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Abstract:Hoh Xil that is situated at the Yushu Tibetan Autonomous Region in southwest Qinghai, is one of the most primitive and well-preserved natural environment in the world. The microbial diversity in this area is largely unknown till date. In the present study an attempt has been made to explore the microbial diversity using both of cultivation-dependent and independent approaches. In the culture-dependent experiment, 19 isolates were obtained from the soil sample IMB08-049. 16S rRNA gene sequence analysis showed that the 19 strains belonged to 8 different genera, including Phyllobacterium, Variovorax, Pseudomonas, Streptomyces, Microlunatus, Kribbella, Promicromonospora and Bacillus. Denaturing gradient gel electrophoresis (DGGE) analysis revealed 17 V3 fragments of 16S rRNA gene from sample IMB08-049. Biodiversity was also assessed by amplified ribosomal DNA restriction analysis (ARDRA), DNA sequencing and phylogenetic analysis, respectively. 19 operational taxonomic units (OTUs) were clustered in the following phyla including Proteobacteria (91%), Actinobacteria (7%) and Firmicutes (2%). Comparison of the results from culture-dependent and culture-independent suggests that some dominant species were not isolated using the above culture-dependent method, and that new isolation method should be developed to discover more bacteria exciting.

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Abstract:To research the effects of growth regulator on the status of mycelia growth during steroid biotransformation, morphology of filamentous microorganisms was characterized in combination with both sedimentary density method in macroscopic view, and using Scanning Electron Microscope (SEM) method in microscopic view during the whole strain growth phases. The results showed that in comparison with strain growing without adding growth regulator, the strains growing, which was added growth regulator, showed relatively lower sedimentation densities during all different stages than the one not added. Through SEM, it can be clearly seen that the bacteria with the regulator presents better conditions such as smooth surfaces, rounded plump as well as excellent shapes. Both methods prove adding regulator are able to promote the exuberant growth of the bacteria.

Abstract:Based on the antimicrobial activity of ε-Polylysine, a 96-well microplate bioassay was established to rapidly determine large numbers of ε-Polylysine samples. The results showed that a high correlation coefficient (R2=0.997 5) was obtained for linear regression in the ε-polylysine concentration range of 100.00?500.00 mg/L with 107?108 CFU/mL indicator strain (Micrococcus luteus) and the incubation time of 4 h. Using these parameters, the microplate bioassay has showed good precision and accuracy, and the relative standard derivation (RSD) and relative derivation (RD) were lower than 3.5% and 3.0% respectively. Compared with the results by Itzhaki method and HPLC assay, the microplate bioassay shows no considerable difference with HPLC, but can directly indicate the inhibition activity and more suitable for rapid determination of numerous samples, which can be used for ε-polylysine quantification in fermentation process and high throughput screening from large numbers of mutants.

Abstract:Located in the 20s loop of mandelate racemase from Pseudomonas putida, Val22 is one of the amino acids related with substrate binding. In this study, a 75.9% decrease of activity was observed after Val was substituted by Arg. In addition to the reduction of hydrophobic interaction between enzyme and substrate, electrostatic repulsion could also lead to the decrease of activity. Molecular dynamic was utilized and showed that the electrostatic potential of substrate and amino acid of No. 22 increased from 0.036 kJ/mol to 0.124 kJ/mol. This result indicated that the increased activity in the mutant was mainly attributed to the shift of side chain polarity and the electrostatic repulsion between side chain and substrate. An increase of 283 kJ/mol in the potential energy of system suggested that the changes of side chain polarity and electric property brought about the increase of potential energy in binding state and the dramatical decline of activity. Therefore hydrophobic interaction and electrostatic interaction should be considered with steric bulk when rationally designing the binding pocket of mandelate racemase.

Abstract:Maar Lake is a unique type of lake in geological structures, and little is known about microbial diversity in various Maar Lakes worldwide. In order to understand the community structures of culturable bacteria in Huguangyan Lake, a typical Maar lake in South China, BOX-PCR was introduced as a screening technique for bacterial isolates that were obtained from three water depths in winter and summer through the general R2A plates and in situ lake-water agar medium. The results indicated that the changes of bacterial abundance were consistent along water column and on the two types of medium: 5 m>1 m>13 m for summer and 1 m>5 m>13 m for winter, with Proteobacteria as the dominant bacteria followed by Actinobacteria, Firmicutes, and Bacteroides. BOX-PCR profiles for cultured bacteria on both mediums were similar, but their community structures were not identical. The bacterial diversity was higher in winter than that in summer, in consistent with the trends found in other similar primitive lakes. Our results show that using different media may have different limitations in cultivating microbes from natural environments. This study may contribute to the understanding of significant relationship between microbial diversity and environmental characteristics in unique Maar Lakes and to the isolation of novel functional bacteria.

Abstract:The influence of radish (Raphanus sativus L.) seeds, seedlings, root exudates, and seedling extracts on the mycelium growth of Cenococcum geophilum Fr. (Cg) was investigated in vitro. The mycelium growth of Cg strains (CgSO1, CgSB2, CgO5, SPOP2 and Cg5#) was promoted, when Cg strains were co-cultured with radish seedlings, or cultured with root exudates, and water extract of radish seedlings. However, Cg mycelium growth was significantly improved by the autoclaved (treated under high temperature) root exudates or autoclaved water extract of radish seedlings, while the autoclaved seedlings segments did not. Results showed that the maximum mycelium dry biomass of the 5 strains of Cg was given in the medium added the autoclaved water extract of the seedlings. The single colony dry maximum biomass of each Cg strain (CgSO1, CgSB2, CgO5, SPOP2 and Cg5#) was reached to 54.8, 45.8, 63.9, 41.2 and 50.5 mg respectively.

Abstract:A newly isolated fungus JMUPMD-2, capable of growing with parathion-methyl as the sole phosphorus source, was identified based on morphological charaterisitics and rDNA ITS sequences analysis, and its capability of degrading parathion-methyl was investigated by determining the changes of parathion-methyl concentration in the broth of the isolate. JMUPMD-2’s colonic and microscopic morphologies were identical with and its sequences of rDNA ITS was 99% similar to Apspergillus oryzae. Consequently, it was identified as Aspergillus oryzae. When cultured with an artificial medium containing parathion-methyl as the sole phosphorus or parathion-methyl-K2HPO4 mixed phosphorus, concentration of parathion-methyl respectively reduced 189 μg/L and 28 μg/L after inoculating for 200 hours. JMUPMD-2’s intracellular extract had obvious parathion-methyl degrading enzyme activity.

Abstract:In this study, PCR and clone library methods were used to analyze the composition and potential functions of integrons in the ecosystem of a lab-scale quinoline-degrading bioreactor. The results showed that the integrons carried gene cassettes of sizes between 200 bp and 1 000 bp. Some gene cassettes could not find any analogue in database. But some of them are similar with the sequences of integrons previously found in industrial wastewater, such as FldF gene, which might be related to degradation of aromatic compounds. Many cassettes had aminoglycoside resistance genes, such as aadA gene, which might encode antibiotic-resistance protein. To verify this result, we measured the antibiotic resistance pattern of 34 isolated strains, which were dominant in the quinoline-degrading bioreactor. Results indicated 44.1% of the isolates were resistant to antibiotics, and the incidence of multiple antibiotic resistances was 29.4%. The ratio of resistance to ampicillin, kanamycin, chloromycetin, streptomycin of the isolates were 29.4%, 23.5%, 20.6%, 23.5%, respectively. There barely exists antibiotic stress in the quinoline wastewater bioreactor and the environment where seed sludge came from. However, the dominant bacterial strains were generally resistant to antibiotic and many integrons carried resistance gene in first gene cassette. This phenomenon so far has not been reported and its mechanism needs further study.

Abstract:The aim of this study was to establish and optimize standard laboratory techniques for cultivation and fermentation of a strain of Beauveria bassiana (B. bassiana AT17), which has shown potential biological activity against ticks. This will provide practical and experimental supports for the further research on preparation of biocontrol agents. Condition optimization was conducted by single factor experiment. The factors studied were basic culture medium, temperature, pH, carbon sources, nitrogen sources, microelements and liquid fermentation medium. The results showed that B. bassiana AT17 could grow well on PDA, PPDA, PDAY, SDAY and SMAY medium. Comparatively, B. bassiana AT17 had the fastest growth on PDA and the highest spore yields on PDAY. Temperature was the major factor that affects on growth speed and yields of B. bassiana AT17. It showed that 25 °C was the optimal temperature for both hyphal growth and the sporulation. B. bassiana AT17 could grow at pH 4?10 with the fastest growth in alkali condition, and produce optimal sporulation at pH 5?6. Based on all of fungal indexes, glucose and yeast were the best carbon and nitrogen sources. The best solid fermentation medium was wheat bran+indian meal+rice flour mixed with 3/4 SDAY containing suitable K+ in proportion of 2:1.

Abstract:Jiaocheng Jun jujube (Zizyphus jujube cv. Jiaocheng Junzao) was selected as the material to isolate endophytic bacteria. 18 strains of endophytic bacteria were obtained and their antibiotic activity against pathogenic fungi and Oomycetes were evaluated in this study. The antagonistic effects showed that 5 of them expressed antibiotic activity on Colletotrichum gloeosporides, Alternaria alternata, Fusarium oxysporium, Alternarial longipes, Pyricularia oryzae, Rhizoctonia solani, Sclerotinia schinseng and Bipolaris sorokiniana to some extent. And morphological performance, physiological and chemical characteristics of 5 antibiotic bacteria were observed.

Abstract:The purpose of this study is to investigate the partial characteristics (spectrum of antibacterial activity; sensitivity of enzymes, pH and temperature; mode of action) of the bacteriocin produced by Lactobacillus brevis KLDS1.0373 which was isolated from “Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia. The bacteriocin was partially purified by ammonium sulfate precipitation followed by sequential gel filtration chromatography and the antibacterial activity of the bacteriocin was heat tolerant and was found to be stable after incubation for different pH values. The slight increase in activity was recorded after 30 min at 100 °C or 121 °C. The bacteriocin was sensitive to several proteinases, but insensitive to α-amylase. The apparent molecular weight of the partially purified bacteriocin was estimated at approximately 3.8 kD by Tricine-SDS-PAGE. The bacteriocin has a broad inhibitory spectrum against Gram-positive and Gram-negative bacteria. The mode of action of the bacteriocin was identified as bactericidal.

Abstract:Polyacrylamide gel electrophoresis was used for studying the protein and several isoenzymes of biomass in two kinds of photosynthetic bacteria (PSB) transforming mistletoe culture fluid (PSBT), pure PSB culture fluid as control. The proteinogram and the zymograms demonstrated that proteins, esterase isoenzyme (EST) and peroxidase isoenzyme (POD) of PSB in PSBT changed, i.e. the expression of some proteins, EST and POD of PSB were inhibited, while some new proteins, EST and POD of PSB in PSBT appeared. The expression of superoxide dismutase (SOD) of PSB in PSBT did not alter evidently. The results showed that PSB could be induced to generate new protiens, EST and POD by mistletoe, and which might take part in biotransformation of mistletoe. The research provided basis for studying the biotransformation mechanism of mistletoe by PSB and the further application of mistletoe to tumor therapy.

Abstract:The solid fermentation conditions, including fermentation medium, fermentation time, initial pH, as well as additional carbon and nitrogen source for borrelidin produced by Streptomycete rochei ATCC10739 were investigated. Under the optimal fermentation conditions (ISP-2 medium supplemented with 1% glycerol; initial pH 6.0; culture temperature 30 °C; fermentation time, 7 days), the highest borrelidin production level was achieved as 1.336 mg/L media. Borrelidin was separated and purified by organic extraction, followed by silica gel chromatography and semipreparative high performance liquid chromatography (HPLC).

Abstract:In order to express soluble foreign proteins in E. coli, a bicistronic translation-coupled expression vector pDICT was constructed by using thioredoxin as a molecular chaperone. In this study, the expression vector pDICT was built by inserting thioredoxin gene into the pET22b vector between Nde I and EcoR I sites, and adding ribosome binding site before the stop codon of thioredoxin. Lumbrokinase gene F238 was cloned into this vector, and transformed into E. coli BL21(DE3), then the recombinant protein was induced by IPTG. SDS-PAGE results showed that the Lumbrokinase F238 was soluble expressed. The fibrinolytic activity was measured by using artificial fibrin plates, and the result indicated that Lumbrokinase could directly dissolve fibrous protein, and also had fibrinokinase activity which could indirectly dissolve fibrous protein. Construction of the bicistronic translation-coupled expression vector provides a new method to achieve soluble expression of heterologous proteins in E. coli.

Abstract:To investigate the effect of lactobacilli on the Th1/Th2 cytokine and antibody production in sensitized-mice spleen lymphocytes in vitro, BALB/c mice were intraperitoneally injected with β-lactoglobulin, After successfully established the allergic model, the sensitized splenic cells were stimulated with active/heat-killed lactobacilli (107 CFU/mL) at different species in vitro. Supernatants were collected to measure the cytokines (IL-12, IFN-γ, IL-4) and Igs (total IgE, β-Lg-specific IgE, total IgG) production by ELISA assay. In vitro studies, 4 active/heat-killed lactobacilli could differently modulate the production of Th1/Th2 cytokine and antibody, especially the heat-killed Lactobacillus fermentum and Lactobacillus acidophilus were not inferior to the live strains. These two heat-killed Lactobacillus suppressed IL-4 and IL-10 production by stimulating IL-12 and IFN-γ production. Their IFN-γ/IL-4 value, which represents the Th1/Th2 cell balance were higher than that of other strains, and compared with the control were significant (P<0.05). At the same time, these two heat-killed strains significantly decreased the secretion of total IgE, β-Lg-specific IgE and total IgG (P<0.05). These data indicate that lactobacilli may be effective in decreasing the antibody production in sensitized-mice splenocytes by increasing the ratio of IFN-γ/IL-4 and changing the dominant of Th2. Four strains may differ in their respective ability to improve Th1/Th2 cell balance toward Th1 dominance.

Abstract:Lipase A gene of Candida Antarctica (cala) was displayed for the first time on the surface of Saccharomyces cerevisiae using a-agglutinin as an anchor protein, recombinant strains were screened on minimal dextrose plates. Results of immunofluorescence microscopy assay revealed that the CALA-a-agglutinin fusion proteins were successfully anchored on the S. cerevisiae cell surface. Furthermore, yeast displayed CALA produced halo on the MD plate containing tributyrin, suggesting that CALA was active on the cell wall. Yeast displayed CALA displayed maximal activity after 72 h cultivation in YNB-CAA medium, with an activity of 80.4 U/g dry cells. The optimal temperature and pH for yeast displayed CALA were 70 °C and pH 8.0, respectively. It remained 60% of activity after incubation at 50 °C for 2 h and the lipase still remained 70% activity after incubated in buffer containing 30% dimethyl sulfoxide for 2 h. Those properties make yeast displayed CALA great potential for industrial application.

Abstract:Vibrio parahaemolyticus is a foodborne pathogenic bacteria which could be widely found in coastal areas, salt lakes and seafoods, it could cause large-scale food poisoning. tlh (Themolabile hemolysin) and tdh (Thermostable direct hemolyticus) are two of the most important virulence genes of Vp, we can compare the virulence of strains which in different stress conditions, as well as differences strains by comparing the expression levels of tlh and tdh indirectly. In this paper, three kinds of Vp cultured under different conditions, total RNA were extracted from them, 16S rRNA as an internal standard gene, using real-time quantitative PCR (RT-PCR) detect Vp’s tlh and tdh gene expression under different conditions. The results showed that gene tlh and tdh expression difference were significant in different strains and in same strains that under different condition, tlh optimal expression conditions were 5% salinity and 20 °C; tdh optimum expression conditions were 1% salinity and 25 °C. The use of SPSS software for statistical analysis of experimental results show that: factor that affect the expression of tlh is strain greater than salinity greater than temperature; factor that affect the expression of tdh is strain greater than temperature greater than salinity.

Abstract:In order to study the effect of Armillaria mellea beverage on mice organic function, the model of immunosuppression was produced by cyclophosphamide (CY) modeling method. The immune function was studied by feeding different doses of Armillaria drinks. Meanwhile the total number of bacteria, E. coli, Lactobacillus and Bifidobacterium was measured. And the effects of Armillaria drinks on intestinal microflora were analyzed. Finally the toxicity test of the drinks was detected. Results showed that Armillaria drinks could significantly enhanced immune function, including promoting the development of spleen and thymus, spleen and thymus index, promoting phagocytic activity of macrophages. Meanwhile it could also promote the growth of beneficial flora, improve the intestinal microbial flora environment. Armillaria drinks did not produce any toxic effects on mice. Armillaria drinks enhanced not only immune function but also bowel function, maintaining and promoting the health of the body.

Abstract:Xylanase B from thermophile bacteria Thermotoga maritima MSB8 has extreme-thermostability, which has potential widely application for feed, papermanufacture, energy, food and medicine industries. Recombinant pET28a(+)-xynB64 was induced and expressed in E. coli BL21(DE3), and the activity of recombinant XynB64 was very low. E. coli BL21-CodonPlus(DE3)-RIPL and Rosetta(DE3) both harbouring rare tRNAs were used to replace E. coli BL21(DE3) and the activity of recombinant XynB64 increased by 197% and 277%, respectively. However, some inclusion body was formed in E. coli Rosetta(DE3). Next, pET32a(+), pET42a(+), pET43.1a(+) and pMAL-c2X, which has the Trx, GST, Nus and MBP fusion tag respectively were used to replace pET28a(+) with E. coli Rosetta(DE3) as host. The activity of recombinant XynB64 produced by Rosetta(DE3)/pMAL-c2X-xynB64 was highest, which was equivalent to 88% of counterparts of Rosetta(DE3)/pET28a-xynB64. Meanwhile about 40 percent whole cell proteins of former were recombinant XynB64 with little inclusion body.

Abstract:Microbial fuel cells (MFCs) are devices that use microorganisms as the catalysts to oxidize organic and inorganic matter and generate current. Previous researches indicate that the internal resistance is an important factor to limit the power output of MFCs. So, the researches on the internal resistance of MFCs at home and abroad were summed up and the definition, constitution and the common determine methods of the internal resistance are systematically introduced in this review. The influencing factors of MFCs’ internal resistance were analyzed in detail in terms of reactor configuration, substrates, microorganisms and operating conditions. Based on the previous results, the feasible methods are proposed to reduce internal resistance and improve power output of MFCs.

Abstract:Problem-based learning is a problem-based and student-centered instructional model which takes professional ability as goal in teaching process. We have introduced it into the teaching of Food Microbiology in higher vocational school. As a result, it is evident that PBL does have a large impact on improving students’ motivation and subjectivity. It is also good for enhancing teachers’ leading station during teaching. At the same time, many other abilities have been greatly improved with the application of PBL.

Abstract:The problems in teaching of graduate student course were discussed by analyzing orientation of advanced microbiology course for graduate student, learning purpose of student, diversity of the knowledge background on microbiology, and the problems in teaching. The satisfying teaching effects were achieved through teaching reform on optimization of teaching contents, construction of teaching material, reform of teaching method, improvement of teaching staff quality, and enhancing communication between teacher and student.

Abstract:Biochemical engineering is an important professional subject in bioengineering major of colleges and universities. In order to catch up the fast development of biotechnology, the bilingual teaching is commonly used in this subject. In this paper, we analyzed the existing problems in the “biochemical engineering” bilingual teaching, some strategies, including reform of teaching and examination modalities, as well as supplement of teaching contents and comprehensive experiments, were put forward to improve the teaching results.

Abstract:Aiming at the problems related with the bilingual teaching in collages, it was discussed five questions including permitting choice bilingual teaching, making full use of textbook material, various measures lightening study pressure and so on in this paper, in order to improve the bilingual teaching effect of Food Microbiology.

Abstract:A new method was developed to screen for antibiotic microorganism producing strains from soil using metagenomics. Six different environmental soils metagenomics DNA were extracted with liquid nitrogen freezing method and purified by polyvinylpolypyrrolidone (PVPP) column chromatography. Soil DNA of 32.25?61.25 μg with DNA length about 23.1 kb can be extracted from 1 g sample. 16S rDNA can be amplified from the purified soil DNA which proved that the extracted soil DNA is suitable for following experiments. In addition, the sensitivity and feasibility of this screening method is evaluated by addition of Herbimycin producing strain into a negative soil. The result showed that when the soil contains 103 CFU/g of Herbimycin producing strain, the Herbimycin biosynthesis gene can be detected by PCR and the incorporated known strains can also be isolated. These results suggest that soil metagenomics can be applied for screening certain antibiotic microorganism producing strains which is sensitive and rapid, and is more efficient than the traditional methods.