Biosurfactant production by Bacillus subtilis using cassava-processing effluent

A cassava flour-processing effluent (manipueira) was evaluated as a substrate for surfactant production by two Bacillus subtilis strains. B. subtilis ATCC 21332 reduced the surface tension of the medium to 25.9 mN/m, producing a crude biosurfactant concentration of 2.2 g/L. The wild-type strain, B. subtilis LB5a, reduced the surface tension of the medium to 26.6 mN/m, giving a crude biosurfactant concentration of 3.0 g/L. A decrease in surfactant concentration observed for B. subtilis ATCC 21332 seemed to be related to an increase in protease activity. The biosurfactant produced on cassava effluent medium by B. subtilis LB5a was similar to surfactin.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Biological Activities of Thermo-tolerant Microbes from Fermented Rice Bran as an Alternative Microbial Feed Additive

To evaluate the commercial potential of new microbial feed additive, Issatchenkia orientalis Y266 and Bacillus subtilis B266 from commercial fermented rice bran were tested for their tolerance or resistance to pH, bile, oxgall, and temperature. It was found that the strains grew very well up to pH 3.0 and resistant to relatively high concentrations of bile salt and oxgall. I. orientalis and B. subtilis are extremely tolerant in range of 70–90°C in solid medium. B. subtilis B266 also has excellent tolerant property up to 90°C in liquid medium. The health indexes (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the fermented rice bran with these strains (0.25% addition to diet) in comparison with the Avilamycin (20 mg/kg diet)-fed group (p < 0.05). The fermented rice bran-fed group showed a better microbial flora in the small intestines. Accordingly, it would appear that the fermented rice bran with these strains may be a potential candidate for an alternative microbial feed additive.     

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.     

Inactivation by Pulsed Light of Bacillus subtilis Spores with Impaired Protection Factors

The resistance to pulsed light (PL) of spores of Bacillus subtilis strain 168 and of strains with mutations increasing sensitivity to UV‐C or affecting spore structure was evaluated and compared to resistance to continuous UV‐C and moist heat, in order to reveal original mechanisms of inactivation by PL. Spores of B. subtilis strain 168 (1A1) and eight mutant strains (sspA, sspB, sspAB, cotA, gerE, cotE, uvrA and recA) were exposed to PL (up to 1.77 J cm^?2), continuous UV‐C (up to 147 mJ cm^?2) and moist heat at 90°C. Spores of the strains lacking proteins linked to coat formation or structure (cotA, gerE and cotE) were markedly more sensitive to PL than 1A1, while their sensitivity to continuous UV‐C or to moist heat was similar to the one of strain 1A1. Coat proteins had a major contribution to the resistance of B. subtilis spores to PL irradiation characterized by short‐time and high‐energy pulses of white light in the wavelengths 200–1100 nm. In contrast the role of coat proteins to UV‐C or to moist heat resistance was marginal or null.     

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.     

Poly(N-phenyl-2-hydroxytrimethylene amine): Its blends with poly(ϵ-caprolactone) and water-soluble polyethers

A new polymer with pendant hydroxyl groups, namely, poly(N-phenyl-2-hydroxytrime-thylene amine) (PHA), was synthesized by a direct condensation polymerization of aniline and epichlorohydrin in an alkaline medium. The new polymer is amorphous with a glass transition temperature (T_g) of 70°C. Blends of PHA with poly(ϵ-caprolactone) (PCL), as well as with two water-soluble polyethers, poly(ethylene oxide) (PEO) and poly(vinyl methyl ether) (PVME), were prepared by casting from a common solvent. It was found that all the three blends were miscible and showed a single, composition dependent glass transition temperature (T_g). FTIR studies revealed that PHA can form hydrogen bonds with PCL, PEO, and PVME, which are driving forces for the miscibility of the blends. © 1997 John Wiley & Sons, Inc.     

NONRECIPROCAL SYNERGISTIC LETHAL INTERACTION BETWEEN 365-nm and 405-nm RADIATION IN WILD TYPE and uvrA STRAINS OF ESCHERICHIA COLI*

Abstract— Lethality by 405-nm radiation in three repair-proficient and two uvrA strains of Escherichia coli that belong to two isogenic series was greatly enhanced by prior exposures to 365-nm radiation at fluences greater than 1 times 10₆Jm_-2. Fluences at 365 nm that yielded a surviving fraction of 0.10 (>1 times 10₆ Jm_-2) in the 5 strains tested resulted in the following 405-nm fluence enhancement factors (FEF, ratio of the 405-nm F₃₇ in the absence of a prior 365-nm irradiation to that in the presence): strain K.12 AB1157 (wild type), 8.7; strain B/r (wild type), 52; strain WP2 (wild type), 25; strain WP2s (uvrA), 13; strain K.12 AB1886 (uvrA), 15. The maximal 405-nm FEF value obtained after a prior 365-nm irradiation at greater fluences was 83 in the wild-type strain B/r. Enhancement of anoxic 405-nm radiation after a prior aerobic 365-nm exposure was not detectable, suggesting that prior aerobic irradiation at 365-nm increased the effects of damage produced at 405 nm by means of an oxygen-dependent process. Single-strand breaks (or alkali-labile bonds) were produced by 405-nm radiation at 3.0 times 10_-5 breaks per 2.5 times 10₉ daltons per Jm_-2 in the polA strain P3478; pyrimidine dimers were not detected by biological assay (photoreactivation) at 405 nm. Although the introduction of different DNA lesions produced by 365- and 405-nm radiations cannot be ruled out, we propose that the strong synergistic effect of 365-nm irradiation on 405-nm lethality is the consequence of pronounced inhibition by 365-nm radiation of components of the DNA-repair systems that can mend or bypass damage produced by 405-nm radiation.     

<Emphasis Type="Italic">Ferula gummosa</Emphasis> Fruits: An Aromatic Antimicrobial Agent

Ferula gummosa Boiss. (Apiaceae) fruit volatile oil was analyzed by GC/MS. Seventy-three components (96.89%) were identified, and the major components were β-pinene (43.78%), α-pinene (27.27%), and myrcene (3.37%). The antimicrobial activity of the oil was tested on three strains of Gram positive bacteria (Staphylococcus aureus, S. epidermis, and Bacillus subtilis), three strains of Gram negative bacteria (Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa), and two strains of fungi (Candida albicans and C. kefyr). The essential oil remarkably inhibited the growth of the tested microorganisms. The results indicate that the fruits have potential for use as an aromatic antimicrobial agent.__________Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 252–254, May–June, 2005.     

Mechanism of Disulfide–Disulfide Interchange in Polysulfide Polymer Melt Blend by Electron Ionization Mass Spectrometry. II

Abstract

The degree of randomization, q, of structural units in melt blends of the polysulfide homopolymers A(PS1) and B(PS2), wherein the disulfide equivalents D _A/D _B = 1, were studied by electron ionization mass spectrometry. Over the temperature range of 207–219°C, the relaxation process, due to the dominant disulfide–disulfide interchange reactions, is postulated to follow an associative reaction mechanism. These intermolecular disulfide–disulfide interactions promote a transient enhancement of the sulfur rank in the activated complex resulting in formation of the randomized co‐polymer AB. The mass spectrometric experimental design enabled measurement of concentrations of reactants A(PS1) and B(PS2), as well as the randomized copolymer AB, by monitoring the abundance of dimer units a₂, b₂, and ab, respectively as a function of time. The degree of randomization, q, was observed in the absence of catalysts or solvents, notwithstanding the solvent/solute and solute/solvent characteristics of the polymer melt blend. The mechanism of this randomization process, was rationalized on the basis of the properties of sulfur, aided by the observation of macrocyclic monomeric and dimeric units during the retro‐polymerization reactions under the EI/MS conditions employed. The model polysulfide polymers A(PS1) and B(P52), used in this study were synthesized from bis(2‐chloroethyl)ether and bis(2‐chloro ethoxy)methane, respectively.     

The effect of composition of parenteral solution on the thermal resistance of Bacillus stearothermophilus and Bacillus subtilis spores

Large-volume parenteral solutions were submitted to heat treatments after being inoculated with Bacillus stearothermophilus ATCC 7953 (T _r =121°C) and Bacillus subtilis ATCC 9372 (T _r =104.5°C) spores. The average decimal reduction time for B. stearothermophilus ranged from a D _121°C value of 1.31 to 3.14 min, in glucophysiologic and Ringer’s solutions respectively. For B. subtilis, D _104.5°C value increased from 0.69 to 1.37 min, in Ringer’s (pH=5.91) and 50% glucose (pH 3.05) solutions respectively. The z value ranged from 7.95°C (20% mannitol solution) to 13.14°C (50% glucose solution), corresponding to an activation energy (Ea) of 81.48 and 49.30 kcal/mol, respectively.     

Synthesis of a highly transparent poly(o‐hydroxyamide) in the i‐line region and its application to photosensitive polymers

A novel poly(σ‐hydroxyamide) (PHA) based photosensitive polymer that exhibits high transparency at 365 nm wavelength (i‐line) has been developed. Time‐dependent density functional theory (TD‐DFT) calculations using the B3LYP hybrid functional were performed to predict the transparencies of various hydroxyamides in the i‐line region. Based on the calculations, 4,4′‐sulfonylbis(σ‐aminophenol) (SAP) was prepared and polymerized with 4,4′‐oxybis(benzoyl chloride) (OBBC), and the resulting PHA, which is abbreviated as PHA‐S, showed a high transparency comparable to that of PHA derived from 4,4′‐(hexafluoroisopropylidene)bis(σ‐aminophenol). Positive‐type photosensitive PHA was then formulated based on PHA‐S with a crosslinker 1,3,5‐tris[(2‐vinyloxy)ethoxy]benzene (TVEB) and a photoacid generator (5‐propylsulfonyloxyimino‐5H‐thiophen‐2‐ylidene)‐2‐(methylphenyl)acetonitrile (PTMA) (17:3:1 in weight ratio), and demonstrated photosensitivity and contrast of 14 mJ/cm² and 2.7, respectively, when the resist film was prebaked at 120 °C for 5 min, irradiated by i‐line, post exposure baked at 120 °C for 5 min, developed with an 2.38 wt% TMAH solution for 5 s. A clear positive image featuring 10‐μm line‐and‐space was also printed in a film which was exposed to 50 mJ/cm² of i‐line by contact‐printing. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2527–2535, 2005     

Synthesis and antimycobacterial screening of new 4-(4-(1-benzyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-3-yl)quinoline derivatives

A new series of 4-(4-(1-benzyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-3-yl)quinoline ( 6a-t ) have been synthesized by a click reaction of 4-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)quinoline ( 4a-d ) with a substituted benzyl azide ( 5a-e ). The starting alkyne derivatives 4a-d are obtained from Bestmann-Ohira reaction of 1-phenyl-3-(quinolin-4-yl)-1H-pyrazole-4-carbaldehyde and dimethyl(1-diazo-2-oxopropyl)phosphonate. The newly synthesized compounds are screened against M. tuberculosis H37Ra dormant and active, Escherichia coli, Pseudomonas fluorescence, Staphylococcus aureus and Bacillus subtilis strains at 30 μg/mL concentration. Most of the screened compounds showed good to moderate antibacterial activity against S. aureus, B. subtilis, and Mycobacterium tuberculosis H37Ra strains. The synthesized derivatives of quinolinyl-pyrazole-4-carbaldehyde and quinolinyl-pyrazole-4-ethyne reportd good to moderate activity against both strains of M. tuberculosis H37Ra. Ten derivatives of quinolinyl-pyrazole presented good activity against B. subtilis. These results suggested that further optimization and development of quinolinyl-pyrazolyl-1,2,3-triazole moeity could serve as lead compounds for antimycobacterial activity.     

Investigation on the eigenvalue-equation problem of molecular crystals and polymers

One-dimensional periodic system, such as molecular crystal and polymer, can be expressed as … ABABAB … structure, where A and B stand for a complete molecule or a part of a molecule. When (AB) is taken as a structure unit, one can obtain the complex generalized eigenvalue-equation H^AB (k) - C^AB (k) - S^AB (k) C^AB (k) E^AB (k); and if (BA) is taken as a structure unit, the corresponding eigenvalue-equation is H^BA (k) C^BA (k) - S^BA (k) C^BA (k) E^BA (k). The relationship between the two equations has been investigated. The results of theoretical analysis are $ E_m^{{\rm BA}} (k) = E_m^{{\rm AB}} (k);\quad C_{j_{\rm a} m}^{{\rm BA}} (k) - C_{j_{\rm a} m}^{{\rm AB}} (k) \cdot \exp (i\Delta \phi) $ and $ C_{j_{\rm b} m}^{{\rm BA}} (k)\quad C_{j_{\rm b} m}^{{\rm AB}} (k) \cdot \quad \exp [i(k.R_{ - 1} + \Delta \phi)] - C_{j_{\rm b} m}^{{\rm AB}} (k) \times \exp (i\Delta \phi) $ where j_a and j_b are the index number of atomic orbitals within (A) and (B) respectively, and m stands for the index number of crystal or polymer orbitals. This result has been verified by the concrete calculation of three periodic systems: (1) hydrogen-molecular chain, …H (A) H (B) … H (A) H (B) …, (2) polyphenylene, … (A) (B) (A) (B) …, where (A) and (B) stand for =C (CH=)₂ and ( HC)₂ C= respectively, and (3) the TCNQ molecular column, … TCNQ (A). TCNQ (B) … TCNQ (A). TCNQ(B) … . The results can be generalized to two- and three-dimensional systems straightforwardly.     

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

Antibacterial mechanism of houttuyfonate homologues against Bacillus subtilis

In this study the effects of houttuyfonate homologues (HOU-C_n) on the surface and shape, the lipid fluidity and the protein conformation, the fatty acid compositions and fatty acid synthase II (FAS II) of B. subtilis were studied to elucidate the antibacterial mechanism of HOU-C_n against Bacillus subtilis. The scanning electronic microscope (SEM) results showed that the glycocalyx on the surface of B. subtilis disappeared and the cell became smaller after being treated with HOU-C_n. During the co-incubation of HOU-C_n and B. subtilis, HOU-C_n was decomposed into alkyl acyl aldehyde and sodium sulfite rapidly. Then alkyl acyl aldehyde was congregated onto cell membrane and inserted into lipid bilayer, further increased the fluidity of membrane and changed the conformation of membrane protein by hydrophobic binding. Subsequently, HOU-C_n inhibited the FAS II activity, and decreased the synthesis of fatty acids, especially increased the percentage of saturated fatty acid. HOU-C_n showed a strong antibacterial activity against G⁺-bacteria by a multi-target antibacterial mechanism.     

Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates

Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism. Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs. The final dried cell mass and specific polymer production of A. latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, responctively. These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.     

Synthesis of multiblock hyperbranched‐linear poly(ether sulfone) copolymers

Hyperbranched poly(ether sulfone) was prepared in the presence of an oligomeric linear poly(ether sulfone) to generate multiblock hyperbranched‐linear (LxHB) copolymers. The LxHB copolymers were prepared in a two‐step, one‐pot synthesis by first polymerizing AB monomer to generate a linear block of a desired molecular weight followed by addition of the AB₂ monomer in a large excess (19:1, AB₂:AB) to generate the hyperbranched block. NMR integration analysis indicates that AB₂:AB ratio is independent of the reaction time. Because the molecular weight still increases with reaction time, these results suggest that polymer growth continues after consumption of monomer by condensation into a multiblock architecture. The LxHB poly(ether sulfone)s have better thermal stability (10% mass loss > 343 vs. 317 °C) and lower T_g (200 vs. > 250 °C) than the hyperbranched homopolymer, higher T_g than the linear homopolymer (<154 °C), while little difference in the solubility character was observed between the two polymers. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4785–4793, 2008     

Synthesis and antibacterial activities of copper(II) with [(2-hydroxy-3,5-diiodo-benzylidene)-amino]-acetic acid

A new tridentate ligand, [(2-hydroxy-3,5-diiodo-benzylidene)-amino]-acetic acid (HDBA), has been synthesized from 3,5-diiodosalicylaldehyde and glycine in ethanol. A 1-D coordination polymer has been synthesized by reaction of HDBA with Cu(Ac)₂ · H₂O in ethanol–water. The complex was characterized by UV, IR, ESI–MS, elemental analyses, and X-ray crystallography. The central copper(II) is five-coordinate by one nitrogen and two oxygens from HDBA, one oxygen from water, and one oxygen from another HDBA. The complex was assayed for antibacterial (Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter cloacae) activities by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. Complex 1 showed favorable antimicrobial activity with minimum inhibitory concentrations of 6.25, 6.25, 3.125, 6.25, 6.25, and 3.125 µg mL^?1 against B. subtilis, S. aureus, S. faecalis, P. aeruginosa, E. coli, and E. cloacae, respectively.     

Antimicrobial properties of modified and electrospun poly(vinyl phenol)

Antimicrobial polymeric systems were prepared from poly(vinyl phenol) (PVP). Four systems were prepared, two of these based on the modification of the poly(vinyl phenol) by sulfonation with fuming sulfuric acid (SPVP 100k) or by formation of lithium salt of the sulfonated poly(vinyl phenol) brought about by its reaction with lithium hydroxide (LiSPVP 100k). The other two systems were prepared by the electrospinning of poly(vinyl phenol) with molecular weight 20 × 10³ (PVP 20k spun) and 100 × 10³ (PVP 100k spun). The antimicrobial activity of the polymers was examined against different test microorganisms. The plug-cutting technique revealed the potency of SPVP 100k and LiSPVP 100k as antimicrobial agents. SPVP 100k was inhibitory to the growth of gram-negative bacteria (E. coli and Salmonella choleraesius) and gram-positive bacteria (B. subtilis and S. aureus). On the other hand, LiSPVP 100k had antifungal activity against A. niger, T. rubrum and C. albicans. Generally, it was found that polymer morphology and molecular weight affect the activities against test microorganisms. For example, PVP 20k and PVP 100k in their powder form showed no antimicrobial activity. However the results showed that PVP 20k spun has antibacterial activity against B. subtilis, and there is no growth of the tested microorganisms on the electrospun fibers of PVP 100k spun, revealing its property of being a self-sterilizing material (SSM).

Growth inhibition of different concentrations of polymer SPVP. Inoculation: 6 × 10⁴ cells · ml⁻¹, B. subtilis, S. choleraesius, S. aureus and E. coli.