Journal of Microbiology

Journal Article

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus: Guomin Han , Qian Shao , Cuiping Li , Kai Zhao , Li Jiang , Jun Fan , Haiyang Jiang , Fang Tao; J. Microbiol. 2018;56(5):356-364. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7349-3

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Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ~ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (~ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

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Research Support, Non-U.S. Gov't

Statistical experimental design optimization of rhamsan gum production by Sphingomonas sp. CGMCC 6833: Xiao-Ying Xu , Shu-Hao Dong , Sha Li , Xiao-Ye Chen , Ding Wu , Hong Xu; J. Microbiol. 2015;53(4):272-278. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-3662-2

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Abstract

Rhamsan gum is a type of water-soluble exopolysaccharide produced by species of Sphingomonas bacteria. The optimal fermentation medium for rhamsan gum production by Sphingomonas sp. CGMCC 6833 was explored definition. Single-factor experiments indicate that glucose, soybean meal, K2HPO4 and MnSO4 compose the optimal medium along with and initial pH 7.5. To discover ideal cultural conditions for rhamsan gum production in a shake flask culture, response surface methodology was employed, from which the following optimal ratio was derived: 5.38 g/L soybean meal, 5.71 g/L K2HPO4 and 0.32 g/L MnSO4. Under ideal fermentation rhamsan gum yield reached 19.58 g/L ?1.23 g/L, 42.09% higher than that of the initial medium (13.78 g/L ? 1.38 g/L). Optimizing the fermentation medium results in enhanced rhamsan gum production.

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Journal Articles

Optimization of Antifungal Lipopeptide Production from Bacillus sp. BH072 by Response Surface Methodology: Xin Zhao , Ye Han , Xi-qian Tan , Jin Wang , Zhi-jiang Zhou; J. Microbiol. 2014;52(4):324-332. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3354-3

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Abstract

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH4Cl, urea, and ammonium citrate); and metal ions (Zn2+, Fe3+, Mg2+, Mn2+, Ca2+, and K+). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg2+ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg2+. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.

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Optimization of Water Absorbing Exopolysaccharide Production on Local Cheap Substrates by Bacillus Strain CMG1403 Using One Variable at a Time Approach: Muhammadi , Muhammad Afzal; J. Microbiol. 2014;52(1):44-52. Published online January 4, 2014; DOI: https://doi.org/10.1007/s12275-014-2622-6

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Abstract

Optimum culture conditions, and carbon and nitrogen sources for production of water absorbing exopolysaccharide by Bacillus strain CMG1403 on local cheap substrates were determined using one variable at a time approach. Carbon source was found to be sole substrate for EPS biosynthesis in the presence of yeast extract that supported the growth only and hence, indirectly enhanced the EPS yield. Whereas, urea only coupled with carbon source could enhance the EPS production but no effect on growth. The maximum yield of EPS was obtained when Bacillus strain CMG1403 was grown statically in neutral minimal medium with 25% volumetric aeration at 30°C for 10 days. Under these optimum conditions, a maximum yield of 2.71±0.024, 3.82±0.005, 4.33±0.021, 4.73±0.021, 4.85±0.024, and 5.52±0.016 g/L culture medium was obtained with 20 g (sugar) of sweet whey, glucose, fructose, sucrose, cane molasses and sugar beet the most efficient one respectively as carbon sources. Thus, the present study showed that under optimum culture conditions, the local cheap substrates could be superior and efficient alternatives to synthetic carbon sources providing way for an economical production of water absorbing EPS by indigenous soil bacterium Bacillus strain CMG1403.

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Research Support, Non-U.S. Gov't

Influence of Culture Conditions and Medium Composition on the Production of Antibacterial Compounds by Marine Serratia sp. WPRA3: Mahtab Jafarzade , Nur Ain Yahya , Fatemeh Shayesteh , Gires Usup , Asmat Ahmad; J. Microbiol. 2013;51(3):373-379. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2440-2

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Abstract: This study was undertaken to investigate the influence of culture conditions and medium components on production of antibacterial compounds by Serratia sp. WPRA3 (JX020764) which was isolated from marine water of Port Dickson, Malaysia. Biochemical, morphological, and molecular characteristics suggested that the isolate is a new candidate of the Serratia sp. The isolate showed strong antimicrobial activity against fungi, Gram-negative and Gram-positive bacteria. This bacterium exhibited optimum antibacterial compounds production at 28°C, pH 7 and 200 rev/min aeration during 72 h of incubation period. Highest antibacterial activity was obtained when sodium chloride (2%), yeast extract (0.5%), and glucose concentration (0.75%) were used as salt, nitrogen, and carbon sources respectively. Different active fractions were obtained by Thin-Layer Chromatography (TLC) and Flash Column Chromatography (FCC) from ethyl acetate crude extracts namely OCE and RCE in different culture conditions, OCE (pH 5, 200 rev/min) and RCE (pH 7/without aeration). In conclusion, the results suggested different culture conditions have a significant impact on the types of secondary metabolites produced by the bacterium.

Journal Article

Chitinase Production by Bacillus thuringiensis and Bacillus licheniformis: Their Potential in Antifungal Biocontrol: Eman Zakaria Gomaa; J. Microbiol. 2012;50(1):103-111. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1343-y

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Abstract

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na+, Mg2+, Cu2+, and Ca2+ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn2+, Hg2+, and Ag+. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.

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Research Support, Non-U.S. Gov't

Optimization and High-level Expression of a Functional GST-tagged rHLT-B in Escherichia coli and GM1 Binding Ability of Purified rHLT-B: Xingyuan Ma , Wenyun Zheng , Tianwen Wang , Dongzhi Wei; J. Microbiol. 2006;44(3):293-300.; DOI: https://doi.org/2383 [pii]

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Abstract: The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL21 (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GM1-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92% purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.

Journal Article

Optimization of Lactic Acid Production in SSF by Lactobacillus amylovorus NRRL B-4542 Using Taguchi Methodology: Pyde Acharya Nagarjun , Ravella Sreenivas Rao , Swargam Rajesham , Linga Venkateswar Rao; J. Microbiol. 2005;43(1):38-43.; DOI: https://doi.org/2140 [pii]

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Abstract: Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO_4 . 7H_20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO_4 . 7H_20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.

Optimization of culture conditions for production of pneumococcal capsular polysaccharide type I: Kim, Su Nam , Min, Kwan Ki , Kim, Seung Hwan , Choi, In Hwa , Lee, Suhk Hyung , Pyo, Suhk Noung , Rhee, Dong Kwon; J. Microbiol. 1996;34(2):179-183.

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Abstract: Streptoccus Pneumoniae (pneumococcus), the most common cause of bacterial pneumonia, has an ample polysaccharide (PS) capsule that is highly antigenic and is the source of PS vaccine. This investigation was undertaken to optimize the culture conditions for the production of capsulard PS by type 1 pneumococcus. Among several culture media, brain heart infusion (BHI) and Casitone based media were found to support luxuriant growth of pneumococcus type 1 at the same level. Because BHI medium is rather expensive and more complex than the Casitone based media, the Casitone based media was uwed to study optimization of the culture condition. The phase of growth which accomodated maximum PS production was logarithmic phase. Concentrations of glucose greater than 0.2% did not ehnahce growth or PS production. Substitution of netrogen sources with other resources or supplementation of various concentrations of metal ion (with the exception of calcium ion) had adverse affects on growth and PS production. On the other hand, low level aeration was beneficial for increased PS production. Addition of 3 mg/l concentration of methionine, phenylalanine, and threonine were found to enhance growth and PS production. The synerigistic effect of all the favorable conditions observed in pneumococcal growth assays provided a two-fold cummulative increase in capsular PS production.

Production of lipocortin-1_1-185 using a recombinant of escherichia coli: Lee, Kyung Il , Oh, Kyung Hee , Lee, Jung Hyun , Na, Do Sun , Lee, Kye Joon; J. Microbiol. 1997;35(2):123-126.

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Abstract: The aim of the present study was to optimize culture condition for the expression of lipocortin 1_1-185 in a recombinant of Escherichia coli using batch system. Plasmid (pHT22) carrying lipocortin-1_1-185 gene was well maintained in the recombinant with the addition of amplicillin as a selection pressures. Optimum temperature was 28℃ for seed culture and 40℃ for main culture and the optimum pH was 7.0. The production of Lipocortin-1_1-185 was closely associated with cell growth and related to plasmid amplification.

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