Abstract:[Objective] To improve xiamenmycin yield from Streptomyces xiamenensis 318 we optimized fermentation medium and doubled biosynthetic gene cluster. [Methods] Glucose-yeast extract-maltose medium (GYM) was optimized by a series of single-factor experiments with variation of carbon source (rhamnose and gluconic acid), nitrogen source (KNO3), trace element (ScCl3) and A-Factor analogous (γ-butyrolactone). Subsequently, the biosynthetic gene cluster xim was doubled by site-specific integration to obtain a genetic engineering strain S. xiamenensis 318Pls1 and orthogonal experiment was applied to optimize the medium. [Results] The single-factor experiments improved xiamenmycin yield in wild-type strain 318 from 15 mg/L to 20 mg/L. The yield in biosynthetic gene doubling strain 318Pls1 attained to 35 mg/L in GYM and was increased to 76.15 mg/L in the optimized medium 9# by orthogonal experiment. [Conclusion] This study shows that the medium optimization combined with biosynthetic gene cluster doubling enhanced xiamenmycin yield and provides an opportunity for further improvement of high productivity strain.

Abstract:[Objective] In order to construct a biocatalytic system to synthesize 4-hydroxyl isoleucine (4-HIL), L-isoleucine (L-Ile) dioxygenase (IDO) from Bacillus subtilis expressed in recombinant Escherichia coli was purified and characterized. [Methods] The recombinant IDO was purified by Ni-NTA affinity chromatography from recombinant Escherichia coli BL21/pET28a-ido. The enzyme was the characterized by using L-Ile as the substrate. As the necessary cofactor of IDO, the concentration of α-ketoglutaric acid (α-KG) in the enzymatic system was further optimized to improve the catalytic efficiency. [Results] Kinetic parameters of the enzyme were obtained as Km 0.247 mmol/L, kcat 1.260 s?1, and kcat/Km 5.101 L/(mmol·s). Compared with other homologous enzymes, the recombinant IDO had higher substrate affinity and catalytic efficiency. The recombinant IDO was more active at 20 °C and pH 7.0, and more stable at the temperatures below 35 °C. The optimal concentration of Fe2+ was 1 mmol/L in the catalytic system. The recombinant IDO was active to a variety of L-amino acids, of which L-isoleucine, L-norleucine, and L-methionine were more suitable for the IDO catalyzing hydroxylation. By optimization of α-KG concentration in enzymatic catalysis, 4-HIL with the yield of 66.20% was achieved from 70 mmol/L L-Ile by adding 30 mmol/L α-KG in the reaction system. Thus, the addition of α-KG as the reaction-coupled cofactor had a significant impact on the reaction efficiency of recombinant IDO-mediated L-Ile hydroxylation. [Conclusion] This study provides the basis for enzymatic conversion of 4-HIL and other hydroxylated amino acids.

Abstract:[Objective] This paper aims at screening marine actinomycetes with broad-spectrum and producing efficient antimicrobial active substance, to lay the foundation for the development of new antibiotics. [Methods] Using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Listeria monocytogenes as indicator bacteria, target bacteria from sea mud samples was primarily screened with agar block method, and further screened with punching diffusion method; twenty indicator bacteria were used to examine the antimicrobial spectrum of the new isolates Y10, Y11, Y15, Y16 and Y21; taxonomic status of the strain Y15 was determined by morphologic observation and 16S rRNA gene sequence analysis; mode of action of antimicrobial active substance to Listeria monocytogenes was studied according to indicator bacteria OD600 value; the antimicrobial activity was used as an index to study the physical and chemical properties of antimicrobial active substances produced by Y15. [Results] A total of 12 strains showing antimicrobial activities to antagonistic strains were isolated; wherein Y15 had the widest spectrum, and could be against 18 of the 20 kinds of indicator strains, and on four media it could produce antimicrobial active substances. The 16S rRNA gene sequence analysis showed that Y15 had closest relationship with Micromonospora endolithica. Activity of Y15 antimicrobial metabolites reached a maximum of 480 AU/mL in 144 h, maintained 320 AU/mL from 168 h to 216 h. The action mode of Y15 antimicrobial active substance to Listeria monocytogenes was sterilization. Antimicrobial activity of Y15 antimicrobial active substances remained stable from ?20 °C to 60 °C, and gradually declined from 80 °C to 120 °C, but still retained 37.5% at 120 °C for 30 min; antimicrobial activity remained stable from pH 7.0 to 10.0, and from pH 2.0 to 6.0 and pH 11.0 to 12.0 antimicrobial activity declined; Y15 antimicrobial substances processed in the UV and four kinds of enzymes (Proteinase K, Trypsin, Papain and α-Amylase) were stable, there were non-protein and non-peptide antimicrobial substances. [Conclusion] The active substances produced by Y15 showed good antimicrobial spectrum and activity, and were relatively stable, indicating a high application value in the future.

Abstract:[Objective] We optimized degradation conditions for diethylstilbestrol (DES) by Aspergillus oryzae M-4. [Methods] Medium components and culture conditions were screened by using Plackett-Burman method. The degradation conditions were then optimized through Box-Bohnken design. [Results] The optimal medium was composed of 1.3% peptone, 0.045% CaCl2, 0.5% glucose, 0.15% K2HPO4, 0.05% KH2PO4, 0.05% NaCl, 0.2% Tween 80 and 44 mg/L diethylstilbestrol. The optimal culture conditions were as follows: initial pH 7.5, inoculum age 72 h, rotation rate 140 r/min, culture temperature 28 °C, culture time 72 h. [Conclusion] Under the optimal conditions, the degradation rate of diethylstilbestrol was 83.89%, 1.38-fold over the value before optimization (60.98%), showing a significant difference (p<0.01).

Abstract:[Objective] The ability of accumulation of Zn and Cd between the hyperaccumlating and non-hyperaccumulating ecotypes of Sedum alfredii differed significantly, which might be related to their endophytic bacteria communities. Thus, the objective of this study is to investigate and compare the endophytic bacterial communities between two ecotypes of S. alfredii. [Methods] By using high-throughput sequencing technique, a research on the diversity of endophytic bacterial communities in leaves and stems samples of the hyperaccumulating and non-hyperaccumulating of S. alfredii was conducted. The samples of the hyperaccumulating of S. alfredii were collected from an old lead-zinc mine located in Quzhou city, the west of Zhejiang province, and the non-hyperaccumulating of S. alfredii were collected from a tea garden located in Jiuxi, Hangzhou city, the north of Zhejiang province. [Results] A total of 366 783 sequences and 39 948 operational taxonomic units (OTUs) were obtained from the four samples (at the level of 97%). The index of Shannon indicated that endophytic bacterial biodiversity in the leaves of two ecotype of S. alfredii were consistenly higher than in the stems, endophytic bacterial biodiversity in the leaves of hyperaccumulator ecotype of S. alfredii were higher than in the leaves of non-hyperaccumulator ecotype of S. alfredii, while the reverse was true for the stem samples. Endophytes in the leaves and stems samples of the hyperaccumulator ecotype of S. alfredii included 26 and 21 phyla, 123 and 117 families respectively, while the non-hyperaccumulator ecotype of S. alfredii included 43 and 22 phyla, 175 and 83 families respectively. In addition, the most abundance associated with the four sample libraries were consistently found to be related to Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Cyanobacteria. On the genus level, the most abundance bacteria associated with the leaves and stems of hyperaccumulator ecotype of S. alfredii were Synechococcus and Plesiomonas, while Pseudomonas and Dechloromonas were for the leaves and stems of non-hyperaccumulator ecotype of S. alfredii. [Conclusion] Diverse communities endophytes inhabit leave and stems of the two ecotype of S. alfredii, while the most endophytes diversity of the four samples was the leave of hyperaccumulator ecotype of S. alfredii.

Abstract:[Objective] In order to study the community diversity of degraded grassland caused by overgrazing, 6 soil samples collected from Xinyuan county were investigated. [Methods] Bacterial diversity and community structure of the samples were analyzed via high throughput sequencing technology Illumina HiSeq 2500. [Results] The mean value Chao1 and Shannon index were 2 118.27 and 8.84, respectively; The bacterial community mainly belong to 35 phylum including Actinobacteria, Proteobacteria, Firmicutes, Gemmatimonadetes, etc., and 220 family including phingomonadaceae, Gemmatimonadaceae, etc., and 329 genera including Sphingomonas, Rubrobacter, etc. Nine genera of nitrogen fixing bacteria were tested, the most high abundance of which were Arthrobacter, Methylobacterium belong to azotobacter, and Bradyrhizobium, Rhizobacter were relatively high abundance bacteria belong to rhizobia. [Conclusion] The bacterial diversity is abundant in the six samples from degraded grassland, but many species were at low abundance, perhaps they declined toward extinction.

Abstract:[Objective] The study is aimed to research the effect of adding sediment extract or not on the cultivable bacterial diversity in the sediment of Taihu Lake. [Methods] By using R2A medium and sediment extract R2A medium to isolate and culture the bacteria in the sediment. Phylogenetic analysis based on the 16S rRNA gene was used to identify the bacterial diversity. [Results] Results showed that the amount of the bacteria cultured on sediment extract R2A increased up to 1.6 times more than that of on R2A medium. Analysis based on the similarities of 16S rRNA gene sequences of isolates presented effects on the dominant bacteria. On the R2A medium, the dominant bacterial groups were Firmicutes (52%), Actinobacteria (24%), Proteobacteria (20%) and Bacteroidetes (4%), and most of the bacteria isolated from R2A were closely related to genera Bacillus, Pseudomonas and Archrobacter. However, the dominant bacterial groups isolated from sediment extract R2A were Proteobacteria (40%), Actinobacteria (35%), Firmicutes (22.5%), and Bacteroidetes (2.5%). They are closely related to Sphingomonas, Bacillus, Paracoccus and Arthrobacter. [Conclusion] Adding the original nutrient factors of the sediment extract could increase the diversity of bacteria in the sediment and help to increase the cultivable efficiency.

Abstract:[Objective] The purpose of this study was to investigate the effects of carbon sources on cell membrane properties (phospholipid fatty acids composition and fluidity) and microbial community of activated sludge. [Methods] Atomic force microscopy (AFM), phospholipid fatty acids (PLFA) and fluorescence recovery after photobleaching (FRAP), and Miseq analysis technology were used to investigate the effects of the different substrates, glucose, sodium acetate, peptone, glucose?peptone (1?1), sodium acetate?peptone (1?1), on the differences of microbial surface adhesion force, PLFA composition and fluidity, and microbial community in five Sequencing Batch Reactors (SBRs). [Results] The involvement of peptone led to an increase of cell membrane phospholipid fluidity, PLFA composition present a consistency with the content of 18?1ω9c, 15?0iso and 17?0iso increased 53.1%?354.7%, 135.6%?407.9% and 88.1%?264.3%, respectively. The dominant phylum response to carbon sources present inconsistent rules: with glucose and sodium acetate as solo substrate, the dominant phylum were Actinobacteria and Proteobacteria, and the dominant genus were Nakamurella and Flavobacterium, diversity index were 3.65 and 4.25, respectively. At the appearance of peptone, the content of Candidatus Saccharibacteria increased obviously, and the diversity index was in the range of 4.96?5.09. [Conclusion] Principal component analysis (PCA) indicated that the involvement of peptone led to similar PLFA composition. Redundancy analysis (RDA) suggested that carbon sources changed the microbial community, which further influenced the composition of PLFA.

Abstract:[Objective] Compared to traditional Bt strains, Bacillus thuringiensis strain LM1212 can differentiate into spore-formers and crystal-producers. In this study, we tried to reveal the effect of plasmid curing on the cell differentiation of LM1212 strain. [Methods] The endogenous large plasmids of LM(p35'Z) carrying cry35-like gene promoter and lacZ gene fusion plasmid p35'Z were cured by high temperature treatment. The mutants were selected at HCO plates with X-gal and identified by using primers of cry genes. We extracted plasmids of mutants and analyzed them by pulsed field gel electrophoresis (PFGE). Observation by laser confocal scanning microscope and optical microscope, calculation of spore formation rate, SDS-PAGE and LC-MS/MS (Q-TOF) mass spectrometry were used to determine the effect of plasmid curing on the cell differentiation and expression of cry genes in LM1212 strain. [Results] Two plasmid curing mutants LM(p35'Z)-W and LM(p35'Z)-DB strains were obtained. The colony of LM(p35'Z)-W was white and LM(p35'Z)-DB was dark blue on HCO plates containing X-gal. It indicated that the activity of cry35-like gene promoter was affected in these two strains. Cell morphology observation showed that more crystal-producers were found in LM(p35'Z)-DB than in both LM(p35'Z)-W and LM(p35'Z). Crystal protein production in LM(p35'Z)-DB increased, but not in LM(p35'Z)-W. [Conclusion] Plasmid curing affected cell differentiation of LM1212 strain Cry protein expression. This will provide the excellent genetically materials for better revealing the regulation mechanism of cell differentiation in LM1212 and genetic modification of Bt strain.

Abstract:[Objective] To screen exopolysaccharide from lactic acid bacteria for inhibiting melanin synthesis. [Methods] Exopolysaccharide-producing lactic acid bacteria were screened by observing the ropy appearance in skim milk media. Effects of exopolysaccharides on melanin synthesis and cell viability were measured on B16 melanoma cell. Exopolysaccharide was purified by chromatography columns of DEAE-52 cellulose and Sepahdex G100. Monosaccharide composition and basic structural information were studied by PMP-HPLC and FT-IR method. Tyrosinase inhibiting activity and antioxidant activity were studied. [Results] Exopolysaccharide form Lactobacillus rhamnosus HLAB122 (LR122EPS) could inhibit melanin synthesis of B16 melanoma cell. Compared with the control, melanin production decreased to 32.7% at 5 g/L of LR122EPS, cell viability was not changed within 96 hours. Purified LR122EPS was composed of rhamnose, glucose, and galactose in a molar ratio of 1:5.44:5.37. LR122EPS could not inhibit tyrosinase activity and its antioxidation was weak. [Conclusion] Exopolysaccharide LR122EPS had the potential in products toinhibit melanin synthesis.

Abstract:[Objective] Geobacter metallireducens, a Gram-negative bacterium, can directly transfer electron to acetoclastic methanogens such as Methanosaeta harundinacea and Methanosarcina barkeri for reducing carbon dioxide to methane. Our previous results showed that Methanosarcina mazei and Geobacteraceae formed aggregates in an iron(III)-reducing enrichment culture indicating direct interspecies electron transfer. However, the capability of direct electron transfer with methanogens for Gram-positive iron(III)-reducing bacteria such as Clostridium spp. is still unknown. [Methods] In this further study, methanogenic isolates (S6) was achieved from the iron (III)-reducing enrichment by roll-tube (Hungate) method with ethanol as the sole electron donor. We used terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis to investigate the community of S6 and used electrochemical method such as cyclic voltammetry (CV) to confirm the electroactivity of isolates. [Results] Clone library analysis of 16S rRNA gene showed that Clostridium spp. (close to C. tunisiense) and Methanosarcina barkeri was predominant in the bacterial and archaeal community, respectively. Interestingly, addition of G. metallireducens into S6 did not increase the ability of both iron(III) reduction and methanogenesis, indicating that Clostridium spp. may play a similar role in direct interspecies electron transfer from G. metallireducens to M. barkeri. Furthermore, current generation of the S6 suspension dramatically decreased when contact between the organisms and the electrodes was prevented by dialysis bag, and CV showed no obvious redox peaks. [Conclusion] These results suggested that there was direct electron transfer in the methanogenic isolates. This work demonstrate that the dominant Gram-positive Clostridium spp. can potentially directly transfer electron to M. barkeri in methanogenic isolates.

Abstract:[Objective] Effects of signal peptides and chemical penetrators on extracellular secretion of cyclodextrin glycosytransferase (CGTase) in recombinant Escherichia coli strain BL21(DE3) was observed in this study in order to lay foundation for fermentation technology of CGTase. [Methods] The CGTase gene was cloned by PCR with genomic DNA of Geobacillus sp. CHB1 as template. Four recombinant plasmids were constructed which included the signal peptide of Geobacillus sp. CHB1, OmpA, PelB and no signal peptide. The recombinant plasmids were respectively transformed into E. coli BL21(DE3) and then induced to express. The extracellular enzyme activities of the target proteins were analyzed and measured to screen the best signal peptide. Chemical penetrators like glycine, SDS, Triton X-100 and Tween 80 were added into the culture to determine the effect of chemical additives on extracellular secretion of recombinant CGTase. [Results] The results showed that these four recombinant plasmids could successfully express CGTase in E. coli BL21(DE3) and OmpA was the optimal signal peptide. Extracellular enzyme activity induced by OmpA could reach 7.44 U/mL which were 2.04- and 11.27-folds of PelB and CHB1, respectively. No extracellular CGTase activity was detected in E. coli BL21(DE3) with the no signal peptide. Extracellular enzyme activities induced by OmpA were 9.27 U/mL and 9.75 U/mL by addition of 0.6% glycine and 0.3% Triton X-100, respectively, and it could reach 14.27 U/mL by synergistic action of glycine and Triton X-100 after 48 h. However, SDS and Tween 80 had obvious inhibition on extracellular CGTase activity. [Conclusion] The extracellular CGTase activity in E. coli BL21(DE3) induced by OmpA could reach the highest by addition of 0.6% glycine and 0.3% Triton X-100.

Abstract:[Objective] Lipopeptides (LPs) are important biosurfactants produced by microorganisms and have significant influences on bacterial biological functions and broad-spectrum antagonistic abilities towards many botanic and human pathogens. LPs produced by Pseudomonas chlororaphis are not reported as yet. [Methods] This study predicted the composition and sequence of the amino acids within the LP produced by P. chlororaphis HT66. The LP biosynthetic genes deletion strain HT66Δclp was constructed, and subsequently the LP produced by strain HT66 was analyzed by the UPLC/QTOF-MS according to the lost metabolite in the mutant. At last, the roles of the LP on bacterial growth, phenazine-1-carboxamide (PCN) production, biofilm formation and swarming motility were studied. [Results] The amino acid sequence of the LP produced by strain HT66 was predicted to be L-Leu–D-Glu–D-allo-Thr–D-Val–L-Leu–D-Ser–L-Leu–D-Ser–L-Ile and the LP was verified to be viscosin via comparing the mass spectra data of the metabolites in the wild type and the mutant. Deficiency of LP was found to have no distinct effect on bacterial growth of strain HT66, however, the reduction in PCN production, biofilm formation and swarming motility were observed in the mutant. [Conclusion] The LP produced by strain HT66 is viscosin and it regulates several biological functions including metabolism, biofilm formation and motility. This study reported the structure and function of an LP produced by P. chlororaphis, which may be helpful in learning its synthesis and regulation mechanism and lay a foundation for its development and application.

Abstract:[Objective] In order to identify the diversity and community structure of the cultivable bacteria inhabiting the intestinal tract in worker adults of honeybee from herlthy Apis mellifera ligustica and Apis cerana cerana Fabricius populations. [Methods] 16S rRNA gene PCR-DGGE (Denaturing gradient gel electrophoresis) molecular methods was employed to study the bacterial diversity. In addition, the cultivable bacteria were identified by colony morphology, physiological and biochemical characteristics tests. [Results] A total of 200 culturable bacteria from aboved two populations were classified into 18 unique phylotypes. All sequenced bacteria strains were grouped into three families: Enterobacteriaceae, Vibrionaceae and Enterococcaceae. The Enterobacteriaceae was dominated in all the populations. The closely related sequences (>97% sequence similarity) which had been retrieved from culturable bacteria of two populations were grouped as one common species. We identified the cultivable bacteria: eight strains of bacteria as Enterobacter, one strain of Klebsiella, two strains of Enterococcus, and one strain of Aeromonas. [Conclusion] The present study significantly contributes to the available information on bacterial isolates from worker adults of honeybee.

Abstract:[Objective] To analyze the endophytic bacteria in leaves of Huanglong disease affected and healthy Gannan navel oranges, and to compare the bacterial community under different culture conditions. [Methods] Candidatus Liberibacter was verified by PCR. Based on 16S rRNA gene high throughput sequencing, the diversity of endophytic bacteria in the leaves of diseased and healthy plants (each five plants) cultured in different media was analyzed. [Results] Thirteen strains existed in diseased plants and seven of them were also found in all healthy plants. The dominant bacterial population of both diseased and healthy plants was mainly from Defluviicoccus sp. and Granulicella sp., whose average contents in healthy plants were higher than that in diseased ones. The similarity of diseased and healthy samples had obvious boundary. The strains obtained after cultivation in different samples and media were different. Enterobacter sp., Curtobacterium sp., Pseudomonas sp. and Pantoea sp. got a lot of enrichment, whereas the enrichment of 9 genera including Acinetobacter sp. and Serratia sp. was less. Besides, the amount of unclassified bacteria in the samples had obvious differences. [Conclusion] There were obvious differences between endophytic bacteria in disease and healthy Gannan navel oranges. The existence of Ca. Liberibacter changed the bacterial community structures. Only direct detection from the living plant tissue could obtain the real endophytic bacterial distribution. It is expected to find the bacteria associated with Ca. Liberibacter growth by analyzing the community difference of bacteria.

Abstract:[Objective] The purpose of this study was to separate and purify the pectinase of Valsa mali, and explore the corresponding enzymatic properties. [Methods] Valsa mali was cultured in MS medium containing 0.5% starch, and maintained for different days. The pectinase was extracted using ammonium sulphate gradient fractionation and purified by Sephacryl S-100 gel fitration and DEAE-Sepharose Fast Flow ion exchange chromatography. The purity was examined by SDS-PAGE. The properties of rhamnose galacturonic acid enzyme were studied using biochemical techniques. [Results] The result showed that the activity of the pectinase was highest after 10 days’ fermentation. The pectinase was identified to be rhamnose galacturonic acid enzyme. The properties including the molecular mass, the isoelectric point, the optimal temperature and pH were 58.83 kD, 6.03, 40 °C and 3.5, respectively. The rhamnose galacturonic acid enzyme was stable when its pH varied from 2.0 to 5.5. Ca2+, Li+, Co2+ could active the rhamnose galacturonic acid enzyme, while the metal ions of K+, Fe2+, Pb2+, Zn2+, Cu2+, Mn2+, Ni+ could inhibit it, especially Ba2+ and Mg2+. Besides, the kinetic constants was also detected, and the Km and Vm values were 3.600 g/L and 0.162 7 g/(L·min), respectively. [Conclusion] The rhamnose galacturonic acid enzyme was isolated from the fermentation liquor of Valsa mali, and enzymatic properties were explored. The results will lay the foundation for the preparation of antibody to pectinase and studies of cytochemistry.

Abstract:[Objective] To study the mutation phenotype of the methyl-accepting chemotaxis protein Tlp1 (transducer-like protein) located in the upstream of the gene cluster of Azorhizobium caulinodans ORS571, and to explore its functional mechanism. [Methods] The tlp1 mutant strain was constructed by the homologous recombination and triparental conjugation. Growth of the wild-type strain ORS571 and the tlp1 mutant was compared by measuring OD600 over time of cultures in TY medium. The semi-solid plate assay was used to test the ability of chemotaxis. The production of extracellular polysaccharide and secondary metabolites was measured through Congo red solid culture medium. The acetylene reduction assay was used to measure nitrogen fixation ability. [Results] Compared with the wild type, the growth of the mutant in rich media was the same as that of the wild type. The tlp1 mutant was impaired in chemotaxis to glycerol, and the complemented strain’s chemotactic capability could be partially complemented. The production of extracellular polysaccharides secreted from tlp1 mutant and wild type has no difference, but one of the secondary metabolites — the melanin tlp1 mutant appeared earlier than the wild type. The nitrogenase activity of tlp1 mutant strain was weaker than that of the wild type, and the complemented strain could complement part of the nitrogen fixation ability. [Conclusion] The methyl-accepting chemotaxis protein Tlp1 of A. caulinodans ORS571 showed a certain ability of chemotaxis to glycerol, and also affected the secondary metabolites and the nitrogenase activity in ORS571.

Abstract:[Objective] Strain of tartary buckwheat by solid-state fermentation was screened for high peptides and their fermentation product liquid had antimicrobial and antioxidant activity. [Methods] Aspergillus oryzae, Aspergillus sojae, Actinomucor repens and Rhizopus oligosporus were respectively applied in tartary buckwheat by solid-state fermentation. The protease activity, degree of hydrolysis, soluble peptide yield, inhibition rate, and radical scavenging rate in vitro were regarded as evaluation indexes to verify target strains. [Results] Aspergillus oryzae was the optimal fermentation strain, the highest soluble peptides rate was 38.83%±1.18%, the inhibition rate of E. coli and S. aureus were 96.62%±1.66% and 97.54%±0.54%, the fermentation product liquid prepared the hydroxyl free radical (·OH) scavenging rate and the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) scavenging rate were 55.65%±1.25% and 10.84%±1.03%, respectively. According to the different molecular weight distributions and activity analysis of liquid fermentation product of Aspergillus oryzae of 2 days demonstrated that different components molecular segments had an impact on antimicrobial and antioxidant activity. [Conclusion] Among these strains, Aspergillus oryzae was the most optimal strains which can be applied in tartary buckwheat by solid-state fermentation to produce peptides, and its fermentation product liquid had antimicrobial and antioxidant activity.

Abstract:[Objective] To study the serotypes, phylogeny of surface protective antigen A gene (spaA), and molecular subtyping of Erysipelothrix rhusiopathiae isolated between 2012 and 2015 in Jianghuai area. [Methods] We collected 42 clinically isolated Erysipelothrix rhusiopathiae strains. Agar gel precipitation tests, PCR and sequence analysis methods, and pulsed-field gel electrophoresis were used to investigate the serotypes, phylogeny of spaA gene, and PFGE typing, respectively. [Results] All 42 strains belonged to serotype 1a. spaA gene was 98.5% to 100% homologous to the reference strains. The sequences showed that the mutations occurred from T to G and C to A at base 609th and 769th respectively, and the corresponding amino acid showed mutations from Ile to Met as Met-203-type, and Leu to Ile as Ile-257-type strains; PFGE revealed 8 different genotypes, with 88.8% to 100% similarity. ER2 (54.8%) was the dominant PFGE genotype, and erysipelas attenuated vaccine strains (G4T10 and GC42) were the same genotype strains. [Conclusion] Erysipelothrix rhusiopathiae serotype 1a was the prevalent serotypes in Jianghuai area. The homology of the spaA was high and had small variations. All the isolates came from one clone strain. Met-203-type and Ile-257-type strains had high pathogenicity and were the most prevalent pathogens type in Jianghuai area.

Abstract:[Objective] In order to determine interaction between outer membrane protein U (OmpU) of Vibrio mimicus and the proteins of intestinal tissue of grass carp. [Methods] By construction of recombinant expression plasmid pET-32a-OmpU, we obtained the soluble fusion His-OmpU protein through IPTG induction and affinity purification. Western blot analysis showed the expressed His-OmpU protein could be combined by polyclonal antibody of OmpU. The proteins of intestinal tissue of grass carp interacting with OmpU was screened by His-tag pull down assay and identified by LC-MS/MS followed by SDS-PAGE. Based on searches of bioinformatics by Mascot software and protein database. [Results] Five proteins were verified which can interact with the OmpU in vitro, including α-actin 2, Filamin A, α-actinin 4, Transgelin-2, Calponin-2. [Conclusion] GO annotation indicated that these proteins are cytoskeletal protein and regulatory protein of cytoskeletal which play an important role in adhesion of pathogens, internalization and pathogenicity. These results provided a basis for studies on future development of adhesion antagonist and exploration the molecular pathogenesis of V. mimicus.

Abstract:Arsenic (As) and antimony (Sb) belong to the same group in the Periodic Table of the Elements and show similar chemical behaviors. Both elements occur widely in the environment and are generally recognized as toxic metalloids. With the development of economy, the pollution of As and Sb becomes a serious environmental issue worldwide and threatens the health of human beings. Microorganisms are well known to play important roles in the transformation and geological cycles of arsenic as well as antimony in nature, especially microbiological oxidation that converts toxic As(III) or Sb(III) to less toxic As(V) or Sb(V). Thus microbial mediated oxidization of As(III) and Sb(III) can be a potential bioremediation for metalloid contamination. This review focuses on the advances of microbial diversity, regulatory mechanism of microbial oxidation of As and Sb and application of arsenite oxidizers and antimonite oxidizers. We try to provide insights into microbial mediated biogeochemical cycles of arsenic and antimony and the development of bioremediation strategies.

Abstract:Bioaerosols are particles of biological origin that are suspended in the air and cover a wide size range. A large number of bioaerosols are emitted during the process of sewage and solid waste treatment. With an increasing attention towards bioaerosols, their generation, transmission and potential hazards to human health and environment have been extensively investigated. Various sampling technologies and devices have been developed for the collection of bioaerosols in the past 150 years. Each one of these methods and devices has its unique characteristics and suitable application areas. Sedimentation, inertial sampling and filtration are three typical techniques used for bioaerosol collection. This paper summarizes the operational principles, the collection efficiencies and the application scope of these techniques, which will serve as the scientific bases for bioaerosol investigations in future.

Abstract:Infectious uterine diseases are mainly caused by bacteria invasion following parturition in dairy cows. A wide range of bacteria population were revealed in the postpartum uterus of cattle, including recognized uterine pathogens such as Escherichia coli, Arcanobacterium pyogenes, Fusobacterium necrophorum; potential pathogens such as Clostridium perfringens, Klebsiella pneumoniae, Micro Streptococcus; opportunist pathogens such as Peptostreptococcus, Staphylococcus aureus. Recently, phyla of Proteobacteria, Fusobacteria, Firmicutes, Bacteroidetes, Tenericutes and a group of uncultured bacteria were observed in the uteri using molecular biology techniques. A positive correlation were found between Bacteroides, Fusobacterium and uterine diseases. G? bacteria, E. coli as representative, and G+ bacteria, A. pyogenes as representative can be detected by Toll like receptors in the endometrial cell membrane and subsequently resulted in inflammation responses, such as changes in prostaglandins type, small size of follicle and corpus luteum, reduction in serum concentration of estrogen and progesterone. Finally, this led to anestrus and anovulation, prolonged calving interval, lowing milk yield and the number of calves, which largely reduced economic profit. This review outlines the dominant microbial community in uterus after calving, correlations between bacterial species and the uterus status, recognitions of pathogen in endometrium and innate immunity, effects of uterine diseases on ovary and uterus function.

Abstract:Microbe-mineral interfacial interaction plays an important role in bioleaching process. It could be affected by the multi-influential factors among the metabolic characteristic of the microbes, state of the mineral surface structure, chemical speciation and the environmental factors. Researches on selective adsorption of microbes, conversion in mineral surface chemical speciation and passivation layer structure, microbial community and iron/sulfur oxidizing activities, and the composition and properties of extracellular polymer substance can help us to better understand the microbe-mineral interaction mechanism accompanying with its key influential factors and influential mechanism. This researches can also provide scientific base for optimization of the bioleaching techniques. In order to achieve these purposes, the progress in methodology and techniques for in situ (micro) analysis of the interfacial interaction is also pivotal. In this article, we reviewed the progresses on research of microbe-mineral interaction and interfacial micro-analysis in the recent years.

Abstract:Aflatoxin, with strong toxicity, is a group of secondary metabolites produced by the Aspergillus flavus, Aspergillus parasitieus and some other fungies. Aflatoxin can lead to liver enlargement, diseases and even cancer, showing a great threat to humans and animals health. This article introduces the molecular structure, physical and chemical properties, pollution situations of aflatoxin B1. Research progress about the biological detoxication especially microbiological degradation of aflatoxin B1 and application are discussed.

Abstract:In order to let the lower classman to have the opportunity to learn the basic experimental techniques and participate in research experiments in the way of low-cost, less class hours and high individuality, the modular independent-designed research experiment based on basic experimental techniques in Microbiology was conceived. The main task for teachers is to propose a series of thematic modular experimental subjects, while student’s role is to choose topics independently, and to think, design, operate their experiment and analyze experiment results on their own. The work was performed with four cycles in three years. The design ideas, implementation approach and the behavior and performance of teachers and students in the research experiments were described in detail. Meanwhile, questionnaires containing 34 questions related to the experimental teaching from three classes were analyzed, and the deficiencies and improvements related to the experimental teaching also were discussed deeply. The teaching practice shows that the experiments approach does not lead to the obvious increase in the number of teachers, experimental costs and class hours. These make it easy to practice and operate in large-scale teaching. Average score for each question from 144 student feedback questionnaires was 4 points or more (out of 5), which indicated that this way could help to improve the students quality and make students to receive initial training of basic capacity and quality in scientific research. Most of students (88%?96%) suggested that it is an effective approach. The form in the research experiment, student initiative, scientificity of the experimental design, discussions and analysis about experimental results and other aspects need to be improved.

Abstract:[Objective] In the present study, an indirect ELISA was developed using the purified recombinant SpaA protein for detection of the antibodies against Erysipelothrix rhusiopathiae. [Methods] Using gene recombination technology, the SpaA gene was cloned and inserted into prokaryotic expression vector pGEX-6P-1 and the recombinant expression vector was identified with PCR, restriction enzyme digestion and sequencing. The positive recombinant plasmid was then transformed into E. coli Rosetta(DE3) and induced by IPTG. The recombinant protein was detected by SDS-PAGE and Western-blot. An indirect ELISA was developed with the purified protein at different concentrations and the optimal antigen concentration and serum dilution were determined by phalanx titration. The other assay conditions were also optimized. [Results] The optimal coating concentration of SpaA and serum dilution were 1.0 mg/L and 1:100, respectively. Both the intra- and inter-coefficient of variation were lower than 10%, suggesting its reproducibility and repeatability. The developed indirect ELISA method was compared with the ELISA kit supplied by TSZ and Western blot, the coincidence rate was 92.20% and 92.59% respectively. [Conclusion] The developed ELISA had good specificity, reproducibility and sensitivity. It could provide a reliable method for the clinical detection of Erysipelothrix rhusiopathiae and epidemiological investigations．