Abstract:[Objective] Research on the fermentation medium to produce low-temperature lipase strain CZW001. [Methods] Based on single factor test results, the liquid fermentation medium for the production of cold-active lipase by CZW001 was optimized using Plackett-Burman, Box-Behnken and response surface method under follow conditions: temperature 20 °C, pH 8.0, stirred velocity 160 r/min and fermentation time 2 d. [Results] The optimal fermentation medium of the strain is as follows (g/L): glucose 7.68, olive oil 21.93, (NH4)2SO4 2.0, KH2PO4 1.0, MgSO4·7H2O 0.27, CaCl2 0.3, NaCl 20.0, Tween-80 1.0. Under these conditions, the highest total enzyme activity (62.8 U/mL) was obtained and increased by 3.14 times compared with the original value before optimization. [Conclusion] The study on optimizing the fermentation medium to produce low-temperature lipase strain CZW001 obviously improved the lipase enzyme activity at low temperature.

Abstract:[Objective] The aim of this study was to analyze the seasonal characteristics of bacterial diversity and community structure in sediments of the Shuang Taizi estuary. [Methods] Four seasonal samples, sitting in Shuang Taizi estuary, were collected in April, July, October and December, 2009, respectively. PCR-DGGE method was employed to analysis the bacterial diversity. [Results] By sequening, the bacteria in sediments of Shuang Taizi estuary fell into five known phyla, which were Proteobacteria (52.6%), Actinobacteria (15.8%), Bacteroidetes (10.5%), Acidobacteria (5.3%) and Chloroflexi (5.3%). Besides, a part of unidentified bacteria (10.5%) was detected. The Proteobacteria (52.6%) were dominant species, in which the δ-Proteobacteria gained the uppermost position. In addition, the results also indicated that the values of Shannon-Wiener index for bacterial community structure ranged from 1.84 to 2.79, the values of Shannon-Wiener index in spring and summer were higher than that of in autumn and winter. [Conclusion] The bacterial diversity in the sediments of Shuang Taizi estuary was in accordance with the bacterial diversity characteristics of the typical estuarine. Low-temperature can lead to a loss of the microbial diversity. This work presented a certain reference for the preliminary understanding of the bacterial species and composition in this estuary. At the same time, it provided a scientific evidence for monitoring of the marine environment, as well as preserving of the biological resources in this area.

Abstract:[Objective] The study aims to screen microorganisms capable of degrading poly lactic acid (PLA), improve the degradation rate and identify degrading enzymes. [Methods] Gelatin was used as sole carbon source to screen PLA degrading microorganisms. To increase the PLA degradation rate, concentration of gelatin, weight of PLA added and metal ions were optimized. By investigating and comparing metabolites and enzyme involved, we determined the category of PLA degradation enzymes. [Results] A PLA degrading strain was screened and identified as Lentzea waywayandensis. After optimization, 84.8% loss of PLA was achieved (25 d). Lactic acid as metabolite was detected during cultivation. Moreover, only obvious protease activity was observed in vitro experiments. [Conclusion] Gelatin was applicable for screening PLA degrading microorganisms. Besides acting as carbon source, gelatin could also be used as an inducer to strengthen L. waywayandensis to degrade PLA. Furthermore, the results obtained herein suggested that protease played an important role on PLA degradation.

Abstract:[Objective] Using culture-independent method to analyze the diversity of actinomyces from Yellow River Delta coastal wetland. [Methods] Actinobacterium-specific 16S rRNA gene clone library was constructed. Representative clones selected based on the RFLP analysis were sequenced and placed into operational taxonomic unit (OTU) groups according to the 16S rRNA sequence similarity. Diversity statistics were analyzed using SPADE analysis software. [Results] 324 sequences were placed into 46 OTUs. 58.7% of the OTUs belonged to Actinobacteridae and 7 suborders of Actinomycetales. The dominant actinobacteria was Frankineae, which accounted for 7 OTUs. 30.4% of the OTUs belonged to subclass Aci-dimicrobidae and suborder Acidimicrobineae. 10.9% of the OTUs shared no close relationship with published sequences. Some sequences which formed a distinct clade in phylogenetic tree may represent new taxonomical groups of actinomyces. [Conclusion] Yellow River Delta coastal wetland harbors abundant and diverse actinomyces. The result also implied that there may be large numbers of unknown actinobacterial groups.

Abstract:[Objective] To gain a better understanding of the synergic interactions between chemoautotrophic bacteria Acidithiobacillus ferrooxidans and heterotrophic bacteria Acidiphilium acidophilum that usually occurred in bioleaching system and acid mine drainage (AMD). For suggestion on AMD bio-remediation and research foundation on ecology functions of acidophilic heterotrophic bacteria in bioleaching system and AMD environment. [Methods] The biomass dynamics of At. ferrooxidans and its natural co-culture with Aph. acidophilum in 9K-ferrous iron media with or without glucose were measured respectively by quantitative real-time PCR; meanwhile, the pH and Fe2+ oxidation were monitored. [Results] Whether the glucose was added in culture media or not, the Fe2+ oxidation efficiency of At. ferrooxidans is higher in co-culture than that in pure culture. When the concentration of glucose is 5 g/L, pure culture of At. ferrooxidans couldn’t oxidize Fe2+ while the co-culture could finish the Fe2+ oxidation in 100 h, and the pH is higher when more glucose was added in both cultures. Without glucose, the cell number ratio of At. ferrooxidans to Aph. acidophilum in co-culture was about 100:1, which suggested the usual cell number ratio between autotrophic bacteria and heterotrophic bacteria in AMD environment. Both in pure and co-culture, the cell number of At. ferrooxidans decreased and the lag phase prolonged with the increase of glucose concentration; while in the case of Aph. acidophilum in co-culture, the cell number and lag phase showed a reverse trend. [Conclusion] For efficient Fe2+ oxidiation, the proper cell number of At. ferrooxidans should be 100 times higher than that of Aph. acidophilum. Because Aph. acidophilum facilitated the Fe2+ oxidation by At. ferrooxidans and reduced the inhibition by glucose, the addition of organic compounds such as glucose could not be a good AMD bio-remediation strategy.

Abstract:[Objective] To obtain the best produce methane of microbial strains, twelve biogas slurry samples were collected from the biogas digesters in Zhangjiakou, Chengde and Handan. [Methods] Biogas fermentation experiment were produced with different biogas slurry samples by decreasing the operating temperature form 16 °C to 5 °C. DGGE analysis was taken on samples (HL2, ZG2, CW1, HLA, ZGB and CWB). [Results] The results showed the ZG2 with biogas yield, gas-production rate and methane content were significantly superiority at the same temperature stage among the inoculation treatments. In addition, DGGE chart were observed that there were abundant microbial diversity and it was significantly different bands among the dominant archaea. Through 16S rDNA cloning sequencing analysis, the dominant archaea were composed of Methanosarcina, Methanosaet and Methanocorpusculum in the samples. [Conclusion] Furthermore, Methanosarcina major existed only in ZG2 and the same dominant archaea as ZGB. In conclusions, Methanosarcina were closely related to effectively gas under low temperature condition.

Abstract:[Objective] In this study, we aimed to observe the detailed features of microbes within white spots adhered to the surface of ancient wall paintings in Cave 98, Mogao Grottoes; meanwhile, we explore microbial community composition and structure characteristics, analyze factors that induce the explosion of microbial diseases on murals; thereafter, our results could serve with scientific support for cave conservation and tourism management. [Methods] Samples of white spots on murals were carefully collected by sterile scalpel and sealed in eppendorf tubes. A part of samples was used for observation of microbes by the scan electronic microscope (SEM); another part was used for the total genomic DNA extraction, followed steps were the bacterial 16S rDNA amplification, clone library construction, sequencing, and phylogenetic analysis, afterwards, the microbial community composition and structure characteristics were clarified. [Results] A number of objects that similar to microbes were observed in white spots of wall paintings, which shaped short rod or ovoid, with the volume ranged among 3.0 μm?5.5 μm multiply by 1.5 μm?2.5 μm. About 111 appropriate sequences were retrieved from clone library in this study. All sequences were similar to members of Enterobacteriaceae and Pseudomonadaceae that both belong to phylum of γ-proteobacteria. Results of community analysis indicated that sequences were mainly affiliated to genus Enterobacter, Escherichia, Azotobacter, Serratia, and Klebsiella; Enterobacter (46.8%) and Escherichia (35.1%) were dominant in our study, both of them are ubiquitous microbes in nature, and human pathogenic bacteria as known. [Conclusion] The white spots on murals in Cave 98 were caused by colony formation of bacterial growth. Members of proteobacteria dominated in the clone library of our study sites. As a result, we speculated that explosion and spread of microbes in Cave 98 was related to long-term tourism in this cave previously.

Abstract:[Objective] A hydrocarbon-degrading strain HL-6 was isolated from oil contaminated soil of Xinjiang Oil Field and identified as Rhodococcus sp., which could use hydrocarbon as sole carbon source to produce biosurfactant. The bacterial cell hydrophobicity and biosurfactant production were studied. [Methods] Through cell adhesion, surface tension and emulsifying activity, the biosurfactant was studied. [Results] The strain could produce biosurfactant in both hydrophilic and hydrophobicsubstrate. The surface tension was reduced from the initial 62.487 mN/m to 30.667 mN/m when substrate was hydrophobic. Diesel could be well emulsified by strain HL-6 and the emulsion toleratedsalinity, alkaliand temperature. In addition, strain HL-6 could grow well in medium containing 30% of diesel and had 44% emulsification. [Conclusion] Strain HL-6 had a strong cell-surface hydrophobicity, which contributed to uptake hydrocarbons. This strain could use hydrocarbon to produce biosurfactant, significantly reduce the culture surface tension and had good emulsification of petroleum hydrocarbons. So strain HL-6 was able to adapt to the environment of the marine shoals oil pollution, especially serious oil pollution.

Abstract:[Objective] To investigate airborne extended-spectrum β-lactamase (ESBLs) producing Escherichia coli (E. coli) infection situation in the student dormitory in Chinese university and to analysis genetic similarities between different strains, and then provide a practical basis for preventing and controlling infectious diseases. [Methods] The air samples were collected by FA-1 six-stage microbial samplers in the different traditional student dormitory, E. coli strains were isolated and identified from the air samples, and then the enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) fingerprints of ESBLs-producing E. coli were analyzed. [Results] 194 strains of E. coli were isolated from 300 air samples, 30 strains of ESBLs-produing E. coli were founded and the resistant rate was 15.46%. The genetic similarity index of ESBLs-produing E. coli was 50%?100%. [Conclusion] The air in the student dormitory in university was contaminated by ESBLs-producing E. coli. The results stress the need for surveillance of the antibiotics resistance of E. coli in the air.

Abstract:[Objective] Function of PlnA in bacteriocin production of Lactobacillus paraplantarum L-XM1 and the influences of environmental conditions on its inducing effect were studied. [Methods] The function of PlnA was determined by adding synthesis PlnA to Bac? cultures of L. paraplantarum L-XM1. Induction activity of PlnA was detected at different temperature, pH, NaCl and ethanol concentrations for investigating the effects of environmental conditions on PlnA. [Results] Variabilities for induction activity of PlnA were detected for different temperature and pH value, higher incubation temperature or relatively higher pH is helpful for its induction activity. No significant differences in PlnA induction activity were found among different concentrations of NaCl. The presence of ethanol was found to reduce the induction activity of PlnA, and at higher concentration of ethanol, its activity is inhibited. The inhibitory of bacteriocin production by 6% ethanol could be attenuated by simultaneously addition of high concentraion of PlnA (700 μg/L), while restoring bacteriocion production in medium containing 8% ethanol need up to 1 000 μg/L PlnA. [Conclusion] The induction effects of PlnA could be affected by environmental factors, the inhibition effects of bacteriocin synthesis by ethanol could be eliminated by increasing PlnA concentrations.

Abstract:[Objective] In order to study the effect of soil bacteria against vegetable Botrytis cinerea, we isolated 9 strains from 56 samples obtained from greenhouses in Liaoning and Shandong Province in China. [Methods] The plate-confrontation method and antibacterial circle method were used for preliminary screening and antimicrobial testing in vitro, respectively. The effect of strain CNY-04 against vegetable Botrytis cinerea was measured in detached condition. The taxonomic identification of strain CNY-04 was studied by analyzing its morphology characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence. [Results] Strain CNY-04 exhibited genetic stability and strong antagonistic ability on vegetable Botrytis cinerea. From the taxonomic identification result, this bacterium was identified as Serratia grimesii. Strain CNY-04 inhibited Botrytis cinerea on tomato by 69.23%, close to that of 75.39% by carbendazim 50 WP. The incidences of Botrytis cinerea in groups inoculated with strain CNY-04 were 40.0% at 24 h and 51.1% at 48 h. [Conclusion] Strain CNY-04 is a potential biological control agent for vegetable Botrytis cinerea.

Abstract:[Objective] To isolate and identify biocontrol bacterial strains from Ginkgo biloba and evaluate their inhibition to Phytophthora capsici in vitro and control efficacy against fruit phytophthora blight of pepper. [Methods] The method of dual culture on agar plate was used for screening the antagonistic endophytic bacteria against P. capsici, and the inhibition effect of the volatile substances from a better control effect strain on P. capsici was tested by two-sealed-base-plates method. Control efficacy against pepper phytophthora blight was evaluated by spraying bacterial cultures on capsicum fruits. The identification of bacterial strains was based on morphological, physiological, biochemical characteristics and phylogenetic analysis of 16S rDNA and gyrA gene sequences. [Results] Nine endophytic bacterial strains were isolated from healthy stems and leaves of Ginkgo biloba. Among them strain W5 was selected for further study, which had antagonistic action against P. capsici, Pyricularia grisea, Rhizoctonia solani, Fusarium oxysporum, Peronophythora litchii, Geotrichum candidum on agar plates, especially to P. capsici, P. grisea and P. litchi, the inhibitory rates were prominent with 88.9%, 86.3% and 90.2%, respectively. The volatile substances produced by strain W5 could significantly inhibit the mycelia growth of P. capsici. The control results of fruit phytophthora blight of pepper by strain W5 showed that it could get better protective efficacy when the spore suspension of the pathogen was inoculated 24 h after spraying inoculation with strain W5. And spraying strain W5 could also prolong the fresh period of pepper fruits to 2?3 d than the control. The strain W5 was identified as Bacillus amyloliquefaciens. [Conclusion] An antagonistic B. amyloliquefaciens strain W5 was obtained, and it had potential for the biological control of fruit phytophthora blight of pepper and other pathogenic fungal diseases.

Abstract:[Objective] The aim of this study was to screen antimicrobial fungal strains and to investigate the main active constituent of endophytic fungi isolated from the medicinal plant Forsythia suspensa. [Methods] Fungal endophytes were isolated with routine tissue separation methods, and the antimicrobial activities of their fermentation products were detected by filter paper method against 3 bacterial strains. Metabolites of active fungal strains were analyzed by TLC, HPLC and HPLC-MS methods. The final active fungal strains were identified based on morphological characteristics and ITS sequence analysis. [Results] Among the 24 isolated strains whose inhibition zone diameters more than 10 mm, group of fermentation broth had 11 (45.8%), group of mycelia had 19 (79.2%). Active fungal strains were mainly isolated from fruit, and G5, G7, G10 showed stronger antibacterial activities. Phillyrin was detected from the intracellular products of isolate G10, which was identified as Colletotrichum gloeosporioides. [Conclusion] The endophytic fungi in F. suspensa were important source for the natural antimicrobial substances.

Abstract:[Objective] Helicobacter pylori (H. pylori) was not only the pathogen of gastritis and peptic ulcer, but a potential normal flora. We detected the effect of honokiol (HK), a kind of Chinese herbal active ingredient from Mangnolia officinalis commonly used in clinical treatment, on H. pylori growth and the expression and activity of VacA to demonstrate the potential effect of HK on down-regulating the toxicity of H. pylori. [Methods] We detected the minimum inhibitory concentration (MIC) of HK and further identified the toxic effect of VacA secreted from H. pylori by neutral red uptake test, and the mRNA and protein levels of VacA in H. pylori and its supernatants by RT-PCR and Western blot, after treated with a low concentration HK (0.09 g/L) without bacteriostatic effects. [Results] The results showed that the high concentration HK (0.75 g/L) can inhibit the growth of H. pylori, and HK could also inhibit the expression and activity of VacA effectively in the concentration far below the MIC. [Conclusion] HK could down-regulate the toxic effect of H. pylori potentially, and which provided a hopeful research prospect for intervention treatment based on transforming the pathogenic H. pylori to the non-pathogenic.

Abstract:[Objective] To provide a certain basis, for the relationship between the ecology of endophytic bacteria and plant as well as the resources of endophytic bacteria. [Methods] Use tradational cultural methods isolated 54 endophytic bacteria from the fruit of Focus hispida through the restriction enzyme analysis (ARDRA) told have 16 operational taxonomic units, 16s rDNA sequence system development analysis on 16 representative strains. [Results] The results showed that 16 strains were found the highest similarity of strains from GenBank, the identities are from 95% to 100%. six endophytic bacreia were classed to Bacillus genera, the identities are from 95% to 98%, was the dominant genus among the endophytic bacteria in Focus hispida fruit. Three were classed to Staphylococcus genera, one were classed to Pseudomonas genus, one was similar to uncultured bacterium, one were classed to Kocuria genus, one were classed to Delftia genus. [Conclusion] The phylogenic tree showed that the 16 endophytic bacteria clustered two main branches. the inhibition experiment of 14 endophytes bacteria, 13 strains have different degrees of inhibition effect. Especially the swx15 of Bacillus genus and the swx25 of Acinetobacter genus had the biggest inhibition spectrum.

Abstract:The Phaf?a rhodozyma has found commercial application as natural source of astaxanthin, a known antioxidant with large importance in the aquaculture, pharmaceutical, and food industries. To satisfy the growing demand for astaxanthin, there are strong efforts to develop economically viable bioprocesses alternative to the current chemical synthesis. However, up to now, natural astaxanthin from P. rhodozyma has not been cost competitive with chemically synthesized astaxanthin, and the knowledge about the carotenogenesis regulation is still limited in comparison to other carotenogenic fungi. The aim of this short review is to examine the published studies concerning the biosynthetic pathway of astaxanthin and the related genes in P. rhodozyma, and describe their applications to improve the bioprocess of astaxanthin production. It also mentions some gaps and opportunities in this field that should be addressed or exploited in the near future.

Abstract:Biofiltration method has proved to be a cost-effective and environment friendly alternative for the removal of pollutants from waste gases stream, and therefore has emerged as a promising technology in the area of air pollution control. This review summaries the characteristics of various bioreactors, the key parameters that should be controlled properly during the biofiltration are described in detail. At last, the hot topics in future researches and development were also presented.

Abstract:With the development of molecular biology, genetic engineering of microorganisms has been increasingly applied in microbial breeding, clinical medicine and environmental bioremediation. However delivery of DNA into the cell can be one bottleneck in efficient genetic engineering. Ultrasound-mediated DNA delivery into microbial cells, with a number of advantages that include in situ operation, spatial scalability, non-invasiveness, high-throughput and low consumable costs, has been under rapid development. Ultrasound can produce a variety of nonthermal effects via acoustic cavitation, and the cavitation bubbles generated during this process can cause transient cell membrane permeability. In this review the basic principles of the ultrasound-based DNA delivery were discussed and its current applications in prokaryotes were summarized. Furthermore, we discussed the advantages and challenges of the technique, using the transformation of Gram-positive bacteria in our laboratory as an example.

Abstract:Aiming at the target to train the practical talents and the present status that general experimental accomplishment is rather low in local college, microbiology experiment teaching system was divided into three types: basis, synthetical advancing and research innovation. Gradual research-oriented teaching pattern was conducted by transition from students’ exploring guided by teacher to students’ exploring independently. The pattern prompted students to master the basic experimental operating technology and cultivated students’ ability of practice and comprehensive quality; synthetic and competency-based evaluation system was established to give an overall investigation on comprehensive ability of students and promote the teaching. In the system, both process and effect were taken into account, various evaluation subjects were involve.

Abstract:The cultivation aim of bio-engineering major is compound talent training of the innovation and engineering type. This paper discusses some approaches in reforming experimental teaching. By optimization of experiment teaching system, reformation of production practice and graduation thesis, construction of extracurricular experiment and so on, an innovative experimental teaching system whose aim is to promote the student’s innovation ability, engineering practical ability and solving question ability was constructed. The novel experimental teaching system makes undergraduates have a good knowledge of the theory and current focuses of bioengineering. The quality of the experimental teaching was significantly improved.

Abstract:[Objective] Rice false smut caused by Villosiclava virens (Cooke) Tak. is a serious fungal disease of rice. This study aimed to construct a high molecular weight bacterial artificial chromosome (BAC) library of UV-2 genome. The BAC library will help identify patho-genicity-associated genes and provide genome resource for map-based cloning and compara-tive genomic analysis. [Methods] High molecular weight genomic DNA was isolated from young mycelia of UV-2 strain, digested with restriction enzyme Hind III and size selected by PFGE. Large genomic DNA fragments were recovered and ligated to the pIndigoBAC536-S vector. The ligation product was used to transform the Escherichia coli strain DH10B T1 Phage-Resistant cells and the transformants were selected on a blue-white selection medium. White colonies were individually picked into 384-well microtiter plates and stored at ?80 °C. [Results] We constructed a high quality deep coverage BAC library of UV-2 strain. The BAC library consisted of 10 368 clones with an average insert size of 124.4 kb and an empty clone rate of lower than 1%, covering 36.8-fold of the UV-2 genome. [Conclusion] We established a method for preparation of fungal high molecular weight genomic DNA, and constructed the first BAC library of V. virens genome successfully. The BAC library has been opened to re-searchers as a public genomic resource (http://GResource.hzau.edu.cn).

Abstract:[Objective] A novel method to differentiate viable/dead cells of Ralstonia solanacearum was established by using a DNA dye of ethidium monoazide bromide (EMA) in combination with the real-time polymerase chain reaction (EMA-qPCR). [Methods] Samples were pre-treated with EMA prior to DNA extraction. DNA from dead cells was bound by EMA, so that only DNA from viable R. solanacearum cells can be amplified by real-time PCR. [Results] A final concentration of 2.0 mg/L EMA was demonstrated to completely inhibit the PCR amplification from DNA derived from 1.0×107 CFU/mL dead cells, but no inhibition to viable and viable but non-culturable (VBNC) cells. A standard curve was generated relating the Ct values of the EMA-qPCR to the log number of genomic targets per PCR. A linear range of DNA amplification was observed from 5.0×100 to 5.0×104 genomic targets per PCR. EMA-qPCR method was used to evaluate the survival rate of R. solanacearum treated with different temperatures for a short time, compared with the method of plate count. The results indicate that samples can be stored for a short time under room temperature and 4 °C. [Conclusion] The EMA-qPCR method established in this work can effectively avoid false positive and false negative results of the R. solanacearum detection.

Abstract:

Abstract:[Objective] Reductive biotransformation of 2,4-dinitrotoluene (2,4-DNT) by Shewanella oneidensis MR-1 was evaluated under anaerobic conditions. [Methods] Four systems were set up where 2,4-dinitrotoluene (2,4-DNT), S. oneidensis MR-1 and flavins were used as electron acceptor, degrading bacteria and extracellular mediator, respectively. Concentrations of 2,4-DNT and its metabolites were monitored by time temporal sampling. Growth of S. oneidensis MR-1 under different concentrations of 2,4-DNT and biotransformation of 2,4-DNT under different concentrations of flavins were also studied. [Results] S. oneidensis MR-1 could efficiently biotransform 2,4-DNT to 4-amino-2-nitrotoluene (4A2NT) or 2-amino-4-nitrotoluene (2A4NT), both of them were further reduced to 2,4-diaminotoluene (2,4-DAT). The presence of flavins could accelerate the biotransformation. [Conclusion] S. oneidensis MR-1 showed a high capacity for biotransforming 2,4-DNT and could be further applied in the in situ bioremediation of nitrobenzene contaminations in the environment.

Abstract: