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.

[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.

[Effects of Nutrients on the Growth of Microcystis aeruginosa and Bacteria in the Phycosphere].

The "bacteria-algae" system plays an important role in water ecosystems. The effects of bacteria in phycospheres on the growth of Microcystis aeruginosa under in-situ nutrient stimulation were studied to explore the bacteria-algae interaction during a cyanobacteria bloom. The results showed that LB medium could inhibit the growth of M. aeruginosa, and the algicidal rate was 86.49%. Sodium acetate, glucose, and sodium citrate could promote M. aeruginosa, and the growth rate was more than 50%. The addition of nutrients in M. aeruginosa could have changed the biocoenosis in the phycosphere and increased the species richness by 16S rRNA gene sequencing, and the number of bacteria in the phycosphere increased dramatically in the LB medium and peptone groups. The physiological and biochemical responses showed that algae suffered serious lipid peroxidation, and superoxide dismutase (SOD) and catalase (CAT) activities first increased significantly and subsequently decreased under the oxidative stress of LB medium or peptone. Scanning electron microscopy (SEM) indicated that the surface of algae cells appeared wrinkled, invaded, and atrophied under LB medium stimulation, whereas bacteria in the phycosphere significantly increased. Furthermore, six strains of algicidal bacteria were isolated from the LB medium and peptone groups, and the algicidal rate of Bacillus sp. A1 was 97.55%, which confirmed that the phycosphere of M. aeruginosa included algicidal bacteria. Therefore, appropriate external nutrient stimulation can produce algicidal bacteria in situ to prevent cyanobacterial blooms.

[Pollution Characterization and Comprehensive Water Quality Assessment of Rain-source River: A Case Study of the Longgang River in Shenzhen].

Rain-source urban rivers are an important part of the urban ecosystem. Due to the small water environment capacity and the rapid development of the regional economy and society, they are vulnerable to serious pollution. The goal of this study was to identify the main pollution characteristics of river water quality and to carry out a scientific comprehensive water quality assessment. Water samples from 12 sampling locations of the Longgang River in Shenzhen, a typical rain-source urban river, were collected from January to December in 2018. According to the Environmental Quality Standard for Surface Water (GB 3838-2002), 22 water quality indicators were analyzed, and the water quality of Longgang River was comprehensively evaluated using the single-factor assessment method, comprehensive pollution index method, and principal component analysis method. The results of the single-factor assessment method showed that water quality of all sampling sites of the Longgang River met the Class V of the Environmental Quality Standard for Surface Water (GB 3838-2002), and the Tiaojiao Shui and Longxi River met the Class Ⅳ and Class Ⅲ of the Environmental Quality Standard for Surface Water (GB 3838-2002), respectively. The results of the comprehensive pollution index method showed that the water quality of 12 sampling sites was clean or relatively clean. Both the results of the comprehensive pollution index and principal component comprehensive score showed that the water quality of Longxi River, Nanyue River, and Tianjiao Shui were the best among all sampling sites. There is still room for improvement in the Wutongshan River, Dakang River, Ailian River, Dingshan River, and Huangsha River, and significant consideration should be given to parameters such as nutrients (TN, TP, and NH4+-N), organic matter (COD and BOD5), fecal coliform, and anionic surfactants. The three methods were a combination of qualitative and quantitative evaluation. The results of each method were not identical. Thus, it is very necessary to explore the comprehensive water quality assessment using various methods for making scientific and reasonable water pollution control strategies.

[Microbial Community Structure of Soil Methanogens and Methanotrophs During Degradation and Restoration of Reed Wetlands in the Songnen Plain].

Wetlands are an important global source and sink of methane. However, human activities and climatic conditions are causing serious degradation of wetlands in China. In response to this, the relevant departments have progressively carried out wetland restoration projects over the past few years. To investigate the response of microbial communities of bacteria, methanogens, and methanotrophs during degradation and restoration of wetlands, soil samples were collected from undegraded reed wetlands, degraded reed wetlands, and restored reed wetlands in the Songnen Plain. Microbial diversity and community composition were studied by high-throughput sequencing based on the 16S rRNA gene of bacteria, the mcrA gene of methanogens, and the pmoA gene of methanotrophs. The results indicate that the degradation of reed wetlands results in a decrease in bacterial and methanogenic α-diversity and an increase in methanotrophic α-diversity. Bacterial α-diversity and methanogenic α-diversity were both significantly positively correlated with soil water content. At different taxonomic levels, higher relative abundances of Rhizobiales and Methanobacteriaceae were detected in the undegraded wetland soils. Wetland degradation decreased the relative abundance of Rhizobiales but increased that of the pathogenic bacteria Burkholderiaceae and microorganisms resistant to harsh and extreme environments including Sphingomonas, Rubrobacter, Methylobacter, Methylomonas, and Methylococcus. In the restored wetland soils, the relative abundances of Bacillus, Methanosarcinaceae, Methanomicrobiaceae, and the type Ⅱ methanotroph Methylocystis were higher. Therefore, different wetland conditions can indirectly change soil properties and, consequently, change the community structure of methanogens and methanotrophs.

[Effects of Nano-membrane on Aerobic Composting Process and Odor Emission of Livestock Manure].

Aerobic composting is an important approach to treat livestock manure; however, traditional composting has some problems, such as low efficiency, or odorous pollution. In order to speed up the composting process and reduce malodorous gas emissions, this study explored the mechanism of nano-membrane for improving the efficiency of livestock manure composting. A trough aerobic composting experiment was set up to evaluate the physicochemical properties, enzyme activities, and emission of odorous gases. The results showed that covering with nano-membrane could accelerate the temperature rise; reduce the pH, organic matter(OM), and ammonia nitrogen(NH4+-N); increase electrical conductivity(EC); enhance the activities of urease, protease, cellulase, xylanase, and peroxidase; while the total cumulative emissions of NH3, H2S, and TVOC were reduced by 58%, 100%, and 61%, respectively. The correlation analysis showed that most enzyme activities were easily affected by temperature(T), EC, OM, and C/N. The emission rate of NH3 was positively correlated with T and negatively correlated with pH, and TVOC was significantly correlated with various physicochemical properties. This experiment showed that covering nano-membrane could accelerate the compost maturity and reduce the emission of odorous gases. This approach has no health risks and produces low malodorous gas, which may effectively solve the problem of pollutant emission caused by livestock manure compost fermentation, promoting the green and sustainable development of the breeding industry. In addition, it facilitates livestock manure fertilizer application, and provides technical support for the development of resource utilization of biomass waste.

[Biodegradation of Expanded Polystyrene Foams in Zophobas morio: Effects of Gut Microbiota].

Polystyrene (PS) foam is one of the main plastic materials dispersed in the environment. In this study, we observed that the insect-super mealworms (Zophobas morio), which belong to a species of the Tenebrio genus, are able to consume and degrade PS foam. Individual Z. morio consumed an extracellular polymeric substance (EPS) over 50 days with high survival rates. Analyses of the frass egested using fourier transform infrared spectroscopy (FTIR) confirmed the formation of a new oxygen-bearing functional group in the EPS. Gel permeation chromatography (GPC) analyses indicated that the depolymerization of ingested EPS with decreases in both Mw and Mn was observed, suggesting that the EPS was biodegraded. We also used 16S rRNA gene sequences to analyze the gut bacteria of Z. morio under three different feeding conditions, including with polystyrene, bran, and bran plus polystyrene. Under different dietary conditions, the gut microbiota of Z. morio showed significant differences, such as Klebsiella and Citrobacter becoming significantly enriched. In vitro studies using 90-days gut microbial culturing experiments indicated that gut microbiota contributed to PS degradation. Our research demonstrates that intestinal bacteria played an essential role in the degradation of PS by Z. morio, and provides a new theoretical basis and application ideas for the biodegradation of PS.

[Bacillus amyloliquefaciens Biofertilizer Mitigating Soil Ammonia Volatilization].

In this study, we investigated the effectiveness and microbial mechanism of Bacillus amyloliquefaciens biofertilizer on reducing ammonia volatilization in farmland soil. Pot experiments were carried out to explore the effects of B. amyloliquefaciens biofertilizer (BB) and chemical fertilizer on soil ammonia volatilization, crop yield and quality, and soil microbial community. Four fertilization strategies were tested, namely no fertilizer (CK), 100% chemical fertilizer (C), 50% BB and 50% chemical fertilizer (B1), and 100% BB (B2). The dynamic flow-through chamber method was used to determine the soil ammonia volatilization flux after fertilization. The soil bacterial community during the peak period of ammonia volatilization was analyzed using 16S rDNA high-throughput sequencing. The results showed that the amount of ammonia volatilization in B1 and B2 decreased by 79.5% and 84.8%, respectively, as compared with treatment C. B2 had the lowest nitrate content and the highest yield; the yield of B2 increased by 50.5% and 12.3% as compared to that of CK and C, respectively. B1 had the highest content of vitamin C, which was 67.6 mg ·kg-1. The application of BB improved the diversity and richness of soil bacterial community, especially the relative abundance of Bacillus and Nitrospira. This shows that BB plays an important role in preventing air pollution and improving nitrogen utilization.

[Effects of Wetland Reclamation on Soil Microbial Community Structure in the Sanjiang Plain].

Excessive reclamation leads to rapid degradation of wetland ecosystems. Microbial changes in wetland soils under the influence of human activities can sensitively indicate degradation of soil quality and ecosystem functions. To study the effects of different land use patterns on microbial community structure of wetlands, the Sanjiang Wetland Protected Area of Fuyuan, Heilongjiang Province, was selected as the research area. Soil samples were collected from replanting legume crop area, rice wetland, and primitive peat wetland. Then, the bacterial community structure in the soil was investigated with high-throughput sequencing based on the 16S rRNA gene. The relationship between bacterial community and environmental factors was further explored. The results indicated that, based on the bacterial phylum, there are no significant differences between the microbial community structures of soils under different land use patterns. Nevertheless, at the genus level, higher abundance of Blastocatella, Coxiella, and Rickettsia were detected in the legume rhizospheric soil. In the paddy soil, the relative abundances of Massilia, Nitrosomonas, and Bradyrhizobium are higher, while in the peatand soil, the higher contents are of Rhizomicrobium, Arthrobacter, and Bacillus. The results of Chao1 and Shannon index indicate that the microbial diversity of the paddy soil was higher than in the legume rhizospheric soil and peatland soils. However, no significant differences on bacterial diversity between the legume rhizospheric soil and peatland soils were observed. The results of the correlation analysis indicate that soil reclamation triggers a shift in microbial community mainly because of its influence on soil pH, moisture, and nutrients.

Screening and identification of the levoglucosan kinase gene (lgk) from Aspergillus niger by LC-ESI-MS/MS and RT-PCR.

A protein of 75,000 Daltons with levoglucosan kinase activity was purified from Aspergillus niger. After in-gel digestion by trypsin, a 14-mer peptide was sequenced and analyzed by LC-ESI-MS/MS. Using a primer derived from the 14-mer peptide in combination with Oligo-(dT)18, a cDNA fragment was obtained by RT-PCR. A search of the GenBank database indicated that the protein had not been identified before. A similar protein named hypothetical protein FG07802.1 (EAA77996.1) was found to exist in Gibberella zeae by Blastx search. Using a primer derived from the protein, a cDNA fragment of second RT-PCR was cloned into plasmid pAJ401, which was transformed to Saccharomyces cerevisiae H158 and expressed. Two positive levoglucosan assimilating recombinants were selected. The lgk gene was screened and identified.

[Analysis of Microbial Community in the Membrane Bio-Reactor (MBR) Rural Sewage Treatment System].

Uncontrolled release and arbitrary irrigation reuse of rural wastewater may lead to water pollution, and the microbial pathogens could threaten the safety of freshwater resources and public health. To understand the microbial community structure of rural wastewater and provide the theory for microbial risk assessment of wastewater irrigation, microbial community diversities in the Membrane Bio-Reactor (MBR) process for rural wastewater treatment was studied by terminal restriction fragment length polymorphism (T-RFLP) and 16S rDNA gene clone library. Meanwhile, changes of Arcobacter spp. and total bacteria before and after treatment were detected through real-time quantitative PCR. The clone library results showed that there were 73 positive clones included Proteobacteria (91. 80%), Firmicutes (2. 70%), Bacteroidetes (1. 40%), and uncultured bacteria (4. 10%) in the untreated wastewater. The typical pathogenic genus Arcobacter belonging to e-Proteobacteria was the dominant component of the library, accounting for 68. 5% of all clones. The main groups and their abundance in different treatments were significantly distinct. The highest values of species abundance (S), Shannon-Wiener (H) and Evenness (E) were observed in the adjusting tank, which were 43. 0, 3. 56 and 0. 95, respectively. The real-time quantitative PCR results showed that the copy number of Arcobacter spp. was (1. 09 ± 0. 064 0) x 10(11) copies.L-1 in the untreated sewage, which was consistent with the result of 16S rDNA gene clone library. Compared to untreated wastewater, bacterial copy number in the treated effluent decreased 100 to 1 000 times, respectively, suggesting that MBR treatment system could remove the microbial quantity in such scale. In the recycled water, the physicochemical parameters and indicator bacteria met the water quality standard of farmland irrigation. However, further research is needed to estimate the potential health risks caused by residual pathogenic microorganisms in future.

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.

[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.

[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.

[Characterization of inductive synthesis of levoglucosan kinase by a combined strategy of enzymological and fast atom bombardment mass spectrometric analysis].

Levoglucosan is the main product derived from pyrolysis of cellulose. A mutant Aspergillus niger CBX-209 could grow on levoglucosan well fermenting it into citric acid with a yield comparable to that on glucose. Levoglucosan hydrolase was absent by measuring glucose formation with the glucose oxidase and peroxidase coupling system. Cell extracts were partly purified by ammonium sulfate fractionation and ion-exchange chromatograph. Direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl2 was observed when it was reacted with partly purified enzyme by a combined strategy of enzymological and fast atom bombardment mass spectrometric analysis. These data showed that the mutant used a novel enzyme, levoglucosan kinase, to convert levoglucosan into glucose 6-phosphate. Levoglucosan kinase was an inductive enzyme.