High-yield spore production from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> by solid state fermentation

Bacillus licheniformis was grown for 48 h at 37°C in solid state fermentation; a maximum of 1.7 × 10¹¹ spores/g dry substrate were obtained using rice straw powder (300 g/kg) and wheat bran (700 g/kg) supplemented with glucose (40 g/kg), peptone (20 g/kg), yeast extract (20 g/kg), KH₂PO₄ (10 g/kg) and CaO (5 g/kg) with an initial moisture content of 65%.     

Production of haloduracin by <Emphasis Type="Italic">Bacillus halodurans</Emphasis> using solid-state fermentation

Bacillus halodurans was cultivated on wheat bran as a solid-state substrate and produced haloduracin, a bacteriocin, at about 245 AU per wheat bran. Supplementation of the bran with Lauria-Bertani broth decreased haloduracin production. However, production was stimulated by addition of Mg₂SO₄ and K₂HPO₄. The highest production was achieved at a wheat bran/moisture ratio of 1:1.8 and in the presence of 10% (w/w) Na₂CO₃. Under optimum conditions, the organism produced about 3,000 AU per gram dry bran.     

<Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> S-layer protein toxicity against <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

The main toxicity mechanism of Lysinibacillus sphaericus, which is used in the control of mosquitoes, is its binary toxin produced during sporulation; additionally the Mtx1, Mtx2 and Mtx 3 toxins are expressed in vegetative cells. Mosquito larvicidal potency of the S-layer protein that is expressed in vegetative cells has been determined. The protein is similar to other S-layer proteins of mosquitocidal L. sphaericus strains. The LC50 values of the S-layer protein of the L. sphaericus OT4b25, OT4b26, and III(3)7 strains against third-instar larvae of Culex quinquefasciatus were 8.7, 24 and 0.68 μg/ml, respectively. To our knowledge this is the first study showing the mosquito larvicidal potency of the S-layer protein from Lysinibacillus sphaericus.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Production and optimization of polygalacturonase from mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) peel using <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in solid state fermentation

Mango peel is one of the major wastes from fruit processing industries, which poses considerable disposal problems and ultimately leads to environmental pollution. The objective of the current research was to determine the significant parameters on the production of polygalacturonase from mango peel which is a major industrial waste. Solid state culture conditions for polygalacturonase production by Fusarium moniliforme from dried mango peel powder were optimized by Taguchi’s L-18 orthogonal array experimental design methodology. Eight fungal metabolic influencing variables, viz. temperature, mango peel, inoculum, peptone, ammonium nitrate (NH₄NO₃₎, magnesium sulphate (MgSO₄), zinc sulphate (ZnSO₄) and potassium dihydrogen phosphate (KH₂PO₄) were selected to optimize polygalacturonase production. The optimized parameters composed of temperature (30°C), mango peel (6.5%, g, w/v), inoculum (8%, ml, v/v), peptone (1%, g, w/v), NH₄NO₃ (0.60%, g, w/v), MgSO₄ (0.05%, g, w/v), ZnSO₄ (0.06%, g, w/v) and KH₂PO₄ (0.4%, g, w/v). Based on the influence of interaction of fermentation components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. The temperature, inoculum level, mango peel substrate and KH₂PO₄ showed maximum production impact at optimized conditions. From the optimized conditions the polygalacturonase activity was maximized to 43.2 U g⁻¹.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

Ethanol production from hydrolysed agricultural wastes using mixed culture of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis. Acid hydrolysis of fruit and vegetable residues gave 49–84 g reducing sugars l⁻¹ and 29–32 g ethanol l⁻¹ was then obtained. Alkaline hydrolysis did not give significant amount of reducing sugars. Enzymatic hydrolysis of fruit and vegetable residues yielded 36–123 g reducing sugars l⁻¹ and 11–54 g ethanol l⁻¹.     

Efficient Expression of the Mosquito Larvicidal Binary Toxin Gene from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The binary toxin gene encoding BinA (42 kDa) and BinB (51 kDa) from Bacillus sphaericus strain 2297 was cloned and expressed in E. coli. Low expression level was found when both proteins were expressed from a single operon. High expression was observed when the gene encoding an individual protein was placed downstream of the T7 promoter. The expression level of BinB was not different when expressed alone (non-fusion) or as a fusion form with T7 peptide (T7-BinB). Both forms of BinB were equally stable. Unlike BinB, the non-fusion form of BinA was less stable than T7-BinA. The mosquito larvicidal test showed that BinA or BinB alone was not toxic to mosquito larvae, but high toxicity was found when both BinA and BinB were applied. The results suggest that a short peptide of T7 linked to the N-terminus of either BinA or BinB does not affect their toxicity, but may make the toxin, especially BinA, more stable.     

High-productivity lipid production using mixed trophic state cultivation of <Emphasis Type="Italic">Auxenochlorella</Emphasis> (<Emphasis Type="Italic">Chlorella</Emphasis>) <Emphasis Type="Italic">protothecoides</Emphasis>

A mixed trophic state production process for algal lipids for use as feedstock for renewable biofuel production was developed and deployed at subpilot scale using a green microalga, Auxenochlorella (Chlorella) protothecoides. The process is composed of two separate stages: (1) the photoautotrophic stage, focused on biomass production in open ponds, and (2) the heterotrophic stage focused on lipid production and accumulation in aerobic bioreactors using fixed carbon substrates (e.g., sugar). The process achieved biomass and lipid productivities of 0.5 and 0.27 g/L/h that were, respectively, over 250 and 670 times higher than those obtained from the photoautotrophic cultivation stage. The biomass oil content (over 60 % w/DCW) following the two-stage process was predominantly monounsaturated fatty acids (~82 %) and largely free of contaminating pigments that is more suitable for biodiesel production than photosynthetically generated lipid. Similar process performances were obtained using cassava hydrolysate as an alternative feedstock to glucose.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

High-Level Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>, Purification and Mosquito-Larvicidal Activity of the Binary Toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis>

The mosquito-larvicidal binary toxin produced by Bacillus sphaericus consists of two polypeptides: BinA and BinB. Both proteins function together, and maximum toxicity is obtained when both are present in equimolar ratio. Cloning and expression of each component separately in heterologous hosts led to low toxicity of the crystal proteins. To improve the expression level, the purification process, and the activity of the binary toxin, the binA and binB genes were separately cloned in Eschericia coli. Each gene was fused in frame to the glutathione S-transferase (GST) gene to be expressed as GST-fusion protein (GST-BinA and GST-BinB). A high expression level was observed from both constructs, and the fusion proteins exhibited high toxicity to Culex quinquefasciatus larvae. High-purity toxin could be obtained by affinity chromatography. The result suggests that GST moiety facilitates high protein production and enables better solubility of the toxin inclusions inside the larval gut, leading to higher toxicity of the fusion protein.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.