Abstract:[Background] Enzymatic property of the MIO-dependent (methylidene-imidazol-5-one) enzyme is significantly affected by the flexibility of the special loop that includes the catalysis Tyr (Tyr78-loop). [Objective] The effect of Tyr78-loop on activity of phenylalanine ammonia-lyase (PAL) from Anabaena variabilis was investigated to improve PAL catalytic activity. [Methods] Tyr78-loop was genetically modified. Positive mutants with enhanced activity were selected, followed by characterization. [Results] The specific activities of S73N, E95V, E95K and S73N/E95K mutants were improved by 34%, 30%, 18% and 35%, respectively, compared with that of the wild type at 37 °C and pH 8.5. According to protein structure simulation, the amino acid sites Asn73, Val95 and Lys95 in the mutants S73N, E95V and E95K, which located at the junction of α-helix and Tyr78-loop, had fewer hydrogen bonds with the nearby amino acids. This would increase the flexibility of Tyr78-loop and result in enhancement of the enzyme activity. [Conclusion] Catalytic activity of AvPAL could be improved by increasing the flexibility of Tyr78-loop through mutation on the Ser73 and Glu95 sites.

Abstract:[Background] Raw starch digesting amylase can directly degrade raw starch granules below the gelatinization temperature. Hence, it has been wildly used in the production of bioethanol, Chinese spirit, yellow rice wine and vinegar. [Objective] This study aims at isolation and identification new raw starch digesting amylase-producing strain, to provide enzyme resource that can be used in the process of fermentation with uncooked materials. [Methods] With tapioca starch as sole carbon source, raw starch digesting amylase-high producing strain was isolated from soil of cassava field. Then, it was identified by analysis of colony morphology, photomicrographs, and 16S rRNA gene sequence. Single factor experiment was used to optimize the medium component and fermentation conditions. The enzyme was purified and characterized. [Results] Strain GEL-09 was isolated and identified as a species of the genus Bacillus, designated as Bacillus sp. GEL-09. The raw starch digesting amylase production was optimized by varying culture conditions. On the optimal culture condition, the maximal enzyme production was 430.6 U/mL, which was 2.8 folds of that before optimization. The enzyme showed highest activity at 50 °C and pH 7.0. Its raw starch digestion ability value was 62.3%, which was higher than that of the α-amylase, maltogenic α-amylase or β-amylase. [Conclusion] Bacillus sp. GEL-09 has great development potential and application prospects in the production of raw starch digesting amylase.

Abstract:[Background] Laccase and manganese peroxidase (Mnp) are major enzymes in lignin biodegradation and they act synergistically. The activity of Mnp depends on the Mn2+, however, Mn2+ is the inhibitor to most laccases. [Objective] The aim of our study is to obtain bacterial laccase with Mn2+ tolerance for lignin degradation. [Methods] We constructed the metagenomic library with environmental DNA isolated from sewage river sediment of Xuchang, and identified a gene encoding a bacteria laccase, lac1542, from the metagenomic library via activity-based functional screening. Then, we overexpressed Lac1542 heterologously as soluble active enzyme in Escherichia coli, and characterized the recombinant enzyme after purification subsequently. Finally, we further investigated the lignin degradation ability of the complex enzyme systems containing Lac1542. [Results] Sequence analysis revealed that lac1542 encoded a protein of 513 amino acids. The purified Lac1542 exhibited maximal activity at 75 °C and pH 4.0 with ABTS as substrate, and this enzyme was stable in the pH range of 3.0–6.5 and at the temperature below 70 °C. Interestingly, the enzymatic activity was increased after addition of 100 mmol/L Mn2+. Also, the optimal substrates were in the order of ABTS>Syringaldazine>catechol>2,6-DMP>guaiacol based on the kinetic parameters. In addition, the degradation percentage of Lac1542/Mnp toward lignin was 47.8%, 25.4% higher than that (22.4%) of Mnp. The degradation percentage of Lac1542/Mnp/Coprinus cinereus Peroxidase (CIP) toward lignin was reached up to 71.5%, 22.6% higher than that (48.9%) of Mnp/CIP. These results suggested that the degradation percentage of the complex enzyme system toward lignin was distinctly improved after addition of Lac1542. [Conclusion] The highly soluble expression, tolerance to high concentration of Mn2+, and thermostability of Lac1542 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and cellulosic ethanol production, and dye decolorization.

Abstract:[Background] The bamboo slips should be kept in water for a long time before dehydration treatment, while the microbe might decompose it during this period. [Objective] In order to preserve bamboo slips effectively, the actinobacterial community structure features and function of soak solution which were collected from Han dynasty tomb located in Laoguanshan of Chengdu were studied. [Methods] The PCR-DGGE (Denaturing gradient gel electrophoresis) and culture-dependent methods were employed to reveal the diversity and function of actinobacteria in soak solution. [Results] The PCR-DGGE and culture-dependent results were showing a certain degree of consistency. The PCR-DGGE result revealed that Richness (S), Shannon-Wiener index (H′) and Simpson index (D) of actinobacteria of soaking bamboo slips were different. 15 DGGE bands were excised and sequenced, the major community of actinobacteria in soak solution were belonged to 10 genera. The genera Promicromonospora, Cellulosimicrobium, Blastococcus and Streptomyces widely distributed in soak solution, furthermore genus Promicromonospora was detected in each samples. 12 strains were isolated by culture-dependent method and belonged to Streptomyces and Promicromonospora. Congo red medium was employed to evaluate their carboxymethyl cellulase activity. The results showed five strains possessed cellulolytic ability. [Conclusion] The analysis of the actinobacterial community and function revealed the potential actinobacteria species may decompose bamboo slips during waterlogged period.

Abstract:[Background] Virioplankton plays an important role in organic carbon cycle. [Objective] We studied the abundance of virioplankton and bacteria in different seasonal water samples and soil samples in Napahai wetland, and analyzed the relationship between the abundance of virioplankton and soluble organic carbon in different seasons. [Methods] The abundance of virioplankton and bacteria in water samples of different seasons was detected by Epifluorescence Microscopy. The abundance of virus and bacteria in soil samples of different seasons was detected by Flowcytometry. [Results] The abundance of phytoplankton bacteria and virus was 3.38×106/mL and 4.38×107/mL in rainy season. The abundance of phytoplankton bacteria and virus in the dry season was 8.85×105/mL and 9.66×106/mL, the average annual abundance of phytoplankton bacteria and virus was 2.13×106/mL and 2.67×107/mL. There were significant differences of phytoplankton bacteria and virus abundance in different seasons. The yield of bacterial carbon was 8.01 μg C/(L·h) in the rainy season, and 10.30 μg C/(L·h) in the dry season. The DOC contributed by the virioplankton accounted for 32.38% to 76.38% of the total DOC library in the rainy season, while it was 8.23% to 47.87% in the dry season. [Conclusion] Virioplankton plays an important role in the organic carbon cycle of the Napahai wetland.

Abstract:[Background] Pingdingshan is rich in hot spring resources. The study of microbial diversity and community structure in the spring water will help us to better exploit these resources. [Objectives] The bacterial diversity of Five Hot Spring (SHANGT, ZHONGT, XIAT, WENT and SHENT) in Henan Lushan was investigated and analyzed. [Methods] The bacterial 16S rRNA V3?V4 genes were sequenced by Illumina Hiseq2500 PE250 technology. The OTUs was sorted and counted using Uparse, Mothur, Silva, MUSCLE and Qiime software. Finally, the bacterial abundance and diversity in Five Hot Spring was analyzed. [Results] It was obtained 56 017, 68 319, 65 247, 59 340, 68 825 effective tags, and acquired 670, 287, 337, 598, 381 OTUs from the SHANGT, ZHONGT, XIAT WENT, and SHENT respectively. The analysis of bacterial classification showed that the bacteria from Five Hot Springs could be classified as more than 40 phyla. Proteobacteria accounted for 84.12% was the dominant bacteria. In the genera level, bacteria from Five Hot Springs could be classified into more than 239 genera. The dominant bacteria of Five Hot Springs (SHANGT, ZHONGT, XIAT, WENT and SHENT) respectively is Acinetobacter, Tepidimonas, Vogesella and Acinetobacter, Sphingopyxis and Novosphingobium, Aquabacterium. [Conclusion] Five Hot Springs in Lushan are rich in microbial resources and may have the potential for the development of such resources.

Abstract:[Objective] The aim of this study was to investigate actinobacterial diversity in the sediment of Xinjiang saline lakes: Taitema, Barkol and Qijiaojing, and the relationship between the actinobacterial community and ionic composition. [Methods] From every lake we collected 5 samples and mixed, to extract total DNA by using SDS-CTAB. We used primers for the class Actinobacteria for actinobacterial l6S rRNA gene amplification and then constructed a clone library for the sediment sample. In total 220 clones were randomly selected from each sample, from which the positive clones were screened on the basis of Hae III digestion patterns, and then these positive clones were sequenced. Eight major chemical ions were detected in each sample, and then the correlation between chemical ions and actinobacterial community was analyzed. [Results] In total 143 Operational Taxonomic Units (OTUs) were obtained from 381 clones which belonged to 37 actinobacterial genera (Taitema: 24, Barko: 16, Qijiaojing: 14). The common Actinobacteria of the three lakes were Aciditerrimonas, Aquihabitans, Demequina, Dietzia, Ilumatobacter and Amycolatopsis. Barkol was closer to Qijiaojing at the actinobacterial community composition, but Taitema had huge difference from the other two. There were plenty of unknown Actinobacteria in the Sediment of these Lakes, it reached 47.59% in the whole Actinobacteria at Barkol, 53.07% at Qijiaojing and 51.53% at Taitema. Redundancy analysis was used to analyze the relationship between chemical ions and actinobacterial community, and the results showed that Mg2+, Ca2+, SO42-, HCO3- and CO32- had closer ties with the actinobacterial community than Na+, Cl- and K+. [Conclusion] These lakes contained plenty of unknown Actinobacteria which were worth to exploring and different ions influenced unique Actinomycetes community.

Abstract:[Background] Bacillus-like bacteria can form endospores with strong resistance and thus, can survive in a variety of extreme environments. Their multiple metabolic products make them have important research value in many fields. Because glacier has the low temperature and oligotrophic unique environment, Bacillus-like bacteria in this environment would be specific, which could be expected to enrich the Bacillus-like resources and discover new functional genes. [Objective] The aim of this study is to understand the diversity of the Bacillus-like resources in the Hailuogou glacier, Sichuan Province, providing a foundation for mining new Bacillus-like resources. [Methods] Bacillus-like strains were isolated by the culture-dependent method and identified using 16S rRNA gene sequence. Subsequently, physiological-biochemical characteristics of the typical strains selected were determined and further performed cluster analysis by the group-average method with the Euclidean distance model. [Results] In total, 44 strains were obtained and 36 strains belonged to Bacillus-like bacteria. The 16S rRNA gene phylogenetic analysis showed that they could be assigned to 19 species within 4 genera including Bacillus (11 species 24 strains), Paenibacillus (2 species 3 strains), Brevibacillus (4 species 5 strains) and Lysinibacillus (2 species 4 strains). Thus, the genus Bacillus was dominant. The physiological-biochemical characteristics showed that: i) only 3 strains could survive at 4 °C, 7 strains could grow at 50 °C, whereas most strains could grow well at 30 °C; ii) 74% of the Bacillus-like strains had alkali resistance; and iii) 37% of them could grow without NaCl. According to the physiological and biochemical results, those strains could be divided into 3 groups by cluster analysis using the group average method and Euclidean distance models. The first group could hydrolyze esculin and utilize glucose. The second groups could not hydrolyze esculin and utilize glucose, and the third group could only utilize glucose. [Conclusion] Hailuogou glacier soil is rich in the Bacillus-like resources, providing a basis for the mining of them.

Abstract:[Background] Zygomycotan fungi are widely distributed in nature. While some members are harmful, more zygomycotan fungi play an important role in industry, food, medicine, biological control, and so on. They have just been reported sporadically in the Tibet and lack a systematic survey. A large number of potential species need to be isolated, identified, understood, preserved and exploited. [Objective] To understand the species diversity of zygomycotan fungi in the Tibet, and lay a foundation for the control of harmful zygomycotan fungi and the utilization of beneficial ones in this area. [Methods] Zygomycotan fungi isolated with plate dilute method from 701 samples in 19 representative counties all over the seven prefectures of Tibet, were identified morphologically and molecularly, with SSU, ITS and LSU rDNA as markers. Based on these data, the biodiversity, dominant and rare taxa of Tibetan zygomycotan fungi were analyzed. [Results] Tibetan zygomycotan fungi obtained here were classified into 10 genera and 26 species. Among these species, five have been reported in the Tibet, while 21 species are new to Tibet; Four species are even new to China, which are Mortierella amoeboidea, M. globalpina, Mucor brunneogriseus and M. fuscus. The diversity index analysis of Tibetan zygomycotan fungi showed significant differences in the number and composition of species among regions. The most diverse four counties are Bome, Mainling, Dangxung and Baxoi. Analyses of species and genera revealed two dominant zygomycotan genera in the Tibet, Mortierella and Mucor, as well as three dominant species Mortierella alpina, Mucor hiemalis, and Rhizopus stolonifer. Eight common genera are Absidia, Actinomucor, Cunninghamella, Rhizomucor, Rhizopus, Syncephalastrum, Umbelopsis and Zygorhynchus. Common and rare species are 9 and 14, respectively. [Conclusion] The zygomycotan fungi in the Tibet are rich and significantly different in biodiversity among regions. Rare species account for more than half, implying the importance of protecting the natural environment of Tibet.

Abstract:[Background] Photorhabdus sp. is preserving in the intestine of Heterorhabditis sp., and it can produce variety efficient and broad spectrum insecticidal proteins and toxins. In recent years, it’s the novel research object on new insecticidal protein and gene after Bacillus thuringiensis. [Objective] To clone Txp40 toxin gene of Photorhabdus luminescens (NLK-1) and analyze the differences on gene sequence, protein composition, physicochemical properties and conformation similarity to other symbiotic bacteria. Txp40 toxin gene was cloned in prokaryotic expression vector of Escherichia coli and induced its to express, then utilized Galleria mellonella larvae to preliminary detect its insecticidal activity. [Methods] The primary symbiotic bacteria were isolated from the infected larvae by Heterorhabditis bacteriophora, and the primers were designed according to the reported sequences. The target gene was amplified and sequenced by the cloning plasmid pMD19-T. The Expasy Online ProtParam Tool to predict its basic physical and chemical properties parameters, NPS@-Network Protein Sequence Analysis online tools for secondary structure prediction. Txp40 toxin gene was detected by cloning, digestion and ligation on pET28a and transformed into Escherichia coli BL21 then induced by IPTG, and identified by blue-white blotting. Utilizing Photorhabdus luminescens (NLK-1) Txp40 protein crude extract to test the insecticidal activity to Galleria mellonella larvae by fed and hemocole injection. [Results] Txp40 toxin gene open reading frame is 1 008 bp (335 amino acid), and the similarity to the known gene was 94%. The similarity with the known 40 kD related protein was 99%, molecular weight was 37.9 kD, pI 8.37, and it was mainly composed of Alpha helix 35.71%, Random coil 54.46%, Extended strand 9.52% by secondary structure predicting. The expression of pET28a-Txp40 was detected by SDS-PAGE, which showed that there were specific band at 38 kD, and the molecular weight of protein was similar with prediction, and the expression was relatively single and the level was higher. The insecticidal activity tests showed that Photorhabdus luminescens (NLK-1) Txp40 protein had high virulence to Galleria mellonella larvae, and the lethality reached 100% at 48 h under 5 μL Txp40 protein crude extract per larvae, but oral toxicity was not detected. [Conclusion] Photorhabdus luminescens (NLK-1) Txp40 toxin gene was successfully obtained, and the physicochemical properties of the protein were analyzed by comparison to the known gene, and secondary structure were predicted and analyzed to other known protein. At the same time, the prokaryotic expression vector was constructed, the preliminary insecticidal test showed Photorhabdus luminescens (NLK-1) Txp40 protein had high hemocole toxicity to Galleria mellonella larvae. The research laid the foundation for the further study and discovery of insecticidal genes and toxic protein from Photorhabdus luminescens (NLK-1).

Abstract:[Background] Quaternary ammonium compounds, especially benzalkonium chloride (BC), are widely used in food industry, leading to decreased sensitivity to BC in Listeria monocytogene. Efflux pumps are recognized as an important mechanism for BC tolerance. [Objective] To investigate the role of efflux pump MdrL in tolerance to BC in Listeria monocytogenes. [Methods] The mutant strain ΔmdrL was constructed from the wild-type strain EGD-e by homologous recombination. The differences in BC tolerance, growth in sub-lethal concentration of BC (2 μg/mL), and survival in lethal concentration of BC (16 μg/mL) between the mutant strain ΔmdrL and the wild-type strain EGD-e were investigated. [Results] The mutant strain ΔmdrL and its complemented strain were constructed in this study. The mutant and wild-type strains showed the same minimum inhibitory concentrations to BC. The results show that compared with the wild-type strain EGD-e, the lag-phase duration of the mutant strain ΔmdrL was prolonged and the mean maximum growth rate and the mean maximum optical density were decrease in the presence of sub-lethal concentration of BC. Significantly difference was observed in the serial diluted culture between the mutant strain ΔmdrL and the wild-type strain EGD-e on the plates in the presence of BC (4 μg/mL). Compared with the wild-type strain EGD-e, two log decrease were observed in the mutant strain ΔmdrL in the presence of the lethal concentration of BC. The results of electron microscopy showed that the mutant strain ΔmdrL became longer when exposed to BC. Besides, no differences were observed in the accumulation and efflux to ethidium bromide (EB) between the mutant strain ΔmdrL and the wild-type strain EGD-e. [Conclusion] Efflux pump MdrL plays an important role in tolerance to BC in Listeria monocytogenes, however, this efflux pump is not associated with efflux of EB.

Abstract:[Background] Plant growth-promoting bacteria (PGPB) have good potential in assisting phytoremediation of heavy metal pollution due to their advantages in promoting the growth and heavy metal-resistance of plants. In heavy metal-contaminated soils, indigenous PGPB can better colonize and exert their plant growth-promoting abilities. [Objective] Our aim is to isolate the indigenous heavy mental-resistant PGPB from the Le’an River-Poyang Lake Wetland, which has been polluted by acid mine drainage for many years, thus to provide superior microbial resources for bacterial assisted phytoremediation of heavy metal polluted wetland soil. [Methods] Cu, Zn and Pb-resistant strains were isolated from heavy metal polluted wetland soils and water in Dai cun, which is located in Le’an River. The plant growth-promoting characteristics (IAA (Indole acetic acid), phosphate solubilization, siderophore and ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity) of the studied isolates were measured, and the isolates with good growth-promoting characteristics were selected and identified using 16S rRNA gene sequencing. The resistant characteristics (heavy metal, antibiotics, acid and alkali, salt) of the selected strains were also determined. [Results] A total of 22 isolates were able to grow in the presence of Cu 50 mg/L, Zn 400?mg/L and Pb 800 mg/L. Among them, 10 strains showed good plant growth-promoting characteristics. According to 16S rRNA gene sequencing, 4 strains belonged to the genus Ralstonia, 3 strains belonging to the genus Burkholderia and the other 3 strains belonged to the genera Cupriavidus, Stenotrophomonas and Novosphingobium, repectively. The numerical classification based on bacterial resistant characteristics was mostly consistent with their phylogenetic position. [Conclusion] The isolation and identification of multiple heavy mental-resistant PGPB could provide good microbial resources for in situ remediation of heavy metal contaminated soils.

Abstract:[Background] In recent years, the ruminal methane mitigation potential of nitrooxy compounds has attracted a wide attention, however, limited information is available for the effect of these compounds on anaerobic fungi, an important fibrolytic microorganism in the rumen. [Objective] To investigate the effects of nitroglycerin (NG) on the activity and metabolism of anaerobic fungi and methanogens. [Methods] The fungal mono-cultures (Piromyces sp. F1) and co-cultures with methanogens (Piromyces sp. F1+Methanobrevibacter sp.) were used. A series of doses of NG (0.0、6.6、13.2 and 19.8 μmol/L) were added in these cultures and the metabolic profile, fibrolytic activities and microbial abundances were comparably analyzed. [Results] In co-cultures, the dose of 6.6 μmol/L NG completely inhibited the methane production, resulting in the accumulation of hydrogen and formate (P<0.05). And the acetate production and the activities of xylanase and carboxymethyl cellulase decreased (P<0.05), while the changes of the abundances of anaerobic fungi and methanogens were not observed (P>0.05). In fungal mono-cultures, the dose of 6.6 μmol/L NG decreased the production of formate, acetate and ethanol as well as the activities of xylanase and carboxymethyl cellulase (P<0.05), while did not affect the total gas and hydrogen production (P>0.05). In the two culture systems, as the dosage of NG increased, the inhibitory effect on anaerobic fungi and methanogens was enhanced. [Conclusion] NG had inhibitory effect on methanogens, but also had negative effect on anaerobic fungi.

Abstract:[Background] Associative nitrogen-fixing bacteria are non-host specific, and widely distributed in soil and phyllosphere environments, playing an important role in nitrogen supply for ecosystem. They also can promote plant growth indirectly via producing hormone for host plants or protecting plant from disease attacking, therein have great potential for agriculture application. However, the application of microbial inoculant into soil is limited owing to the unstable efficiency in field, due to the competition from indigenous microorganisms and inhibition effect of soils. Compared to soil habitat, phyllosphere is relatively simple and constitute large areas. Foliar application of associative nitrogen fixing bacterial inoculant provides an alternative way instead of soil application in agriculture. [Objective] To optimize the liquid inoculant of an associative nitrogen-fixing bacteria strain W12 by adding different additives, and to track its colonization on the phyllosphere of maize after foliar application. [Methods] Firstly, the 16S rRNA and nifH gene of strain W12 were sequenced to identify its taxonomy. The effects of carboxymethyl cellulose (CMC) and glycerol (Gly) used as liquid inoculant additives on the growth and nitrogenase activity of strain W12 were evaluated in liquid culture and the liquid inoculant was prepared based on the optimized condition, and then the colonization of strain W12 on the phyllosphere of maize after spraying of liquid inoculant were estimated by plate counting on a low-nitrogen agar. After harvest, the grain yield and nitrogen content of grain, stem and leaf of maize in field were measured. Meanwhile, we also inspected the shelf life of inoculant under different storage conditions. [Results] The phylogeny analysis based on 16S rRNA and nifH gene both suggested that strain W12 is closely related to the species Klebsiella variicola. CMC and glycerol addition in liquid media showed no significant influence on the growth of strain W12, but remarkably improved the nitrogenase activity by 3.7 and 24.2 times, respectively. After the foliar application of liquid inoculant via leaf spraying, the recovered strain W12-similar colonies on low-nitrogen media were up to 1.7×105 and 4.3×105 CFU/g leaf from maize phyllosphere in pot and field experiments, significantly higher than the control without inoculation, while the nitrogen content of grain, stem and leaf of maize in inoculation treatment were higher than those treatments without inoculation. The number of viable strain W12 decreased over the storage time while storage at 4 °C maintained much higher viable bacteria than at room temperature, with the viable bacteria number more than 1.0×108 CFU/mL after storage at 4 °C for 90 days. [Conclusion] CMC and glycerol used as additives of liquid inoculant are favorable for bacteria adhesion on leaves and could remarkably improve nitrogenase activity of strain W12. Storage at 4 °C can sustain long shelf life of strain W12 liquid inoculation, compared with room temperature. After foliar application in field, strain W12 could successfully colonize in the phyllosphere and inoculation significantly improved the nitrogen content of maize grain and plant. Our results exhibit new insights on the preparation and application of foliar inoculant, and set a good example for field application of nitrogen fixing bacteria to reduce the nitrogen fertilizer application and stabilize crop yield.

Abstract:[Background] Endophytic actinomycetes are ubiquitous in all the plant species. The use of plant growth-promoting endophytes has become of great interest as a way to enhance plant growth. [Objective] Endophytic actinomyces that could effectively promote the growth of plant were isolated, screened and identified from the tomato root, laying a foundation for the development of biological fertilizer. [Methods] Endophytic actinomyces in the tomato root were isolated with tissue sand culture method and actinomyces isolation medium; bacterial strains with strong growth promotion characteristic were screened through Salkowski colorimetric method, Mo-Sb colorimetric method and CAS plate detection method, and their growth promotion effect was verified through the pot experiment of tomato and cucumber seedling. Bacterial strains were identified through analyses of morphology, physiological biochemistry, 16S rRNA gene sequence similarity and phylogeny. [Results] Endophytic actinomyces NEAU-D1 with an Indole-3-acetic acid (IAA) yield of 25.56 mg/L was isolated and screened, which could produce siderophore and had good dissolving effect on various insoluble phosphate. 16S rRNA gene sequence analysis showed that the bacterial strains were Streptomyces. Pot experiment of tomato and cucumber seedling showed that, compared with the control group, the root length, plant height, fresh weight and dry weight of the tomato seedling were significantly increased by 9%, 23%, 47% and 92% respectively, while those of the cucumber seedling were significantly increased by 43%, 47%, 134% and 58% respectively. [Conclusion] Streptomyces NEAU-D1 can be used as a potential growth promoting endophytic actinomyces for the research and development of multifunctional biological fertilizer for facility vegetables.

Abstract:[Background] N2O is a powerful greenhouse gas with a 265-fold stronger warming potential than CO2. Fertilization plays an important role in affecting N2O emission from soils driven by bacterial community, and denitrification is the major source of N2O under anaerobic conditions. [Objective] To investigate N2O emission and greenhouse soil bacterial community response to the overuse of nitrogen fertilizer. [Methods] Robot system was used to monitor the denitrifying gas (N2O and N2) kinetics of soils during anaerobic incubation, and compare the difference of N2O emissions between traditional fertilization and reduced nitrogen fertilization. The soil microbial community structure was analyzed by sequencing the 16S rRNA gene V3?V4 region using Illumina MiSeq. [Results] The nitrate concentration in conventional nitrogen fertilization soil (CNS) was about two folds higher than that in reduced nitrogen fertilization soil (RNS). CNS showed higher N2O accumulation and emission rate during earlier anaerobic-incubation stage although the nitrate content was adjusted to the same level in both types of soil. Traditional fertilization significantly changed the bacterial community structure, and decreased the microbial diversity. Although Rhodanobacter was the most abundant genus both in CNS and RNS, it was enriched by traditional fertilization. However, relative abundance of denitrifying functional genes (narG, nirK, norB, nosZ) had little response to the overuse of fertilizer. [Conclusion] Traditional fertilization reshaped the bacterial community in soil. Overuse nitrogen fertilizer influenced N2O emission from soil via changing the microbial community including the microbiota related with nitrogen transformations.

Abstract:[Background] Lung pathogenic Escherichia coli (LPEC) belonging to extraintestinal pathogenic Escherichia coli was one of the important pathogens of zoonosis. [Objective] To establish a BALB/c mice challenge model of LPEC from forest musk deer for the study of pathogenicity of LPEC O78. [Methods] BALB/c mice were infected by LPEC O78 that was preserved in the laboratory, and the infection dose of model was determined by intraperitoneal inoculation to calculate LD50 (median lethal dose). The effect of mice model was evaluated based on monitoring the changes in bodyweight, biochemical indexes and bacterial colonization, and further evaluated by isolation of bacteria and histopathological examination. [Results] The LD50 of LPEC O78 in BALB/c mice was 3.6×108 CFU/mL. After infection, the challenge group showed that depressed, anorexic and sluggish symptom. Lung and liver enlarged, intestine hemorrhage was observed by necropsy after 3 h post-challenge. The bodyweight of mice decreased by 3.2 g within 24 h post-challenge, and then slowly increased. The biochemichel indexs of ALT, AST, TP, ALB, IP, Ca, Mg, TBIL, URIC, UREA, GLU, CHE and LDH in the challenge mice were higher than the mock mice (P<0.05), except the uric acid content (P>0.05). All organs had bacterial colonization, in which heart, spleen and kidney reached the highest at 24 h post-challenge whereas the time for highest colonization in liver, lung and intestine was 12 h post-challenge. Histopathological examination revealed different degree of inflammatory cell infiltration and necrosis. [Conclusion] This study successfully established the mice challenge model of LPEC O78 and laid a foundation for the future research of pathogenesis and pathophysiology of LPEC O78 from forest musk deer.

Abstract:[Background] Using traditional antibiotics as growth promoters in the pig industry leads to the richness and transmission of bacterial resistant genes. Thus, the identification and development of the emerging growth promoters as alternatives to antibiotics represents tremendous economic potential in feed additive industry. Probiotic cultures are interesting candidates as feed additives for food-producing animals. [Objective] This work had as main objectives to isolate Bacilli from the feces of free-range swine and then to characterize bacteriocins produced by Bacilli showing inhibition of typical gastrointestinal bacterial pathogens, in order to evaluate their potential as new growth promoters. [Methods] Using the dilution plate separation method and coated plate method, we screened culturable Bacilli. Strains presenting inhibitory properties for gastrointestinal bacterial pathogens were determined by Oxford cup method. The bacteriocin-producing Bacillus isolates were identified by 16S rRNA gene sequencing. [Results] Screening of 116 candidate strains isolated from feces samples resulted in the selection of six isolates presenting markedly inhibitory properties for bacterial pathogens, of which two strains were identified as Bacillus velezensis, three as Bacillus subtilis and one as Bacillus licheniformis. Among of them, B. licheniformis strain DY7 and B. subtilis strain FX4 exhibited the most effective inhibitory activity against the indicator pathogens, except for Salmonella enterica CICC10420, and were susceptible to Cefotaxime and Erythromycin. Additionally, both bacteriocins were stable over a wide range of pH 3.0?9.0 and temperatures 50?100 °C. [Conclusion] The effective inhibitory activity against the indicator pathogens and the stability may lead to the assumption that the strains DY7 and FX4 may be applied as pig growth promoters to control pathogens.

Abstract:[Background] Cordyceps pruinosa is a traditional medicinal fungus, which has many kinds of pharmacological effects such as anti-radiation. Cordycepin is an important bioactive compound of this fungus. [Objective] The influencing factors of cordycepin extracted from Cordyceps pruinosa were studied, so as to provide reference for the preparation of cordycepin from the fungus. [Methods] With extraction yield of cordycepin as the target, the response surface methodology was used to optimize the ultrasonic extraction process. The effects of ultrasonic time, liquid-to-solid ratio and ultrasound power on the extraction efficiency were studied using single factor experiment, then a three levels of response surface methodology experiments was chose to optimize the extraction process. [Results] The results showed that the optimum extraction conditions were: ultrasonic time 606 s, liquid-to-solid ratio 45.9:1, ultrasound power 400 W. A cordycepin yield of 6.136 mg/g was obtained under this condition, which was close to the model predicted value and showed the fitting equation was good. [Conclusion] In this experiment, the extraction conditions of cordycepin in Cordyceps pruinosa were studied for the first time, which would provide reference for the production of cordycepin from Cordyceps pruinosa and be helpful for deep use of this fungus.

Abstract:Gene assemblies are new technologies developed in recent years in the field of synthetic biology. They are based on large-scale analysis of genome sequences to find new or cryptic gene clusters which synthesize bioactive substances. The silent or cryptic gene clusters in microorganisms can be activated to synthesize potential valuable compounds by these technologies. We aim to elucidate the principle, the key strategy and the application of in vivo and in vitro gene assemblies. These technologies play an important role in synthetic biology, metabolic engineering and functional genomics and they will be of great value to efficiently produce bioactive compounds.

Abstract:Medical Microbiology is an important basic medical course, with the characteristics of numerous knowledge points and dispersive contents. Young teachers with high academic qualifications are becoming the principal teaching staffs of colleges and universities. How to improve the teaching quality of Medical Microbiology by young teachers is an important urgent problem to be solved. This paper discussed the solutions to this problem from the aspects of laying a solid foundation in the basic teaching skills of young teachers, enhancing teaching abilities, flexibly using various teaching methods including digital teaching measures, and well coordinating the relationship between teaching and scientific research.

Abstract:[Background] Flammulina velutipes is an important edible mushroom cultivated in China with an annual output of more than 2 500 000 tons and the cultivation scale became first place in the world. Spawn preservation is the foundation of cultivation and breeding of new varieties. However, weak research about spawn preservation has become a bottleneck that restricts the further development of F. velutipes industry in China. [Objective] To establish an efficient, low-cost and easy method for short- and medium-term preservation of F. velutipes spawn by evaluated the preservation factors. [Methods] The orthogonal experiments were carried out with temperature, glycerol, trehalose, mannitol and the volume of protectant as the experimental factors. After 12 months of preservation, the growth rate of mycelia on sawdust medium was investigated. Range analysis and regression analysis were used to detect the effect of preservation factors. [Results] The results showed that temperature, trehalose, glycerol and mannitol had very significant influences on the short- and medium-term preservation of F. velutipes spawn, while the effect of protectant volume was not significant. Temperature was the most important factor, and the interaction effects of temperature and other 4 factors were all very significant. The proper short-term preservation temperature was 20 °C, and the proper medium-term preservation temperature was ?80 °C. The interaction between osmotic and nonosmotic protectant had a significant effect on spawn preservation. The interaction effect between osmotic protectants (trehalose and mannitol) was not significant. High concentrations of trehalose, glycerol and mannitol were not conducive to the short- and medium-term spawn preservation of F. velutipes. The best protectant was a mixture of 10% glycerol and 0.3 mol/L mannitol. [Conclusion] The spawn preservation method established by this study filled the blank of F. velutipes industry, and the results can also provide important reference value for short- and medium-term spawn preservation of other macro fungi.

Abstract:[Background] The future development of pertussis vaccine is acellular pertussis (component) vaccine, which could significantly reduce the incidence of adverse events after inoculation. This new type of vaccine needs to remove the endotoxin in the process of production. [Objective] In our study, the removal process of the endotoxin from pertussis filamentous hemagglutinin (FHA) was investigated by optimized chromatography purification with response surface methodology. [Methods] The process of chromatographic purification was verified by the single factor experiment, to ensure the range of response surface methodology; the method of response surface analysis was adopted according to the CCD experimental design principles by using Minitab software: the variable factors were pH, Cond, and Mass; Response values were considered the concentration of FHA protein and recovery of FHA, and finally combined the value of endotoxin. [Results] These results showed that the optimum conditions for chromatographic process of endotoxin removal from pertussis filamentous hemagglutinin were as follows: pH 5.3, Cond 9.6, and Mass 3.0. [Conclusion] The optimum processes of chromatographic purification on endotoxin removal were screened successfully by response surface methodology. This method has advantages of higher efficient and lower energy consumption, which will lay a good foundation for the subsequent biological process of expanded reproduction.