Abstract:

Abstract:[Objective] The influence mechanism of the algicidal bacterial strain L7 on nitrogen metabolism of Anabaena flos-aquae were investigated to understand the interaction of cyanobacteria-bacteria. [Methods] The algicidal bacterial strain L7 and Anabaena flos-aquae with different ratio were inoculated into BG11 liquid medium. The initial concentration of the algicidal bacterial strain L7 were 1.75×107 and 1.75×108 CFU/mL responding to the same initial concentration of Anabaena flos-aquae (1.21×108 cells/L), respectively. The treatment without the algicidal bacterial strain L7 was set up as the control. The number of cyanobacterial cells, the heterocyst frequency, activities of the nitrate reductase (NR), glutamine synthelase (GS), glutamate synthetase in cyanobacterial cells, and the protein and malondialdehyde (MDA) contents in cyanobacterial cells were measured for 7 days after incubation. [Results] Lower concentration of the algicidal bacterial strain L7 stimulated the growth of the cyanobacteria and increased the heterocyst frequency. On the 7th day, the concentration of cyanobacterial cells and the heterocyst frequency were 1.58 times and 66.67% more than that in the control, respectively. Higher concentration of the algicidal bacterial strain L7 had opposite effects to Anabaena flos-aquae. The cyanobacterial cell concentration was 98.84% less than that in the control and the heterocyst frequency decreased to 0 on the 7th day. As to activities of important enzymes relating to nitrogen metabolism in cyanobacterial cells, activities of NR and GOGAT in two treatments with the algicidal bacterial strain L7 were significantly higher than that in the control between the 2nd and 5th day (P<0.01). In the first 5 days, GS activity was significantly higher than that in the control (P<0.01) in the treatment with higher concentration of the algicidal bacterial strain L7, while lower concentration of the algicidal bacterial strain L7 had opposite effects for most of time. During the entire experiment, the protein content in the treatment with lower concentration of the algicidal bacterial strain L7 was significantly higher than that in the control (P<0.01). However, the protein content in the treatment with higher concentration of the algicidal bacterial strain L7 was always less than that in the control (P<0.01), except on the 5th day. During the period between the 2nd day and the 4th day, the MDA content in the treatment with higher concentration of algicidal bacteria strain L7 exhibited a rising trend and were significantly higher than that in the other treatments (P<0.01). [Conclusion] Lower concentration of the algicidal bacterial strain L7 could stimulate nitrogen metabolism of Anabaena flos-aquae by enhancing its demand for nitrogen and accelerating protein synthesis. Higher concentration of the algicidal bacterial strain L7 could induce membrane lipid peroxidation of Anabaena flos-aquae, thereby inhibiting protein synthesis and nitrogen metabolism.

Abstract:

Abstract:[Objective] Small RNAs (sRNAs) regulate various genes expression, which involved in primary and secondary metabolism, quorum sensing system and a variety of toxins factors in various Pseudomonas spp.. Through studying the different role of RsmY on regulation of the two antibiotics phenazine-1-carboxylic acid (PCA) and Pyoluteorin (Plt) in the plant rhizosphere growth-promoting Pseudomonas aeruginosa M18, the results were to shed light on the pathways involved in secondary metabolism and provided some theoretical basis for building high-yield engineering strains. [Methods] The rsmY inactivated mutant M18RY was constructed by homologous recombination, and gene over-expression experiment and lacZ gene fusion report analysis were carried out to investigate the regulatory mechanisms involved in RsmY. [Results] PCA and Plt production were respectively assayed in wild type M18 and mutant M18RY strains. Compared with that in wild type M18, PCA production in rsmY mutant had a 5-fold increase, but Plt production in M18RY had an 8-fold decrease. The results could be confirmed identified by Gene rsmY over-expression experiment. Moreover, lacZ gene fusion report analysis further clarified that RsmY strongly up-regulated PCA biosynthesis via phzA2-G2 gene cluster. [Conclusion] Small RNA RsmY distinctively regulates the PCA and Plt biosynthesis.

Abstract:[Objective] To isolate a novel β-glucosidase-producing strain from soil, to clarify the taxonomic status, and to study the enzymatic characteristics of the β-glucosidase. [Methods] A β-glucosidase-producing strain was isolated from soil sample using esculin and 4-nitrophenyl-β-D-glucopyranoside (PNPG) coloration methods; the taxonomic status of strain ZF-6C was clarified by morphological, physiological, chemotaxonomic characteristics and 16S rDNA analysis; the β-glucosidase was isolated and purified by ultra-filtration, hydrophobic interaction chromatography, anion chromatography, molecular sieve chromatography; with PNPG as the substrate, the optimal reaction pH and temperature of β-glucosidase were determined, the Michaelis constant Km of β-glucosidase against different substrates were determined by Lineweaver-Burk plot. [Results] A strain, which produced β-glucosidase at high level, was isolated from soil and identified as Bacillus korlensis, the molecular weight of β-glucosidase isolated from Bacillus ZF-6C was 90 kD, the optimum reaction conditions for this enzyme were pH 7.0 and 40 °C. The enzyme was active against a wide range of β (1,4) linked disaccharides, the optimal substrate was o-nitrophenyl-β-D-glucopyranoside, the Km value was 0.73 mmol/L. Metal ions Ca2+ and Pb2+ enhanced enzyme activity, Cu2+ and Fe2+ inhibit the enzyme activity. [Conclusion] This is the first report of isolation and identification of β-glucosidase from Bacillus korlensis. The β-glucosidase from Bacillus korlensis is widely different to known enzymes at molecular weight, optimum reaction conditions and substrate specificity aspects. This β-glucosidase may have novel structure and high catalytic efficiency.

Abstract:[Objective] to investigate the influence of acidophilic heterotrophic bacteria on Acidithiobacillus ferrooxidans in extremely acidic environment such as acid mine drainage (AMD) and bioleaching system. [Methods] a co-culture consists of Aph. acidophilum and At. ferrooxidans was separately exposed to four metal ions (Cd2+, Cu2+, Ni2+ and Mg2+) to test its stability. This co-culture was also applied to bioleaching of pyrite and low grade chalcopyrite. [Results] In the metal resistance experiment, heterotrophic bacteria Aph. acidophilum facilitated the ferrous iron oxidation by At. ferrooxidans and improved its efficiency of energy utilization. The maximum tolerant concentration (MTC) of At. ferrooxidans to Cu2+ was improved from 2.0 g/L to 5.0 g/L by Aph. acidophilum, and the cell density of co-culture in 5.0?g/L Cu2+ was almost the same with purely cultured At. ferrooxidans in 2.0 g/L Cu2+. In addition, the MTC of co-cultured At. ferrooxidans to Mg2+ was also improved from 12.0 g/L to 17.0 g/L by Aph. acidophilum. In bioleaching experiment, the pyrite bioleaching efficiency of co-culture increased by 22.70% as compared with that of purely cultured At. ferrooxidans. While in the low grade chalcopyrite bioleaching system with few iron, the bioleaching efficiency of both At. ferrooxidans and its co-culture with Aph. acidophilum were lower than 33%. In the low grade chalcopyrite bioleaching system with pre-added 2 g/L Fe2+, the bioleaching efficiency of At. ferrooxidans and its co-culture with Aph. acidophilum were raised to 41.27% and 52.22%, respectively. [Conclusion] Results in this study demonstrated that At. ferrooxidans and Aph. acidophilum in co-culture could maintain their physiological stability and sustain their ecological function under environmental stress. The bioleaching results suggested that acidophilic heterotrophic bacteria Aph. acidophilum should be applied to the bioleaching system with high iron concentration, in which it could collaborate with iron oxidation bacteria to improve the bioleaching efficiency.

Abstract:[Objective] Microbe resource research in extreme environment. [Methods] Eight Bacillus strains isolated from Chaidamu extreme dry-sand region in Qinghai Province, were identified by polyphasic molecular taxonomy methods including rep-PCR (BOX-PCR and ERIC-PCR) fingerprints, as well as gyrB and 16S rDNA partial sequence analysis. Antagonistic activity detection and dual culture method screening were used to check antagonistic activity and bio-control efficacy of isolates. MALDI-TOF-MS were used to analyze lipopeptide compound of strains. [Results] Eight isolates were identified as Bacillus amyloliquefaciens (6 isolates), Bacillus axarquiensis (1 isolate), Bacillus atrophaeus (1 isolate). All of the isolates presented distinct antagonistic activity and bio-control efficacy to Sclerotinia sclerotiorum. MALDI-TOF-MS results showed that the strain DGL1 (B. amyloliquefaciens) produced Fengycin, strain DGL6 (B. axarquiensis)produced Surfactin, BacillomycinsD and Fengycin, while strain DCD1 (B. atrophaeus) produced Surfactin and Fengycin. [Conclusion] The research provided Bacillus resources for further developing and application of bio-control strains adjusting in dry-sand extreme environment of Qing-Tibet Altiplano.

Abstract:[Objective] Iron sulfur cluster are one of the most ancient and ubiquitous redox centre in almost all living organism and play an important role in photosynthesis, respiration and nitrogen fixation. [Methods] In this study, three key proteins (IscS, IscU, IscA) involved in iron sulfur cluster assembly from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli. [Results] IscS is a cysteine desulfurase, IscU is a scaffold protein, IscA has strong iron binding activity which acts an iron chaperon. [Conclusion] The Fe-IscA can provide iron for the assembly of transient iron sulfur cluster in IscU in the presence of IscS and L-cysteine in vitro.

Abstract:[Objective] Lactic acid bacterium with aflatoxin B1 detoxification abilities was isolated and evaluated from chicken gut for the present study. [Methods] A special enrichment and isolation technology was designed for aflatoxin B1 detoxified lactic acid bacteria, and the aflatoxin B1 detoxification strain was further determined for its taxonomical position using morphological, physiological and biochemical characteristics combined with phylogenetic analysis. [Results] A lactic acid bacterium strain, which designated LAB-10, displayed aflatoxin B1 detoxification ability was isolated by the present technology. The detoxification analysis results displayed that strain LAB-10 was a aflatoxin B1 degradative bacterium and there was 63.4% of aflatoxin B1 could be decomposed by the strain under the concentration of 14 μg/L, within 48 hours. On the base of taxonomical analysis results, the new isolated strain LAB-10 was identified as Lactobacillus fermentum. [Conclusion] The isolate LAB-10 of the present study was Lactobacillus fermentum and the bio-safety characteristics implied that the degradative stain has potential values for the application of aflatoxins detoxification.

Abstract:[Objective] The complete sequence of a β-glucosidase gene from a Trichoderma longibrachiatum strain GM2 previously isolated in the laboratory, namely bgl?, was amplified and expressed. [Methods] The gene of bgl? was amplified via homologous cloning. The bgl? sequence corresponding to the mature peptide was subcloned into plasmids pET-32a(+) and pPICZα-B, respectively. [Results] Sequencing results showed that the bgl? gene was 2 369 bp in size encoding 744 amino acids, interrupted by two introns. The bgl? protein expressed in E.?coli BL21(DE3) existed mostly in inclusion bodies, and there was no detectable β-glucosidase activity in the soluble proteins. The expression vector pPIZα-B-bglI was transformed into Pichia pastoris GS115 by electroporation, and the recombinant protein with the molecular weight around 78 kD, consistent with the expected protein size, was secreted. Under the fermentation conditions of 9% initial inoculum, initial pH of 5.5, 30 °C and 1% methanol induction, after shaking for 96 h, the β-glucosidase activity of 60 U/mL was obtained. Enzyme property analyses demonstrated that the optimum pH and the optimum temperature for the recombinant bgl? were 5.0 and 70 °C, respectively; furthermore, this bgl? exhibited good stability at pH between 3.0 and 10.0 and the temperature range of 40 °C?60 °C. [Conclusion] The gene of bgl? was expressed in P. pastoris with β-glucosidase activity.

Abstract:[Objective] To isolate and identify the endogenous bacteria, which caused tissue culture contamination of Dendrocalamus aspera. [Methods] Using the improved NA culture medium to isolate and identify the strain, then identify the bacterial species by morphology observation, physiological and biochemical tests and the sequence analysis of the 16S rDNA. [Results] The morphological characteristics of the strain SWFU01, together with related physiological and biochemical tests, match the descriptions of Bacillus amyloliquefaciens (Fukumoto) Priest et al.. The sequence analysis of 16SrDNA indicates that this strain has the same embranchment with the Bacillus amyloliquefaciens JS in the Phylogenetic tree, the homology is 99.28%. [Conclusion] By combining the traditional morphology observation and physiological and biochemical tests, the SWFU01 strain is identified as Bacillus amylolique-faciens.

Abstract:[Objective] To explore culture conditions for chicken hepatocellular carcinoma cell line (LMH) and growth pattern of fowl adenovirus typeⅠ-AV208 strain (FAVⅠ-AV208) on LMH cells. [Methods] Cells were cultured with different concentration of serum and propagated at different subcultivation ratios. Cytopathic effect and growth pattern of FAVⅠ- AV208 on LMH cells were studied at an optimal MOI. Relation between virus concentration and plaque number was discussed by a standard plaque assay. [Results] Culturing with 10% FBS is suitable for LMH cells and a subcultivation ratio of 1: 5 is recommended. FAVⅠ- AV208 could be propagated efficiently in LMH cells and high virus titer up to 107.5 TCID50/0.1 mL was reached. Obvious CPE was observed after 72 h post- infection at an MOI of 0.01. The nomal cobblestone- like pattern cells grew into larger and round, they aggregated into irregularly botryoidal appearance and finally disintergrated. A linear relation was proved between virus concentration and plaque number. [Conclusion] LMH cell line is a suitable virus culture system and provides an excellent tool for constrution of recombinant fowl adenovirus typeⅠ.

Abstract:[Objective] Clavulanic acid (CA) is a β-lactamase inhibitor produced by Streptomyces clavuligerus. Since urea is a byproduct of CA biosynthesis, we investigated the potential inhibitory effect of urea on CA biosynthesis. [Methods] We designed urea addition, ammonium addition, urease inactivation and pH gradient experiments to research effect of urea on CA production. [Results] Addition of urea inhibited CA production in the wild type, and the production was completely abolished when the concentration of urea reached 20 mmol/L. To prevent urea hydrolysis, the genes encoding urease were disrupted and the corresponding mutant was analyzed: which accumulated higher concentration of urea, reaching 10mmol/L, but this did not inhibit CA production. Therefore, urea itself was excluded as the factor inhibiting CA biosynthesis. So the inhibitory effect of urea on CA production in the wild type may be due to the elevated ammonium concentration or pH resulting from urea hydrolysis by urease. To test these possibilities, ammonium salt were added in the fermentation medium, which showed no inhibitory effect on CA production. Furthermore, the gradient experiment of pH confirmed that different pH had a large influence for CA production. [Conclusion] pH increase due to urea hydrolysis was identified as the real reason to inhibit CA production in the wild type strain. This study laid the foundation for further elucidation of the regulatory mechanisms controlling CA biosynthesis in S. clavuligerus.

Abstract:[Objective] To clone squalene?synthase (SS) gene from Penicillium?minioluteum P116-1a isolated from Eleutherococcus senticosus. [Methods] Using rapid amplification of cDNA 5￠ ends (5￠ RACE) method, we isolated completed cDNA and DNA sequence of SS from P. minioluteum. The gene was analyzed and corresponding structure and functions were predicted by the bioinformatic method. Expression of SS was detected by RT-PCR and SDS-PAGE. [Results] The results showed that SS gene had 4 exons and 3 introns. The open reading frame was 1?416?bp encoding a protein of 471 amino acid residues. The predicted secondary structure composition for the protein contained about 67.73% α helixes, 5.31% extended strand, 2.97% β turns and 23.99% random coil. SS had conserved binding regions of squalene synthase and phytoene synthase and located in endoplasmic reticulum membrane. The SS amino acid sequence of P. minioluteum showed more than 90% homology with that of P. marneffei and Talaromyces stipitatus. Expression of P. minioluteum P116-1a SS gene varied in different tempreture. [Conclusion] The SS gene of P. minioluteum form E. senticosus was successfully cloned for the first time, providing a stable foundation for studying on mechanism of improving eleutheroside content by P. minioluteum P116-1a.

Abstract:With the increasing of energy shortage and environmental problems caused by burning fossil fuels, hydrogen, as a clean and renewable energy has gained special attention. Compared with thermochemical and electrochemical hydrogen production, bio-hydrogen production has a lot of advantages, such as ambient reaction conditions, low consumption and environmentally friendly. Therefore, it was considered as a promising technology. Bio-hydrogen production usually undergoes two stages, i.e., dark fermentation and light fermentation. In dark fermentative stage, waste organic materials can be converted to hydrogen by bacteria, and leave some by-products, such as organic acids. The photosynthetic bacteria can decompose organic acids and release hydrogen by using light energy and nitrogenase in a photo fermentation process. Thus, combining these two kinds of hydrogen production methods can improve the utilization rate of waste organic material. This review is based on the development of dark and photo two-stage fermentation in recent years. It was elaborated mainly from the following aspects: mechanism of two-stage hydrogen production, main influence factors, kinds of two steps bio-hydrogen production (sequential dark and photo-fermentation & combined dark and photo-fermentation), and the facing challenges.

Abstract:Study on Salmonella typhimurium (S. typhimurium) pathogenesis in vivo is significant for control of food poisoning, gastroenteritis, typhoid fever and other intestinal infectious diseases. Because it is difficult to detect dynamic changes for S. typhimurium in living hosts, the strategy for preventation and treatment of Salmonellosis as well as the development of drug and vaccine production are restricted. In recent years, small animal imaging technology is widely used to track bioluminescent Salmonella typhimurium (S. typh-lux) in vivo. The present article summarizes the merit and shortage of the application of small animal imaging.

Abstract:The work-study combination personnel training mode has been approved in higher vocational education and teaching reform. At present, the key issue is how to implement the combination of production and learning and the cooperation between colleges and enterprises in the daily teaching. We propose the means of carrying out the work-study combination training mode in the teaching of biochemical technology based on our long-term teaching practice. We aim to explore a new path for the implement of the combination of production and learning by combining the teaching contents with the employment orientation, combining school classrooms with workshops, combining teachers in colleges with workers in enterprises, combining teaching process with production process and combining scientific research of colleges with factories.

Abstract:[Objective] The current study investigates the microbial quality of drinking water using various microbial detection methods. [Methods] We analyzed the microbial quality of still bottled drinking water using flow cytometric cell count, ATP assay, AOC (assimilable organic carbon) assay and cell viability test. [Results] We found that the flow cytometry allowed fast and accurate discrimination of the live/dead bacteria and detection of the micro-scale AOC. Our results also showed that ATP is a better indicator than the heterotrophic plate counts of the actual active microorganism content in still bottled drinking water. [Conclusion] The results suggested that the flow cytometric cell count and ATP assay are more suitable methods for assessing microbial quality in bottled drinking water than the conventional heterotrophic plate count method.

Abstract:[Objective] To develop a Lactobacillus genus-specific T-RFLP technique for differentiating 14 Lactobacillus strains. [Methods] A Lactobacillus genus-specific primer (LAB-rev) was firstly designed based on the sequence variation of bacterial 16S-23S space region, which was then labeled with 6-Carboxyfluorescein (6-FAM), followed by PCR with a 16S universal primer (7f). The PCR amplicon was then digested by restriction enzyme Hae III and HhaI before terminal sequencing by AB3730 Gene Scan to achieve T-RFLP profiles. [Results] Results showed that this technique is more specific than the traditional T-RFLP using universal primers, which can be used to detect and quantitate Lactobacillus of 14 different species in this study. [Conclusion] This technique has a great potential of application in investigating lactobacillus communities, evaluating the probiotic function of functional food, dairy drinks and medicine on intestinal micro-ecological environment.

Abstract:[Objective] To establish the flow cytometry (FCM) method for detecting multi-vibrio based on anti-OmpW monoclonal antibody. [Methods] With the rate of cell stainning as an indicator, optimize the reactived conditions of anti-OmpW mAb concentration and reaction time for detecting Vibrio parahaemolyticus by flow cytometry. Evaluate the accuracy, detection limits and precision of flow cytometry by comparing the munber of bacteria calculated by FCM with that in the culture. Based on the obove flow cytometry method, analysize and identify the mAb specificity to the five vibrios. [Results] The optimized mAb concentraion was 20 mg/L, and the optimized reaction time of mAb was 60min when Vibrio parahaemolyticus was detected by flow cytometry. The FCM method could provide high reliability which could specifically recognize the five vibrios in the appropriate cell concentration of 104?107cells/mL. To detect different cell concentration samples repeatedly, the coefficient of variation was less than 7%. [Conclusion] The established flow cytometry method based on anti-OmpW mAb could quickly and accurately detect multiple vibrios.