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Aneurinibacillus thermoaerophilus CCM 8960 is a thermophilic bacterium isolated from compost in Brno. The bacterium accumulates polyhydroxyalkanoates (PHAs), a biodegradable and renewable alternative to petrochemical polymers. The bacterium reveals several features that make it a very interesting candidate for the industrial production of PHA. At first, due to its thermophilic character, the bacterium can be utilized in agreement with the concept of next-generation industrial biotechnology (NGIB), which relies on extremophiles. Second, the bacterium is capable of producing PHA copolymers containing a very high portion of 4-hydroxybutyrate (4HB). Such materials possess unique properties and can be advantageously used in multiple applications, including but not limited to medicine and healthcare. Therefore, this work focuses on the in-depth characterization of A. thermoaerophilus CCM 8960. In particular, we sequenced and assembled the genome of the bacterium and identified its most important genetic features, such as the presence of plasmids, prophages, CRISPR arrays, antibiotic-resistant genes, and restriction-modification (R-M) systems, which might be crucial for the development of genome editing tools. Furthermore, we focused on genes directly involved in PHA metabolism. We also experimentally studied the kinetics of glycerol and 1,4-butanediol (1,4BD) utilization as well as biomass growth and PHA production during cultivation. Based on these data, we constructed a metabolic model to reveal metabolic fluxes and nodes of glycerol and 1,4BD concerning their incorporation into the poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) structure. KEY POINTS: • Aneurinibacillus sp. H1 was identified as Aneurinibacillus thermoaerophilus. • PHA metabolism pathway with associated genes was presented. • Unique monomer composition of produced PHAs was reported.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-β-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

A β-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

Phytases hydrolyze phytate to release inorganic phosphate, which decreases the requirement for phosphorus in fertilizers for crops and thus reduces environmental pollutants. This study analyzed microbial communities in rhizosphere sediment, collected in September 2012 from Shenzhen Bay, Guangdong, China, using high-throughput pyrosequencing; the results showed that the dominant taxonomic phyla were Chloroflexi, Firmicutes, and Proteobacteria, and the proportion of the beneficial bacteria, Bacillus, was 4.95 %. Twenty-nine culturable, phytase-producing bacteria were isolated, their phosphorus solubilization capacity was analyzed, and they were taxonomically characterized. Their phylogenetic placement was determined using 16S ribosomal RNA (rRNA) gene sequence analysis. The result shows that most of the isolates are members of the order Bacillales, although seven strains of Enterobacteriales, two strains of Pseudomonadales, and one strain of Oceanospirillales were also identified. The phytase gene was cloned from SPC09, Bacillus cereus, which showed the highest phosphorus solubilizing ability among the isolated strains. The gene encoded a primary translation product of 335 amino acids. A construct including the 1005-nt ORF fragment, Bc-phy, was transformed into Escherichia coli. The recombinant phytase was produced and purified, which revealed the temperature optima at 60 °C and pH optima at 6.5. The assessment by quantitative PCR (qPCR) showed an abundance of bacteria containing the Bc-phy gene; the level was generally higher in the mangrove forest than in the tidal flats and in surface soil compared to bottom soil, and the highest value was obtained in June. Herein, we report on the cloning, characterization, and activity of a novel phytase isolated from a mangrove system.

A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg

Sporolactobacillus inulinus is a superior d-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for d-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and d-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na+ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO3. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na+-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the d-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na+-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient d-lactic acid production with easy subsequent purification.

A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

3-Phenyllactic acid (PLA) is an antimicrobial compound with broad-spectrum activity against bacteria and fungi that could be widely used in the food industry and livestock feeds. Notably, d-PLA exhibits higher antibacterial activity, which gains more attention than l-PLA. In this report, the d-lactate dehydrogenase DLDH744 from Sporolactobacillus inulinus CASD was engineered to increase the enzymatic activities toward phenylpyruvate by protein structure-guided modeling analysis. The phenylpyruvate molecule was first docked in the active center of DLDH744. The residues that might tightly pack around the benzene ring of phenylpyruvate were all selected for mutation. The single site mutant M307L showed the highest increased activity toward bulkier substrate phenylpyruvate than the wild type. By using the engineered d-lactate dehydrogenase M307L expressed in Escherichia coli strains, without coexpression of the cofactor regeneration system, 21.43 g/L d-PLA was produced from phenylpyruvate with a productivity of 1.58 g/L/h in the fed-batch biotransformation process, which ranked in the list as the highest production titer of d-PLA by d-lactate dehydrogenase. The enantiomeric excess value of produced d-PLA in the broth was higher than 99.7 %. Additionally, the structure-guided design of this enzyme will also provide referential information for further engineering other 2-hydroxyacid dehydrogenases, which are useful for a wide range of fine chemical synthesis.

During operation of mobile air conditioning (MAC) systems in automobiles, malodours can occur. We studied the microbial communities found on contaminated heat exchanger fins of 45 evaporators from car MAC systems which were operated in seven different regions of the world and identified corresponding volatile organic compounds. Collected biofilms were examined by scanning electron microscopy and fluorescent in situ hybridization. The detected bacteria were loosely attached to the metal surface. Further analyses of the bacteria using PCR-based single-strand conformation polymorphism and sequencing of isolated 16S rRNA gene fragments identified highly divergent microbial communities with multiple members of the Alphaproteobacteriales, Methylobacteria were the prevalent bacteria. In addition, Sphingomonadales, Burkholderiales, Bacillales, Alcanivorax spp. and Stenotrophomonas spp. were found among many others depending on the location the evaporators were operated. Interestingly, typical pathogenic bacteria related to air conditioning systems including Legionella spp. were not found. In order to determine the nature of the chemical compounds produced by the bacteria, the volatile organic compounds were examined by closed loop stripping analysis and identified by combined gas chromatography/mass spectrometry. Sulphur compounds, i.e. di-, tri- and multiple sulphides, acetylthiazole, aromatic compounds and diverse substituted pyrazines were detected. Mathematical clustering of the determined microbial community structures against their origin identified a European/American/Arabic cluster versus two mainly tropical Asian clusters. Interestingly, clustering of the determined volatiles against the origin of the corresponding MAC revealed a highly similar pattern. A close relationship of microbial community structure and resulting malodours to the climate and air quality at the location of MAC operation was concluded.

Bacillus megaterium was used for production of the lysozyme-specific recombinant scFv D1.3 antibody fragment. Key process parameters like the temperature and the hydromechanical stress play a very important role for significant product formation during process development or scale-up. In this study, the influence of these two variables on growth and recombinant antibody fragment production in a 2-L lab-scale bioreactor system was investigated using a central composite design. Especially a significant influence of the hydromechanical stress on antibody fragment production was detected in batch cultivations. While volumetric power inputs of about 0.5 kW/m(3) (agitation rates around 500 min(-1)) are usually employed in batch cultivations, in this work maximal product concentration was found at a volumetric power input of about 0.06 kW/m(3) (agitation rate around 250 min(-1)) and at a high cultivation temperature of 41 °C. The influence of the two process variables at single-cell level was estimated using flow cytometry too. The characterization was done by estimating the membrane potential giving a hint on bioprocess productivity and secretion capability: the best production was obtained through big cells with low specific membrane potential, which grew at low volumetric power inputs and high cultivation temperatures.

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.

Highly efficient D-lactate production by Sporolactobacillus sp. strain CASD was demonstrated in this study. Peanut meal was found to be a better nutrient than yeast extract, soybean meal, soybean peptone, corn steep, liquor beef extract, and ammonium sulfate in the production of D-lactate. To improve the utilization of peanut meal, the material was enzymatically hydrolyzed and simultaneously utilized as the nitrogen source in D-lactate fermentation. Very high D-lactate production (207 g/L) was obtained using 40 g/L of peanut meal in 30-L fed-batch fermentation, with the average productivity of 3.8 g/(L·h) and optical purity of 99.3%. The production of such a high concentration of optically pure D-lactate by strain CASD, with the simultaneous enzymatic hydrolysis of peanut meal and fermentation, represents a new cost-efficient and integrated method for D-lactate production using agricultural by-products.

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na+ and K+ in the concentration range of 1–100 mM as well as the divalent metal cations Mg2+, Mn2+, and Ca2+ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.

Recombinant Escherichia coli harboring the L: -arabinose isomerase (BLAI) from Bacillus licheniformis was used as a biocatalyst to produce L: -ribulose in the presence of borate. Effects of substrate concentration, the borate to L: -arabinose ratio, pH, and temperature on the conversion of L: -arabinose to L: -ribulose were investigated. L: -Ribulose production was efficient when pH was higher than 9 and temperature was higher than 50 degrees C. Borate addition to the reaction mixture was essential for high conversion of L: -arabinose to L: -ribulose as it resulted in an equilibrium shift in favor of the product. Under the optimal conditions determined by response surface methodology, the E. coli harboring BLAI produced 375 g l(-1) L-ribulose from 500 g l(-1) L: -arabinose at a reaction time of 60 min, corresponding to a conversion yield of 75% and productivity of 375 g l(-1) h(-1). When the resting recombinant E. coli cells were recycled, 85% of the yield was obtained even after seven cycles of reuse. The productivity and final concentration of L: -ribulose obtained in the present study were the highest yet reported.

Marine microbes are a rich source of bioactive compounds, such as drugs, enzymes, and biosurfactants. To explore the bioactive compounds from our marine natural product library, an oil emulsification assay was applied to discover biosurfactants and bioemulsifiers. A spore-forming bacterial strain from sea mud was found to produce bioemulsifiers with good biosurfactant activity and a broad spectrum of antimicrobial properties. It was identified as Bacillus velezensis H3 using genomic and phenotypic data analysis. This strain was able to produce biosurfactants with an optimum emulsification activity at pH 6.0 and 2% NaCl by using starch as the carbon source and ammonium sulfate as the nitrogen source. The emulsification-guided isolation and purification procedure led to the discovery of the biosurfactant components, which were mainly composed of nC(14)-surfactin and anteisoC(15)-surfactin as determined by NMR and MS spectra. These compounds can reduce the surface tension of phosphate-buffered saline (PBS) from 71.8 to 24.8 mN/m. The critical micelle concentrations (CMCs) of C(14)-surfactin and C(15)-surfactin in 0.1 M PBS (pH 8.0) were determined to be 3.06 x 10(-5) and 2.03 x 10(-5) mol/L, respectively. The surface tension values at CMCs for C(14)-surfactin and C(15)-surfactin were 25.7 and 27.0 mM/m, respectively. In addition, the H3 biosurfactant exhibited antimicrobial activities against Staphyloccocus aureus, Mycobacterium, Klebsiella peneumoniae, Pseudomonas aeruginosa, and Candida albicans. Thus B. velezensis H3 is an alternative surfactin producer with potential application as an industrial strain for the lipopeptide production.

Corynebacterium glutamicum was genetically engineered to produce L-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD+ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of L-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l(-1) of L-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.

Improvement of acid tolerance and production of D-lactic acid by Sporolactobacillus inulinus ATCC 15538 was performed by using recursive protoplast fusion in a genome shuffling format. The starting population was generated by ultraviolet irradiation, diethyl sulfate mutagenesis, and pH-gradient filter and then, subjected for the recursive protoplast fusion. The concentration of lysozyme, time, and temperature for enzyme treatment were optimized by response surface methodology based on the central composite design. Based on contour plots and variance analysis, the model predicted a maximum Y (multiply protoplasts formation ratio by protoplasts regeneration ratio), 60.4%, and the corresponding above used values were 7.75 mg/ml lysozyme, 1.59 h, and 38 degrees C. A pH-5-resistant recombinant, F3-4, was obtained after three rounds of genome shuffling and its production of D-lactic acid reached 93.4 g/l in a 5 L bioreactor, which was increased by 39.8% and 119% in comparison with that of UV generated strain and the original strain S. inulinus ATCC 15538, respectively. The subculture experiments indicated that F3-4 was genetically stable.

MAN5, the main extracellular saccharide hydrolase from Bacillus sp. MSJ-5, is an endo-beta-mannanase with a demand of at least five sugar moieties for effective cleavage. It has a pH optimum of 5.5 and a temperature optimum of 50 degrees C and is stable at pH 5-9 or below 65 degrees C. MAN5 has a very high ability to hydrolyze konjac flour, 10 U/mg of which could completely liquefy konjac flour gum in 10 min at 50 degrees C. HPLC analysis showed that most glucomannan in the konjac flour was hydrolyzed into a large amount of oligosaccharides with DP of 2-6 and a very small amount of monosaccharide. With the culture supernatant as enzyme source, the optimum condition to prepare oligosaccharides from konjac flour was obtained as 10 mg/ml konjac flour incubated with 10 U/mg enzyme at 50 degrees C for 24 h. With this condition, more than 90% polysaccharides in the konjac flour solution were hydrolyzed into oligosaccharides and a little monosaccharide (2.98% of the oligosaccharides). Konjac flour is an underutilized agricultural material with low commercial value in China. With MAN5, konjac flour can be utilized to generate high value-added oligosaccharides. The high effectiveness and cheapness of this technique indicates its potential in industry.

In this work, two biosurfactant-producing strains, Bacillus subtilis and Bacillus licheniformis, have been characterized. Both strains were able to grow at high salinity conditions and produce biosurfactants up to 10% NaCl. Both extracted-enriched biosurfactants showed good surface tension reduction of water, from 72 to 26-30 mN/m, low critical micelle concentration, and high resistance to pH and salinity. The potential of the two lipopeptide biosurfactants at inhibiting biofilm adhesion of pathogenic bacteria was demonstrated by using the MBEC device. The two biosurfactants showed interesting specific anti-adhesion activity being able to inhibit selectively biofilm formation of two pathogenic strains. In particular, Escherichia coli CFT073 and Staphylococcus aureus ATCC 29213 biofilm formation was decreased of 97% and 90%, respectively. The V9T14 biosurfactant active on the Gram-negative strain was ineffective against the Gram-positive and the opposite for the V19T21. This activity was observed either by coating the polystyrene surface or by adding the biosurfactant to the inoculum. Two fractions from each purified biosurfactant, obtained by flash chromatography, fractions (I) and (II), showed that fraction (II), belonging to fengycin-like family, was responsible for the anti-adhesion activity against biofilm of both strains.

The original publication is available at www.springerlink.com, Article, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 82(4):741-748 (2009)

Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth and has toxicity against a wide spectrum of lepidopteran insects. Unlike insecticidal crystal proteins, which are produced as parasporal crystal proteins within the cell during sporulation, VIP is secreted into the culture medium. Here, we show that Vip3Aa7 protein can be relocated into the mother cell of B. thuringiensis by altering its synthesis using cry1C promoters, combined with a cry1C transcription termination sequence at the 3' region and a STAB-SD sequence from cry1C promoters at the 5' region of the gene. Further, when the carboxy-terminal half of Cry1C was included in the construct, the synthesis of Vip3Aa7 markedly increased, and its expression was relocated into the mother cell in the form of inclusion bodies. The expression of Vip3Aa7 with higher yields in the form of inclusion bodies demonstrated here would facilitate the development of a suitable formulation for the application of this class of insecticidal protein in the field, and the described system offers an additional method for potentially improving the efficacy of insecticides based on B. thuringiensis.

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g(-1) dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l(-1)). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root-bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.

A novel dichloromethane (DCM)-degrading bacterial strain named WZ-12 (GenBank accession no. EF100968) was isolated and identified as Bacillus circulans based on standard morphological and physiological properties and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. DCM dehalogenase from B. circulans WZ-12 was purified to 8.27-fold with a yield of 34.83%. The electrophoretically homogeneous-purified enzyme exhibited a specific activity of 118.82 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified DCM dehalogenase gave a distinct band with an estimated molecular mass of 20,000 +/- 1,000.

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 mug/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.

In this work, a 2(2) factorial design was employed combining with response surface methodology (RSM) to optimize the medium compositions for the production of alkaline beta-mannanase by alkaliphilic Bacillus sp. N16-5 isolated previously from sediment of Wudunur Soda Lake in Inner Mongolia, China. The central composite design (CCD) used for the analysis of treatment combinations showed that a second-order polynomial regression model was in good agreement with experimental results, with R (2) = 0.9829 (P0.05). The maximum activity was obtained at NaCl concentration (84.4 g l(-1)) and sodium glutamate (3.11 g l(-1)) and a high medium pH around 10.0. Under such conditions, the activity of alkaline beta-mannanase achieved 310.1 U/ml in the scale of 5-l fermenter, which was increased nearly twice compared with the original. Through optimization, the substrates shifted from the expensive substrates, such as locust bean gum and peptone, to the inexpensive ones such as konjac powder, soymeal, and sodium glutamate. The experiment results also suggested that the environmental conditions of high salinity and high alkalinity, as well as the inducer substrates, play very important roles in the production of the alkaline beta-mannanase by alkaliphilic Bacillus sp. N16-5.

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R(f) value of 0.52 from B. subtilis C9 or authentic surfactin (R(f) = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.

The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb+ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb- strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb+ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb- strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.

An agar-degrading bacterium, strain LGH, was isolated and identified as Cohnella sp. This strain had a capability of utilizing agar as a sole carbon source for growth and showed a strong agarolytic activity. A novel endo-type β-agarase gene agaW, encoding a primary translation product of 891 amino acids, including a 26 amino acid signal peptide, was cloned and identified from a genomic library of strain LGH. The AgaW belonged to the glycoside hydrolase (GH) GH50 family, with less than 39 % amino acid sequence similarity with any known protein, and hydrolyzed agarose into neoagarotetraose as the major end product and neoagarobiose as the minor end product through other neoagarooligosaccharide intermediates, such as neoagarohexaose.

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of approximately 38 kDa having activity against Colorado potato beetle (CPB). An oligonucleotide probe based on the N-terminal sequence of the purified Sip1A protein was used to isolate the sip1A gene. The sequence of the Sip1A protein, as deduced from the sequence of the cloned sip1A gene, contained 367 residues (41,492 Da). Recombinant B. thuringiensis and Escherichia coli harboring cloned sip1A produced Sip1A protein which had insecticidal activity against larvae of CPB, southern corn rootworm (Diabrotica undecimpunctata howardi), and western corn rootworm (Diabrotica virgifera virgifera).

Sclerotinia sclerotiorum causes serious yield losses in oilseed crops worldwide. Bacillus subtilis Tu-100 significantly reduced (Por =0.05) the incidence of disease caused by S. sclerotiorum on oilseed rape at harvest in two trials conducted in fields artificially infested with this pathogen. Mean plant dry weight was significantly greater (Por =0.05) and mean plant length was significantly greater (Por =0.07) at the seven-true-leaf stage with the Tu-100 treatment than with the control. Mean seed yield per 120 plants at harvest was significantly greater (Por =0.05) in the second field trial with treatments containing isolate Tu-100. B. subtilis Tu-100 also promoted the growth of hydroponically grown oilseed rape. Plants were approximately 15% greater in dry weight (Por =0.0001) and 6% greater in length (Por =0.0025) when grown in the presence of isolate Tu-100 in Hoagland's solution, compared with the noninoculated control. In gnotobiotic studies, the lacZ-tagged strain B. subtilis Tu-100(pUC18) was detected within all roots of oilseed rape. Isolate Tu-100 did not persist in the ectorhizosphere of oilseed rape. Populations of this isolate decreased from 8.5x10(8) colony-forming units (CFU) per seed to approximately 10(2) CFU in the plant ectorhizosphere within 30 days of sowing in autoclaved soil.

To improve the production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633, two genetic engineering strategies were followed. Firstly, additional copies of subtilin self-protection (immunity) genes spaIFEG have been integrated into the genome of the producer strain. Their expression significantly enhanced the subtilin tolerance level, and concomitantly, the subtilin yield 1.7-fold. Secondly, a repressor of subtilin gene expression, the B. subtilis general transition state regulator protein AbrB, was deleted. A sixfold enhancement of the subtilin yield could be achieved with the abrB deletion mutant; however, the produced subtilin fraction predominantly consists of succinylated subtilin species with less antimicrobial activity compared to unmodified subtilin.

Bacillus subtilis C9 effectively degrades aliphatic hydrocarbons up to a chain length of C19 and produces a lipopeptide-type biosurfactant, surfactin, yet it has no genetic competency. Therefore, to obtain a transformable surfactin producer, the sfp gene cloned from B. subtilis C9 was integrated into the chromosome of B. subtilis 168, a non-surfactin producer, by homologous recombination. The transformants reduced the surface tension of the culture broth from 70.0 mN/m to 28.0 mN/m, plus the surface-active compound produced by the transformants exhibited the same Rf value as that from B. subtilis C9 and authentic surfactin in a thin-layer chromatographic analysis. The integration of the sfp gene into the chromosome of B. subtilis 168 was confirmed by Southern hybridization. Like B. subtilis C9, the transformants readily degraded n-hexadecane, although the original strain did not. It was also statistically confirmed that the hydrocarbon degradation of the transformants was highly correlated to their surfactin production by the determination of the correlation coefficient (r2 = 0.997, P0.01). Therefore, these results indicate that the surfactin produced from B. subtilis enhances the bioavailability of hydrophobic liquids.

A convenient and rapid method for screening and identifying rod mutants of Bacillus subtilis is described. At the restrictive temperature (45 degrees C), all rod mutants of B. subtilis screened lost their ability to sporulate. The morphology and colour of mutant colonies grown on sporulation agar plates differed from those of rod+ cells, which were able to sporulate even at elevated temperature. These characteristics provide an alternative approach for the identification of rod mutants in B. subtilis culture by streaking the cells onto a minimal glucose agar plate and incubating at the restrictive temperature. After 30 h of incubation at this temperature, rod mutants are easily identified. This method will facilitate the screening and isolation of rod mutants of B. subtilis.

Chromium-resistant bacteria (CRB) isolated from soils can be used to reduce toxic Cr(VI) from contaminated environments. This study assessed in vitro reduction of hexavalent Cr using a cell-free extract (CFE) of CRB isolated from soil contaminated with dichromate. One isolate, ES 29, that substantially reduced Cr(VI) was identified as a Bacillus species by 16S rRNA gene-sequence homology. The isolate reduced Cr(VI) under aerobic conditions, using NADH as an electron donor and produced a soluble Cr(VI)-reducing enzyme stimulated by copper (Cu2+). The CFE of the bacterial isolate reduced 50% of Cr(VI) in 6 h. The Cr(VI)-reduction activity of the CFE had a K m of 7.09 μM and a V max of 0.171 μmol min−1 mg−1 protein. Mercury inhibited the enzyme, but not competitively, with a V max of 0.143 μmol min−1 mg−1 protein, a K m of 7.07 μM and a K i of 1.58 μM. This study characterizes the enzymatic reduction of Cr(VI) by Bacillus sp. ES 29 which can be used for the bioremediation of chromate.

The degradation of alpha and beta isomers of endosulfan by a two-member bacterial co-culture was studied. Results were similar whether the two isomers were present individually or together, as in technical endosulfan. The degradation of both isomers was accompanied by the formation of endosulfan diol and endosulfan lactone. Accumulation of the metabolite, endosulfan sulfate was, however, not observed during the reaction with either of the isomers. The microbial degradation of endosulfan isomers was also accompanied by a decrease in its toxicity to the test organism Tubifex tubifex Müller.

A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang. This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+. It was judged to be a variety of B. subtilis and designated B. subtilis (chungkookjang). L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized. The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate. The D-glutamate content was over 50% in every PGA produced under the conditions used. During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply. Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl. B. subtilis (chungkookjang) harbors no plasmid and is the first B. subtilis strain reported with both naturally high PGA productivity and high genetic competence.

A new engineering strain, Bacillus pumilus c172-14 (pBX 96), was obtained by introducing the pBX 96 plasmid, which carries the alpha-amylase amy gene, into the host strain of alkalophilic Bacillus pumilus c172 via transformation. The newly constructed strain was found to express the amy gene and could use starch instead of glucose or starch hydrolysate as carbon source for its fermentation of alkaline protease. The pBX 96 plasmid in the new host was found to be segregationally and structurally stable. The expression of amy gene did not affect the host strain's resistance to bacteriophages. Moreover, the level of alkaline protease was improved significantly compared with the parent strain. The constructed strain gave a maximum alkaline protease activity of 14,014 U/ml in shaking flask after 48 h cultivation when growing in a medium containing 6% corn meal, 4% soybean flour, 0.4% Na2HPO4, 0.03% KH2PO4, 0.02% MgCl2, 0.3% CaCl2, 0.25% Na2CO3, 0.1% glucose, and 20 microg/ml kanamycin (pH 7.0). The optimal pH value and temperature of the alkaline protease were 11.0 and 40 degrees C, respectively. This enzyme was stable over a pH range of 8-11. Its residual activity remained at 100% when treated under a temperature of less than 45 degrees C for 30 min. The corresponding residual activity reduced to 65% of its optimal value at 60 degrees C for 30 min. The alkaline protease was a kind of serine protease, which was demonstrated by the complete inactivation by PMSF (1 mM). This newly constructed strain will be useful in the alkaline protease industry.

The thermostable esterase from the moderate thermophile Bacillus circulans was purified to homogeneity using a four-step procedure. Esterase activity was associated with a protein of molecular mass 95 kDa, composed of three identical subunits of 30 kDa. The esterase activity was thermostable with a maximum activity at 55 degrees C using initial rate assay. The half-inactivation temperature was 71 degrees C after a 1-h treatment, which compared favorably to that of other enzymes. Activity at temperatures of 30-37 degrees C was high (about half of maximum), making this new enzyme very attractive for applications in this moderate temperature range. The esterase also showed high activity at a rather alkaline pH (higher than 10). The specificity pattern showed a marked specificity for mid-chain-length fatty acids (3-8 carbon atoms), which classified the enzyme as a carboxylesterase.

An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated, while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures grown on locust bean gum.

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth.

The conversion of soluble starch to cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD) and cyclomaltononaose (delta-CD) by cyclodextrin glycosyltransferases (E.C. 2.4.1.19) from Bacillus spp. and bacterial isolates was studied. The results show that delta-CD was formed by all the enzymes investigated in the range of 5%-11.5% of the total amount of alpha-, beta-, gamma-, and delta-CD produced.

A response-surface methodology was used to study the effect of carbon:nitrogen ratio (C:N) and initial concentration of total solids (C TS) on insecticidal crystal protein production and final spore count. Bacillus thuringiensis var. kurstaki HD-73 was grown in a stirred-tank reactor using soybean meal, glucose, yeast extract, corn steep solids and mineral salts. Soybean meal and glucose were added according to a central composite experimental design to test C:N ratios ranging from 3:1 to 11:1 and C TS levels from 60␣g/l to 150 g/l. Cry production was quantified using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The response-surface model, adjusted to the data, indicated that media with a C:N of 7:1 yielded the highest relative Cry production at each C TS. The spore count was higher at low C:N ratio (4:1) and high C TS (near 150 g/l). Specific Cry production varied from 0.6 to 2.2 g Cry/1010 spores. A 2.5-fold increase in C TS resulted in a six-fold increase of protoxin production at a 7:1 C:N ratio. It is concluded that the best production conditions for Cry and for spores are different and optimization of B. thuringiensis processes should not be done on a spore-count basis but on the amount of Cry synthesized.

2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TTMP) were produced using Bacillus subtilis IFO 3013 grown on soybeans. Solid-state cultivations were carried out either in 100-ml bottles or in a fixed-bed column reactor, both systems being at 27 °C. Optimization studies showed that the best way to produce the two above aroma compounds involved two separate processes. 2,5-DMP was obtained using soybeans enriched with 75 g threonine/kg initial dry weight (i.d.w.), giving 0.85 g metabolite/kg i.d.w. after 6 days. TTMP production involved addition of 90 g/kg i.d.w. acetoin to soybeans, and 2.5 g/kg i.d.w. was recovered after 14 days. These results demonstrated the suitability of solid-state cultivation for production of high-added-value compounds.

A chemically defined medium has been developed to support the growth and the production of mosquito larvicidal factor(s) (MLF) of Bacillus sphaericus 1593M. On the basis of the data of steady-state continuous cultures, it has been understood that acetate can serve as a sole carbon and energy source for B. sphaericus 1593M. Utilization of acetate by B. sphaer-icus 1593M and the production of MLF are further enhanced by the addition of glutamate at low concentrations, both in steady-state continuous as well as in total-cell-retention cultures (TCRC). A two-step TCRC procedure resulted in better biomass and MLF production by B. sphaericus 1593M. It was also found that glutamate can serve as a carbon source as well as a growth factor in the presence of acetate and hence is a partially substitutable carbon source.

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10(-5)-10(-7) exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50% of larvae (LC50) being 2.1 x 10(5) and 1.3 x 10(5) cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth.

The effect of some culture variables in the production of beta-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), beta-galactosidase production was partially growth-associated, as 40%-60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, beta-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific beta-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-1 fermenter scale, a three-fold increase in volumetric beta-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration.