparent trend. Bacterial community potential function was mainly influenced by redox potential and available phosphorus.

[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.

Bioaugmentation of oil reservoir indigenous Pseudomonas aeruginosa to enhance oil recovery through in-situ biosurfactant production without air injection.

Considering the anoxic conditions within oil reservoirs, a new microbial enhanced oil recovery (MEOR) technology through in-situ biosurfactant production without air injection was proposed. High-throughput sequencing data revealed that Pseudomonas was one of dominant genera in Daqing oil reservoirs. Pseudomonas aeruginosa DQ3 which can anaerobically produce biosurfactant at 42 °C was isolated. Strain DQ3 was bioaugmented in an anaerobic bioreactor to approximately simulate MEOR process. During bioaugmentation process, although a new bacterial community was gradually formed, Pseudomonas was still one of dominant genera. Culture-based data showed that hydrocarbon-degrading bacteria and biosurfactant-producing bacteria were activated, while sulfate reducing bacteria were controlled. Biosurfactant was produced at simulated reservoir conditions, decreasing surface tension to 33.8 mN/m and emulsifying crude oil with EI24 = 58%. Core flooding tests revealed that extra 5.22% of oil was displaced by in-situ biosurfactant production. Bioaugmenting indigenous biosurfactant producer P. aeruginosa without air injection is promising for in-situ MEOR applications.

[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.

[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.

[NO3-/NO2- inhibits sulfate-reducing activity of the enrichment culture of sulfate-reducing prokaryotes from an off-shore oil reservoir at Bohai Bay, China].

Long-term injection of sulfate-rich water into oil reservoirs stimulates the proliferation of sulfate-reducing prokaryotes (SRP) therein and results in production of a great amount of H2S, leading to souring in oil reservoirs and related environmental problems. In this study, we first, using modified API RP 38 medium, enriched SRP present in production water from a producing well at Bohai Bay, China, and then examined the inhibitory effects of nitrate or nitrite on sulfate reduction activity of the SRP. Results showed that the enriched SRP culture exhibited a high sulfate reduction activity as indicated by a sulfate-reducing rate of 10.4 mmol SO4(2-) x d(-1) x g(-1) dry cell. In presence of 0.4, 0.8, 1.8, and 4.2 mmol x L(-1) nitrate, sulfate reduction was inhibited for 5, 9, 20, and over 35 days, respectively. With the addition of 0.6, 0.9, 1.4, 2.6 and 4.6 mmol x L(-1) of nitrite, the inhibitory period lasted 3, 12, 22, and over 39 days, respectively. The SRP enrichment culture could dissimilatorily reduce nitrate to ammonium. When sulfate, nitrate and nitrite coexisted, nitrate or nitrite was preferentially used over sulfate as electron acceptor by the enriched SRP. This competitive use of electron acceptor and the strong inhibitory effect of nitrite possibly accounted for the suppression of nitrate and nitrite on the sulfate-reducing activity of the enriched SRP cultures from offshore oil reservoir at Bohai Bay.

[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.