Microbial production of L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone by Ketogulonicigenium vulgare DSM 4025

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 degrees C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.     

Metabolome profiling reveals metabolic cooperation between Bacillus megaterium and Ketogulonicigenium vulgare during induced swarm motility

The metabolic cooperation in the ecosystem of Bacillus megaterium and Ketogulonicigenium vulgare was investigated by cultivating them spatially on a soft agar plate. We found that B. megaterium swarmed in a direction along the trace of K. vulgare on the agar plate. Metabolomics based on gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) was employed to analyze the interaction mechanism between the two microorganisms. We found that the microorganisms interact by exchanging a number of metabolites. Both intracellular metabolism and cell-cell communication via metabolic cooperation were essential in determining the population dynamics of the ecosystem. The contents of amino acids and other nutritional compounds in K. vulgare were rather low in comparison to those in B. megaterium, but the levels of these compounds in the medium surrounding K. vulgare were fairly high, even higher than in fresh medium. Erythrose, erythritol, guanine, and inositol accumulated around B. megaterium were consumed by K. vulgare upon its migration. The oxidization products of K. vulgare, including 2-keto-gulonic acids (2KGA), were sharply increased. Upon coculturing of B. megaterium and K. vulgare, 2,6-dipicolinic acid (the biomarker of sporulation of B. megaterium), was remarkably increased compared with those in the monocultures. Therefore, the interactions between B. megaterium and K. vulgare were a synergistic combination of mutualism and antagonism. This paper is the first to systematically identify a symbiotic interaction mechanism via metabolites in the ecosystem established by two isolated colonies of B. megaterium and K. vulgare.     

Development of chemically defined media supporting high cell density growth of Ketogulonicigenium vulgare and Bacillus megaterium

The immediate precursor of L-ascorbic acid, or vitamin C, is 2-keto-l-gulonic acid (2-KLG). This is commonly produced commercially by Ketogulonicigenium vulgare and Bacillus megaterium, using corn steep liquor powder (CSLP) as an organic nitrogen source. In this study, the effects of the individual CSLP components (amino acids, vitamins, and metal elements) on 2-KLG production were evaluated, with the aim of developing a complete, chemically defined medium for 2-KLG production. Forty components of CSLP were analyzed, and key components were correlated to biomass, 2-KLG productivity, and consumption rate of L-sorbose. Glycine had the greatest effect, followed by serine, biotin, proline, nicotinic acid, and threonine. The combination of 0.28 g L⁻¹ serine, 0.36 g L⁻¹glycine, 0.18 g L⁻¹ threonine, 0.28 g L⁻¹proline, 0.19 g L⁻¹nicotinic acid, and 0.62 mg L⁻¹biotin in a chemically defined medium produced the highest maximum biomass concentration (4.2 × 10⁹ cfu mL⁻¹), 2-KLG concentration (58 g L⁻¹), and yield (0.76 g g⁻¹) after culturing for 28 h.     

混合培养中巨大芽孢杆菌对氧化葡萄糖酸杆菌的作用

为查明维生素Ｃ二步发酵混合培养中巨大芽孢杆菌与氧化葡萄糖酸杆菌间的关系,通过生长曲线测定、静息细胞实验及摇瓶发酵实验研究了巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产生２－酮基－Ｌ－古龙酸作用的影响;采用超滤分离、凝胶层析及聚丙烯酰胺凝胶电泳技术对巨大芽孢杆菌胞外液中具有促进氧化葡萄糖酸杆菌产酸作用的活性物质进行了分离和纯化。结果表明,大菌胞内液和胞外液均可促进小菌生长,大菌胞外液中具有该作用的组分分子     

一株芽孢杆菌在维生素C二步发酵中对小菌的促进作用

从土壤中分离到1株能更好促使小菌生长和产酸的芽孢杆菌B601,作为伴生菌与巨大芽孢杆菌相比,在生长过程中,发酵液中B601活菌数小于巨大芽孢杆菌,而其芽孢数则多于巨大芽孢杆菌。对B601组成菌系的发酵条件进行优化,得到如下结果：100g／L L-山梨糖、6g／L尿素、10g/L玉米浆、培养温度30℃和发酵周期44h。与巨大芽孢杆菌组成菌系相比其底物,L-山梨糖质量浓度提高了25％,尿素下降了50％．玉米浆质量浓度下降了33％,温度提高了2℃,发酵周期缩短了4h。结果表明：B601作为伴生菌,与巨大芽孢杆菌相比,该菌株明显提高了发酵效率。     

Partial degradation of p-aminoazobenzene by a defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia

A defined mixed culture of Bacillus subtilis and Stenotrophomonas maltophilia was used to accomplish the partial biodegradation of the azo-dye p-aminoazobenzene (pAAB). Kinetic experiments were conducted, under aerobic conditions, to study the mineralization of p-aminoazobenzene by the above-defined mixed culture, under aerobic conditions. The combination of two previously developed models, (Zissi et al., 1997), which describes pAAB biodegradation by Bacillus subtilis into aniline and p-phenylenediamine, and (Zissi and Lyberatos, 1999), which describes aniline biodegradation by Stenotrophomonas maltophilia, is shown to predict well the anticipated mixed culture growth and partial biodegradation of pAAB. In previous work (Zissi et al., 1997) it was observed that pphenylenediamine was unstable during the experiments therefore the fate of p-phenylenediamine was not studied. The overall kinetic model of the defined mixed culture was then used to study the behavior of the mixed culture system in a range of operating conditions in the chemostat. The partial degradation of pAAB (regarding one of the two products, aniline) was described by an interaction between the two bacteria with competitive and commensalistic elements. The two bacteria are shown to coexist in a CSTR for some ranges of the operating variables.     

Crystallization and structural analysis of 2-hydroxyacid dehydrogenase from Ketogulonicigenium vulgare

l-2-Hydroxyacid dehydrogenase (HDH) from Ketogulonicigenium vulgare Y25 was cloned and overexpressed in Escherichia coli. The protein was purified and crystallized by the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as precipitant. The crystal structure of HDH was determined at 1.64 Å resolution using the molecular replacement method with the crystal structure of hydroxyl (phenyl) pyruvate reductase from Coleus blumei Benth as the search model. The overall structure of HDH was similar to that of hydroxyl(phenyl)pyruvate reductase, consisting of two compact domains separated by a deep active cleft. The most significant structural divergence is located around the pocket gate comprising residues A210, T211 and R212, which is located on top of the catalytic triad.     

A high throughput method to screen companion bacterium for 2-keto-l-gulonic acid biosynthesis by co-culturing Ketogulonicigenium vulgare

Bacillus megaterium is widely used as companion bacterium in the two-step biosynthesis of 2-keto-l-gulonic acid (2-KLG) by Ketogulonicigenium vulgare. To screen efficiently target companion strains from large numbers of random mutants, a screen method based on spectrophotometry and 24-well microtiter plates was developed and validated on an integrated library of 450 transposon random insertional mutants and two sporulation-defective mutants. The co-culture processes were classified into three groups (low, intermediate and high performance) by K-mean clustering analysis. In addition, mutant m71 was successfully screened out from the library. The substrate conversion ratio of m71 and K. vulgare co-culture process after 72 h was decreased by about 38% compared with that of the wild-type co-culture process in 750 ml flasks. These results indicated that the proposed high throughput method is feasible for screening target companions for the co-culture process of 2-KLG biosynthesis.     

Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement

An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319. A temperature-dependent suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene. One mutant B. megaterium MS941 was selected for further characterization. Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B. megaterium. Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme.Data presented in this contribution are part of a doctoral thesis of the Naturwissenschaftliche Fakultät Münster, Germany (KDW)     

Pressure measurement to evaluate ethanol or lactic acid production during glucose fermentation by yeast or heterofermentative bacteria in pure and mixed culture

A rapid and simple technique to follow CO2 release during fermentation of glucose by heterofermentative bacteria or yeasts was used in order to evaluate ethanol and lactate production in pure and mixed cultures of yeast and bacteria. In pure cultures, good correlations were found between gas pressure variations (deltaP) and ethanol or lactate production by yeasts or heterofermentative bacteria, and ratios between deltaP and ethanol or lactate produced could be established. In mixed cultures, ratios between maximal deltaP and total amount of glucose consumed were determined. It was thus possible to evaluate the amount of glucose that was consumed by each strain and then deduce the bacterial lactate production. Good results were obtained for mixed cultures of yeast and homofermentative bacteria. This technique may be useful to evaluate the activity of strains in mixed cultures of yeast and lactic acid bacteria.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

Comparative analysis of PHAs production by Bacillus megaterium OUAT 016 under submerged and solid-state fermentation

In view of risk coupled with synthetic polymer waste, there is an imperative need to explore biodegradable polymer. On account of that, six PHAs producing bacteria were isolated from mangrove forest and affilated to the genera Bacillus & Pseudomonas from morpho-physiological characterizations. Among which the potent PHAs producer was identified as Bacillus megaterium OUAT 016 by 16S rDNA sequencing and in-silico analysis. This research addressed a comparative account on PHAs production by submerged and solid-state fermentation pertaining to different downstream processing. Here, we established higher PHAs production by solid-state fermentation through sonication and mono-solvent extraction. Using modified MSM media under optimized conditions, 49.5% & 57.7% of PHAs were produced in submerged and 34.1% & 62.0% in solid-state fermentation process. Extracted PHAs was identified as a valuable polymer PHB-co-PHV and its crystallinity & thermostability nature was validated by FTIR, ¹H NMR and XRD. The melting (Tm) and thermal degradation temperature (Td) of PHB-co-PHV was 166 °C and 273 °C as depicted from DTA. Moreover, FE-SEM and SPM surface imaging indicated biodegradable nature, while FACS assay confirmed cytocompatibility of PHB-co-PHV.     

Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.     

Microbial transformation of glycyrrhetinic acid derivatives by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741

Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process, which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them, olean-12-ene-3β,7β,15α,19α,30-pentol (16) and olean-12-ene-3β,7β,15α,27,30-pentol (17) showed significant inhibitory effect with IC₅₀ values of 0.64 and 0.07 μM, respectively.     

Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923

Abstract Growth of Bacillus sp. DSM 2923 on nicotinic acid in mineral medium was dependent on the concentration of sodium molybdate added. Addition of increasing amounts of tungstate to the medium resulted in an inhibition of growth on nicotinic acid or 6-hydroxynicotinic acid as sole source of carbon and energy. Chlorate-resistant mutants were isolated which were not able to degrade nicotinic acid and 6-hydroxynicotinic acid nor to reduce nitrate. Additionally, enzyme activities of nicotinic acid dehydrogenase and 6-hydroxynicotinic acid dehydrogenase increased with increasing concentrations of molybdate (10⁻⁸ to 10⁻⁶ M) added to the medium, and decreased with increasing amounts of tungstate (10⁻⁶ to 10⁻⁵ M) in the medium.     

Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551

We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning. Received: 28 October 1996 / Received revision: 29 December 1996 / Accepted: 4 January 1997     

Production of propionic acid by mixed bacterial fermentation

Summary VariousPropionibacteria andLactobacilli have been grown in starch based media in pure and mixed cultures to ascertain the optimum conditions for propionic acid production. A system has been identified using.Propionibacterium freudenreichii ssshermanii grown in mixed culture withLactobacillus amylophilus that yields approximately 20g/l propionic acid from a medium consisting of wheat flour and corn steep liquor. Simple processing of fermentation broths results in a product containing approximately 30% (w/w) propionic acid that may be suitable for use as a food preservative.