Abstract:[Objective] This study aimed to get more information about sequence and protein structure of type IV lanthipeptide synthetases. [Methods] Eight potential type IV lanthipeptide synthetases were chosen from Bacillus subtilis, Streptomyces collinus Tu 365, Lactobacillus iners LactinV, Streptococcus pneumoniae, Lactobacillus delbruecki, Bifidobacterium longum, Amycolatopsis azurea and Finegoldia magna, and their physicochemical properties, domains, secondary structures were analyzed and predicted using different softwares. By using the Neighbor-joining method of Molecular Evolutionary Genetics Analysis software, a dendrogram was obtained based on genetic distances. [Results] All eight proteins were hydrophilicity and there was no signal peptide in them. The proteins in S. collinus Tu 365, B. subtilis, S. pneumoniae, Lactobacillus delbruecki, Bifidobacterium longum, Amycolatopsis azurea and Finegoldia magna were acidic whereas the others were alkaline. The proteins in S. collinus Tu 365, B. subtilis and S. pneumoniae were unstable whereas the others were stable. The evolutionary analysis showed that B. subtilis had the closest genetic relationship with S. pneumonia. The evolutionary relationships of LANC-like domains were similar to total synthase, which was different from STYKc/S_TKc domains. Alpha helix and coil were the basic secondary structure. All those proteins contained LANC-like domain. [Conclusion] All that eight synthetases have their conservative structure in different bacteria, therefore, they can play the similar biological functions. All these results will provide information of the type IV lanthipeptide synthetase for further study, especially for improving the bio-control value of B. subtilis.

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Abstract:[Objective] We cultivated Corynebacterium glutamicum under 0, 30 and 50% dissolved oxygen (DO) to study the change of metabolism. [Methods] The change of substance metabolism was studied by detecting the content of organic acids and amino acids, the activity of key enzymes and expression of their coding genes under different DO level. Meanwhile, the effect of different DO level on energy metabolism was analyzed by detecting the content of cellular reducing power and ATP. [Results] DO level affected metabolism in C. glutamicum and the content of organic acids and amino acids was also changed. Especially under low DO level, cellular oxidative phosphorylation was weakened, resulting in the decrease of ATP provided for life activity. Therefore, the excessive substrate-level phosphorylation was needed to generate ATP for meeting the demand of life activity. In this case, the accumulation of cellular NADH increased; the flow of TCA cycle decreased and switched to the glycolysis pathway and glyoxylate cycle. As a result, a variety of organic acids and amino acids was generated, including lactate, valine and leucine and so on, which can further influence the production of target product under zero dissolved oxygen concentration. [Conclusion] The results are of certain guiding significance for taking further measures to optimize the control strategy of dissolved oxygen and improve product yield in fermentation of Corynebacterium glutamicum.

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Abstract:[Objective] We constructed a recombinant Kluyveromyces lactis GG799 strain to constitutively produce adenosine monophosphate (AMP) deaminase. [Methods] The codons of AMP deaminase gene derived from Streptomyces murinus were optimized and used as template. We designed primers and amplified the opt-AMPD gene. The opt-AMPD gene was cloned into the expression plasmid pKLAC1. The recombinant expression plasmid was linearized by Sac II and transformed into K. lactis GG799 by electrotransformation. We determined the AMP deaminase activity of positive transformants. The AMP deaminase was purified by His TrapTM HP and we preliminary optimized the fermentation medium of K. lactis GG799. [Results] The codons of AMPD gene were optimized and a recombinant K. lactis GG799/pKLAC1-opt-AMPD was constructed to constitutively produce AMP deaminase. The AMP deaminase activity reached 586±50 U/mL after optimizing codon. The purified AMP deaminase showed asingle band on SDS-PAGE and the molecular weight by SDS-PAGE was about 60 kD. The preliminary optimized medium contained 40 g/L glucose, 20 g/L peptone, 15 g/L yeast extract, 8 g/L NaCl, 10 g/L KCl, 2 g/L MgSO4. The activity of AMP deaminase reached 2 100±60 U/mL cultured in flask after 120 h at 30 °C with agitation 200 r/min. [Conclusion] These codons of AMPD gene were optimized and AMP deaminase was constitutively produced in the K. lactis GG799. It is a valuable exploration about high efficiency recombinant expression and production of AMP deaminase.

Abstract:[Objective] By studying the distribution and origin of clustered regularly interspaced short palindromic repeats (CRISPR) in 51 methanogenic archaea genomes, material exchanges and interaction within methanogenic archaea and other microorganism were inferred, and the genomic divergence among the genomes of methanogenic archaea was characterized. [Methods] we identified all potential CRISPR arrays in the methanogenic archaea by applying the CRISPRdb and CRISPRFinder, and then the components of each CRISPR array were analyzed, where repeats were classified by BLASTClust and spacers were aligned to Refseq viral genome, Refseq plasmid genome and Refseq methanogenic genome respectively to retrieve both taxonomic and functional annotation. [Results] Among the 51 methanogenic archaea, in total 196 CRISPR arrays with 4 355 spacers were identified. The distribution of CRISPR arrays in methanogenic archaea was not even, and the number of spacers in one strain was not proportional to the number of CRISPR arrays in the strain. After clustering on repeat sequences of CRISPR arrays, we found that Mclu1 was the most diverse and representative repeat sequence in methanogenic archaea. Among 4 355 spacers, 388 spacers were assigned with taxonomic information and 266 spacers were assigned with functional annotation. By inferring the origin of each spacer sequence, we found that methanogenic archaea might be attacked by viruses belonging to Poxviridae, Siphoviridae or Myoviridae family. Moreover, and t exchanges of genetic materials among these archaea were observed. [Conclusion] Differences in the distribution and origin of CRISPR arrays in methanogenic archaea genomes were observed and characterized, and these differences might result from the interactions and conditions of their living environment. In this study, we could also infer from CRISPR arrays characterize the genomic divergence among methanogenic archaea.

Abstract:[Objective] To reveal the cultivable diazotrophic communities in the rhizosphere of three relict shrubs of Tetraena mongolica, Ammopiptanthus mongolicus and Nitraria tangutorum in Western Ordos and the bacterial capabilities for nitrogen-fixation and siderophore-producing, and finally to provide basis for understanding and utilizing the desert plant growth-promoting rhizobacteria. [Methods] Using Ashby medium nitrogen-free, the nitrogen-fixation bacteria strains were isolated and purified by the plate spread and streak technique; 16S rRNA gene was applied to classify the isolates and reveal the community composition. The nitrogenase activities of rapid-growing strains were analyzed through acetylene reduction assay. To screen siderophore-producing strains and their capability of siderophore production, chrome azurol S (CAS) blue plate and spectrophotometer were used for qualitative screening the strains and quantitative determination of their products, respectively. [Results] A total of 22 rhizospheric diazotrophs were isolated and classified into 3 phyla and 9 genera and they were predominated by the phylum Proteobacteria (82%) and the genus Pseudomonas (27.27%). The genus Rhizobium and Bacillus was solely found associated with Ammopiptanths mongolicus and Teraena mongolica respectively, whereas Enterobacter, Stenotrophomonas and Paenibacillus only with Nitraria tangutorum. Ten strains could rapidly grow on the nitrogen-free medium and they possessed nitrogenase activity in the range of 871.71 to 3 383.09 nmol C2H4/(H?culture). Seven of the 10 strains could produce siderophore and the producing capability (As/Ar value) was ranged from 0.35 to 0.79. [Conclusion] the cultivable rhizospheric diazotroph in the rhizosphere soil of the relict shrubs in Ordos desert was highly diverse and markedly different between the shrubs. Most of these diazotrophs have both high nitrogen-fixation and siderophore-producing capability. Thus, they could be useful as plant growth-promoting rhizobacteria.

Abstract:[Objective] In order to explore new potential agricultural biocontrol actinobacteria strains and discover drug lead compounds, the diversity of endophytic actinobacteria isolated from medicinal Melia toosendan Sieb. et Zucc. were researched. [Methods] 148 endophytic actinobacteria were isolated from Melia toosendan Sieb. et Zucc. collected in Ziyang, Suining of Sichuan Province and Chongqing by four isolation medium. Based on morphological characteristic, 60 isolates were selected and analyzed by 16S rRNA-RFLP, the representative strains were further sequenced. Antibacterial activity of 60 strains were evaluated by three bacteria and six pathogenic fungi strains, respectively, and the genes coding for polyketide synthases (PKSI, PKSII), nonribosomal peptide synthetase (NRPS) and halogenated enzyme gene (Halo) were amplified. [Results] 60 strains of actinobacteria were divided into 10 clusters based on 16S rRNA-RFLP analysis, 25 strains of representative strains belonging to 7 genera: Streptomyces, Micromonospora, Planotetraspora, Streptosporangium, Nocardiopsis, Prauseria, Microbispora, the genus Streptomyces accounted for 73.3%. An evaluation of antimicrobial activity showed most isolates possessed activity towards pathogenic bacteria and fungi. The frequency of PCR amplification of 4 synthesis genes was 10%?55%. [Conclusion] This study indicated that endophytic actinobacteria associated Melia toosendan Sieb. et Zucc. are abundant and the distribution is influenced by sampling sites and plant organs, which possessed potential diverse bioactivities and can be further explored.

Abstract:[Objective] Microbial prospecting for hydrocarbons is based on the hydrocarbon microseepage, the natural phenomenon that hydrocarbon gases of subsurface petroleum accumulations migrate upward by reservoir pressure. The detection of the activity and distribution of these highly specialized populations can be used to forecast the existence of oil and gas deposits. However, the hydrocarbon-oxidizing bacterial population is usually not predominant in soil samples above the typical onshore oil and gas reservoirs. It is hard to assess the abundance, distribution, and community composition of the hydrocarbon-oxidizing bacteria, neither to assess the functional activity and distribution of different groups. [Methods] In this study, the hydrocarbon-oxidizing bacteria will be studied under simulated environment. Furthermore, hydrocarbon-oxidizing bacteria above the typical onshore oil and gas reservoirs in Puguang gas field, were studied. [Results] A coincidence existed between elevated CH4 oxidation activity and the methanotrophic community structure with Lacibacter cauensis, Methylococcaceae, and Methylophilaceae, whereas unculture sulfur oxidizing bacteria had a coincidence with butane oxidation activity. Analysis of the biotechnology profile data for hydrocarbon-oxidizing bacteria was used to extend genetic information for the bacteria and to contribute to a comprehensive understanding of the molecular mechanisms involved in specific biological processes above the typical onshore oil and gas reservoirs. [Conclusion] The profiling data of hydrocarbon-oxidizing bacteria will provide a comprehensive insight at different areas of onshore oil and gas reservoirs and lay the foundation for the study of optimizing the technology of microbial prospecting at the biotechnology level.

Abstract:[Objective] In order to identify peat bacterial diversity and community structure in Gahai Lake wetland in Gan’nan. [Methods] By Illumina miseq high-throughput DNA sequencing platforms, we studied the bacterial diversity from three samples of peat colonies. [Results] We obtained 108 096 non-redundant sequences by comparing the silva databases. The preponderant bacterium from three Gahai Lake wetland peat samples were Anawerodineaceae-uncultured, Micromonospora, Brevundimong and Nocardioides. [Conclusion] Peat bacterial diversity and community structure in Gahai Lake wetland can contribute to the development and utilization of functional bacteria.

Abstract:[Objective] With the defection in starch synthesis in mutated Chlamydomonas reinhardtii CC-4326, the effects on the acyl profiling variation of glycerides were inspected with or without nitrogen deficiency stress. It provided basic information to understand glyceride metabolism. Wild type strain CC-137 was chosen as the control. [Methods] Cultivations were carried out in both shake flasks and 500 mL cylindrical bubbling photobioreactors under nitrogen rich or deficient conditions. Triacylglycerols were purified by thin-layer chromatography. After transesterified with methanol, acyls were quantified by gas chromatography with an internal standard and the abundance of acyls in glycerides and triacylglycerols were compared. [Results] The amount of C16:4 and C18:3 in each strain accounted for about 45% of total acyls under nitrogen rich condition in both shake flask and photobioreactor cultivation. With nitrogen supply, CC-4326 showed no difference in the abundance and changing pattern of the above two major acyls in glycerides, whereas the wild type showed higher increase and higher abundance of these two acyls in shake flasks than in photobioreactors. However, without nitrogen supply, the difference in the accumulation of triacylglycerols was observed between strains. In photobioreactors, CC-4326 accumulated 0.5 fold more triacylglycerols than CC-137, whereas in shake flasks, the difference was not significant. As an indicator, C18:1 was increased significantly in glycerides and triacylglycerols of both strains. And the same as triacylglycerol accumulation, C18:1 increased faster in photobioreactors than in shake flasks. As contrast, CC-137 showed similar triacylglycerol level in the matter of weight percentage of biomass, however, with higher monounsaturated fatty acids in shake flasks than in photobioreactors. It suggested that triacylglycerols can be more effectively induced in the shake flasks than in photobioreactors. But the highest triacylglycerol accumulation achieved in CC-4326 with 12.8% in dry weight, which were cultured in the photobioreactors under nitrogen depletion. [Conclusion] By inhibiting the starch synthesis, comparing to CC-137, CC-4326 obtains the high efficient triacylglycerol accumulation in photobioreactors with nitrogen depletion regulation.

Abstract:[Objective] To explore the relationship between AURI gene sequences in Botrytis cinerea and its mutants with aureobasidium A-resistance and the activity of Inositol phosphorylceramide (IPC) synthase. [Methods] Molecular-biology methods were adopted to determine the AUR1 gene sequences of the wild type and the mutants. The activity of IPC synthase was analysed by HPLC-FLD. The content of ceramide was used the method of benzene formylation. [Results] The results showed that 4 different mutant strains resisted to inhibitor AbA of IPC synthase. Their mutant types were: (1) AUR1 gene lost an intron; (2) AUR1 gene lost an intron and one amino acid mutated (P155S); (3) AUR1 gene lost an intron and one amino acid mutated (V33A); (4) AUR1 gene lost an intron and three amino acids mutated (F237L, S177P, and P155S). Both the mutant with the AUR1 gene lack of an intron and the mutant with the AUR1 gene lack of an intron and the mutant of P155S had stronger resistance to AbA. The determination of ceramide content showed that IPC synthase of wild type was inhibited to cause ceramide accumulation, and the mutants could resist to AbA inhibition on IPC synthase. [Conclusion] The intron of AUR1 gene plays an important role in the regulation of IPC synthase. AbA inhibites IPC synthase from inducing ceramide accumulation, IPC synthase is a key enzyme in sphingolipid metabolism.

Abstract:[Objective] To clone the gene ST0929 encoding maltooligosyl trehalose synthase from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7 and characterize the enzyme. [Methods] The ST0929 gene was amplified by PCR based on Sulfolobus tokodaii strain 7 ST0929 gene sequence and cloned into the expression vector pET-15b. The recombinant plasmid was transformed into E. coli BL21. After induced by IPTG (isopropyl-β-D-1-thiogalactoside), the bacterial pellet was sonicated and purified by affinity chromatography. The enzymatic properties were then measured. [Results] SDS-PAGE analysis showed that the molecular mass of the enzyme was about 83 kD. The optimal temperature was at 75 °C and pH at 5.0. Moreover, the enzyme exhibited notable pH and thermal stability and was resistant to additives and mental ions. Substrate specificity analysis showed that the enzyme could use maltodextrin and maltooligosaccharide as substrates but could not use maltose, chitooligosaccharide. [Conclusion] The recombinant enzyme described in this study suggest its potential in industrial production of trehalose.

Abstract:[Objective] Some naturally diseased Apocheima cinerarius pupae were collected in poplar forests in Hongtong, Shanxi province, China. The strain of YHT01 was isolated and purified from these naturally diseased pupae, and its classification status was determined. [Methods] A. cinerarius inoculation was conducted with conidial suspension (1.0×108 conidia/mL) of strain YHT01. The morphological characteristic was observed using scanning electron microscopy and the molecular identification was conducted by ITS sequence determination and phylogenetic analysis. [Results] White mycelia emerged between body segments after inoculated by strain YHT01. The colonies of the strain in PDA culture medium showed oval shape, white and fluffy, while the back of the colonies showed orange yellow. The coremium of the strain were like brooms, and the conidium was small (2.1–3.4) μm×(1.1–1.8) μm, oval to ellipsoid, smooth observed by scanning electron microscope. The comparability between the rDNA-ITS sequence of strain YHT01 and Isaria farinosa (Holm: Fr.) Fr. (AB233337) in Genbank is 99%, and these two strains are gathered in one branch in the phylogenetic tree. [Conclusion] Strain YHT01 is a pathogenic fungus of A. cinerarius, and identified as I. farinosa.

Abstract:[Objective] Isolation and identification of a strain with antagonistic activity against Fusarium graminearum, and the antibiotics from the target strain were identified and their bio-characteristics, antifungal activity was determined. [Methods] The inhibition activity of antibiotics was analyzed using oxford cup method and light growth chamber assay. Phylogenetic tree was constructed using the Neighbor-Joining method based on the 16S rRNA gene sequences, the antibiotics related genes were amplified by PCR, then the bioinformatics were determined using online software (Pro Param tool, TMHMM). [Results] The inhibitory zone diameter of 7M1 strain and antibiotics were 16.33±0.13 mm and 15.43±0.21 mm, light growth chamber assay showed that the control efficacy of wheat scab by 7M1 antibiotics was 76.41%. The 7M1 strain was found closely related to Bacillus amyloliquefaciens HQ840415.1 (98% similarity) based on the 16S rRNA gene sequence analysis. The antibiotics showed strong resistance to high temperature and a clear inhibition zone (5.33±0.32 mm) was still obtained after intense heating treatment of 90 °C for 30 min. The inhibitory activity was partially sensitive to proteinase K, pepsin and trysin. The antifungal activity of antibiotics was sustained throughout the pH range from 5.0 to 10.0. Along with the time of ultraviolet irradiation extending, the inhibitory activity was reduced. The gene essential for bacilysin, iturin and bacillomycin D synthetase was present in strain 7M1, which was naturally clustered from Bacillus sp. in GenBank, the amino acid sequence of the encoded product of the bacilysin, iturin and bacillomycin D synthetase gene exhibited high similarity up to 99% to that of iturin, bacilysin and bacillomycin D. bacAB encoded protein and ituC encoded protein were stable protein, bamD encoded protein were unstable protein. In addition, there was no obvious transmembrane structure present in the encoded product. [Conclusion] The antibiotics extracted from 7M1 had an obvious inhibiting effect against Fusarium graminearum and the antagonistic activity was resistant to some extent against temperature, pH proteinase and ultraviolet irradiation. These results suggested that 7M1 antibiotics were a promising material to control wheat scab.

Abstract:[Objective] To investigate the bacterial communities structure in rhizosphere and phyllosphere of different varieties of wine grapes. [Methods] By using samples of rhizosphere soil and phyllosphere leaf of different varities of wine grapes as the analysis materials, it was used the method of the terminal restriction fragment length polymorphism (T-RFLP) to study the microbial communities in the rhizosphere soil and phyllosphere, and analyzis the diversity of bacterial communities. [Results] The dominant microflora of wine grapes in rhizosphere soil is belonging to Proteobacteria, the dominant microflora of wine grapes in phyllosphere leaf is belonging to Actinomycetales. The order of Rhizosphere simpson index is Folle Blanche>Gewurztraminer>Chenin blanc>Semillon>Ugni Blanc>Muller-Thurgau>Sauvignon Blanc, Shannon index is keeping consistent with Simpson index. The largest of Simpson index in phyllosphere is Chenin blanc, the smallest is Muller-Thurgau, which is basically consistent with the index of Shannon. [Conclusion] the diversity indices of bacterial communities structure is keeping consistent with grape varitiesand, and the habitat is related closely to their colonization parts on plants.

Abstract:[Objective] Nitrogen-fixing microorganisms are the main component of biological nitrogen fixation agent, and the breeding of nitrogen-fixing-bacterium provides a solid foundation for the investigation and application in biological nitrogen fixation field. A nitrogen-fixing bacterium was identified and furthermore its nitrogenase activities were determined. [Methods] Ashby medium is used for the isolation and purification of nitrogen-fixing-bacterium. Morphological, physiological and biochemical analyses were conducted combining with 16S rRNA gene sequencing and genomic scanning to identify the bacteria. The ammonia-secreting capacity of bacterial cells was measured by indophenol blue spectrophotometric method and the nitrogenase activity was determined using the acetylene reduction assay (ARA). [Results] Kosakonia radicincitans GXGL-4A, a gram-negative nitrogen-fixing bacterium with the size of 1.5 μm×0.5 μm was finally isolated from maize root tissues. The strain were observed to show single cell distribution and joint of two tandem bacterial cells under a microscope. Phylogenic analysis based on 16S rRNA gene sequence revealed that GXGL-4A shares 95% of DNA homology with the Kosakonia oryzae strain Fo8A1d. Eventually, the isolate was identified as Kosakonia radicincitans and the stain was named as K. radicincitans GXGL-4A. The nitrogen-fixing gene, nifH was successfully amplified by PCR technique. The content of ammonia nitrogen secreted by GXGL-4A bacterial cells was assayed at 2.5 mg/L through indophenol blue spectrophotometric method. The results showed that this strain has a good capacity in ammonium secretion. The nitrogen-fixing bacterium GXGL-4A grown on nitrogen-free media can effectively restore acetylene, and its nitrogenase activity expressed in ethylene production capacity was evaluated at 232.94 nmol C2H4/(mL·h). [Conclusion] K. radicincitans GXGL-4A will benefit for the research of microbial nitrogen fixation.

Abstract:[Objective] In order to study the mechanism of vancomycin intermediate resistance Staphylococcus aureus development, strains were treated with vancomycin in vitro and mutations in critical genes were analyzed. [Methods] Thirteen vancomycin susceptible S. aureus were treated by low dose of vancomycin in vitro. Minimum inhibitory concentrations were determined by the agar dilution and E-test methods. The complete sequences of rpoB, vraS, graR and graS associated with drug-resistance development were compared with those in the parental strains. [Results] Through a 60 d vancomycin treatment in vitro, 6 vancomycin intermediate resistance strains were generated, whereas 7 strains remained vancomycin susceptible. Sequence analysis revealed that 3 vancomycin intermediate resistance strains contained L466S and H481N mutations in rpoB gene and R232K mutation in graS gene. [Conclusion] Evolution of vancomycin intermediate resistance S. aureus could be achieved by long-term vancomycin treatment in vitro. Our results suggest that rpoB and graS play crucial roles in vancomycin non-susceptibility development, whereas vraS and graR are less important.

Abstract:In recent years, aerobic denitrification researches focused on three fields: isolation and performance of aerobic denitrification strains, potential applications of aerobic denitrification microorganisms, and aerobic denitrifying mechanism. Aerobic denitrification strains are widely distributed in several environments. Functional species were mostly selected from Pseudomonas sp., Alcaligenes sp. and Paracoccus sp. In lab-scale tests, some aerobic denitrification microorganisms showed excellent psychrotolerant or haloduric property, and other strains had great potential on biodegradation of toxicant, or N2O emission reduction. Mechanism result of aerobic denitrification indicated that the competitiveness of nitrate was weaker than oxygen as an electron acceptor, whereas denitrification process could still occur as an assistant electron transport pathway to prevent the overload of NAD(P)H. Therefore, nitrate and oxygen could simultaneously participate in metabolism of microorganisms. However, researches on mechanisms and practical bioaugmentation of aerobic denitrification microorganisms are still insufficient, and more researches and process tests should be carried out in the future.

Abstract:Legionella pneumophila secretes a large number of effectors into the host cytosol via its Dot/Icm type-IVB secretion system. Those effectors efficiently recruit important host proteins that participating in the process of host vesicular trafficking, thereby “kidnap” the intracellular vesicular trafficking in the host. This process ensures L. pneumophila’s intracellular replication and avoids host lysosome clearance. Effectors involved in the process including SidM, LidA, LepB, AnkX, Lem3, SidD, RalF, VipD et al. According to the studies of identification, functional detection and crystal structural analysis of those effectors, their sophisticated molecular mechanisms have been gradually revealed. Here we summarized the structures and mechanisms of well-known important effectors exploiting host vesicular trafficking, in favor of the comprehensively understanding of the intricate pathogen-host interaction process.

Abstract:Nowadays, most of Chinese oil reservoirs are at the high-moisture later stage. In this case, microbial enhanced oil recovery (MEOR) technology attracts more and more attention in oil industry by its special advantages such as low cost and environmental friendliness. Until now, MEOR has been developed as one of the most affective techniques for petroleum exploitation. High pressure is an important abiotic feature of oil reservoirs which makes an influence on survival and activity of microorganisms in oil reservoirs. In this paper, we reviewed the main features of oil reservoir and its microbial community, the pressure adaptation mechanisms of microorganisms, and the metabolic characteristics of hydrocarbon-degrading microorganisms under high pressure. In details, we introduced the microorganisms resources in oil reservoirs, microbial community structure both in oil and water phases, how microorganisms emulsified petroleum, microbial cooperation patterns in anaerobic environment, microbial community structure was affected by temperature and pressure, as well as a successive of MEOR examples. Further on, detailed discussions were given about the pressure adaptation mechanisms of microorganisms in oil reservoirs, in which cells may modify cell membrane structure, storage lipid composition, and special intracellular enzymes expression to adapt well in the high-pressure environment. Finally, special attention was paid to hydrocarbon-degrading microorganisms grown at high pressure. Our review provides that the metabolic rates of microorganisms were lower at high pressure than that at atmospheric pressure, and piezotolerant and piezophilic microorganisms have significantly different hydrocarbon-degrading rates.

Abstract:[Objective] To develop a noninvasive, rapid, reliable and cost-saving method for the identification of Pseudomonas aeruginosa. [Methods] Photoacoustic spectroscopy trace analyzer (PASTA) was adopted to analyze bacterial volatile compounds (VCs), the volatile metabolites of Pseudomonas aeruginosa and Escherichia coli. Both bacteria were cultured at 35 °C and measurements of VCs were performed at 24 h. After that the results were analyzed using the reverse thinking strategy. [Results] Volatile metabolites of Pseudomonas aeruginosa and Escherichia coli were analyzed by PASTA. A large amount of HCN was found in the VCs of Pseudomonas aeruginosa whereas none was detected in that of Escherichia coli. PASTA could be used for the identification of Pseudomonas aeruginosa. [Conclusion] Photoacoustic spectroscopy provided simple, rapid and cost-saving detection and identification of Pseudomonas aeruginosa.

Abstract:[Objective] In order to evaluate the VITEK2 Compact automatic microbial analysis system in identifying Staphylococcus in raw milk. [Methods] We isolated 52 strains of Staphylococcus from milk samples from Xining, Baiyin and Xi’an, and these strains were identified by VITEK2 Compact. Then, we compared the results obtained from VITEK2 Compact to the results from Gram's staining, catalase test, plasma coagulase test and 16S rRNA gene sequence analysis. Furthermore, the stability of this system was tested by identifying the coagulase negative and positive Staphylococcus at different concentrations. [Results] With 16S rRNA gene sequence analysis as the reference standard, the coincidence rate of this system reached 96.15% for Staphylococcus at “Genus” level. With plasma coagulase test as the reference standard, the coincidence rate of this system was 92.86% for the coagulase negative Staphylococcus while 50% for the coagulase positive Staphylococcus. When it came to “Species” level, the coincidence rate of this system was only 44.23%. In stability test, coincidence rate was 100% and the reliability of appraisal result did not change by bacteria liquid concentration. [Conclusion] In conclusion, VITEK2 Compact automatic microbial analysis system could be used to identify Staphylococcus in milk at “Genus” level.

Abstract:[Objective] This study aimed at establishment of a rapid specific quadruple PCR assay for detecting Vibrio parahaemolyticus and its virulence genes. [Methods] Four pairs of primers were designed based on toxR, tdh, trh and tlh genes sequences of V. parahaemolyticus. Optimized the concentration of four pairs of primers and the annealing temperature to obtain the best ratio of primer and amplification conditions, the rapid quadruple PCR detection of pathogenic V. parahaemolyticus was established. The specificity and sensitivity of the quadruple PCR method were also evaluated. [Results] The expected sizes of amplification bands were 115 bp, 244 bp, 418 bp and 759 bp for toxR, tdh, trh and tlh, respectively. The highly specificity of this method was evaluated by using 74 V. parahaemolyticus strains and 37 non-targeted bacteria. The detection limits of the multiplex PCR for DNA was 50 μg/L. Detection sensitivity of V. parahaemolyticus in pure culture condition was 6.7×103 CFU/mL. Four targeted fragments were detected in artificially contaminated seafood with 1.36 CFU/g V. parahaemolyticus after 6 h enrichment. [Conclusion] The multiplex-PCR method can simultaneously detect V. parahaemolyticus with toxR, tdh, trh and/or tlh. It is of certain significance to carry out the detection of pathogenic V. parahaemolyticus.

Abstract:[Objective] Rapid detection of ochratoxin A-producing Aspergillus niger. [Methods] A PCR procedure has been developed for the rapid detection of ochratoxin A-producing A. niger. Two specific primers were designed based on the nucleotide sequence of the acyl transferase (AT) domain of the polyketide synthase encoded by An15g07920 from A. niger CBS 513.88. [Results] Specificity was confirmed by testing primers towards purified DNA from 72 Aspergillus genus strains, including A. niger, A. carbonarius, A. ochraceus, A. petrakii, A. parasiticus and A. tubingensis. Two specific primers can amplify a unique band from OTA-producing A. niger, but not from other OTA-producing strains. However, the use of the primer pairs also allowed amplification of the DNA from three OTA-non-producing A. niger. The quantitative real-time PCR found that the part of AT homeotic gene of An15g07920 can be normally expressed under the OTA-producing condition in OTA-non-producing A. niger. Therefore, the reason of false positive was not the gene can not express. The detection limit of the developed PCR protocol was 25 pg for DNA templates and about 4.0×104–4.0×105 spores/g when it was evaluated directly on artificially inoculated food. [Conclusion] The developed PCR procedure could be used for rapid detection of OTA-producing A. niger and is a promising tool in the prediction of potential ochratoxigenic risk for A. niger in foods.