Abstract:

Abstract:[Objective] A molecular-based approach, terminal restriction fragment length polymorphism (T-RFLP), for community analysis of ruminal methanogens was developed and used to assess the effects of transfer frequency on the community of methanogens co-cultured with anaerobic fungi. [Methods] The specific primers for mcrA genes were used to amplify the mcrA genes of methanogens and the amplicons were then digested with restriction enzymes. The size of each of the individual resulting terminal fragments were detected using DNA sequencer. [Results] With the analysis by Msp I, the dominant methanogens co-cultured with anaerobic fungi were those with terminal fragment of 470 bp and those of 130 bp and 200 bp were also dominant in the 15th transfers of 3-day co-cultures. Comparative study with Taq I showed that the dominant methanogens both in rumen digesta and 3-day co-cultures were those with terminal fragment of 70, 100, 200, 270, 300, 330 and 470 bp. The methanogens of 70, 100, 270 and 470 bp changed dramatically during in vitro transfers. Subsequently, the effects of transfer frequency on community of methanogens co-cultured with anaerobic fungi were assessed by Taq I and results showed that the community of methanogens in 3-day co-cultures was similar with rumen digesta, while they are significantly different from those in 5- and 7-day co-cultures, which was resulted from the shift of methanogens with terminal length of 100, 70 and 270 bp. [Conclusion] The molecular-based approach, T-RFLP, was suitable for analysis of ruminal methanogens. The community of methanogens co-cultured with anaerobic fungi was significantly affected by transfer frequency of the co-cultures, and that in 3-day co-cultures was similar to that in the rumen digesta.

Abstract:

Abstract:[Objective] The metabolism of 1,3-propanediol in Citrobacter freundii was studied. [Methods] Transcriptional fusion reporter genes of GSR-lacZ, PDO-lacZ, and GL-lacZ were constructed. Based on this, the mariner transposon libraries were constructed. [Results] Six mutants were isolated. The expression level of the corresponding key enzymes was increased from 1 to 11 folds in the mutants, and the corresponding 1,3-propanediol production was increased from 3% to 50%. Transposon insertion sites were analyzed and the results show that the β-lactamase gene was inactivated in 5 mutants and the dihydrolipoamide acyltransferase gene was inactivated in 1 mutant. Furthermore, the expression levels of glycerol dehydrogenase and glycerol dehydratase were increased significantly in the β-lactamase mutant, whereas no increase of the 1,3-propanediol oxidoreductase expression was observed. In the diydrolipoamide acyltransferase mutant, 1,3-propanediol oxidoreductase expression level was significantly improved, whereas the other two enzymes expression level remained unchanged. [Conclusion] The results provide insight into constructing engineering strains producing a high level of 1,3-propanediol.

Abstract:[Objective] NAD-dependent D-lactate dehydrogenase (D-LDH) catalyzed the conversion of pyruvate to D-lactic acid. However, the weakness of thermostability of D-LDHs reported to date hindered the recombinant strain construction for high-temperature fermentation process. Finding a novel thermostable D-LDH would lay the foundation for constructing the efficient D-lactate producer under high-temperature fermentation conditions and thus will reduce the operation cost of producing D-lactic acid. [Methods] D-LDH was cloned from Lactobacillus jensenii strain and then expressed in Escherichia coli to determine its optimal reaction temperature, optimal reaction pH, kinetic parameter, thermostability and thermal inactivation, which is compared with the mesophilic D-LDH from Lactobacillus plantarum ssp. plantarum. [Results] D-LDH from Lactobacillus jensenii strain had higher optimal reaction temperature (45 °C), better thermostability and 3 times higher catalytic activity (kcat/Km) than those of D-LDH from Lactobacillus plantarum ssp. plantarum (optimal reaction temperature was just 30 °C). [Conclusion] D-LDH from Lactobacillus jensenii strain had better thermostability and higher catalytic activity, which is useful component for industrial applications.

Abstract:[Objective] The aim of the present study was to isolate and identify hyperthermophilic archaea around the hydrothermal vent on the Mid-Atlantic Ridge, and to establish a foundation for further understanding and characterization of the microbial species in this ecosystem. [Methods] The hydrothermal vent seawater samples were enriched with YTSV media and strain TVG2 was purified from the enrichment cultures by the means of dilution-to-extinction. Strain TVG2 was characterized using morphologic, physiologic and biochemical analysis and preliminarily identified through the molecular biological methods. [Results] Strain TVG2 was an obligate anaerobic hyperthermophilic archaeon. Cells were regular cocci with 1.0 μm in diameter. It grew optimally at 82 °C (range 50?88 °C), at pH 6.5 (range 5.0?9.0) and with NaCl of 2.5% (range 1.0%?4.0%, w/v). Element sulfur was not indispensable for strain TVG2, but notably promoted its growth. Sodium pyruvate could significantly facilitate the growth of strain TVG2, whereas glucose had the opposite effect. Based on the morphological, physiological and biochemical characteritics and the 16S rRNA gene sequence analysis, strain TVG2 was regarded as belonging to Thermococcus. [Conclusion] Hyperthermophilic archaeon strain TVG2 was isolated from Atlantic Ridge hydrothermal vent samples with the YTSV medium and it was a member of the genus Thermococcus.

Abstract:[Objective] In order to evaluate the application potential of white-rot fungi Irpex lacteus XX-5, Trichaptum abietinum 1302BG, Ceriporia lacerata P2 and Bjerkandera adusta XX-2 in treating Microcystis aeruginosa. [Methods] Batch experiments were carried out to study the effect of pH, temperature, concentration of M. aeruginosa, metal ions, nitrogen source and phosphorus source on inhibition of M. aeruginosa by whiter-rot fungi. [Results] The results showed that under different exogenous conditions, four white fungi all showed good inhibition effect, achieving 60% of inhibiting rate. C. lacerata P2 and B. adusta XX-2 strains were less effected by environmental factors, and the inhibiting rates of both strains were over 70% and 60% respectively. The algal inhibiting rate of T. abietinum 1302BG and I. lacteus XX-5 could be slightly affected by experimental factors, however the inhibiting rates were still over 60%. [Conclusion] Therefore, the four white-rot fungi, especially for C. lacerata P2 and B. adusta XX-2, had a good potential for inhibiting M. aeruginosa.

Abstract:[Objective] The aim was to isolate new hydrolytic enzymes from themetagenomic library from mangrove soil. [Methods] To isolate new hydrolytic enzymes, we constructed a metagenomic library from mangrove soil and screened clones with lipolytic activities by a function-driven approach based on a tributyrin hydrolysis. The identification of Phop1413 was based on deduced amino acid sequence comparison and phylogenetic analysis. [Results] One new phospholipase A1 gene phop1413 (GenBank KF767097) was finally identified from the library by functional screening. The result of BLASTp analysis revealed that phop1413 consisted of an open reading frame of 1 413 bp and encoded a protein of 470 amino acids. The protein showed 42% amino acid identity to phospholipase from Pseudomonas (WP 018928790.1). According to the phylogenetic analysis of Phop1413, Phop1413 belonged to FAMILY VI of lipase. Phop1413 was then subcloned to the express vector pET-32a(+), and overexpressed in E. coli BL21 with 1 mmol/L IPTG induction at 30 °C. The overexpressed protein revealed a molecular weight of 51.7 kD. A detailed analysis of the enzyme’s substrate spectrum with eight different substrates revealed that Phop1413 could hydrolyze a wide variety of substrates. Phop1413 showed the highest activity with p-Nitrophenyl caproate (C6). The optimum temperature of Phop1413 was detemined to be 54 °C, and the recombinant enzyme showed an optimum pH of 7.8. The enzyme retained 44% initial activity after incubating at 50 °C for 1.5 h. It indicated that Phop1413 had a good thermostability. [Conclusion] A new phospholipase A1 gene phop1413 was screened by function-driven approach from metagenomic library. The characterization of Phop1413 is good enough to apply to industry.

Abstract:[Objective] The degradation characteristics of phenol by a 3-phenoxybenzoic acid degrading strain-Sphingomonas sp. SC-1 were studied. [Methods] The influence factors of phenol degradation were investigated by determining the concentrations of phenol in microbial degradation systems by HPLC. We analyzed the intermediate products of phenol by analyzing the HPLC chromatograms of the mixed samples taken from phenol degradation systems at different time. [Results] The results showed that SC-1 could grow well by using phenol as sole carbon source in mineral basal medium. Strain SC-1 could degrade phenol entirely within 24 hours while cultured in the medium (pH 7.0) containing 100 mg/L phenol at 30 °C. The strain’s growth and phenol degradation were inhibited when the training systems were treated with some heavy metal ions such as Cu2+, Ba2+, Mn2+, etc. Catechol was an intermediate product of phenol and strain SC-1 could degrade catechol (100 mg/L) within 48 hours. [Conclusion] This research indicated that strain SC-1 could degrade phenol and its intermediate product catechol effectively, and this work could provide data reference for the thorough degradation of 3-phenoxybenzoic acid and the degradation of phenol containing wastewater or phenol containing pesticide residues.

Abstract:[Objective] This study chose healthy adult volunteers of Bai ethnic group at Yunnan Province as the research object, investigating the relationship between the structural intestinal flora and human health. [Methods] This study recruits 43 healthy adult volunteers of Bai ethnic group living in Kunming urban and Er Yuan rural county Dali Bai autonomous prefecture at Yunnan Province as the research object, analyzed the dominant bacterium of intestinal flora by specificity primer qualitative PCR and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), and measured the content of short chain fatty acids (SCFAs) in the feces. [Results] Results in present study showed that although the average of SCFAs content in Rural is higher than that of Urban, the difference was not significant. The qualitative PCR results showed that, for the diversity of Lactobacillus and Bifidobacterium, these two groups have obviously differences, Unweighted pair-group method with arithmetic means (UPGMA) based on optical density was performed on the base of 16S rRNA V3 region PCR-DGGE profile, showing samples were divided into two groups as urban and rural groups. We extracted the typical bands for further cloning and sequencing, and constructing phylogenetic tree diagram the results showed that the Bifidobacterium, Enterubacterium and Enterococcus have a high diversity, suggesting that it was predominant in the gut of healthy volunteers living in county of Dali bai autonomous prefecture Er Yuan Xian. [Conclusion] To sum up, the structural intestinal flora of Bai ethnic group living in urban of Kunming and Er Yuan rural county Dali Bai autonomous prefecture at Yunnan Province has the distinguishing trend, but the difference of diversity was not significant. The current study aimed to provide certain reference for exploring the relationship between healthy host and intestinal flora.

Abstract:[Objective] This paper aims to clarify the bacteria associated with the salivary glands of cicada Hyalessa maculaticollis (Motschulsky), and to address if the endosymbiont Candidatus Sulcia muelleri occurrs in the salivary glands. [Methods] We investigated the bacterial community in the salivary glands of cicada H. maculaticollis using 16S rRNA restriction fragment-length polymorphism (RFLP). [Results] We identified seven bacteria in the salivary glands, which belong to the Phyla Proteobacteria and Firmicutes. The bacterial community is dominated by Pseudomonas aeruginosa and Enterobacter sp., each of them accounted for 48.7% in the clone library. In total, the other five bacterial species (Thermomonas brevis, Sphingomonas sp., Methyloversatilis sp., Anaerococcus sp. and Bacillus sp.) together accounted for 2.05% in the clone library. [Conclusion] The results show that the bacterial species composition in the cicada salivary glands of H. maculaticollis was relatively simple, which were dominated by two bacterial species. The endosymbiont Candidatus Sulcia muelleri of Auchenorrhyncha, formerly detected from bacteriomes of related auchenorrhynchan hosts, were not detected in the salivary glands of H. maculaticollis. The bacteria harbored in salivary glands of cicadas probably play an important role in both food ingestion and interactions between the cicadas and their host plants. However, whether the related bacteria are commonly harbored in other cicadas and their certain function need to be further investigated.

Abstract:[Objective] We used roots and rhizosphere soils of ramie (Boehmeria nivea L. Gaud) to isolate plant growth promoting bacteria (PGPB), and then studied the mechanism why they promote growth of ramie in preliminarily. [Methods] Preliminary screening of PGPBs under the premises of bacteria have the abilities of phosphate-and potassium-solubilizing, moreover, the bacteria strains were screened by testing their abilities of siderophore, indole acetic acid (IAA) and ammonia production in vitro. Then, seeds germination and pot experiments were conducted to measure the promoting-growth effects on ramie of the isolates. Finally, PGPBs were classified and identified by combining physiological and biochemical tests and 16S rRNA gene sequences analysis. [Results] Thirteen strains were isolated based on the abilities of phosphate- and potassium-solubilizing, of which four strains (RA-2, RAM-2, RAM-5, and RAM-6) also exhibited plant growth promoting properties like siderophores, IAA and ammonia production. Seeds germination and pot experiments showed that four selected strains could promote growth of ramie, moreover, strain RA-2 and RAM-5 could significantly improve the germination rates, root lengths, plant heights, and root dry weights, both of isolates RA-2 and RAM-5 were identified as Burkholderia sp.. [Conclusion] PGPBs which were isolated from ramie rhizosphere will helpful for developing the specially PGPB microbial inoculants or bio-organic fertilizers of ramie.

Abstract:[Objective] A lytic phage SLMP1 against Salmonella was isolated from the shellfish samples and identified, and its biological properties were assayed as well. [Methods] The lytic phage SLMP1 was isolated by the double-layer agar culture method. The phage plaque was observed and its host range was analyzed. Phage particles of SLMP1 were concentrated by precipitation with PEG8000 and purified by CsCl gradient centrifugation. The morphology of SLMP1 was observed by transmission electron microscopy. The genome of SLMP1 was extracted by phenol-chloroform method and the type of nucleic acid was identified with enzyme digestion analysis. The biological parameters including the thermal stability, pH stability, optimal multiplicity of infection (MOI) and the inactivation effects on the growth of Salmonella were assayed. [Results] The plaques of SLMP1 were round and transparent and their sizes were about 2–3 mm in diameter. SLMP1 can lyse Salmonella enterica subsp. Enteric and Salmonella typhimurium. SLMP1 had an icosahedral head approximately 62 nm in diameter and a long noncontractile tail with 110 nm in length. SLMP1 belongs to family Siphoviridae and is a dsDNA virus. SLMP1 was stable over a wide range of temperature (30–60 °C) and pH (4.0–11.0). The optimal MOI of SLMP1 was 0.001. One-step growth experiment showed that the latent time and burst time were 10 min and 120 min, respectively, and the burst size was 51. SLMP1 showed a good inactivation effects on the growth of Salmonella in broth. [Conclusion] SLMP1 is a dsDNA virulent phage and belongs to family Siphoviridae. The results indicated SLMP1 can serve as a promising biocontrol agent against Salmonella.

Abstract:[Objective] To detect the distribution of CRISPR/Cas system in Shigella clinical strains and to analyze the relationship between CRISPR/Cas system and virulence genes. [Methods] We used 10 pairs of primers of PCR assay for detection of the CRISPR/Cas system including CRISPR1, cas2-cas1 genes, cas6e-cas5 genes, cas7 gene, cse2 gene, cse1-cas3 genes and virulence genes including ipaH, ial, ipaBCD, virA in 57 Shigella strains. The CRISPR sequences were analyzed using CRISPR finder. The relativity between the presence of CRISPR/Cas system and virulence gene was analyzed by χ2 -test. [Results] Among the 57 strains of Shigella, the number of spacers is little and its sequences have similarity among different strains. The positive rate of CRISPR/Cas system is 84.2%. However, 68.8% of Shigella strains analyzed contained insertion sequencese in cse2 gene or cas6e-cas5 gene. The positive rates of ipaH, ial, virA and ipaBCD is 100%, 100%, 98.2% and 87.7%, respectively. No statistical difference was found between the distribution of active-CRISPR/Cas system and the ipaBCD gene. [Conclusion] CRISPR/Cas system was widely distributed in Shigella clinical strains. Insertion sequences were presented in some strains. Our analysis of CRISPR/Cas system did not reveal a potential link between its presence and the virulence genes in Shigella clinical isolates.

Abstract:Halophilic microorganisms can thrive at hypersaline environments, and the mechanisms of salt-tolerance in halophilic microorganisms focused on the following three points: mechanisms of potassium-absorbing and sodium exclusion; accumulation of intracellular compatible solutes; the amino acid composition of halophilic enzyme. This paper reviewed the molecular mechanisms of salt-tolerance in halophilic bacteria and archaea, and discussed the prospects of the application in hypersaline wastewater treatment.

Abstract:Pseudomonas species can synthesize a variety of metabolic products, such as alginate, vitamin B12, cyclic lipopeptide, phenazine, 2,4-Diacetylphloroglucinol, rhamnolipid and polyhydroxyalkanoates. In this review, we summarized recent progress in biosynthesis of these polymers and complex compounds in Pseudomonas species, and discussed further their synthetic mechanism. We also addressed future research direction of these products.

Abstract:The biofilms formed by bacteria can protect them from antibiotics, it also can bring difficulties for the treatment of infectious diseases and the sterilization of clinical machines. Recent research progress showed that bacteriophages and their lysins are potential use for eradication of biofilm, which is of great significance in control of various infectious diseases clinically. Bacteriophages can eradicate abiotic biofilms as well as zoetic ones. The degradation of biofilms caused by lysins such as LySMP, CHAPk and CWHs is probably associated with their activity of direct bacteriolysis and extracellular matrix degradation. In addition, the combined use of antibiotics, cobalt ion or chlorine with bacteriophage may have a better effect to biofilms. The review discussed the action of bacteriophages, their lysins and combination with other materials to eliminate biofilms and we hope they can be used clinically in the future.

Abstract:The mucosal surfaces of the gastrointestinal, respiratory, and urinary tracts as well as the inner ear and the ocular conjunctiva are contiunously exposed to potentially pathogenic microoganisms and represent major dosease sites and entry points for harmful substances. Research has indicated that mucosal vaccination is capable of inducing protective immune responses both in the mucosal and systemic immune compartments, thereby preventing adhesion, invasion and colonization of disease pathogens.Many mucosal vaccines have been developed and evaluated, only a few of which were approved for human or animal use based on the safety, stability and efficacy of these vaccines. Application of adjuvants or vaccine delivery systems is able to efficiently improve these deficiencies. Here we will review current strategies for improvement mucosal immunization.

Abstract:Microbially enhanced coalbed methane (MECoM), which can produce new coalbed methane (CBM) and efficiently release the stress of energy needs, is the hot point in the study on the exploitation and enhancement of CBM. The mechanism of MECoM is the production of methane via anaerobic biodegradation of coal. Stimulation experiments have been performed to investigate the ability of indigenous and exogenous microorganisms to produce methane by degrading coal. And the influential factors have been analyzed in laboratory as well. Diverse methanogenic flora have been obtained through enrichment or isolation methods by some CBM companies. The methods of field implementation have also been investigated. The abilities of microorganisms to produce methane by coal biodegradation could be improved by developing new methods of coal pretreatment, and structuring new microbial flora with high potential to degrade coal, which would promote the application of MECoM.

Abstract:Talent cultivation mode in universities must fit with the cooperation of production, teaching and research. To cultivate the ability of actual application and professional analysis of students in application-oriented universities, the reform and innovation should be kept during the setting process of talent cultivation mode and construction of base for production-teaching-research for Biological Engineering specialty. Taking Xuzhou Insititute of Technology for example, this paper analyzed past education expeience in production-teaching-research and problems in talent cultivation mode, indicated that production-teaching-research for biological engineering specialty in application-oriented universities should be taken into practice.

Abstract:[Objective] To establish a fast and accurate quantification method for Cylindrocarpon destructans causing root rot disease in the rhizosphere of Panax notoginseng, and to reveal the correlation of C. destructans abundance with plant growth and AM fungal colonization. [Methods] Based on the rDNA IGS sequence of C. destructans obtained from GenBank, specific primer pair CDU2 and CDL2b was designed. Molecular quantification of C. destructans by real-time PCR with SYBR Green I was established. IGS fragment copies of C. destructans in the rhizosphere of Panax notoginseng in field was detected with the newly established method and its relationships with plant biomass and AM fungal colonization were statistically analyzed. [Results] C. destructans was more abundant in rhizosphere of infected P. notoginseng than that of healthy plants (P<0.05). IGS fragment copies of C. destructans was negatively correlated with shoot biomass and mycorrhizal colonization rate, but the correlation between IGS fragment copies of C. destructans and root biomass and arbuscule abundance were insignificant. [Conclusion] The quantification method for C. destructans based on real-time PCR could well reflect the population dynamics of C. destructans in the rhizosphere of P. notoginseng in relation to plant growth and AM fungal colonization.