Differential proteomics research of Bacillus amyloliquefaciens and its genome-shuffled saltant for improving fengycin production

Abstract In the previous study, we used genome shuffling to improve fengycin production of the original strain Bacillus amyloliquefaciens ES-2-4. After two rounds of genome shuffling, a high-yield recombinant FMB72 strain that exhibited 8.30-fold increase in fengycin production was obtained. In this study, comparative proteomic analysis of the parental ES-2-4 and genome-shuffled FMB72 strains was conducted to examine the differentially expressed proteins. In the shuffled strain FMB72, 50 differently expressed spots (p < 0.05) were selected to be excised and analyzed using Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry, and finally 44 protein spots were confidently identified according to NCBI database. According to clusters of orthologous groups (COG) functional category analysis and related references, the differentially expressed proteins could be classified into several functional categories, including proteins involved in metabolism, energy generation and conversion, DNA replication, transcription, translation, ribosomal structure and biogenesis, cell motility and secretion, signal transduction mechanisms, general function prediction. Of the 44 identified proteins, signaling proteins ComA and Spo0A may positively regulate fengycin synthesis at transcriptional level. Taken together, the present study will be informative for exploring the exact roles of ComA and Spo0A in fengycin synthesis and explaining the molecular mechanism of fengycin synthesis.

Lipopeptide production by Bacillus subtilis R1 and its possible applications

Abstract The possible application of a bacterial strain - Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30-45 °C and in presence of up to 7% salt. It significantly reduced the surface tension from 66 ± 1.25 mN/m to 29 ± 0.85 mN/m within 24 h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen - ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group - surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33 ± 1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium.

Optimization of surfactin production by Bacillus subtilis ATCC 6633 in a miniaturized bioreactor

In recent years, biosurfactants due to wide applications in chemical, petroleum, food and pharmaceutical industries, have been widely considered by researchers. Biosurfactants are produced by a series of microorganisms, so it is important to screen culture medium and operating conditions in miniaturized bioreactors prior to scaling up to large bioreactors.In this study, using a kind of miniaturized bioreactor called ventilation flask, optimal production conditions, including filling volume and shaking frequency to produce a surfactin-type biosurfactant by Bacillus subtilis ATCC 6633, were examined. Moreover, the effect of oxygen transfer rate [OTR] on the surfactin production was investigated according to Amoabediny and Buchs model. The results indicated that the maximum biomass and biosurfactant yield which obtained under optimal conditions [filling volume of 15 mL and shaking frequency of 300 rpm] were evaluated 0.3 g/L/h and 0.0485 g/L/h, respectively. Also, at the same conditions, the amount of surface tension decreased from 60.5 mN/m to 31.7 mN/m and the maximum oxygen transfer rate [OTR[max]] obtained as 0.01 mol/L/h

Enhanced production of mosquitocidal cyclic lipopeptide from Bacillus subtilis subsp. subtilis.

Background & objectives: A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. Methods: Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC50 value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC50 dosage requirement for the pupal stage of the above mosquito species determined. Results: The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC50 value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 μl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. Interpretation & conclusions: The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant.