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.

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-).

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.

Microbial community characterization of an UASB treating increased organic loading rates of vitamin C biosynthesis wastewater.

The microbial community of a mesophilic lab-scale upflow anaerobic sludge blanket (UASB) reactor treating vitamin C biosynthesis wastewater at gradually elevated organic loading rates (OLRs) was characterized using 16S rDNA-based polymerase chain reaction-DGGE (denatured gradient gel electrophoresis) analysis. The DGGE fingerprints suggested that the elevated OLRs did not cause any significant changes in the microbial community. The predominant bacterial bands were affiliated with the Firmicutes (Clostridiales, four bands), Proteobacteria (Deltaproteobacteria, six bands), Bacteroidetes, and Synergistetes, respectively. All the archaeal bands were very similar to already known methanogenic species: Methanobacterium formicicum (two bands), Methanomethylovorans hollandica (one band) and Methanosaeta concilli (two bands), which belonged to the divisions Methanobacteria and Methanomicrobia, respectively.

Enhanced treatment of wastewater from the vitamin C biosynthesis industry using a UASB reactor supplemented with zero-valent iron.

The effects of zero-valent iron (Fe0) on the performance of a mesophilic upflow anaerobic sludge blanket (UASB) reactor treating high-strength wastewater from the vitamin C biosynthesis industry (VCW) was investigated during a 200-day period. The results showed that the chemical oxygen demand (COD) removal efficiency, CH4 content in biogas, specific methanogenic activity of sludge, and phosphate removal efficiency were significantly improved up to 81.8-96.1%, 76.5-79.6%, 1.71-2.87 g CH4-COD g(-1) VSS d(-1) and 68.5-85.2%, respectively, at elevated organic loading rates (OLRs) in the Fe0-amended reactor (RFe). In contrast, the corresponding values of 65.3-83.4%, 69.1-70.8%, 1.12-1.95 g CH4-COD g(-1) VSS d(-1) and 1.4-1.6%, respectively, were recorded in the control (R0). Elevated ferrous concentration of nearly 400 mg L(-1) in sludge was detected in RFe, whereas in the effluent of both reactors it was low (< 1.0 mg L(-1)). Batch tests further showed that Fe0 significantly enhanced the biodegradability of the VCW as shown by an increase in BOD/COD ratio from 0.41 to 0.65, and could serve as the electron donor for methanogenesis by anaerobic sludge, which were responsible for the differences between RFe and R0. The results suggest this integrated Fe0-microbial system is promising in facilitating the anaerobic digestion of VCW in UASB reactors.

Warming-driven migration of core microbiota indicates soil property changes at continental scale.

Terrestrial species are predicted to migrate northward under global warming conditions, yet little is known about the direction and magnitude of change in microbial distribution patterns. In this continental-scale study with more than 1600 forest soil samples, we verify the existence of core microbiota and lump them into a manageable number of eco-clusters based on microbial habitat preferences. By projecting the abundance differences of eco-clusters between future and current climatic conditions, we observed the potential warming-driven migration of the core microbiota under warming, partially verified by a field warming experiment at Southwest China. Specifically, the species that favor low pH are potentially expanding and moving northward to medium-latitudes (25°-45°N), potentially implying that warm temperate forest would be under threat of soil acidification with warming. The eco-cluster of high-pH with high-annual mean temperature (AMT) experienced significant abundance increases at middle- (35°-45°N) to high-latitudes (> 45°N), especially under Representative Concentration Pathway (RCP) 8.5, likely resulting in northward expansion. Furthermore, the eco-cluster that favors low-soil organic carbon (SOC) was projected to increase under warming scenarios at low-latitudes (< 25°N), potentially an indicator of SOC storage accumulation in warmer areas. Meanwhile, at high-latitudes (> 45°N) the changes in relative abundance of this eco-cluster is inversely related with the temperature variation trends, suggesting microbes-mediated soil organic carbon changes are more responsive to temperature variation in colder areas. These results have vital implications for the migration direction of microbial communities and its potential ecological consequences in future warming scenarios.

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.

[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.

[Diversity of nirS-type denitrifying bacteria under different nitrogen fertilizer management in wheat soil].

OBJECTIVE: To study the effect of inorganic nitrogen fertilizer on nirS-type denitrifiers' community in the wheat soil. METHODS: We constructed nirS gene libraries of two different treatments of soil: soil fertilized with inorganic nitrogen fertilizer (B-UN) and without nitrogen fertilizer (B-NN). And we used restriction fragment length polymorphism (RFLP) method to analyze the nirS-type denitrifiers' diversity. RESULTS: There were 27 operational taxa units (OTUs) in each treatment after Msp I and Afa I digestion and only nine OTUs existed in both treatments. The ecological indexes such as Shannon-Wiene index, Simpon index, richness index and evenness index of two different soils were nearly equivalent. However, significant difference was found between the OTU clusters from clone libraries of different treatments. Eleven representative clones were sequenced. The similarities of ten sequences were from 73% to 95% to the sequences of the database. There was one sequence which had no similar nucleotide identities in database. CONCLUSION: The application of inorganic nitrogen fertilizer altered significantly the nirS-type denitrifiers' community in the wheat soil.

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.

[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.

Soil pH and plant diversity shape soil bacterial community structure in the active layer across the latitudinal gradients in continuous permafrost region of Northeastern China.

In the permafrost region of northeastern China, vegetation and soil environment have showed response to permafrost degradation triggered by global warming, but the corresponding variation of the soil microbial communities remains poorly investigated. Here, a field investigation in the continuous permafrost region was conducted to collect 63 soil samples from 21 sites along a latitudinal gradient to assess the distribution pattern of microbial communities and their correlation with environmental factors. High-throughput Illumina sequencing revealed that bacterial communities were dominated by Proteobacteria, Acidobacteria, Bacteroidetes and Actinobacteria. Both microbial richness and phylogenetic diversity decreased initially and then increased as the latitude increased. UniFrac analysis of microbial communities detected significant differences among latitudes. Variation partitioning analysis and structural equation models revealed that environmental variables, including geographic factors, plant-community factors and soil physicochemical factors, all played non-negligible roles in affecting the microbial community structures directly or indirectly. Redundancy analysis and boosted regression tree analysis further highlighted the influences of soil pH and plant richness on microbial community compositions and diversity patterns. Taken together, these results suggest that the distribution pattern of soil microbial communities shows distinct changes along the latitudinal gradients in northeastern China and is predominantly mediated by soil pH and plant diversity.

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.

[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.

[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;

[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.

[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.