[QMEC-based Analysis of the Soil Microbial Functional Potentials across Different Tibetan Plateau Glacier Forelands].

Soil microorganisms dominate the biogeochemical cycles of elements in glacier forelands, which continue to expand due to the climate warming. We analyzed the soil microbial functional characteristics among three types of glacier forelands on the Tibetan Plateau: Yulong Glacier (Y), a temperate glacier; Tianshan Urumqi Glacier No.1 (T), a sub-continental glacier; and Laohugou Glacier No.12 (L), a continental glacier. Here, soil microbial functional genes were quantified using quantitative microbial element cycling technology (QMEC). We found that, in the three glacier forelands, the abundances of soil microbial functional genes related to hemicellulose degradation and reductive acetyl-CoA pathway were highest compared with other carbon-related functional genes. The main nitrogen cycling genes were involved in ammonification. The functional genes of the phosphorus cycle and sulfur cycle were related to organic phosphate mineralization and sulfur oxidation. Furthermore, the soils of the temperate glacier foreland with better hydrothermal conditions had the most complex microbial functional gene structure and the highest functional potentials, followed by those of the soils of continental glacier foreland with the driest environment. These significant differences in soil microbial functional genes among the three types of glacier forelands verified the impacts of geographic difference on microbial functional characteristics, as well as providing a basis for the study of soil microbial functions and biogeochemical cycles in glacier forelands.

[Effect of Simulated Warming on Microbial Community in Glacier Forefield].

Glaciers are constantly retreating because of global warming. In this study, three soil samples along the forefield of Urumqi Glacier No. 1 were collected. The effects of warming on the microbial community in the glacier forefield were investigated through a 150-day laboratory experiment. In this experiment, two temperature treatments were performed at 5℃and 15℃. The results showed that with increasing deglaciation age, the concentrations of carbon and nitrogen increased and the abundance and alpha diversity of microbial communities increased in the original samples. The 150-day laboratory experiment indicated that warming insignificantly changed the copy number of archaea and bacteria. Furthermore, it changed the microbial community composition, and the changes varied in different sampling sites. Based on the analysis of abundant OTUs changing significantly with warming, the sampling sites with shorter deglaciation age had stronger response with warming, representing an increase in the abundance of genus Thiobacillus. Furthermore, these results revealed that warming caused different effects on microbes along glacier forefield and thus, it could provide important characteristics of the microbial community with warming in alpine glacier regions.

[Community Characteristics of Cultivable Bacteria in Fine Particles(PM2.5) of Beijing and Baoding].

Fine particles(PM2.5) collected by two middle volume air samplers in the suburbs of Baoding and the urban area of Beijing during Jan 10th to 17th, 2015, were used to compare the community characteristics of cultivable bacteria in the two sites. In this study, we observed the particle morphology of PM2.5 using a field emission scanning electron microscope (FESEM), analyzed the air mass transportation source of the two sampling sites by the NOAA/ARL HYSPLIT-4 backward trajectory model, and measured the concentrations of the major water-soluble ions and heavy metal elements in the PM2.5 samples using ion chromatography, continuous flow analyzer and ICP-MS. The results indicated that, the cultivable bacteria of PM2.5 contained three phyla, which were Firmicutes, Actinobacteria and α-Proteobacteria. Nine genera and 17 species of cultivable airborne bacteria were isolated and identified. The most abundant phylum was Firmicutes. The Gram-positive bacteria accounted for more than 90%. The spore-forming Bacillus which was the dominant species contributed 68.15% and 75% to the total bacteria in urban Beijing and the suburbs of Baoding, respectively. The difference in the community structure of PM2.5's cultivable bacteria in the two sampling areas may be affected by PM2.5's physical and chemical properties and air mass transportation.

[Bioconversion of cellulose to methane by a consortium consisting of four microbial strains].

Cellulose was usually degraded by microbial communities in natural habitats. Construction of a simple cellulolytic consortium is necessary to understand the underlying interaction within microorganisms involved in cellulose conversion. A screening approach was developed to obtain a simple microbial community with the ability of cellulose degradation to methane. This technique was based on the method of enrichment culture accompanying with denaturing gel gradient electrophoresis (DGGE) fingerprint detection technology and roll-tube method. Moreover, a four-strain mixed culture capable of degrading cellulose to methane was isolated from Zoige alpine wetland of the Tibetan Plateau. The results showed that the microbial consortia consisted of three functional groups: the cellulolytic bacterium Clostridium glycolicum, the non-celluloytic bacteria group of Trichococcus flocculiformis and Parabacteroides merdae, and the methanogenic bacterium Methanobacterium subterraneum. This four-strain co-culture can convert cellulose to methane. In the future, the isolated cellulolytic consortia could provide a platform for controlling metabolic pathways and genetic modification involved in methane production from cellulose.

The impacts of different long-term fertilization regimes on the bioavailability of arsenic in soil: integrating chemical approach with Escherichia coli arsRp::luc-based biosensor.

An Escherichia coli arsRp::luc-based biosensor was constructed to measure the bioavailability of arsenic (As) in soil. In previous induction experiments, it produced a linear response (R (2) = 0.96, P < 0.01) to As from 0.05 to 5 µmol/L after a 2-h incubation. Then, both chemical sequential extraction, Community Bureau of Reference recommended sequential extraction procedures (BCR-SEPs) and E. coli biosensor, were employed to assess the impact of different long-term fertilization regimes containing N, NP, NPK, M (manure), and NPK + M treatments on the bioavailability of arsenic (As) in soil. Per the BCR-SEPs analysis, the application of M and M + NPK led to a significant (P < 0.01) increase of exchangeable As (2-7 times and 2-5 times, respectively) and reducible As (1.5-2.5 times and 1.5-2.3 times, respectively) compared with the no fertilization treated soil (CK). In addition, direct contact assay of E. coli biosensor with soil particles also supported that bioavailable As in manure-fertilized (M and M + NPK) soil was significantly higher (P < 0.01) than that in CK soil (7 and 9 times, respectively). Organic carbon may be the major factor governing the increase of bioavailable As. More significantly, E. coli biosensor-determined As was only 18.46-85.17 % of exchangeable As and 20.68-90.1 % of reducible As based on BCR-SEPs. In conclusion, NKP fertilization was recommended as a more suitable regime in As-polluted soil especially with high As concentration, and this E. coli arsRp::luc-based biosensor was a more realistic approach in assessing the bioavailability of As in soil since it would not overrate the risk of As to the environment.

Assessing the effect of phosphate and silicate on Cd bioavailability in soil using an Escherichia coli cadAp::luc-based whole-cell sensor.

An Escherichia coli cadAp::luc-based whole-cell sensor was constructed to measure cadmium (Cd) bioavailability and assess the immobilizing efficiency of phosphate and silicate on Cd. In previous induction experiments, a linear response (R(2) = 0.97, P < 0.01) from 0.1 to 5 µmol L(-1) of Cd was detected by this sensor after a 2 h incubation. The sensor was then used to estimate Cd bioavailability in soils spiked with different amounts of dipotassium phosphate (DKP, K2HPO4) or sodium silicate (SS, Na2SiO3·9H2O). The total Cd in soil-water extracts (TSWE) was determined with ICP-MS, and the bioavailable Cd in soil-water extracts (BSWE) and bioavailable Cd in soil-water suspensions (BSWS) were measured by the E. coli cadAp::luc-based whole-cell sensor. Final results showed that spiked SS (Si : Cd = 2 : 1, mol mol(-1)) reduced the different forms of Cd (TSWE, BSWE and BSWS) from 56.47 mg kg(-1), 42.11 mg kg(-1), and 206.72 mg kg(-1) to 16.63 mg kg(-1), 15.90 mg kg(-1), and 67.57 mg kg(-1), respectively. In other words, SS had 25.68%, 19.5%, and 9.54% better immobilizing efficiency, respectively, compared with DKP. All the results supported SS was more efficient than DKP at immobilizing Cd in soil, and higher soil pH and higher solubility of the immobilizing agents may have been the major factor affecting immobilizing efficiency. In addition, the total and bioavailable Cd in soil-water extracts was only 16.13-35.41% of the sensor contact assay-determined Cd (BSWS), which indicated that the whole-cell sensor-based contact assay was more practical in assessing the risk of Cd in soil after immobilization since it would not overrate the immobilizing capacity of the agents.

[Comparison between transgenic insect-resistant cotton expressing Cry1Ac protein and its parental variety in rhizospheric fungal diversity].

The dynamics of rhizospheric fungal diversity and biomass at different sampling stages associated with two transgenic insectresistant cottons expressing Cry1Ac protein and their control varieties were studied under greenhouse conditions, followed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time polymerase chain reaction (Q-PCR), in order to evaluate the ecological security of planting transgenic cotton expressing Cry1Ac protein. The results indicated that the fungal superior bands in rhizosphere of transgenic Bt cotton were similar with that of control cotton at four sampling stages, the more obvious difference in the blurred bands among transgenic Bt cotton, JM20 and SHIYUAN321 was detected. The rhizospheric fungal biomass of transgenic Bt cotton SGK321 was significantly lower than that of its parental control cotton at seedling stage, while the slight decrease in fungal biomass of transgenic Bt cotton XP188 was detected at boll forming stage, the ill-defined decrease, even growing tendency in two transgenic Bt cottons was detected at other stages. However, the difference of rhizospheric fungal community compositions and biomass was not only existed between transgenic cotton and its control, but also between SHIYUAN321 and JM20, and the same phenomenon was also detected between transgenic Bt cotton SGK321 and XP188. Hence, Bt protein is not the only incentive resulting in the difference in fungal community composition and diversity, the decrease in biomass between transgenic cotton and untransgenic cotton, different cotton varieties has an effect on them.

[Construction and properties of a microbial whole-cell sensor CB10 for the bioavailability detection of Cr6+].

A microbial whole-cell biosensor CB10 for the bioavailability assessing of Cr6+ was constructed by molecular biotechnology. The regulatory gene and promoter of CB10 was from the chromium resistance system of plasmid pMOL28 from Cupriavidus metallidurans CH34, and the reporter gene of CB10 was luc which was derived from Photinus pyralis. Finally, its response characteristic was discussed under different incubation conditions e. g. pH and temperature. The results showed that a microbial whole-cell biosensor CB10 had been successfully constructed which could respond to Cr6+ within 30 min, with a LOD for Cr6+ of 2 micromol x L(-1). When the incubation concentration of Cr6+ was between 20 micromol x L(-1) and 200 micromol x L(-1), the luc activity of the CB10 biosensor was in linear correlation with the concentration of Cr6+. When the concentration of heavy metal was in the range of 10-50 micromol x L(-1), the response of CB10 was relatively more specific. Moreover, high concentrations of Pb2+, Mn2+ and Sb2+ could also induce CB10. By analyzing the response characteristic of CB10 biosensor, we could draw the conclusion that 15-30 degrees C and pH 4-7 were appropriate for CB10, and 30 degrees C and pH 7 were the optimal conditions for the incubation of the CB10 biosensor. The microbial whole-cell biosensor CB10 for the detection of Cr6+ was fast-responding, specific, sensitive and stable under various conditions. In prospective, it could be used in the fast detection of Cr6+ in water and assessment of the bioavailability of Cr6+ in soil.

[Effect of immobilization on biosensor for benzene derivates detection].

A whole cell sensor, Pseudomonas fluorescens A506 (pTS), was immobilized by sodium alginate and the factors of cell density, immobilization time and beads usage were optimized. The performance of the immobilized cells was compared with that of the free cells. After 2 h immobilization,the increasing speed of fluorescent signal of immobilized cells was 2.26 times as high as that of the free cells,and the peak value was 2.23 times as high during the detection time ranging from 1.5 to 6.0 h. The constantly lower growth and density of the immobilized cell implied the enhanced signal intensity of single cells after immobilization. Meanwhile, the cell density decreased as the immobilization time prolonged. Cell density and immobilization time were the dominant factors affecting the detection signal. For benzene at higher concentrations, the immobilized biosensor showed more rapid signal response at the early period of detection.

[Isolation of quizalofop-p-ethyl-degrading bacteria from soil by DGGE-colony in situ hybridization].

Naturally occurring bacteria isolates capable of metabolizing pesticides have received considerable attention because they offer the possibility of both environmentally friendly and in situ remediation. The effect of herbicide quizalofop-p-ethyl on bacterial community in soil was analyzed using the technique of PCR-DGGE for isolating strains biodegrading quizalofop-p-ethyl. Results indicated that the soil bacterial community structures significantly changed after adding quizalofop-p-ethyl. The bacterial diversity of soil showed an increasing-decreasing-increasing trend. The largest changes occurred in the 9th day and then became stabilized. According to the sequencing results of bands in DGGE profiles, it inferred that members of bacterial genera Pseudomonas, Massilia and Burkholderia had tolerance to quizalofop-p-ethyl, and the potential for degradation. These microbial groups could be used to isolate and screen as the indigenous microbial resources to reduce pesticide residues. Digoxigenin-labeled probes had been synthesized based on the sequencing results of bands in the DGGE profiles, and three bacterial strains capable of biodegrading quizalofop-p-ethyl were isolated from soil by colony in situ hybridization technique. The strain named L1 was able to utilize quizalofop-p-ethyl as the sole source of carbon. The strain was identified as Pseudomonas sp., based on the phylogenetic analysis of 16S rRNA. The degrading ability of strain L1 in minimal medium with quizalofop-p-ethyl was investigated by HPLC. The quizalofop-p-ethyl content decreased by almost 50% after 7 days, and the biomass of strain L1 increased while the content of quizalofop-p-ethyl was decreased. This confirmed that the strain L1 had the capacity of degradation. This result provided a basis for future research on degradation mechanism and functional genes.

Ultrasound-intensified laccase production from Trametes versicolor.

An efficient intermittent ultrasonic treatment strategy was developed to improve laccase production from Trametes versicolor mycelia cultures. The optimized strategy consisted of exposing 2-day-old mycelia cultures to 5-min ultrasonic treatments for two times with a 12-h interval at the fixed ultrasonic power and frequency (120 W, 40 kHz). After 5 days of culture, this strategy produced the highest extracellular laccase activity of 588.9 U/L among all treatments tested which was 1.8-fold greater than the control without ultrasound treatment. The ultrasonic treatment resulted in a higher pellet porosity that facilitated the mass transfer of nutrients and metabolites from the pellets to the surrounding liquid. Furthermore, the ultrasonic treatment induced the expression of the laccase gene (lcc), which correlated with a sharp increase in both extracellular and intracellular laccase activity. This is the first study to find positive effects of ultrasound on gene expression in fungal cells. These results provide a basis for understanding the stimulation of metabolite production and process intensification by ultrasonic treatment in filamentous fungal culture.

[Microbial community structure analysis of unexploited oil and gas fields by PCR-DGGE].

Microbial communities of different depths (30, 60, 100, 150, 200cm) from the unexploited oilfield, gas field and control area were studied by PCR-DGGE and sequencing methods. The objectives of this study were to understand the microbial distribution in the regions of unexploited oil and gas fields, and to investigate the potential microbial indicators of oil and gas resources. The results showed that the Dice coefficients between different depths were very low (26-69.9). The microbial communities in the soil of 150 cm and 200 cm depth had greater richness (S > or = 19), diversity (H > or = 2.69) and evenness (E > or = 0. 90). The results of sequencing demonstrated that the bands from oilfield were mainly grouped into alpha-Proteobacteria, gamma-Proteobacteria, Actinobacteria, Acidobacteria with the predominance of gamma-Proteobacteria (75%). Most of the bands were related to oil-associated and hydrocarbon degrading bacteria, such as Methylophaga and Alcanivorax. While the gas field had alpha, beta, gamma, delta-Proteobacteria and Bacteroidetes, and gamma-Proteobacteria accounted for only 24%. More strains showed relativity to methanotrophs, such as Methylocystaceae. Thus, 150 cm and 200 cm were more suitable as the oil-gas exploration sampling depth. Methylocystaceae may act as potential indicators for gas resources, Methylophaga and Alcanivorax for oil.

Effect of lignocellulosic inhibitory compounds on growth and ethanol fermentation of newly-isolated thermotolerant Issatchenkia orientalis.

A newly isolated thermotolerant ethanologenic yeast strain, Issatchenkia orientalis IPE 100, was able to produce ethanol with a theoretical yield of 85% per g of glucose at 42°C. Ethanol production was inhibited by furfural, hydroxymethylfurfural and vanillin concentrations above 5.56 gL(-1), 7.81 gL(-1), and 3.17 gL(-1), respectively, but the strain was able to produce ethanol from enzymatically hydrolyzed steam-exploded cornstalk with 93.8% of theoretical yield and 0.91 gL(-1)h(-1) of productivity at 42°C. Therefore, I. orientalis IPE 100 is a potential candidate for commercial lignocelluloses-to-ethanol production.