The effect of bioindicator preparation and storage on thermal resistance of Bacillus stearothermophilus spores

Paper strips inoculated with spores of Bacillus stearothermophilus ATCC 7953 were conventionally dried (lot 1) and lyophilized (lot 2); stored in defined environments of 32 and 86% relative humidity at 10, 25 and 33°C for 210 d; and submitted to moist heat treatments at 121°C. A significant decrease in thermal resistance from initial starting levels was found for lyophilized bioindicators stored at 86% relative humidity. The respective average D _121°C values were 1.55 ± 0.05 and 1.37 ± 0.10 min for lyophilized bioindicators stored at 32 and 86% relative humidity; and 1.65±0.15min and 1.57 ± 0.11 min for dried bioindicators stored in the same environments.     

Effect of media on spore yield and thermal resistance of Bacillus stearothermophilus

The interference of eight components in the yield of sporulation and thermal resistance to moist heat (121°C) of Bacillus stearothermophilus spores suspended in 0.02 M calcium acetate solution and inoculated on paper strips previously treated with calcium acetate/calcium hydroxide was studied. The spore yield of 1.0×10⁸/mL was developed at 62°C in 17 media containing different concentrations of d-glucose, sodium chloride, l-glutamic acid, yeast extract, peptone, manganese sulfate, potassium phosphate, and ammonium phosphate. The combined effects of yeast extract, peptone, and glucose contributed positively to the spore yield and to the stability of the thermal resistance of both spores in suspension and on strips.     

The effect of pretreatments on surfactin production from potato process effluent by Bacillus subtilis

Pretreatments of low-solids potato process effluent were tested for their potential to increase surfactin yield. Pretreatments included heat, removal of starch particulates, and acid hydrolysis. Elimination of contaminating vegetative cells was necessary for surfactin production. After autoclaving, 0.40 g/L of surfactin was produced from the effluent in 72 h, vs 0.24 g/L in the purified potato starch control. However, surfactin yields per carbon consumed were 76% lower from process effluent. Removal of starch particulates had little effect on the culture. Acid hydrolysis decreased growth and surfactant production, except 0.5 wt% acid, which increased the yield by 25% over untreated effluent.     

Amiodarone interactions with membrane lipids and with growth of Bacillus stearothermophilus used as a model

The thermophilic eubacterium Bacillus stearothermophilus was used as a model to study the effects of amiodarone (2-butyl-3-[3′,5′diido-4′α-diethyl-aminoethoxybenzoyl]-benzofuran) in lipid organization and in bacterial growth. Effects on the structural order of lipids were assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and of the propionic acid derivative 3-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (DPH-PA), probing the outer regions of the bilayer. Amiodarone fluidizes bacterial polar lipid bilayers for temperatures below the phase transition midpoint, and orders the fluid phase of the bacterial polar lipids, as evaluated by DPH and DPH-PA. The ordering and disordering effects, which are concentration dependent, are more extensive when detected by DPH relative to DPH-PA. Growth studies performed in parallel revealed that amiodarone inhibits bacterial growth as a function of concentration. Amiodarone concentrations in the range from 1 to 2.5 μM increased the lag time, decreased the specific growth rate, and decreased the final cell density. Furthermore, 3 μM amiodarone completely inhibited growth. These in vivo effects of amiodarone can be related to its ability to perturb the phospholipid bilayer structure, whose integrity is essential for cell function, viability, and growth.     

Influence of culture conditions on lipopeptide production by Bacillus subtilis

Bacillus subtilis produces various families of lipopeptides with different homologous compounds. To produce “new molecules” with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family. This study focused on two B. subtilis strains cultivated in flasks. Optimized medium for lipopeptide production and Landymedium modified by replacing glutamic acid with other α-amino acids were used. We found that the intensity of production of homologous compounds depends on the strain and the culture medium. Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B. subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium. Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L c15.     

Lipopeptide surfactant production by Bacillus subtilis grown on low-cost raw materials

The production of biosurfactant by Bacillus subtilis ATCC 6633 was investigated using commercial sugar, sugarcane juice and cane molasses, sugarcane juice alcohol stillage, glycerol, mannitol, and soybean oil. Commercial sugar generated the minimum values of surface tension, with the best results (28.7 mN/m, (relative critical micelle concentration [CMC⁻¹] of 78.6) being achieved with 10 g of substrate/L in 48 h. At a pH between 7.0 and 8.0, a higher production of surface-active compounds and a greater emulsifier activity was also observed. Enrichment of the culture medium with trace minerals and EDTA showed maximum yields, whereas supplementation with yeast extract stimulated only cell growth. The kinetic studies revealed that biosurfactant production is a cell growth-associated process; surface tension, CMC, and emulsification index values of 29.6 dyn/cm, 82.3, and 57%, respectively, were achieved, thus indicating that it is feasible to produce biosurfactants from a renewable and low-cost carbon source.     

Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design

Bacillus subtilis S499 is well-known for its ability to produce two families of surfactant lipopeptides: Iturin A and Surfactin S1. Fermentation optimization for this strain was performed to amplify the surfactant production. Ten active variables were analyzed by two successive Plackett-Burman designs, consisting respectively of 12 and 16 experiments to give an optimized medium. The amount of biosurfactant lipopeptides in the supernatant of a culture carried out in this optimized medium was about five times higher than that obtained in nonoptimized rich medium. The analysis of the surfactant molecules produced in such optimized conditions has revealed the presence of a third family of lipopeptides: the fengycins. The time-dependent production of these three families of molecules in bioreactors showed that surfactin S1 is produced during the exponential phase and iturin A and fengycins during the stationary phase.     

Cyclic lipopeptides with fungicidal activityfrom the sea isolate of the bacterium Bacillus subtilis

A mixture of cyclic lipopeptides with fungicidal activity was extracted from the sea isolate of the bacterium Bacillus subtilis (KMM 457). HPLC separation gave two main individual peptides of this mixture (M = 1030 and 1044 Da). According to amino acid analysis and ¹H and ¹³C NMR data, they belong to iturin antibiotics and are cyclic systems composed of the same seven -amino acids (2 Asn, Glu, Tyr, Pro, Thr, and Ser) and one -amino acid (3-aminotetradecanoic or 3-amino-13-methyltetradecanoic acid, respectively). The sequence of amino acids in these peptides was determined for the first time using tandem electrospray ionization mass spectrometry.     

Construction of recombinant Bacillus subtilis for production of polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1, and pBE2C1AB, containing one or two PHA synthse, genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxyde-canoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.     

Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery

Surfactin produced by Bacillus subtilis (ATCC 21332) was used to examine the effect of altering salt concentration, pH, and temperature on surfactin activity (as measured by reductions in surface tension). These parameters are some of the conditions that define oil reservoir characteristics and can affect the application of surfactants. The Biotechnology for Oilfield Operations research program at the Idaho National Engineering and Environmental Laboratory (INEEL) has successfully produced surfactin from potato process effluents for possible use as an economical alternative to chemical surfactants for improved oil recovery. Surfactants enhance the recovery of oil through a reduction of the interfacial tension between the oil and water interfaces, or by mediating changes in the wettability index of the system. We investigated changes in surfactin activity under a range of conditions by measuring surface tension. Surface tension was determined using video image analysis of inverted pendant drops. Experimental variables included NaCl (0–10%), pH (3.0–10.0), and temperature (21–70°C). Each of these parameters, as well as selected combinations, resulted in discrete changes in surfactin activity. It is therefore important to consider the exploration of the studied surfactin as an enhanced oil recovery agent.     

Development of continuous surfactin production from potato process effluent by Bacillus subtilis in an airlift reactor

The biosurfactant surfactin has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery). However, high medium and purification costs limit its use in these high-volume applications. In previous work, we showed that surfactin can be produced from an inexpensive low-solids (LS) potato process effluent with minimal amendments or pretreatments. Previous research has also shown that 95% or more of the surfactin in Bacillus subtilis cultures can be recovered by foam fractionation. In this work, we present the results of research to integrate surfactin production with foam fractionation. Experiments were performed in an airlift reactor, with continuous collection of the foam through a tube at the top of the column. Preliminary results using both purified potato starch and unamended low-solids potato process effluent as substrates for surfactin production indicate that the process is oxygen limited and that recalcitrant indigenous bacteria in the potato process effluent may hamper continuous surfactin production.     

Application of capillary zone electrophoresis to the study of interactions betweenBacillus subtilis tryptophanyl-tRNA synthetase and tRNATrp

Summary The application of capillary zone electrophoresis to the study of interactions betweenBacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) and tRNA^Trp is described. Significant changes in peak shape of tRNA^Trp incubated with TrpRS indicated the occurrence of interactions between TrpRS and tRNA^Trp in pH 8.0 Tris-HCl buffer containing 0.1 mmol L⁻¹ EDTA and 1 mmol L⁻¹−5 mmol L⁻¹ mgCl₂. Addition of Mg²⁺ decreased the electrophoretic mobility of tRNA^Trp, which illustrated that conformation of tRNA^Trp depended on Mg²⁺. The dissociation constant of the TrpRS-tRNA^Trp complex was estimated to be 0.63 μmol L⁻¹ at 25°C in buffer solution.     

Cloning and expression of the limonene hydroxylase of Bacillus stearothermophilus BR388 and utilization in two-phase limonene conversions

A 3.6-kb fragment of Bacillus stearo thermophilus Br388 chromosomal DNA that confers growth on limonene to Escherichia coli has been sequenced, revealing a single open reading frame encoding a single subunit limonene hydroxylase containing 444 amino acid residues. This enzyme proved capable of limonene hydroxylation to a mixture of carveol and perillyl alcohol as well as dehydrogenation of these products to carvone and perillyl aldehyde. Oxygen, FAD, and NADH were found to stimulate the hydroxylation reaction in cell extracts, and NAD⁺ stimulated the dehydrogenase reaction. In two-phase bioconversionsusing viable E. coli cells overexpressing the limonene hydroxylase, perillyl alcohol and carvone were the principal products observed.     

Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium

Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was reali-zed by applying successively ammonium sulfate precipitation, dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration, reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was found that the weight of enzyme was 45 kDa by using SDS-PAGE.     

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 10⁵ Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use. Figure Schematic diagram of the miniature biochip detection system     

Potential Application of Alkaline Pectinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SS in Pulp and Paper Industry

Pectinase production from Bacillus subtilis SS was optimized under solid-state fermentation (5,943 U/g of dry bacterial bran). The pectinase produced was stable in neutral to alkaline pH range at 70 degrees C; therefore, the suitability of this pectinase in pulp and paper industry was investigated. The enzyme pretreatment process was optimized, and a pectinase dose of 5 IU/g of oven-dried pulp (10% consistency) at pH 9.5 temperature 70 degrees C after 150 min of treatment gave the best pretreatment to the pulp. An increase of 4.3% in brightness along with an increase of 14.8 and 65.3% in whiteness and fluorescence, respectively, whereas a 15% decrease in the yellowness of the pretreated pulp were observed. There was a 5.85% reduction in kappa number and 6.1% reduction in permanganate number along with a reduction in the chemical oxygen demand value. Significant characteristics showed by pectinase open new possibilities of application of this cellulase-free enzyme in the pulp and paper industry by reducing the negative environmental impact of chemicals apart from improving the properties of paper.     

The influence of products and by-products obtained by drinking water electrolysis on microorganisms

It is the aim of this work to underline the chemical character of disinfection processes and to present the results of treatment experiments for artificially and electrochemically added chemicals focusing on chlorine components and hydrogen peroxide obtained on IrO₂/RuO₂ anodes. A discontinuous cell with rotating anode placed 4 mm above an IrO₂ expanded mesh cathode of same diameter was used for the generation of electrolysis products. The thermostated experiments were carried out at a temperature of 20 °C and in constant current mode. Artificial solutions prepared using sodium salts and deionised water were electrolysed. Samples were analysed using the DPD (N, N-diethyl-p-phenylendiamine) method. Escherichia coli K-12, Bacillus subtilis DSM 2277 and Saccharomyces cerevisiae Kolin were used as test-microorganisms. Morphological changes were studied using transmission electron microscopy. The results show that electrolysed water had a higher lethal efficiency than Ca(OCl)₂ of the same measured active chlorine concentration. During the killing period mainly inner cell parts of the microorganisms react chemically with the disinfectants. In sufficiently high concentrations and reaction times, the cell material continues to react. Cell walls or membranes can break-through as electron microscopy pictures show. Electrolysed water had a higher lethal efficiency on microorganisms. This behaviour demonstrates the existence of additional by-products.     

Bacillomycin D from the Marine Isolate of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KMM 1922

Strain KMM 1922 of Bacillus subtilis, a producer of the antifungal iturin peptide antibiotic bacillomycin D, was isolated from a specimen of the Kuril sponge Stelletta validissima. The structure of the compound was proved using two-dimensional NMR spectroscopy, tandem electrospray mass spectrometry, and literature data. The peptide was shown to exhibit a pH-dependent cytotoxic activity. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 373–375, July–August, 2005.     

Capacity of Bacillus thuringiensis S-layer protein displaying polyhistidine peptides on the cell surface

S-layer protein of Bacillus thuringiensis strain CTC was used as the carrier protein to display polyhistidine (poly[6His]) peptides on the cell surface. Poly(6His) _n was fused with S-layer protein at two different sites, inserting just downstream of the S-layer protein homologous domain (slh) and replacing the non-slh region of S-layer protein, respectively. The two series chimeric proteins were both expressed by crystal negative B. thuringiensis strain 4Q7 and strain 171, respectively, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant B. thuringiensis cells gained Ni²⁺- and Cd²⁺-binding ability and had a capacity to display up to nine copies of poly(6His). The Cd²⁺ adsorption quantity of the recombinant strain with the strongest adsorption ability was twice that of the host strain.     

The effect ofpH on thermal stability of globular proteins

In this study we try to re-analyze thepH dependence of thermal stability of small globular proteins. From the thermodynamic point of view a long series of calorimetric and spectroscopic investigations has shown that the decreased stability in very acidic conditions can be ascribed to entropic effects. The same conclusion is reached, from a microscopic point of view, by assuming that a binding of protons on equal and noninteracting sites takes place as a consequence of unfolding process. By linking the conformational unfolding equilibrium to the proton binding equilibrium, a model is developed that is able to describe the dependence on thepH of the thermal denaturation processes of small globular protiens. The application of the model to hen lysozyme and T4 lysozyme correctly accounts for the experimental results.