Abstract:Abstract: Microalgae enables aquaculture wastewater recycling through a biological conversion. Recently, many studies have been reported on microalgae cultivation and wastewater treatment, including developing various wastewater treatment technologies such as algae pond, activated algae, immobilized algae and algae photo-bioreactor. In this review, we address the mechanisms, progress and application in the purification of aquaculture wastewater, as well as some research perspectives.

Abstract:Abstract: Fermentative hydrogen production can be improved by electrolysis and electrochemically active microorganisms which are capable of using an electrode as an electron acceptor for the oxidation of organic matter, in particular, volatile acids produced after fermentation. Firstly volatile acids can be completely converted into CO2, electrons and protons on the surface of anode. Then the electrons flow to cathode through anode and wires, and at the same time the protons move to cathode through cation membrane between anode chamber and cathode chamber. Finally the electrons and the protons combine into hydrogen when they meet at the surface of cathode. In such a process, the fermentation barrier and the product inhibition can be avoided to improve the conversion of hydrogen. 8-9mol H2/mol glucose of hydrogen potential can be obtained when glucose is used as substrate. This technology is very likely to be applied to produce hydrogen high efficiently from any energy crops, organic waste and wastewater.

Abstract:Abstract: Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered about 14 years ago. Mobile Group II introns were also found in bacteria recently, and they can either retrohome into cognate alleles that lack the intron or retrotranspose to ectopic sites. We reviewed the main mobility (homing) pathways of bacterial group II introns by describing the relationship between Intron-encoded protein activities and intron mobility. We discussed whether mobility of Cyanobacteria group Ⅱ introns could occur and possible mechanisms of their mobility. Furthermore, the biological implications of groupⅡ introns transferring within a species or among different species are also discussed.

Abstract:Abstract: [Objective] To establish effective methods for selective isolation of acidophilic filamentous actinomycetes from acidic soils, and to investigate their genus and species diversity. [Methods] Four pretreatments and 5 media supplemented with different inhibitors were used for isolation. The best combination of methods was determined according to the number and ratio of actinomycete colonies on plates, and was applied to isolate actinomycetes from 17 acidic soil samples collected in Jiangxi Province. The isolates were grouped based on cultural characteristics. The micromorphology and pH growth range of each group were studied to choose the representative isolates, which were subsequently subjected to 16S rRNA gene analysis to investigate their diversity. [Results] We found the best isolation approach involved Dispersion and Differential Centrifugation (DDC) pretreatment and GTV medium supplemented with cycloheximide, nalidixic acid nystatin (each at 50 mg/L). 369 isolates were obtained, and were assigned to 10 color groups. 6.6% of the isolates were strictly acidophilic actinomycetes, 72.4% were neutrotolerant acidophilic actinomycetes, and 21.0% were acidotolerant ones. The 52 representative isolates belong to 12 recognized genera: 30 of them fall within the genus Streptomyces, 6 belong to Micromonospora, 3 belong to Nocardia, 3 belong to Nonomuraea, 2 belong to Kribbella, 2 belong to Microbispora, and the other 6 strains belong to Actinomadura, Amycolatopsis, Dactylosporangium, Lentzea, Planotetraspora and Streptacidiphilus, respectivley. The Streptomyces isolates formed 12 evolutionary groups in the Streptomyces 16S rRNA gene tree. [Conclusion] The selective isolation approach established here is robust for isolating various acidophilic filamentous actinomycetes from soil. Acidic soils in Jiangxi Province harbor abundant and diverse acidophilic actinomycetes.

Abstract:Abstract: [Objective] We investigated endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang. [Methods] We investigated endophytic bacterial diversity in root of Glycyrrhiza inflata Bat. from Xingjian by culture-independent method. Total DNA genome of Glycyrrhiza sample was extracted using CTAB (Hexadecyl trimethyl ammonium Bromide) procedure with some modifications. A pair of bacterial PCR primers were used for endophytic bacterial 16S rDNA gene amplification and a clone library was constructed for the Glycyrrhiza DNA samples. Clones screened from clone library on the basis of Hae III digestion patterns were sequenced and compared, flowed by constructing Neighbor-Joining tree. [Results] In total 150 clones were grouped to 32 operational taxonomic units, most of the clones showed high similarity to the known cultured bacteria. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, gamma subclasses of the Proteobacteria, Bacteroides, Firmicutes, Actinobacteria, and Uncultured bacteria; 74% of the clones were highly related to the known bacteria in the genus Sphingobium, Phyllobacterium, Hyphomonas, Agrobacterium etc (>96% sequence similarity); whereas 26% of the clones showed lower affiliation with known genus (<96% sequence similarity) and may represent novel taxa. [Conclusion] There was abundant endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang as well as many unknown organisms.

Abstract:Abstract: [Objective] Serpins from pathogens have been implicated in evasion of the host immune system. We identified a new serpin protein (NbSPN106), analyzed its sequences, and detected using Western blotting. [Methods] Nosema bombycis proteins with an expect score less than 1×10-5 were checked against MEROPS database (http://merops.sanger.ac.uk) by Local Alignment Search Tool search and confirmed with Database of Protein Families and HMMS. Multiple sequence alignments were performed using the ClustalX. The sequence encoding the mature protein was amplified by PCR, cloned into the pGEX4T-1 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay. [Results] A new serpin gene, named NbSPN106, was identified form Nosema Bombycis genome. The coding sequence of this gene is 1155 bp length with a putative signal peptide and contains the conserved serpin sequences. A specific band of approximately 45 kDa was recognized by the anti-NbSPN106 serum. [Conclusions] The finding of serpins in Nosema bombycis raises new questions about their possible role in pathogenicity, which deserves further studies.

Abstract:Abstract: [Objective] To analyze gene expression differences of toxigenic and nontoxigenic strains of El Tor Vibrio cholerae growing separately in mannitol and LB (Luria-Bertani) fermentation medium. [Methods] Total RNA was extracted from the mannitol slow-fermenting strain N16961 (toxigenic) and the mannitol fast-fermenting strain 93097 (nontoxigenic) at 1 h of fermentation. The large scale gene expression profiles were detected and compared with high throughout microarray. [Results] By comparing the strains growing in different cultures, we found 142 differentially expressed genes in N16961 and 418 genes in 93097. Most of these genes were grouped into six functional classes. They were mainly related to transport and binding, energy metabolism, protein biosynthesis, and protein fate. [Conclusion] The expression levels of genes in N16961 and 93097 were affected by culture conditions, which can serve as basis for further studying the mechanism of metabolism of mannitol.

Abstract:Abstract: [Objective] To better understand the structure and biological function of rbfCxoo, a gene with the putative function in lipopolysaccharide O-antigen synthesis in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. [Methods] The molecular identification and function analysis of rbfCxoo were performed through gene cloning, sequencing and deletion analysis. [Results] The sequence of rbfCxoo cloned from the genomic DNA of wild-type PXO99A was the same as that of the sequenced strain KACC10331. There were glycosyltransferase domains (Glycos_transf_2) at N and C terminal of RbfCxoo respectively. The deletion mutant △rbfCxoo generated through a double crossover recombination and validated by PCR assay displayed the reduced flagellin glycosylation and no change in lipopolysaccharide O-antigen synthesis compared with PXO99A. Moreover, no significant changes in flagellar mobility, biofilm formation and production of extracellular cellulase and xylanase in vitro were observed in △rbfCxoo compared to PXO99A. Most importantly, the deletion mutation of rbfCxoo resulted in enhanced virulence and gene expression. [Conclusion] RbfCxoo might be related to flagellin glycosylation and virulence expression in Xoo.

Abstract:Abstract: [Objective] Our aim is to analyze the exoproteins of the epidemic strain and nonepidemic strain of El Tor Vibrio cholerae. [Methods] We separated the exoproteins of two El Tor strains, one epidemic strain N16961 and one nonepidemic strain 92-3, by two dimension electrophoresis. All protein spots were identified by MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry and database referencing. [Results] We identified 49 exoproteins from 206 protein spots in epidemic strain and 42 exoproteins from 236 protein spots in nonepidemic strain. Sixty-eight exoproteins were totally identified in two strains, and the proteins with signal peptides were about 55.88% in total. We classified the exoproteins into 10 subgroups according to their functions. Among them were metabolic enzymes, protein maintenance and folding, protein synthesis and signal transduction proteins, which accounted for 36.76% of all the proteins identified. We identified 11 hypothetical proteins and 2 unknown function proteins with signal peptides at the first time, accounting for 19.12%. Transporter, flagellum components, degradation enzymes, outer membrane proteins and toxin accounted for 14.71%, 11.76%, 10.29%, 5.88%,1.47% separately. [Conclusion] We obtained the exoprotein profiles of epidemic strain and nonepidemic strain of El Tor Vibrio cholerae. The reasons of why the flagellum components without signal peptides that are associated closely with virulence could be released out of the bacteria, and why with high hemagglutinin/protease secretion in nonepidemic strains should be analyzed in the future.

Abstract:Abstract: [Objective] In this study, we studied the toxic effect of nitrification substrates and products on photobacterium. [Methods] The acute toxicities of nitrification substrates and products to photobacterium were studied with the 15-min half inhibitory concentration (IC50) as indicator at pH=7.0. [Results] The results of individual toxicity indicated that the toxicity of nitrification substrates and products to photobacterium increased with increasing concentration and there was a linear correlation. The IC50 values of ammonium, hydroxylamine, nitrite and nitrate were 2180.2 mg?L-1, 6.2740 mg?L-1, 1207.2 mg?L-1 and 3140.3 mg?L-1, respectively, and their toxic order was hydroxylamine> nitrite> ammonium> nitrate. The combined effects of substrates and products were assayed by equivalent concentration mixing method. The results showed that the combined effects of ammonium and hydroxylamine, ammonium and nitrite, hydroxylamine and nitrite were additive effects, whereas the combined effects of ammonium and nitrate, hydroxylamine and nitrate, nitrite and nitrate were independent effects. The combined effect of all nitrification substrates and productions was also additive effect. [Conclusion] According to the correlation of the inhibiting concentration to photobacterium and nitrifying bacterium by nitrification substrates and products, the change of luminous intensity of photobacterium can indicate the inhibition from nitrification substrates and products.

Abstract:Abstract: [Objective] We examined the prevalence, molecular characteristics and virulence potential of Listeria monocytogenes isolates from imported fishery products to gain further insights on the public health risk caused by this important food borne pathogen. [Methods] L. monocytogenes isolates, screened from 1275 batches of fishery products imported from July 2007 to November 2008, were studied by lineage classification, serotyping, in vivo virulence assessment in ICR mouse model and multilocus sequence typing (MLST). [Results] Thirty-three batches were contaminated by L. monocytogenes (33/1275, 2.6%), in which serovar 4b dominated (65.2%), followed by serovars 1/2b (17.4%), 1/2a (13.0%) and 1/2c (4.4%). These isolates were all as virulent as L. monocytogenes reference strains. Of 23 selected L. monocytogenes isolates and 9 reference strains, 23 sequence types (STs) were recognized with discrimination index (D.I.) of 0.97, based on the concatenated gene cluster covering one virulent gene actA, two housekeeping genes hisJ and ribC, and one stress-response gene sigB. Remarkably, 3 isolates from American salmon, Argentina squid and Peru squid respectively belonged to ST9 which represented epidemic clone I (ECI). The isolation rate of L. monocytogenes in imported fishery products (2.6%) was similar to that in domestic fishery products (2.7%). Serovar 4b, which was more virulent than other serovars in humans, was dominant in imported fishery products, and even ECI was recognized. [Conclusion] All of these isolates were as virulent as L. monocytogenes reference strains, suggesting inspection and quarantine strategies of imported fishery products should be strengthened effectively.

Abstract:Abstract: [Objective] To study the influence of dissolved oxygen (DO) on bacterial population in a one-step autotrophic nitrogen removal process and to improve the system operation. [Methods] Ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and anaerobic ammonium oxidizing bacteria (ANAMMOX) were amplified directly from the one-step completely autotrophic nitrogen removal reactor by using the specific PCR primers. The purified PCR products were cloned into T-vector and identified as the target fragments of AOB, NOB and ANAMMOX by sequencing. Recombined plasmid was used as standard molecule sample in Real-time PCR for quantification. [Results] High DO was beneficial to AOB and NOB. The population of both AOB and NOB were higher in activated sludge than biofilm samples. High DO had the directly influence on ANAMMOX population whereas low DO had the inhibitory effect of ANAMMOX activity if the system was lack of NO3- and NO2-. [Conclusion] DO of 2.0 (aeration)/ 0.4(non-aeration) mg/L was the best operation concentration. Meanwhile, ANAMMOX population reached the peak in this condition and AOB ，NOB as well as ANAMMOX composes of a steady co-metabolisms system.

Abstract:Abstract: [Objective] To treat wastewater from beer breweries, we used the wastewater for the heterotrophic cultivation of Chlorella. [Methods] Using basal medium containing 10 g/L glucose?H2O we screened 5 Chlorella strains of which 2 strains with high specific growth rate and maximal biomass were used wastewater treatment. [Results] The two strains, Chlorella pyrenoidosa 15-2070 and Chlorella vulgaris 15-2075, were suitable for heterotrophic mass cultivation, and had similar results. For Chlorella pyrenoidosa 15-2070, 5.3 g/L biomass was achieved with basal medium containing 10 g/L glucose?H2O and beer wastewater. In addition, the highest removal efficiencies for CODcr (92.2 %), BOD5 (95.1 %), NO3-_N (98.5 %), NH4+-N (92.3 %) were achieved during the process. [Conclusion]Beer wastewater could be used as a major component of the medium for heterotrophic cultivation. Most of the harmful compounds in the wastewater could be removed during this process.

Abstract:Abstract: [Objective] To obtain the spiroplasma resources and characterize the spiroplasmas from insects in China, as well as study the taxonomy of spiroplasma based on biological characteristics. [Methods] We determined morphology by using dark field and transmission electron microscopy. The biological characteristics of the spiroplasmas were studied by using conventional culture-dependent method and phylogenetic analysis based on 16S rDNA. [Results] Based on morphological characteristics, biological characteristics and phylogenetic evidences, we studied the taxonomy of the strain YY0801 isolated from Phytomia zonata (Diptera: Syrphidae). The isolate grew well in R2 liquid medium and could pass through 0.22 μm and 0.45 μm filtrate membranes. The colony was grain-like in solid medium. Through electron microscopy, the isolate YY0801 exhibited helicity during their exponential growth phase. The isolate YY0801 was able to ferment glucose and D-fructose and to catabolize arginine, but did not to hydrolyse urea. The isolate was resistant to ampicillin (2000 U/mL). The phylogenetic relationships based on 16S rDNA supported YY0801 grouped with the serogroupⅠand was close to S. melliferum. [Conclusion] The result indicated that the spiroplasma isolate YY0801 was close to S. melliferum, but further designation need support of serological analyses.

Abstract:Abstract: [Objective] To provide insights into the role of Suilysin in the pathogenesis of Streptococcus suis by analysing the biological profiles of recombinant Suilysin (rSLY). [Methods] sly gene of S. suis strain SC22 was cloned into pET30a(+) vector and overexpressed in E. coli Rosseta, and rSLY was purified with Ni2+ affinity chromatography, anion exchange chromatography and gel filtration. The hemolytic unit of rSLY was measured with human red blood cells. The cellular toxicity of rSLY to human peripheral white blood cell and cells derived from fetal heart (HEH), liver (HEL), kidney (HEK), lung (HPL) was detected by lactate dehydrogenase (LDH) release assay. The blocking effects of water-soluble cholesterol, serum and specific antibody on the hemolytic activity of rSLY were detected. The amount of proinflammatory cytokines in the serum of mice stimulated by rSLY was measured by Luminex-100. [Results] The hemolytic unit of rSLY was 0.125 nmol/L, but the cellular toxicity of 1 nmol/L rSLY to human peripheral white blood cell, HEH, HEL, HEK and HPF was 20%～25%. The hemolytic activity of rSLY was blocked completely by equal molar of water-soluble cholesterol. Human serum containing 2.8 mmol/L～5.7 mmol/L of cholesterol in physical condition, could merely blocked 1 nmol/L of rSLY to lyse erythrocytes. The human serum with the addition of rabbit anti-rSLY IgG to a final concentration of 15mg/ml decreased the hemolysis by 10nmol/L rSLY from 77% down to 5% successfully, but hemolysis remained 60% when tested with 100nmol/L rSLY. Although IL-1β and TNF-α didn’t display any observable change, the level of interleukin -6 (IL-6) and KC were increased continually in C57BL/6 mice injected intraperitoneumly with rSLY whereas the IL-6 and KC level of mice in the control group only increased slightly and quickly decreased. [Conclusion] The data suggest that rSLY could not only lyse cells but also induce strong inflammatory response and damage immune cells recruited to the sites of inflammation. This finding implies that serum could provide light protection against rSLY, and specific antibody show a concentration-dependent protection.

Abstract:Abstract: [Objective] To evaluate the effects of the fusion gene of ubiquitin (Ub) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) M gene on the immune response in inoculated mice. [Methods] Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-1a strain, respectively, and the M and Ub gene (U-M) was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression. [Results] The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M (P< 0.05), but lower level of antibody (P< 0.05). [Conclusion] Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

Abstract:Abstract: [Objective] In order to overcome the defect of traditional avian influenza vaccine that lacks cross-protection among different serotypes, we developed a universal anti-influenza vaccine. [Methods] Based on the gene analysis for AIV matrix protein 2 and two cytotoxic T-lymphocyte epitopes, we constructed four prokaryotic expression vectors. The target gene was induced by IPTG and the fusion protein was mixed with Freund’s adjuvant; then used to immunize20-day-old chicken by intramuscular injection and boosted 3 weeks later. Blood samples were collected weekly following the primary vaccination. The anti-M2e antibody was detected with ELISA coated by synthesized peptide; the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo; the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken were measured by flow cytometry. [Results] The fusion protein induced immunological reaction, and the antibody bound with the viral M2 protein expressed on the surface of MDCK cells. Serum could only inhibit the replication without neutralizing the virus. Flow cytometry results showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization (P<0.05), which possessed the character of cell immunization. [Conclution] Chimeric peptides kept good immuno-genicity and provided useful probes for the control of avian influenza.

Abstract:Abstract: [Objective] To study the mechanisms of trans-species transmission of influenza virus for developing novel vaccine of influenza in future. [Methods] We rescued H3N2 subtype swine influenza virus strain A/Swine/Henan/S4/01 successfully by a plasmid-base reverse genetics. Eight gene segments were synthesized by reverse transcriptase-PCR and cloned into bidirection expression vector pHW2000. We cotransfected 8 recombinant plasmids into 293T and MDCK cells and got the rescued virus rgH3N2. Then we replaced Hemagglutinin, Neuraminidase of rgH3N2 by Hemagglutinin, Neuraminidase gene from Human influenza virus, Avian influenza virus, Equine influenza virus. [Results] The rescued virus rgH3N2 and the wild type virus shared similar biological properties such as in titers of 50% embryo infective, 50% tissue culture infective dose and stability tests. The rescued virus titer in MDCK cell culture was measured by hemagglutination assay and the maximum virus titre of 1∶64 hemagglutination unit was obtained after infection of MDCK cell for 60 h, The hemagglutination titre was 1:256 after several passages in embryonated eggs. With various combinations of HA, NA genes, we successfully generated high-yield reassortant viruses rgH1N1, rgH4N6 and rgH3N8 in embryonated eggs and MDCK cells. [Conclusion] The successful rescue of reassortment viruses establish the foundation for the molecular mechanism research on how the swine influenza virus breakthrough the intermediate barriers and the function of HA , NA during transmitting among species, Also it is feasible to be used for developing novel vaccine of H3N2 subtype Swine influenza in future.

Abstract:Abstract: [Objective] We developed a sensitive method to detect Escherichia coli O157:H7 based on enhancement with immuno-nanoparticles. [Method] About 10 nm ultrafine immunomagnetic particles were prepared by a co-precipitation method, about 20 nm immuno-colloid-gold was prepared by sodium citrate reduction. The immunosensor was fabricated by using Protein A from Staphylococcus aureus (SPA) for the antibody immobilization. Two immuno-nanoparticles by two antibodies were used to amplify frequency changing. [Result] The most suitable immobilization concentration and time for SPA was 1.2 mg/ml and 40 min, and dose for the antibody was 1.0 mg/ml and 60 min. With the signal amplification of the two immuno-nanoparticles, the detection limit of E. coli O157 : H7 was increased from 104 colony-forming units (cfu) /mL to 101 cfu/mL. [Conclusion] The prepared immuno-nanoparticles might be used to significantly amplify the frequency signal of the piezoelectric immunosensor and improve the detect sensitivity.

Abstract:Abstract: [Objective] To detect low levels of microorganism by bioluminescence assay, the reaction of ATP amplification catalyzed by ADK (adenylate kinase) combined with PPK (polyphosphate kinase) can be employed. However, the endogenous ADP bound to PPK is a background source and interfere the effective detection of low levels of exogenous ATP. We expressed a fusion protein of PPK and ADK and established a new method to decrease the background signal. [Methods] The genes of PPK and ADK were amplified by PCR and cloned into vector pET28a (+) to provide a recombinant expression plasmid pET28a (+)-PPKADK to prepare the fusion protein. Apyrase was immobilized on the surface of magnetic beads coated with polyurethane to provide Beads-apyrase to eliminate background caused by ADP bound to PPK-ADK. The exogenous ATP and microorganism were also detected by using ATP amplification reaction coupled with bioluminescence assay. [Results] The purified fusion protein showed both ADK and PPK activities. Beads-apyrase could eliminate ADP contamination conveniently and effectively, thus less than 1 fmol of ATP was detected by ATP amplification reaction coupled with bioluminescence assay. Using ATP amplification reaction, the sensitivity of bioluminescence assay was 100-fold than that of normal bioluminescence assay without ATP amplification. [Conclusions] Beads-apyrase is an effective tool to eliminate the background of the reaction of ATP amplification. The sensitivity of bioluminescence assay was increased significantly with concomitant use of ATP amplification and bioluminescence assay.

Abstract:Abstract: [Objective] In order to construct a recombinant Bombyx mori Nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli efficiently, a new zero background transposition system was developed. [Method] The new system consisted of a conditional replication donor vector pRADM and an attTn7 site blocked E. coli containing BmNPV-Bacmid. The donor transposon vector pRADM with the replication origin derived from R6Kγ required the factor π encoded by the pir gene to propagate in host cells. Another conditional replication plasmid pBlockA was constructed to block the attTn7 site in host E. coli genome. [Results] Compared with the original vector with ColE1 origin, the transposition efficiency increased from 5.7% to 66% when using conditional replication vector pRADM transposition into original BmDH10Bac. The attTn7 site blocked strain BmDH10Bac△Tn7 resulted in a significant increase from 5.7% to 23% in the efficacy of generating recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10Bac△Tn7 with pRADM resulted in 100% white colonies. [Conclusion] This highly efficient and zero background transposition system provides a simple and rapid way of construction of recombinant BmNPV to express target genes or produce gene-delivery virus particles in silkworm.