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Abstract:In order to improve the productivity of Ectoine, the strain which could resist to higher halostress of NaCl concentration and synthesize higher intracellular Ectoine concentration was adopted. The fermentation conditions and processes of Ectoine were assessed. The impacts of carbon source and concentration of NaCl and yeast extract on the synthesizing Ectoine with Halomonas venusta DSM4743 were investigated. The batch fermentation processe of Ectoine was investigated under the optimum conditions and the Ectoine was produced by “bacterial milking”. As the results showed, the amount of synthesized Ectoine could be enhanced when the medium adopted monosodium glutamate as the sole source of carbon and nitrogen, and at the same time in 1.5 mol/L NaCl and 0.5% yeast extract. Under optimal conditions, batch fermentations were performed in a 10-liter fermentor. The maximum Ectoine concentration and the Ectoine productivity were 3.2 g/L and 2.7 g/(L·d), respectively. Ectoine was produced by Halomonas venusta DSM4743 with “bacterial milking”. The total concentration of synthesized and released Ectoine were 14.7 g/L and 14.3 g/L, respectively. The average release rate of Ectoine was 97% and the Ectoine productivity was 2.1 g/(L·d) after six cycles of osmotic shock. Moderately halophilic bacterial strain Halomonas venusta DSM4743 was tolerant to higher NaCl concentrations and had the higher threshold of intracellular Ectoine concentration. The higher Ectoine productivity was obtained with the optimization of fermentation conditions and “bacterial milking”.

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Abstract:On the solid surface of indoor moist environment bacteria grow to form biofilms easily, which are hard to remove because of its high adhesion. It is of great microbiological safety significance to research bacteria in such kind of biolfilms. Here we isolated bacteria from biofilm growing on solid surface of a public bathroom with Luria Bertani medium (LB) and its 10-fold diluted medium (LB/10). We found that the biodiversity of bacteria growing on LB/10 was better than that of LB. Furthermore one species of bacterium overgrowing other bacteria phenomena was observed on LB but not on LB/10. Eight strains of bacteria isolated with LB1/10 were identified by 16S rDNA analysis. They are clustered with some clinical isolates and uncultured bacteria. Considering the growing environment of these bacteria we also tested their detergent-degrading ability, and found that they could decrease the quantity of foam caused by detergent.

Abstract:The genomic DNA isolated from enriched culture of sedimentary soil samples from Daban Salt Lake in Xinjiang was used as PCR template to amplify 16S rRNA gene and the library carrying 16S rRNA genes of halophilic bacteria was constructed, 100 clones of the library were selected randomly and sequenced for molecular systematic analysis. Phylogenetic analysis indicated that 100 selected clones were clustered into twenty-seven species of nine genera, a lineage of the domain bacteria. Among them, Bacillus was the most dominant genus as represents 48% of the clones, followed by Halobacillus (14%) and Halomonas (13%). Furthermore, ten clones may be potential novel species or genera. The results show that the halophilic bacteria population diversity is abundant in enriched culture of sedimentary soil from Daban Salt Lake in Xinjiang.

Abstract:A strain capable of growth at high rates under nitrogen fixation conditions was isolated from the root of the seagrass (Thalassia hemprichii), which appeared after the degradation of coral reefs in Sanya natural reserves. The bacterium was identified as Pantoea agglomerans, which was also called Enterobacter agglomerans, on the basis of its morphology, physiological and biochemical characteristics, and the sequence analysis of 16S rDNA and the nitrogenase structural gene nifH. It was identified as a gram-negative, smooth, low raised, straight-rodded bacterium, which formed translucent colony with diameter of about 1 mm on the solid agar medium. Compared with the standard strain of the species Pantoea agglomerans (ATCC27155TM), there were high similarity in the carbon sources utility, hydrolysis and the optimal growth temperature and salinity. It could use a series of hydrocarbons as carbon sources, such as D-glucose, L-arabinose, sucrose, raffinose, maltose, rhamnose, D-xylose, D-mannitol, cellobiose, micronesia disaccharide. The results of the arginine dihydrolase, phenylalanine deaminase, lysine decarboxylation, ornithine deaminase were negative. The optimal growth temperature, pH and salinity were 37°C, 8 and 25‰, respectively. It could also grow at 40°C and pH was 10 but could not grow at zero salinity. Phylogenetic analysis of the 16S rDNA gene sequences (EU841417) showed more than 99% similarities to that from other recognized Pantoea agglomerans. The nitrogen fixation rate of G33-1 was 299.16 nmol C2H2/(mL·h).

Abstract:The study was conducted to screen and isolate dissolved organic phosphorus removal bacteria in soils taken from the eutrophic pond by adopting sodium glycerophosphate (NaGly) as exogenous dissolved organic phosphate. By comparing dissolved organic phosphorus removing abilities between five isolated bacteria, the most effective dissolved organic phosphorus removal bacterium named D2 was chose. In liquid media contained 5 mg/L phosphorus glycerophosphate (GP-P), the phosphorus-removal ratio of the strain D2 could reach 99.0%. Based on the 16S rRNA analysis, the strain D2 exhibited high similarity (Almost 100%) with Enterococcus faecium KT4S13 (Accession number: AB481104) and CICC6078 (Accession number: DQ672262), so it was identified as Enterococcus sp.. The growth characteristics and phosphorus removing abilities of the strain D2 were measured. The growth curve of the strain D2 was as follows: lag phase was 0-4 h, log phase was 4-8 h, stationary phase was 8-28 h, decline phase was after 28 h. Additionally, the phosphate-removal ratio of the strain D2 increased gradually before decline phase, and it could maintain the phosphate removal effect stability for 4 hours after the decline phase. Results also exhibited that the strain D2 could grow normally with temperature ranging from 15°C to 40°C, pH from 4.0 to 9.0 and initial GP-P concentration from 5 mg/L to 40 mg/L, meanwhile the optimal temperature and pH for growth varied from 30°C-35°C and 6.0-7.0. Moreover, the GP-P could significantly promote the growth of the strain D2 when its concentration was from 20 mg/L to 30 mg/L. In addition, the 20 mg/L GP-P removing capability was found gradually increased with extension of action time before the coming of decline phase and keep stability from 28 h to 32 h after entry of decline phase. The D2 had the phosphorus removal ability at the condition of temperature of 15°C-40°C, pH of 4.0-9.0 and concentration of 5-40 mg/L. The optimal range of temperature, pH and initial GP-P concentration for the dissolved organic phosphorus removing capability ranged from 25°C-35°C, 6.0-7.0 and 5-10 mg/L , respectively.

Abstract:Single inactivated protoplast fusion technique was adopted in the fusion of Citrobacter freundii and Deinococcus radiodurans, the condition of protoplast preparation and influence factor of fusion process were examined in this paper. With the lysozyme concentration, enzymolysis time increased and temperature rose, an upward trend of protoplast formation rate was showed, while regeneration rate was found gradually decreased in the test. In addition, the results of orthogonal experiment on fusion factors indicated that the optimal fusion condition was 40% PEG (6000) which induced protoplast for 10 minutes at 42°C, pH 8, and the highest fusion rate reached to 2.74 × 10-7. The screened fusants had been stably transferred for ten generations. Furthermore, the tolerance to uranium for fusant was greater than that of Citrobacter freundii under the concentration of 85 mg/L, and biosorption capability of fusant was better than that of parental strains appreciably in the comparison experiment. This works lay foundation for constructing radioresistant engineering strains.

Abstract:In order to study the mechanism of bacteria producing siderophore, B50 was isolated from the cotton rhizosphere. The strain B50 can produce siderophore under iron-limiting conditions. It was identified as Pseudomonas pseudoalcaligenes based on its morphological, physical-chemical characteristics and 16S rDNA sequence. Mutants of B50 were obtained through conjugation. The pyrD gene was cloned from the mutant deficient in siderophores synthesis by TAIL-PCR (Thermal asymmetric interlaced). The pyrD gene is required for the synthesis of dihydroorotate which is a predecessor of deoxyquinoline. Deoxyquinoline was the important part of several siderophores. This is the first report that the Pseudomonas pseudoalcaligenes can produce siderophore.

Abstract:A bacterial strain SB1, isolated from tomato roots, was tested for its antimicrobial activities. Results indicated that strain SB1 showed strong inhibitory activities against various plant pathogenic fungi and pathogenic bacteria, exhibiting wide antimicrobial spectrum. Strain SB1 was identified as Bacillus subtilis subsp. endophyticus based on its morphological, physiological characteristics and 16S rDNA sequence. In addition, properties of antibacterial substances of strain SB1 were examined using Ralstonia solanacearum as an indicator. The results showed that antibacterial substances were heat-stable, water-soluble, alcohol-soluble, and resistant to ultraviolet radiation and protease K. High performance liquid chromatography (HPLC) results further suggested that one of the antibacterial substances was surfactin.

Abstract:In the present study, we utilized a previously characterized N-acetylglucosaminidase (AcmA) to develop a whole-cell catalyst of bacterial superoxide dismutase (SOD) in Lactococcus lactis. The truncated C-terminal domain (cA) and the full-length AcmA from L. lactis wild-type strain MB191, were used as the anchoring motifs to immobilize an Escherichia coli-derived SOD onto the surfaces of L. lactis ATCC11454 cells. The PCR-amplified cA fragment, the signal and the full-length sequences of acmA, were fused with sod to generate the recombinant ss-cA-sod and acmA-sod, respectively, followed by ligating into the expression vector pMG36K, yielding the recombinant strain MB193 (harboring the ss-cA-sod fusion gene) and MB194 (harboring acmA-sod), respectively. SDS-PAGE analysis showed that the substantial expression profile of the fusion enzymes cA-SOD and AcmA-SOD in the recombinant L. lactis MB193 and MB194, with the predicted Mr of 46 kD and 64 kD, respectively. The Mn-SOD activities of MB193 and MB194 cells were determined by using the standard xanthinoxidase assay procedure. It showed that MB193 and MB194 exhibited the higher whole-cell Mn-SOD activities [by (2.63 ± 0.51) U/mL and (3.51 ± 0.64) U/mL, respectively, compared to the control strain MB192 by (1.53 ± 0.38) U/mL] that expressing the intracellular SOD. In addition, the recombinant MB194 cells exhibited the higher cell-surface display efficiency (by 56.4%) compared to MB193 cells (by 41.9%).

Abstract:The bacterial community structure of 5 high and medium temperature Chinese liquor Daqu were investigated using PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) as a culture-independent method. DNA sequencing was proceeded to obtain the dominant bacterial population information. The result of DGGE profile showed that Weissella cibaria, Lactobacillus helveticus, L. fermentum and L. panis were commonly detected in all the five Daqu. Thermoactinomyces sanguinis was detected only in the Jiangqu sample. Compared with culture-dependent method, DGGE was able to detect Staphylococcus xylosus and Klebsiella oxytoca. The correlation between the craftwork of Daqu and the bacterial community structure was obvious. The Daqu bacterial Shannon-Wiener index decreased as the craftwork temperature of Daqu increasing. PCR-DGGE was proved to be a powerful tool for gaining detailed insight into the bacterial diversity of the Chinese liquor Daqu.

Abstract:Rolling-circle amplification (RCA) is an isothermal DNA amplification assay in vitro, which has good specificity and sensitivity and has been extensively applied to microbiological investigations. In this study, five diseased swine samples from certain region of Xinjiang were first detected by differential PCR for porcine circovirus type 2 (PCV2), and then RCA was utilized for amplification of the full-length genomic DNA of two positive samples. Verification with Sac II resticition enzymatic mapping showed that PCV2 specific genome-size bands appeared on agarose gel. The targeted bands were purified and cloned. With the aid of sequencing, the genomes of two viral strains were obtained and proved to be 1768 bp in length. Moreover, the phylogenetic analysis indicated that they were divided into two different genotypes (PCV2a and PCV2e).

Abstract:The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain was constructed in this study. Total RNA were extracted from HPT-8 cells at 20 min, 1 h, 2 h, 3 h and 4 h post infection respectively. With cDNA synthesized by reverse transcriptase, homologous recombination construct infected HPT-8 cell cDNA library. The partial genes were sequenced and classifid by function. The capacity of cDNA library was 1.43 × 106, the recombinant rate was 96.92 percent. The size of the inserted fragments is between 0.2-5 kb. 63 clones of cDNA library were random selected and sequenced. The rates of transcription, energy metabolism, transporter, cytokine are relatively high. The results showed that cDNA library was successfully constructed. The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain may facilitate to identify the receptors associated with the resistance against Brucella in host cells and to cast new light on the mechanism of cellular tropism.

Abstract:Endophytic strain LD5 was isolated from the stems of Gentiana nubigena. The fermentation broth of LD5 showed antibiosis activities against Staphylococcus aureus and Escherichia coli. According to the characteristics of morphology, physiology and biochemistry tests and the comparison of 16S rDNA sequence, strain LD5 was identified as Bacillus amyloliquefaciens. Strain LD5 could produce flavonoids, gentiopicroside, and a kind of analogue of alcoholic extract from Gentiana nubigena.

Abstract:As natamycin-producing strain, Streptomyces gilvosporeus was studied to develop the micro-cultivation system in 96 deep-well microplates for rapid screening from a large number of isolates. Firstly, the stainless steel cover perforated with holes and dressed with eight gauzes was designed to solve water evaporation and cross-contamination in 96 deep-well microplates. The optimal conditions for micro-cultivation were as follows: culture volume 600 μL, shaking speed 300 r/min, shaking diameter 40 mm, and the pattern of microbial growth and natamycin biosynthesis with the above micro-cultivation mode was quite similar with that in shake flasks. It was found that the maximum intra- microplate and inter-microplate difference were 1.93% and 6.62%, respectively. Using the statistic software analysis, the productivity in 96 deep-well microplates were in good linear correlation with those in shake flasks (F = 39.303, P = 0.00 < 0.01) and the rank of production distribution in two different cultivation systems was consistent. Therefore, the micro-cultivation in microplates was proved to be good standardization and parallelization, which was potential to be applied in rapid screening from a large number of strains.

Abstract:The bioinformatics analysis tools of SignalP, PSORT-B, Cell-Ploc, TMHMM, Phobius, LipoP and PRED-TMBB were used to screen putative surface-exposed or exported proteins for vaccine candidate antigens among three Shigella genome sequences of Shigella flexneri 2a strain 301, S. flexneri 2a strain 5b8401 and Shigella sonnei strain Ss046. As results, 96, 158 and 107 ORFs, putatively coding a series of lipoproteins, β-barrel outer membrane proteins and secreted proteins, respectively, were identified from these genome sequences. Among these ORFs, a total of 63 ORFs were conserved in all of these three genomes. In conclusion, bioinformatics methods could be used as an assistant tool in screening candidate antigens for the development of Shigella vaccines. The results might provide an useful basis for further studies of novel Shigella vaccines.

Abstract:Pichia pastoris is easy to genetically manipulate and can be grown to high cell densities, at the same time, P. pastoris is a eukaryote, and thereby has the potential for producing soluble, correctly folded recombinant protein with high yield, so the P. pastoris expression system is an ideal choice for the production of various heterologous proteins. However, at present not all the heterologous proteins can be successfully and efficiently expressed in P. pastoris, as a result, different proteins present different yield levels, bioactivity and stability. Here strategies for reducing proteolytic degradation and improving production of the expressed heterologous proteins are briefly summarized in terms of genetically factors and cultivation level.

Abstract:Brominated flame retardants (BFRs) are widely used in plastics, textiles, and electronics for their excellent ability of preventing fires. However, BFRs cause adverse effects on human health and environment for their bioaccumulation, persistence and biotoxicant. Although it is predicted that microbial remediation will bring bright prospective for the purification of BFRs-contaminated environment, less research has been done about the microbial degradation of BFRs. In this paper, we reviewed the recent progresses on microbial degradation of BFRs, including anaerobic degradation, aerobic degradation and other kinds of microbial degradation. In addition, we discussed the problems and challenges in this filed, and the future exploitation of the anaerobic debromination.

Abstract:Quorum-sensing system is the gene regulation system depending on the population concentrations. A number of different types of QS systems have been discovered. However, a unifying theme is the synthesis of a small signal molecule, often called an autoinducer or pheromone. These systems regulate the expression of a number of genes synchronously across the bacterial population. When an autoinducer accumulates to the threshold concentration in a population density manner, the expression triggers induction or repression of certain sets of genes that co-ordinate the behavior of the bacterial population, including the expression of virulence factors. In addition, the expression of quorum sensing system is influenced by environmental factors. Quorum sensing in P. aeruginosa consists of a complex network. Based on the facts that QS regulates such an array of P. aeruginosa factors and that deletion of QS regulators attenuates P. aeruginosa virulence, it is conceivable that QS would be an ideal target for the inhibition of Pseudomonas infections. Therefore, alternative mechanisms for targeting P. aeruginosa have been the focus of much research. In this review, the roles and the regulation mechanisms of quorum sensing in P. aeruginosa has been disscussed based on the author’s own research and the latest literatures.

Abstract:In order to train students to have innovative consciousness and applied ability, resolve the contradiction between increasing content and decreasing class hours, several kinds of discuss-based teaching methods put in practice on microbiology classroom were proposed in this paper, including debate contest, academic report contest and lecture competition. It has been found that compared with traditional classroom teaching methods of instilling, these new measures had many obvious advantages, such as enlivened atmosphere in classroom and redoubled effect of teaching, students acted as judges so teacher and students mixed together, encouraged students to create something new and developed different individual characters, made up for inadequate class hours and widened contents of curriculum. Qualities of students who were trained were improved significantly.

Abstract:The prolegomena teaching plays an important role in the microbiology integrated teaching. This paper firstly analyzes the goal of the prolegomena teaching in terms of knowledge, ability and emotion, and then emphasizes the importance of assisting students in accumulating their fundamental knowledge, stimulating students’ creative ability, encouraging students to follow research ethics rules strictly, and spurring students’ learning interests and strong spirits in order to achieve the integrated and three-dimensional teaching goal.

Abstract:Medical Microbiology is an important basic course in medical colleges. It is also difficult to teach, especially for foreign students. Faced with the problems we meet when teaching medical microbiology to overseas students at our college, we have made a lot of attempts to overcome language barriers, select textbooks and teaching contents, improve our teaching methods and means, reinforce teaching management and assessment to name some of our initiatives. Now we are discussing and sharing our experiences aiming at enhancing the teaching level of medical microbiology for foreign students.

Abstract:By analyzing the characteristics of medical microbiology and foreign students, the teaching of medical microbiology for foreign students was reformed in choosing teachers, selecting textbooks, applying modern teaching technique, reforming examination managements and strengthening experiment teaching. The quality of teaching was enhanced by employing elicitation method, contrast induction method, typical cases method and PBL method. The exploring will help the teaching reform on medical microbiology for foreign students.

Abstract:Investigation of microbial spoilage in meats is usually hindered by the lack of suitable growth media and protocols to characterize the causative agents. A near-native medium of chicken extract agar (CEA), developed by the addition of sterile chicken extract into conventional plate count agar (PCA), was utilized to detect difficult cultivable bacteria on chicken carcasses. Suitable additive concentration was selected. Bacteria absent on conventional PCA medium but recovered by CEA were identified based on morphology, biochemical features and 16S rRNA gene sequencing. The colony form unit (CFU) was significantly greater (P < 0.05) and microflora more diversified on CEA than on PCA. CEA containing 3.0% (W/V) chicken extract gave the highest microbial enumeration at 64 CFU/g, almost twice that in PCA. Three species of bacteria absent on conventional PCA medium were recovered by CEA. According to their morphology and biochemical features, three bacteria recovered were identified as Enterococcus sp., Rothia sp., and Staphylococcus sp., respectively. The bacterial identification was confirmed by 16S rRNA sequence analysis with the similarity being 99% for both Enterococcus faecalis and Staphylococcus saprophyticus subsp. saprophyticus, and 96% for Rothia mucllaginosa. Three bacteria recovered by CEA are considered as either spoilage (Enterococcus sp.), opportunistic pathogen (Rothia sp.), or pathogen (Staphylococcus sp.) in foods, thus to cause problems associated with food safety.

Abstract:To evaluate the application of reverse staining in proteomic research, the same protein samples were run by two-dimensional electrophoresis, then the gels were dyed by reverse staining and coomassie Brilliant Blue (CBB) staining. The reverse staining and CBB staining gels were compared, eight pairs corresponding protein spots from the two different gels were cut out, digested by trypsin and analyzed by MALDI-TOF/ TOF. Image analysis showed that the reverse staining gel could display more protein spots than the CBB staining one. Seven of eight protein spots on reverse staining gel were effectively identified by MALDI-TOF/TOF, and eight protein spots on CBB staining gel. So, we consider that the sensitivity of reverse staining is higher than the CBB staining, and its compatibility with MALDI-TOF/TOF is good. Therefore, the reverse staining method can be used to set up the reference map of 2-DE, but not suitable to use on the comparative of proteins.

Abstract:In this study, we present the development of a new method for biosurfactants detection. A relation between surface tension and concentration of rhamnolipid was plotted in an x-y scatter by Origin using empirical formula. So the concentration of crude fermentation fluid was determined measuring surface tension of the diluted fermentation fluid with the fitted equation. This method is rapid, simple, accurate and low-cost compared to currently used methods.