Abstract:[Background] Recently, IgA proteases that specifically cleave human IgA1 molecules have been considered as potential drugs for the treatment of IgA nephropathy (IgAN), but its biological activity may be affected by various physical and chemical factors. [Objective] To explore the physicochemical properties of Haemophilus influenzae ATCC49247 IgA protease for determining the optimal conditions and then to observe its decomposition of low glycosylated IgA1. [Methods] IgA protease was isolated and purified from the bacterial culture solution and the hydrolysis activity of IgA protease and its decomposition of low glycosylated IgA1 were detected by SDS-PAGE electrophoresis under various physicochemical conditions. [Results] H. influenzae ATCC49247 IgA protease tolerated a wide range of temperatures and the optimum temperature is 50 °C. IgA protease irreversibly lost stability above 60 °C. IgA protease maintained full catalytic activity between pH 6.0 and 9.0. PMSF of 1 mmol/L and SDS of above 10 mmol/L strongly inhibited the activity of IgA protease, whereas DTT and EDTA had no significant effect on its activity. IgA protease was obviously inhibited by all concentrations of Al3+, Fe3+ and high concentration of Cu2+, Zn2+ and Fe2+, whereas Co2+, Mn2+, Ca2+, Ni2+ and Mg2+ had no significant effect on its activity. By selecting the most suitable conditions for IgA protease, we found that most of the low-glycosylated IgA1 substrate could be degraded by IgA protease. [Conclusion] IgA protease can maintain good enzyme activity under the optimal conditions, when exerting the degradation of low glycosylated IgA1 and the result lays the fundation for the clinical research and the further development of the medicinal value for IgA protease.

Abstract:[Background] Marine microorganisms are important source of pharmaceutical active leading compounds and chemical induction can be used as a convenient approach to mine their secondary metabolic potential. [Objective] Based on a marine-derived fungus Aspergillus terreus C23-3, this study aimed to optimize fermentation conditions for more abundant metabolites (including butyrolactones), and exploit the potential of the strains in drugs targeting Alzheimer’s disease. [Methods] The strain was first cultivated on micro-scale, by taking four basic cultures mediums including seawater potato medium, malt extract medium, rice medium and soybean medium, and adopting six chemical inducers including sodium butyrate, suberoylanilide hydroxamic acid (SAHA), 5-azacytidine (5-azaC), procaine hydrochloride, ZnCl2 and CuCl2. The morphological variation of the mycelia was observed. Then, based on the thin layer chromatography (TLC) fingerprints, chemical coloration and bioautography results, inducing conditions with rich new metabolites and rich anti-acetylcholinesterase and antioxidant products were initially screened out for further routine-scale fermentation. Using the above methods and high-performance liquid chromatography-diode array detector (HPLC-DAD) analysis, the diversity of the total metabolites and of butyrolactones was analyzed for the routine fermentation. [Results] The results indicated that CuCl2, ZnCl2, SAHA and 5-azaC caused remarkable changes in the growth morphology of Aspergillus terreus C23-3 mycelium. Six chemical inducing conditions, e.g. seawater potato medium plus 100 μmol/L sodium butyrate, led to diverse bioactive metabolites both in micro-scale cultivation and routine-scale fermentation. Furthermore, its butyrolactone metabolites also displayed difference under different conditions. [Conclusion] Chemical induction approaches can prompt the Aspergillus terreus C23-3 to produce rich bioactive substances, laying foundation for the discovery of diverse anti-Alzheimer’s disease natural products.

Abstract:[Background] Betula alnoides is a typical mycorrhizal nutritional tree species with both arbuscular mycorrhizae and ectomycorrhizae, and mycorrhizal inoculation is an effective measure to grow its robust seedlings. [Objective] The objectives of this study are to reveal the effects of ectomycorrhizal fungi (ECMF) inoculation on seedling growth performance and nutrient contents of B. alnoides clones, and to screen suitable ectomycorrhizal fungi species as well as to provide scientific evidence for growing mycorrhizal seedlings of these species. [Methods] Seedling height, root collar diameter, biomass and nutrient contents of four B. alnoides clones, BY1, FB4, FB4+ and A5, were studied under inoculations of six ECMF including Cenococcum geophilum, Lactarius deliciosus, Scleroderma flavidum, Scleroderma polyrhizum, Suillus luteus and Xerocomus chrysenteron through a pot cultivation trial, and their differences were analyzed among different ECMF and clones. [Results] Seedling roots of four B. alnoides clones could be colonized by all six ECMF, in particular, S. polyrhizum and S. flavidum could remarkably promote growth and nutrient absorption of B. alnoides seedling after inoculation (P<0.05), inferring that these two ECMF demonstrated stronger affinity for clonal seedlings than other ECMF. There were not significant differences in mycorrhizal colonization rate among four clones, whereas the positive effects of ECMF on seedling growth of FB4 and BY1 were remarkably better than those of the other two clones. [Conclusion] It is recommended that S. polyrhizum and S. flavidum be applied in growing mycorrhizal seedling of B. alnoides.

Abstract:[Background] As a refractory organic pollutant, pyridine is commonly found in wastewater from coking, refining, leather and pharmaceutical industries, causing harm to the environment. [Objective] To treat pyridine contaminated wastewater, bacteria capable of degrading pyridine were screened. [Methods] Pyridine degrading bacteria were isolated from the activated sludge of a wastewater plant in Shijiazhuang by enrichment and selective medium. Bacterium B21-3 was identified by morphological, physiological and biochemical characteristics, (G+C)mol% assay, and 16S rRNA gene phylogenic analysis. The pyridine degradation characteristics were analyzed. [Results] A bacterial strain B21-3 that used pyridine as the sole carbon and energy source was isolated and identified as Paracoccus pantotrophus. The optimal pH and temperature for pyridine degradation were 7.0 and 32 °C, respectively. When the initial concentration of pyridine was 100 mg/L, the degradation percentage of pyridine was 48.50%±0.02%. After acclimation by increasing the initial concentration of pyridine, strain B21-3 tolerated higher concentrations of pyridine and the degradation of pyridine increased significantly. Under the pyridine concentration of 100 mg/L, the degradation percentate of pyridine by domesticated strain B21-3 was 90.26%±1.70%. After the acclimatized strain B21-3 was subcultured on mineral salt plates supplemented with pyridine for 15 generations, the degradation percentage of pyridine was 89.39%±2.03%. [Conclusion] Strain B21-3 had strong pyridine degradation ability with potential for bioremediation of pyridine contaminated wastewater.

Abstract:[Background] Microbial deterioration is a common biological disease in ancient wall paintings, which seriously threaten the long-term safety preservation and exhibition of the wall paintings. As the main source of the harmful microorganisms to wall paintings, airborne microorganisms have attracted extensive attention in the monitoring and preventive conservation of cultural relics in recent years. [Objective] systematic survey of culturable airborne bacteria, including the concentration, community structure and seasonal variation, was carried out in environments of wall paintings preserved at Tiantishan Grottoes and Western Xia Museum, China. [Methods] Bio-aerosol sampler containing R2A agar was used for sampling in all four seasons in 2016. Traditional culture-based method to acquire the airborne bacterial concentration information and purified strains; by the extraction of genomic DNA, amplification of bacteria 16S rRNA gene region, sequencing, and phylogenetic analysis, thereafter the bacteria community composition and distribution characteristics of different study sites were clarified. Combined with environmental monitoring data, to disclosure the main factors which responsible for the dynamic changes of the airborne bacteria at the site. [Results] The concentration of culturable airborne bacteria was in a range from 16.7 to 1 451.8 CFU/m3. There was no significant difference in bacteria concentration between the Cave 18 and Cave 13, namely, the two sites at the Tiantishan Grottoes, with the obvious characteristics of the seasonal variation, briefly speaking, Winter and Spring were higher than Summer and Autumn. Meanwhile, there was significant difference in bacteria concentration between the inside and outside of the Xixia Museum, the square outside of the museum were far more than the inside of the museum at the four seasons, particularly in the winter. A total of 19 bacteria genera that affiliated to four phylum were detected, among which, Acinetobacter, Arthrobacter, Bacillus, Kocuria, Brevundimonas, Carnobacterium, Pseudoclavibacter and Hymenobacter were the dominant ones. [Conclusion] The airborne bacterial community structures of the Tiantishan Grottoes showed a distinct characteristic of the seasonal variation and spatial distribution; RH, temperature and seasonal rainfall were all have influences on the airborne bacteria distribution; some of the genera have potential of causing biodeterioration of the ancient wall paintings at this site; this study could provide supporting information for the preventive protection of cultural relics that preserved at local site and museums.

Abstract:[Background] With the increase of CO2 emissions and global temperatures, thermophilic cyanobacteria are considered as a promising group of organisms to convert CO2 into useful chemicals and biofuels at 45 °C or above. [Objective] This paper describes isolation and identification of thermophilic cyanobacteria collected from Huizhou area, and describes growth characteristics of two strains belonging to the family Leptolyngbyaceae to provide the basis for their subsequent application. [Methods] 16S rRNA gene and phycocyanin alpha chain (PhycoA) gene sequences were used in phylogenetic analysis to determine the taxonomical position of isolates from Huizhou area. Two strains PKUAC-GDTS1-24 and PKUAC-GDTS1-29 were observed morphologically and analyzed for major cellular components (ash, carbohydrate, lipid, protein and pigment). [Results] Twelve strains of thermophilic cyanobacteria were isolated, of which PKUAC-GDTS1-24 and PKUAC-GDTS1-29 were morphologically blue-green globular trichomes. Cells forming trichome formed dense clusters, that mostly attached to each other. Cellular contents of: ash, lipids, and protein were estimated to be 24.41%, 21.40%, 26.64%, respectively in PKUAC-GDTS1-24 strain. Carbohydrates were the major component of the strain at 36.42%. In another strain, PKUAC-GDTS1-29, the cellular contents of: ash, lipids, and protein contents were estimated to be 24.72%, 23.92%, 12.93%, respectively. The carbohydrates once again were the most abundant component with 28.46%. The contents of phycocyanin (PC) in PKUAC-GDTS1-24 and PKUAC-GDTS1-29 were 157.29 and 374.86 mg/g dry weight respectively, and the carotenoids were 65.13 and 18.87 mg/g dry weight, respectively. [Conclusion] Based on the phylogeny, PKUAC-GDTS1-24 and PKUAC-GDTS1-29 belong to the family Leptolyngbyaceae and cluster with many poorly described isolates. These isolates may be a potentially novel phylotype or a new genus of filamentous mildly thermophilic cyanobacteria that are present in the hot springs of Guangdong and Sichuan. The morphological features and cellular composition of both thermophilic Leptolynbya-like strains were similar. To our best knowledge, the two strains are the highest phycobiliprotein producers described to date from this cyanobacterial family, which could be used as potential strains for phycocyanin production, especially PKUAC-GDTS1-29.

Abstract:[Background] Napahai plateau wetland is located in the northwest of Yunnan province. It is a uniquelow-latitude-high-altitude and seasonal semi-closed plateau wetland in China. Fungi play special roles in the maintenance and stabilization of wetland ecosystems. However, little is known about the fungal community composition and diversity in Napahai plateau wetland. [Objective] To systematically analyze the fungal community composition and diversity a in the soils of Napahai plateau wetland in different seasons and locations and their relationship with the environmental factors, promoting the understanding of microbial diversity in plateau wetlands. [Methods] In this study, fluorescence quantitative PCR and high-throughput sequencing were used to analyze quantity, diversity and composition of fungal community in the soils of Napahai plateau wetland in different seasons and sampling sites, and their association with soil environmental factors was further analyzed. [Results] The results showed that about 60% of the fungal sequences did not exhibit similarity to known sequences. The rest belonged to 6 phyla, 17 classes, 37 orders, 53 families and 63 genera, and most belonged to the phylum Ascomycota and the major dominant genus Gibberella. Taxonomic composition, OTU distributions and beta diversity analysis showed that the diversity and composition of fungal community were more affected by soil types than by season changes, presumably due to different rhizosphere effects and species of plants. CCA (Canonical correlation analysis) showed that the diversity of fungi was almost influenced by soil physiochemical factors in different sampling sites. [Conclusion] Our research revealed the fungal community in the soils of Napahai plateau wetland exhibit unique diversity and composition, emphasizing the importance of protecting and restoring the enviroment of Napahai plateau wetland from the perspective of microorganisms.

Abstract:[Background] N-acylhomoserine lactones (AHLs) are used as the main quorum-sensing signaling molecules in many Gram-negative bacteria. [Objective] To screen and identify new quorum quenching (QQ) bacteria from soil organisms. [Methods] We used “washer method” to grow organisms by cultivation in situ. QQ bacteria were screened through “agar slice”, “reporter plate” and the assay of β-galactosidase activity. The phylogenetic status was determined based on 16S rRNA gene homology analysis. [Results] Here, 502 organisms were obtained from the soil samples in situ and screened for AHL-degrading bacteria using A. tumefaciens NTL4 (pZLR4) as a reporter strain. Eleven isolates degraded AHLs within 24 h. Based on their 16S rRNA gene sequences similarities, five isolates (3-49, 3-58, 61, 66, 120) and four isolates (3-59, 151, 3-41, 2-34) were assigned to the genera Pseudomonas and Acinetobacter. Strain 2-63 and 129 were identified as Proteus sp. and Rheinheimera sp., respectively. Most of these strains degraded strongly 3OC12-HSL and were active against 3OC6-HSL and 3OC8-HSL. [Conclusion] This study showed that Proteus sp. and Rheinheimera sp. may degrade AHLs, which might provide new biocontrol resources to control of the quorum sensing-dependent bacterial diseases.

Abstract:[Background] With the rapid development of economy in middle and small cities and the improvement of people’s living standard, Which the discharge of domestic sewage is also magnify, resulting in a worsening trend of water quality pollution. [Objective] To understand the effect of domestic sewage treatment on changes of microbial community diversity in wetland surface water. [Methods] Physical and chemical tests were conducted on 6 points of surface water in reference points (?2 000), 200, 400, 600, 3 000 and 5 000 m in constructed wetlands (Xiantao), Then the microbial community structure was analyzed by high-throughput sequencing, and the effects of microbial community structure change and domestic sewage pollution were also discussed. [Results] When the sewage treated by wetland method reached 5 000 m, Chemical oxygen demand (COD), NH4+N, Total nitrogen (TN) and Total phosphorus (TP) reached the first grade standard of pollutant discharge standard for urban sewage treatment plant, and there was a very significant positive correlation between the physical and chemical indexes. The dominant phyla in the studied surface water were Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Verrucomicrobia, Firmicutes, Acidobacteria and Chloroflexi. Proteobacteria can be used as an indicator microorganism for the discharge of pollutants from domestic sewage. Actinobacteria, cyanobacteria, verrucomicrobia and Chloroflexi can be used as an indicator microorganism for the purification of domestic sewage. [Conclusion] The changes of microbial community diversity in the surface water environment of Xiantao domestic sewage treatment were compared for the first time, which can make people understand the operation mechanism of constructed wetland and the pollution of surface water environment more clearly from the microbiological level.

Abstract:[Background] Single cell culture of fungi is very important to study cell heterogeneity and cell growth characteristics. Therefore, it is necessary to establish a simple and convenient method for culturing and observing fungal single cells. [Objective] To establish a method for capturing and culturing the single cell of fungi based on microfluidic. At the same time, localization and real-time observation of single cell will be done. [Methods] L-edit was used to design the pattern and the plasma bonding was used to produce the microfluidic chip. Rhodotorula glutinis solution and Trichoderma reesei spore solution were injected by syringe pump to capture single cell. Trypan blue was used to determine the survival rate of yeast cells. Germination, growth and reproduction of yeast single cells and spore were observed under microscope. [Results] The microfluidic chip was intact and can be used in single cell capture of yeast or spore. The capture rate of the yeast was 25.00%±1.38%. The budding process of yeast cells was observed at 0, 2, 4 and 6 h. There was no significant difference between the survival rate of yeast cultured in chip and in shaking culture until 48 h. The process of spore germination and mycelium growth was observed at 0, 3, 6 and 9 h. Moreover, the mycelium kept growing until 120 h. [Conclusion] A microfluidic chip was designed and produced to capture, culture and localization the single cell of Fungi. This is the first application of this kind of chip in the fungal single cell culture. The cell can grow normally in this chip for at least 2 days, and 5 days even longer cell culture could be achieved by this way. The microfluidic chip can achieve intuitionistic localization and real-time observation of fungal single cell. It has the potential to study physiological and genetic characters of a variety of microorganism.

Abstract:[Background] Most alginolytic bacteria currently reported are aerobic bacteria and studies on anaerobic ones have not been reported yet. Characterization of alginate lyases isolated from alginolytic bacteria are mainly focused on the endo-type alginate lyases and rarely on exo-type ones. [Objective] To investigate the genes encoding alginate lyases isolated from anaerobic alginolytic bacteria, to characterize the novel alginate lyase, and to elucidate its enzymatic properties, providing a theoretical basis for the diversity of alginate lyases and the mechanism of microbial degradation of alginate. [Methods] The gene encoding SHA-4, an alginate lyase isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52, was cloned and sequenced. Recombinant plasmid PGEX-4T-1-SHA-4 was constructed and heterologously expressed in E. coli. The expressed enzyme was purified and the enzymatic and degradation characteristics were analyzed. [Results] Maximum expression was achieved when induced by 0.1 mmol/L IPTG (Isopropyl-β-D-Thiogalactoside) at 28 °C for 6 hours and the specific activity of the purified enzyme was 21 U/mg. Characterization of the enzyme showed that the optimal condition for SHA-4 includes the temperature at 37 °C, pH at 7.5, and PolyMG (heteropolymeric MG blocks) as a preferred substrate. The activity of this enzyme was inhibited by Na+ and significantly elevated to about 168% by Cu2+. Km and Vmax of SHA-4 when used to catalyze alginate were 2.5 mg/mL and 8.7 mg/(mL·min), respectively. SHA-4 was an exo-type alginate lyase with monosaccharides as the final degradation products. [Conclusion] An alginate lyase SHA-4 isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52 is successfully expressed. Among other PL6 family members, SHA-4 is the first exo-type alginate lyase with PolyMG as a preferred substrate. Along with its relatively high enzymatic activity, SHA-4 may serve as a tool enzyme with promising application and provide new clues for further exploration of the mechanism of alginate degradation.

Abstract:[Background] Heme oxygenase-1 (HO-1) has many physiological effects, such as anti-oxidative stress, anti-apoptosis and anti-fibrosis, and is expected to become a new drug for the treatment of clinical diseases. [Objective] A gene recombinant Escherichia coli was constructed for expressing HO-1, and its culture conditions were optimized to achieve the high yield of HO-1. [Methods] The gene of HO-1 (ho1) was cloned from the cell of Synechocystis sp. PCC 6803, and was recombined with the plasmid of pET-28a. The recombinant plasmid (pET-28a-ho1) was transformed into E. coli BL21(DE3). The single factor experiment was applied to optimize the type of medium, inducer adding time, cultivation time, inducer concentration and cultivation temperature for the expression of HO-1. [Results] A gene recombinant E. coli strain, BL21(DE3)/pET-28a-ho1, was successfully constructed for expressing HO-1. When the strain was cultured in glycerol medium (GY), and as the cell OD600 was about 0.8, IPTG with the final concentration of 0.1 mmol/L was added, the highest yield of HO-1 could be obtained after induction cultivation for 6 h at 30 °C. Separation of HO-1 by Ni-NTA column accounted for 10.9% yield of the total cell protein. [Conclusion] A gene recombinant E. coli strain, and its optimal cultivation conditions were achieved for the expression of soluble HO-1, which laid a foundation for further study on the enzymatic properties and application of HO-1 from Synechocystis sp.

Abstract:[Background] Plant growth-promoting rhizobacteria can promote plant growth and improve stress tolerance. There are special microbial habitats on tea rhizosphere and some beneficial microorganisms with growth-promoting effects could be easily acquired. [Objective] To ascertain taxonomic status and study growth-promoting characteristics of four PGPR strains isolated from tea rhizosphere, and further screen superior PGPR strain. [Methods] These four strains were identified based on morphological, physiological and biochemical characteristics, homology alignments of 16S rRNA gene sequences. The plant growth-promoting characteristics of isolates were studied. Phosphorus contents were analyzed with Mo-Sb colorimetry. ACC (1-Aminocyclopropane-1-carboxylic acid) deaminase activity was measured by colorimetric method. Also, siderophere secreting capacity and IAA content were determined by quantitative test of CAS (Chrome zaurol S) medium and method of Salkowski, respectively. In pot experiments, plant growth-promoting effects were analyzed by measuring shoot heights and fresh weights of tested plants (including Chinese cabbage, swamp cabbage, amaranth and rice). [Results] The strain KKS-6-N1 was identified as Agrobacterium radiobacter, KKS-7-N7 identified as Pseudomonas aeruginosa, GD3 identified as Pseudomonas hunanensis, and GD12 identified as Bacillus flexus, respectively. In these four strains, nitrogen-fixing strains KKS-6-N1 and KKS-7-N7 produced siderophore, KKS-7-N7 also had the abilities of phosphorus-dissolving and IAA secreting as high as 101.29 mg/L. The other two strains, potassium-solubilizing strain GD3 still dissolved phosphorus, its ACC deaminase activity was 8.09 μmol/(mg·h) and relative content of siderophore was 0.31. whereas GD12, which had dissolved potassium and fixed nitrogen, was found to secret ACC deaminase of 14.46 μmol/(mg·h). In pot experiments, shoot heights and fresh weights of Chinese cabbage, swamp cabbage and amaranth were increased obviously by inoculation of these four strains, especially GD3 is the most excellent among them. [Conclusion] Pseudomonas hunanensis GD3, which isolated from tea rhizosphere, could strongly promote plant growth. And it is possible to develop as an excellent microbial fertilizer.

Abstract:[Background] Microbial fertilizers have been widely used in the cultivation of organic crops in China, and their effects on the micro-ecology in soils of organic farm still needs scientific evaluation. [Objective] The high-throughput sequencing technology can be used to analyze soil microbial community accurately. We explained effects of Bacillus subtilis microbial fertilizer on organic farm root-zone soil bacterial and fungal community in the view of structure and diversity. [Methods] Under organic and rotate farming conditions, soil genomic DNA was extracted after application of Bacillus subtilis microbial fertilizer. And PCR amplification was made to establish libraries. Then, the 16S rRNA genes V3?V4 regions of soil bacterium and fungal ITS1 regions were sequenced by Illumina high-throughput sequencing technology on MiSeq platform, and related biological analysis was conducted to explain the changes of soil bacterial and fungal diversities and structures. In addition, soil chemical properties and enzyme activities, nutritional quality of organic Chinese watermelon were determined. [Results] A total of 14 199 bacterial operational taxonomic units (OTUs) and 3 378 fungal OTUs were obtained from 6 organic Chinese watermelon root-zone soil samples. The sequencing coverage of bacterial and fungal libraries were above 98% and 99%, respectively. Bacillus subtilis microbial fertilizer would increase the soil bacterial diversity while reduce the fungal diversity to a certain degree, and increased the abundance of bacteria, but it reduced the abundance of fungi significantly (P<0.05), and reduced the number of bacterial and fungal unique OTU. Proteobacteria, Firmicutes and Actinobacteria were the dominant bacteria, and Ascomycota was dominant fungi. It increased the relative abundance of Chloroflexi and Ascomycota by 46.23% and 10.01%, respectively. And, it reduced the relative abundance of Proteobacteria and Basidiomycota by 11.14% and 74.72%, respectively. It significantly reduced the pH of soil (P<0.05). Wherein, it significantly increased the content of total amino acids and soluble solid in organic Chinese watermelon (P<0.05). [Conclusion] The abundance and diversity of soil bacteria and fungi were changed with Bacillus subtilis microbial fertilizer applied. And soil pH was reduced, nutritional quality of organic Chinese watermelon was increased also.

Abstract:[Background] There are a large number of microorganisms in the intestinal tract of insects, which are necessary for the normal life activities of insects. They promote the synthesis of vitamins, absorption and utilization of fats, carbohydrates and also protect the host against natural enemies, endure high temperature and promote the metabolism of toxins or xenobiotics, and indirectly promote resource development. [Objective] To study the diversity of culturable bacteria isolated from the whole larvae gut of Galleria mellonella. [Methods] A molecular phylogeny of the 16S rRNA gene, the morphology of the colonies and the cells as well as related physiological and biochemical characteristics were used to identify the bacterial species. [Results] A total of 40 culturable bacteria from the intestine of G. mellonella were classified into 16 unique phylotypes. All sequenced bacteria strains were grouped into four families: Bacillaceae, Enterococcaceae, Staphylococcaceae and Moraxellaceae. Among them, Bacillus spp. is the most dominant species of culturable intestinal bacteria. It was determined that the culturable intestinal bacteria included 9 strains of Bacillus, 4 strains of Enterococcus, 2 strains of Staphylococcus, and 1 strain of Acinetobacter. [Conclusion] By studying the composition of bacterial communities that can be cultured from the larvae intestine of G. mellonella, this study provides theoretical basis for the microecology research of the gut of G. mellonella.

Abstract:[Background] Research on rhizobium diversity has paved the way for utilization of rhizobial germplasm resources. [Objective] To research the phenotypic and genetic diversity of endophytic and non-endophytic rhizobia of alfalfa (Medicago sativa L.), and verify the hypothesis that rhizobial symbiotic efficiency differed according to alfalfa variety by comparing their symbiotic difference on five alfalfa varieties. [Methods] Endophytic (seed, flower, leaf, stem, root epidermis, root stele, nodule) and non-endophytic (rhizosphere soil and field soil) bacteria isolates were collected from M. sativa cvs. Longzhong and Qingshui in arid crop area of Huining, Baiyin, M. sativa cv. WL168HQ in irrigated area of Anning, Lanzhou, and M. sativa cvs. Gannong No. 3 and Gannong No. 9 in irrigated area of Liangzhou, Wuwei, Gansu. Numerical analysis, 16S rRNA restriction fragment length polymorphism fingerprinting (RFLP), 16S rRNA gene sequencing, multilocus sequence typing (MLST) of concatenated sequences of atpD, glnII, and recA genes, and sequence analysis of symbiotic genes nodC and nifH were applied to study the phenotypic and genetic diversity of endophytic and non-endophytic rhizobia. A principal component analysis (PCA) was used to investigate their symbiotic differences on five alfalfa varieties as well. [Results] Totally 43 endophytes and 10 non-endophytic isolates were obtained. None were collected from flowers and leaves. The phenotypic diversity of these 53 isolates along with two reference strains (R.GN5 and S.12531) were abundant, with eight phenotypic clusters formed. Twenty-two RFLP patterns were produced after 16S rRNA-RFLP analysis, and the most widespread genotype among the isolates was that designated as genotype Ⅰ (24). Three other genotypes (Ⅻ, ⅩⅤ and ⅩⅨ) occurred less frequently in alfalfa symbionts (five, five and three). There were 16 genotypes specific to a single M. sativa isolate. According to the phylogenetic analyses of 16S rRNA gene and MLST, isolates were further classified into Rhizobium radiobacter, R. rosettiformans, and Ensifer meliloti. The nodC and nifH gene fragments were only amplified and sequenced from seven representative E. meliloti strains and reference strain S.12531, indicating that they were capable of nodulating alfalfa. The nodule number per plant, shoot dry weight and crude protein content of M. sativa cvs. Gannong No. 3 (inoculated with G3L3), Longzhong (inoculated with LP3, LL1 and LL2), Qingshui (inoculated with QL2), and WL168HQ (inoculated with LL1, LL2 and WLP2) were promoted simultaneously. The parameter values of M. sativa cvs. Gannong No. 3, Gannong No. 9, and Qingshui plants inoculated with the E. meliloti isolates clustered together, which ranged from ?1 to 1 in PC1 axis and ?1.5 to 1.5 in PC3 axis. Compared with these three alfalfa varieties, that of M. sativa cvs. Longzhong and WL168HQ plants dispersed greatly and ranged from ?1.5 to 4 in PC1 axis and ?3 to 4 in PC3 axis. [Conclusion] The phenotypic and genetic diversity of endophytic and non-endophytic rhizobia were abundant, and there was no direct relationship between diversity and strains’ origins. Strong mutualistic symbiosis and adaptability were presented between G3L3 and M. sativa cv. Gannong No. 3, LP3, LL1, LL2 and M. sativa cv. Longzhong, QL2 and M. sativa cv. Qingshui, and LL1, LL2, WLP2 and M. sativa cv. WL168HQ. The tested strains exhibited similar symbiotic efficiency when inoculated onto M. sativa cvs. Gannong No. 3, Gannong No. 9, and Qingshui plants, while an obvious symbiotic difference of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants. Their symbiotic efficiency varied according to alfalfa varieties, which manifested that the sensitivity of different alfalfa varieties to rhizobial strains may differ.

Abstract:[Background] Cold-active β-galactosidases are typically characterized by a high lactose hydrolysis activity at low temperature. Such a characteristic makes cold-active β-galactosidase highly attractive for application in low-lactose milk production industry, and how to mine novel cold-active β-galactosidase with enzymatic properties perfectly suitable for lactose hydrolysis in milk solution condition becomes a research focus. [Objective] This study is aiming to screen novel strains producing cold-active β-galactosidase from glacier sediments at China No.1 glacier in Tianshan Mountains and to evaluate if the enzymatic properties of the cold-active β-galactosidase is suitable for further application in low-lactose milk production. [Methods] Primary screening using X-gal culture plate, and secondary screening by assaying the relative β-galactosidase activity at low temperature were carried out. The target strain selected was identified by physiological, biochemical and 16S rRNA gene sequencing. The growth condition and enzyme production condition of the isolate were optimized by single-factor experiment. Enzymatic properties of the partially purified β-galactosidase produced by the isolate were also investigated. [Results] LW106, a strain with high β-galactosidase activity, was identified as Microbacterium sp. LW106. The optimal temperature for LW106 producing β-galactosidase is 25 °C. Soluble starch was the optimal carbon source for LW106 to produce β-galactosidase, the optimum medium initial pH was 7.0, and the optimal inoculum size was 3%. Preliminary study on its enzymatic properties showed that the optimal pH of the β-galactosidase of strain LW106 was 6.0, and the optimal reaction temperature was 35 °C with a relative activity of 78% at 4 °C. The β-galactosidase produced by LW106 showed the best stability at 4 °C and pH 7.0. Metal ion Na+ at a level of 10 mmol/L did not inhibit the activity of β-galactosidase of LW106, while 10 mmol/L of Ca2+ activated the β-galactosidase activity. [Conclusion] The enzymatic properties of β-galactosidase produced by Microbacterium sp. LW106 indicated it may be suitable for lactose hydrolysis at low temperature, showing good application potential in low-lactose milk production industry.

Abstract:[Background] Brucellosis, caused by Brucella, is a significant zoonotic disease distributed and prevalent widely in many countries around the world. As an important disease, Brucellosis re-emerged in China in recent years, posing a great challenge to animal breeding industry and public health. Existing data about infection and epidemiology of Brucellosis in China are not comprehensive and systematic, due to the intensive breeding and big populations of animals in the country. [Objective] To better understand the prevalence of Brucellosis in dairy cows in China and to provide basis for the establishment of control measures and strategies. [Methods] Data were obtained and analysed based on the results of surveillance on 23 381 serum samples collected from dairy cows with miscarriage during 2013?2017. [Results] The positive rate of antibody against Brucella was 44.2%. The epidemic situation of Brucellosis in cows in 2015 was most severe, as evidenced by the findings that the highest levels of individual positive rate and group positive rate up to 55.3% and 93.5%, respectively, were detected. The positive rate of Brucellosis among the miscarriage herds in 5 provinces of the first-class Brucellosis epidemic areas ranged from 13.8% to 57.8%, and that in the second-class Brucellosis epidemic areas was between 54.4% and 86.9%. The incidence rate of Brucellosis in aborted dairy cows correlated well with the epidemic trend of human Brucellosis (r=0.806>0). Severe epidemic situation was observed in the second-class Brucellosis epidemic areas. [Conclusion] Our data presented here enhance our understanding of epidemiology of Brucellosis in China, and underscore the importance of nationwide monitoring of animal Brucellosis. In addition, prevention, accurate diagnosis, and elimination of the diseases on the farms should be strengthen in China.

Abstract:[Background] Streptococcus suis (SS) is an important zoonotic pathogen with 35 serotypes, of which the most serious is Streptococcus suis type 2 (SS2). Suilysin (SLY) secreted by various serotypes of SS may have a better immune protection. Therefore, SLY has a great advantage as a component of SS genetic engineering subunit vaccine. [Objective] to express the suilysin gene (sly) from a highly virulent strain (AH10-8) of SS2 in Anhui Province and to determine the immune protection of the SLY protein. [Methods] The full-length of sly was amplified by using a pair of specific primers with BamH I and Xho I sites. Subsequently, the PCR-amplified sly gene was cloned into the pET-30a vector. The plasmid was transformed into E. coli BL21 and expressed under the IPTG induced promoter. SLY was visualized by SDS-PAGE, and the protein antigenicity was confirmed by Western blotting. Zebrafish and Kunming mice were used to carry out SLY immune attack protection test, and the IgG antibody titer of serum in Kunming mice was detected by indirect ELISA, and the pathological changes and the protective rate were observed. [Results] The sly gene that cloned into pET-30a vector was highly expressed in E. coli BL21. The molecular weight of the SLY protein was about 60 kD and it is in accordance with the expected molecular weight of sly and SLY protein could react with serum from the SS2 infected patients. The serum antibody titers induced by the SLY protein and AH10-8 inactivated whole cells immune to IgG after three times were 1:6 400, 1:204 800 (ultrasound lysate of AH10-8 strain whole cell used as antigen) and 1:102 400, 1:51 200 (the SLY protein used as antigen). The relative protection rates of sly and AH10-8 inactivated total bacteria to Kunming mice and zebrafish were 40%, 80%, 84% and 92%, respectively. There was obvious difference between pathological changes in control group and experimental group. [Conclusion] The expressed SLY protein has a good reactivity and immunogenicity, which can induce a protective immune response. SLY protein can be a good candidate for the development of the SS2 vaccine.

Abstract:Oil reservoirs are often deep subsurface extreme environments with high temperatures, pressures, and salinities. Several physiological and taxonomic groups of thermophilic anaerobic bacteria are present in the oil reservoirs, such as fermentative, methanogenic, sulfate-reducing and Fe(III)-reducing microorganisms. A total of 90 strains of Fe(III)-reducing bacteria have been isolated from oilfield fluids and identified as the genus Thermotoga, Thermoanaerobacter, Deferribacteres, the order Desulfuromonadales within the class Deltaproteobacteria, the order Shewanella within the class Gammaproteobacteria, and Thermococcus within the Euryarchaeota, in the growth temperature range of 4?85 °C and the growth salinity range of 0.1%?10% NaCl. The growth pressure range of Fe(III)-reducing microorganisms in oil reservoirs has not been reported yet. There is strong interaction among the microorganisms, minerals and fluids (oil/water) at the actual reservoir conditions. The clay minerals can serve as carriers for microbial life activities and also provide electron acceptors for microbial metabolism. Here, we present the current status of isolation and characterization of Fe(III)-reducing bacteria in oil reservoirs, describe the environmental applicability of Fe(III)-reducing bacteria, and predict microbial enhanced oil recovery (MEOR) application prospects by using Fe(III)-reducing bacteria.

Abstract:Recently, it is the one of the hot topic for antimicrobial peptides (AMPs) to be produced using recombinant expression system with efficiency and stable protocols. It is particularly crucial for us to know different expression systems we use and their production. For this purpose, we narrate methods to produce AMPs, species, elements and properties of different expression systems, mainly there is an analysis for some difficult factors to construct engineered bacteria. And, we also reviewed the progress of producing antibacterial peptides using engineered bacteria in recent years.

Abstract:Environmental Engineering Microbiology is one of the basic core courses of Environmental Engineering major in higher education in China, with the characteristics of short knowledge refresh period, numerous knowledge points and dispersive contents. In the process of teaching, how to strike a balance between traditional microbiology content and progress in modern microbiology research, choose the teaching content rationally, arrange the experiment project and experiment time reasonably, guide the students to grasp the basic concepts and knowledge points accurately, and train students’ hands-on ability, innovation consciousness and innovation ability are difficult problems for every teacher. Around the above-mentioned problems, our teaching team has taken some measures from the aspects of classroom and experiment teaching contents, teaching methods and extracurricular activities, and achieved better teaching results.

Abstract:In order to strengthen the application of information technology, improve students’ information literacy and comprehensive ability, the microbiology teacher in Dalian University School of Life Science and Technology designed a new assessment method named Informatization Knowledge Contest for the first time. The microbiology course assessment formed a pattern involving “informatization technology of answering” and “receptor test”, the specific steps are as follows: first, students ask and answer question, and then the teacher “audit” assessment. The results showed that this new type of receptor appraisal method has been widely recognized by students. It is beneficial to the cultivation of students? comprehensive ability and improvement of students’ information literacy.

Abstract:This paper probes into the reform of the course of Microbiology under the background of applied universities, so as to highlight the characteristics of applied universities and to train higher applied talents. Taking the undergraduate students majoring in biological engineering in Jing Chu University of Technology as the research objects, the teaching mode of Microbiology has been innovated and reformed through the comprehensive application of teaching methods, such as rearrangement of teaching contents, micro-lesson production, case teaching and flipping classroom. The results of teaching and questionnaire survey in the experimental group and the control group show that, compared to the traditional one, the new teaching model is more conducive to the students' mastery and application of knowledge.