Use of maleic acid by mixed cultures of microorganisms

In a mixed batch culture, Alcaligenes xylosoxidans subsp. xylosoxidans 260 transformed maleic acid into malic acid. Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid. Both bacterial cultures dissociated with the formation of R, S, and M forms. At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A. xylosoxidans and Bacillus subtilis. At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively. Association of bacteria A. xylosoxidans and B. subtilis was not stable under flow conditions water.     

Optimum Conditions for Transformation of Maleic Acid by Immobilized Cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260

Immobilized cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260 transformed 98% of the maleic acid (initial concentration of 5.0 g/l medium) under periodic conditions for 48 h. Free cells transformed only 26% of the substrate in 96 h. Immobilized cells of a selected S-variant ofA. xylosoxidanstransformed the maleate (30.0 g/l) entirely in 96 h during batch cultivation and only 15.0 g/l of the maleate in continuous cultivation at a flow rate of 0.03 h^–1.     

β‐poly(l‐malic acid) production by fed‐batch culture of Aureobasidium pullulans ipe‐1 with mixed sugars

β‐poly(l ‐malic acid) (PMLA) is a biopolyester, which has attracted growing attention due to its potential applications in medicine and other industries. In this study, the biosynthetic pathway of PMLA and the fermentation strategies with mixed sugars were both investigated to enhance PMLA production by Aureobasidium pullulans ipe‐1. Metabolic intermediates and inhibitors were used to study the biosynthetic pathway of PMLA. It showed that exogenous addition of l ‐malic acid, succinic acid, TFA, and avidin had negligible effect on PMLA production, while pyruvic acid and biotin were the inhibitors, indicating that PMLA biosynthesis was probably related to phosphoenolpyruvate via oxaloacetate catalyzed by phosphoenolpyruvate carboxylase. Sucrose was suitable for achieving the highest PMLA concentration, while fructose generated a higher yield of PMLA (PMLA produced per biomass). Furthermore, the fed‐batch culture using fed solution with different sugar mixture for PMLA production was implemented. During the fed‐batch culture with mixed solution, fructose could increase PMLA production. Compared with the batch culture, the feeding with mixed sugar (sucrose and glucose) increased PMLA concentration by 23.9%, up to 63.2 g/L, and the final volume of the broth was increased by 25%. These results provide a good reference for process development and optimization of PMLA production.     

Biodegradation of native feather keratin by Bacillus subtilis recombinant strains

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.     

Influence of pH,malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae

Malic acid consumption by Saccharomyces cerevisiae was studied in a synthetic medium. The extent of malic acid degradation is affected by its initial concentration, the extent and the rate of deacidification increased with initial malate concentration up to 10 g/l. For malic acid consumption, an optimal pH range of 3–3.5 was found, confirming that non-dissociated organic acids enter S. cerevisiae cells by simple diffusion. A full factorial design has been employed to describe a statistical model of the effect of sugar and malic acid on the quantity of malate degraded (g/l) by a given amount of biomass (g/l). The results indicated that the initial malic acid concentration is very important for the ratio of malate consumption to quantity of biomass. The yeast was found to be most efficient at higher levels of malate.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

N‐acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non‐ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors—N‐acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)—and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc‐producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine‐6‐phosphate acetyl‐transferase (GNA1), N‐acetylglucosamine 2‐epimerase (AGE), and N‐acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0‐fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner–Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32‐fold and 2.64‐fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose‐malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.     

Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis

Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l^–1 in a glutamic acid/citric acid medium. Poly(glutamic acid) reached 23 g l^–1 after 30 h.     

Chemotaxis of Azotobacter vinelandii and Bacillus subtilis in a mixed culture

In the mixed culture of Azotobacter vinelandii and Bacillus subtilis, chemotaxis of Azotobacter to glucose remained unchanged, while that of bacilli decreased. Microelectrophoresis demonstrated adhesion of the A. vinelandii polysaccharide on the surface of B. subtilis cells. In the presence of 0.05–1.0 g/L of this biopolymer, the chemotaxis of bacilli to glucose decreased 2.6 to 6.8 times. A. vinelandii polysaccharide molecules adherent on the surface of B. subtilis cells were suggested to block bacillary chemotactic receptors, resulting in a decrease in their directed motility in the mixed culture.     

Enhanced production of succinic acid by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis> with amplified activities of malic enzyme and fumarase

Apfl ldhA double mutantEscherichia coli strain NZN111 was used to produce succinic acid by overexpressing theE. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, thefumB gene encoding the anaerobic fumarase ofE. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducibletrc promoter. From the batch culture of recombinantE. coli NZN111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.     

Transporters involved in uptake of di- and tricarboxylates in Bacillus subtilis

Di- and tricarboxylates found as intermediates in the tricarboxylic acid cycle can be utilized by many bacteria and serve as carbon and energy source under aerobic and anaerobic conditions. A prerequisite for metabolism is that the carboxylates are transported into the cells across the cytoplasmic membrane. Bacillus subtilis is able to metabolize many di- and tricarboxylates and in this overview the available data on all known and putative di- and tricarboxylate transporters in B. subtilis is summarized. The B. subtilis transporters, that are of the secondary type, are discussed in the context of the protein families to which they belong. Available data on biochemical characterization, regulation of gene expression and the physiological function is summarized. It is concluded that in B. subtilis multiple transporters are present for tricarboxylic acid cycle intermediates. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of promoter activities of heterologous and homologous promoters in Bacillus subtilis

Summary We have constructed promoter probe vectors with the Escherichia coli galactokinase monitoring system that can be used in Bacillus subtilis. In vivo studies with these vectors demonstrated that the E. coli trp and tac(trp::lac) promoter regions could be utilized in B. subtilis. These promoter regions and the promoter region for the erythromycin resistance gene originating from Staphylococcus aureus were preferentially utilized during the stationary growth phase of B. subtilis, whereas the B. subtilis P21K and P29K promoters were utilized during the exponential growth phase and decreased rapidly during the stationary phase. The apparent strength of these promoters of E. coli in B. subtilis, in terms of galactokinase units, was comparable with those of the B. subtilis promoters.     

Malate degradation by Schizosaccharomyces yeasts included in alginate beads

Schizosaccharomyces yeasts can be used for deacidification of grape musts. To this aim, we studied malic acid degradation by yeasts included in double layer alginate beads in a bubble column reactor. Use of immobilized micro-organisms allowed a continuous process with high dilution rates giving a deacidification capacity of 0.032 g of malate/hour/dm³/g of beads. The pneumatic agitation was very convenient in this case.List of Symbols D h^–1 Dilution rate for continuous culture - h Residence time for continuous culture - dM/dt kg/(m³ · h) Rate of degradation of malic acid - dS/dt kg/(m³ · h) Rate of consumption of glucose - _max h^–1 Maximal specific rate of growth     

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis

Summary The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens -amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.     

Specific Toxic Effects of 2,4,6-Trinitrotoluene on Bacillus subtilisSK1

Using Bacillus subtilis SK1 as an example, it was demonstrated for the first time that 2,4,6-trinitrotoluene (TNT) transformation pathways change with TNT concentration. The growth of cultured B. subtilis SK1, delayed at 20 mg/l TNT (minimum toxic concentration), was resumed following TNT transformation. Aromatic amines were predominant metabolites detected in the culture medium at early stages of TNT transformation. The culture growth was completely inhibited by 200 mg/l TNT. As this took place, nitrites accumulated in the culture medium.     

Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins

Summary The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH₂-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH₂-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis.Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH₂-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation.     

Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B.intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism.     

Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid,citric acid,and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme. Correspondence to: M. Kunioka     

Biological treatments to control bacterial canker of greenhouse tomatoes

Experiments were conducted to determine the effects of treatments on Clavibacter michiganensis subsp. michiganensis in vitro and on young seedlingsinoculated with the pathogen under greenhouseconditions. Lysozyme was bactericidal at 10 g/l concentration in vitro. Tomato plantstreated with lysozyme at 10 g/l and 100 g/lshowed significantly higher plant heightcompared with the inoculated control plants,and plants in these treatments were as tall asthose observed in untreated uninoculatedcontrol plants. Treatments with B. subtilis (Quadra 136) and Trichoderma harzianum (RootShield®), lysozyme,vermicompostea, Rhodosporidium diobovatum(S33), B. subtilis (Quadra 137) appliedas a spray at 0.3 g/l, 0.6 g/l, 10 g/l,concentrated, 1 × 10⁹ CFU/ml, and 0.5 g/l,respectively, have the ability to prevent theincidence of bacterial canker of tomato plantscaused by C. michiganensis subsp.michiganensis under greenhouse conditions.     

Co-producing iturin A and poly-γ-glutamic acid from rapeseed meal under solid state fermentation by the newly isolated <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain 3-10

The strain 3-10 was isolated from soil and identified as B. subtilis according to morphological and physiological characteristics and nucleotide sequence of 16S rRNA. It co-produced anti-fungal iturin A and fertilizer synergist of poly-γ-glutamic acid (γ-PGA) under solid state fermentation (SSF) with rapeseed meal. The co-production of iturin A and γ-PGA reached 5.3 and 51.3 g/kg-dry weight culture, respectively, and the number of viable cells reached 1.9 × 10¹⁰ CFU/g-dry weight culture. In pot tests, the shoot length and dry weight of watermelon seedlings treated by the SSF culture improved by 48.0 and 30.8%, respectively compared to the control; and its biocontrol effect on watermelon fusarium wilt achieved 89.6%. These results highlight a novel strategy to exploit the low-cost and widely available rapeseed meal as dual-functional bio-organic fertilizer under SSF by B. subtilis.