Mohammad Shahedur Rahman

Production characteristics of lipopeptide antibiotics in biofilm fermentation of Bacillus subtilis.

In biofilm fermentation, from the very early moments, surfactin was produced along with the biofilm development in the lipopeptide antibiotic production medium by using Bacillus subtilis. However, almost no iturin A was produced in its first 24 hours of cultivation and the production of iturin A began much later. Volumes of the nutrient medium and available surface area of the biofilm reactors were found to be important with the relative production of these two antibiotics. Production of iturin A was increased from 12 mg to about 50 mg per reactor when the culture size was increased from 5 mL to 20 mL, as the depth of the medium was increased. The production level was saturated thereafter with larger volumes. On the other hand, surfactin production was remained similar, which was about 10 mg per reactor, from all the 5 mL to 80 mL of biofilm culture. Optimized temperature for iturin A and surfactin production was observed at 25 and 37°C, respectively. In the biofilm fermentation, production of surfactin was increased when the incubation temperature was increased within the temperature range of 25 to 37°C, on the other hand, iturin A production was gradually decreased with the increase of the incubation temperature.

Production of iturin A homologues under different culture conditions.

Iturin A is a cyclic lipopeptide antibiotic and eight different kinds of iturin A have been reported based on its alkyl side chains. As iturin A is a promising biocontrol agent, total production of iturin A was tried to enhance and comparative production of its homologues was investigated by using different nitrogen and carbon sources. When Polypepton S and defatted soybean meal were used, total production as well as the ratio of the iturin A homologues were similar. However, production of iturin A was relatively lower and also the ratio of the iturin A homologues was different when Polypepton was used, where A2 was decreased and A4 was increased. Production ratio of the iturin A homologues was similar for the carbon sources like maltose, mannitol, sucrose and starch but relative production of iturin A2 was much enhanced compared to A3 when lactose or galactose was used. Interestingly production ratio of A4 was increased and A2 and A3 were decreased when no additional carbon source was used, and similar tendency was observed in the homologue ratio with glucose and fructose. Production of iturin A homologue A6 was significantly increased whereas A2 and A3 were decreased when defatted rapeseed cake was used. Utilization of different amino acids did not show significant differences in their production of the iturin A homologues. Oxygen supply found to be the factor affecting the production of iturin A homologues when it was investigated in a varied culture volume size and shaking speed. A2 found to be increased with increased oxygen supply where the production of A3 was affected inversely.

Experimental Investigation of Effect of Low-Energy Pulsed Atmospheric Electron Beam on Bacterial Cells

In this paper, the effect of a pulsed low-energy electron beam on bacteria has been explored. The experiment has been carried out using an electron beam with a pulse duration of 5 µs and an acceleration voltage of 80 kV. It is shown that homogeneous irradiation with an electron beam decontaminates surfaces. Completely Sterilization is achieved at a low concentration of bacteria. With increasing concentration of bacteria or for a wet target, the survivability of a microorganism increases. A spectroscopic experiment and also scanning electron microscopy (SEM) images show that the low-energy electron beam does not break the outer structure of a bacterial cell and that bacteria inactivation occurs through internal chemical or genetic changes.

Bacterial inactivation using low-energy pulsed-electron beam

In this paper, we report experimental results aimed at the quantitative description of bacterial inactivation using a low-energy (<100 kV) pulsed-electron beam. The initial step was to demonstrate the feasibility of a secondary emission electron gun (SEEG) on the area of sterilization/decontamination, which is quantitatively related to the survival characteristics of bacteria. The survival characteristic of most common type of bacterium Escherichia coli JM 109 (E. coli) was studied in an atmospheric pressure decontamination chamber under the increased gun voltage as well as varied pulsed-electron-beam parameters such as current density, pulse width, and repetition rate. A complete inactivation of E. coli was achieved by a single-electron-beam pulse at an accelerating gun voltage of 85 kV in time duration of 5 /spl mu/s or by five electron beam pulses of the same time duration at a voltage of 77 kV. Several inherent advantages including an efficient bacterial inactivation have been provided as a basis for utilization of SEEG (or low-energy electron beam in general) as a decontamination tool in various biological and medical applications.

Biofilm formation and lipopeptide antibiotic iturin A production in different peptone media

Biofilm fermentation is a newly developed promising technique in fermentation technology. In this study no.3 and no.3S media have been used for the lipopeptide antibiotic iturin A production by Bacillus subtilis RB14. The main component of no.3 and no.3S media is Polypepton and Polypepton S, respectively. B. subtilis RB14 produces thick stable biofilm and high amount of iturin A in no.3S medium. Whereas, impaired biofilm formation and lower iturin A production was observed in no.3 medium. From the analytical information it was observed that the amounts of metal ions, such as K(+), Ca(2+) and Mn(2+), cysteine and cellulose are lower in Polypepton compared to the Polypepton S. To investigate their effect on biofilm formation and iturin A production cysteine, cellulose, K(+), Ca(2+) and Mn(2+) were added respectively into the no.3 medium at similar amount that Polypepton S contains. It was observed that individual addition of K(+), Ca(2+), cysteine and cellulose had no effect on biofilm formation, cellular growth induction or iturin A production. However, when Mn(2+) was supplemented in no.3 medium, biofilm development was restored with an improved production of iturin A. Finally, combined addition of investigated substances into the no.3 medium resulted with highly folded, thick biofilm with high cellular growth and iturin A production compared to the original no.3 medium.

Production of surfactin using pentose carbohydrate by Bacillus subtilis

Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, mass production possibility, selectivity, performance under extreme conditions and potential applications in environmental protection. In this study two pentose sugars (xylose and arabinose) were investigated for the submerged fermentation (SmF) of Bacillus subtilis in surfactant production medium for bio-surfactant surfactin production. An excellent vegetative growth of B. subtilis (× 10(10) CFU/mL) was observed for xylose and arabinose containing medium which were comparable to glucose supplemented medium. Low growth (× 10(8) CFU/mL) was found when medium was not supplemented with any of the sugars. Surfactin production in xylose, arabinose and glucose containing medium was 2700, 2600 and 2000 mg/L, respectively, whereas, medium without any sugar showed low surfactin (700 mg/L) production. These results clearly indicate the effect of pentose sugars on production of surfactin. Gradual depletion of the xylose and arabinose were confirmed by HPLC analysis during the growth phase of the strain that ultimately produced the surfactin.

Inactivation of Cells and Spores of Bacillus subtilis Using Low Energy Pulsed Electron Beam

In this work, we present inactivation kinetics of cells and spores of Gram-positive bacteria, Bacillus subtilis following treatment with a high-power and low-energy pulsed electron beam. We observed a high-degree of bacterial inactivation in both the vegetative cells and spores, when they were subjected to the electron beam of energies 60 to 90 keV at the beam current density of 28 mA/cm2. Interestingly, the survival characteristics showed almost a similar response in both the cases as they are plotted against the energy of the electron beam. We believe that our experimental result is one of the first step towards utilizing this technique as a potential tool for inactivation of spores, which is very much demanding in food industries and biomedical field.

Application of malt residue in submerged fermentation of Bacillus subtilis.

Malt residue is a common waste or byproduct from beer industries after brewing and milling of malted barley. In this work, Bacillus subtilis RB14 was used to study the microbial growth and production of secondary metabolites like lipopeptide antibiotic iturin A in the malt residue for its effective recycling. B. subtilis RB14 could grow in submerged fermentation of malt residue and significant growth (10(9) CFU/mL) was observed without any supplementation. In submerged fermentation iturin A production using malt residue was about 170 mg/L, which was found to be higher than its production in No.3 (Polypepton, glucose, KH2PO4, MgSO4·7H2O) medium where production was about 120 mg/L. More than 600 mg/L of iturin A production was observed when malt residue was combinedly used with No.3 medium. This production was significantly higher than their summation of their individual production. However, the growth of B. subtilis in combined medium was found to be similar to that of the submerged fermentation in simple malt residue. Therefore, the remarkable enhancement in production of iturin A in supplemented malt residue was attributed to the nutrients supplied from No.3 medium.

Improvement of production of lipopeptide antibiotic iturin A using fish protein.

To enhance the production of lipopeptide antibiotic iturin A, nutrient contents of the culture mediums were investigated in both submerged and biofilm fermentations. As a carbon source maltose and as nitrogen source, fish protein was used. In submerged fermentation maltose uptake was found lower (12%) compared to biofilm fermentation (15%) that was associated with higher cellular growth in biofilm. However, requirement of nitrogen (fish protein) concentration was found similar in both submerged and biofilm fermentations. Production of iturin A in submerged fermentation with 12% maltose and 5% fish protein was 4450 mg/L, and in biofilm fermentation it was 5050 mg/L when 15% maltose and 5% fish protein was used.

Study of Submerged and Biofilm Fermentation of Bacillus subtilis using Fish Protein for Production of Lipopeptide Antibiotic Iturin A

Iturin A is an environmentally safe biocontrol agent produced by Bacillus subtilis as a secondary metabolite. Generally iturin A is produced in conventional submerged fermentation. Recently, B. subtilis has received a huge interest for its nature to develop into biofilm as it shows significantly independent genetic and morphological development in biofilm compared to its planktonic culture. In this study it was attempted to compare the production of iturin A in submerged with that in biofilm fermentation using novel marine fish protein as a medium component. When fish protein was compared with commercially available peptones, it was observed that the microbial growth and iturin A productions were similar to those in the medium containing Polypepton S (originated from soybean) and higher than those in the medium containing Polypepton (originated from casein). Quicker cellular growth and secondary metabolite production was observed in submerged fermentation whereas slower but higher cellular growth and iturin A production was found in biofilm fermentation.