Comparative P solubilizing efficiencies of five acid soil bacteria incubated with calcium, aluminium and iron phosphates

As acid soils of Odisha have been facing a major constraint in phosphorus availability, application of native P solubilizing bacteria could be promising as well as ecofriendly step towards sustainable P availability for crop growth and development. To address the problem of P availability in acid soil of Bhubaneswar, Odisha, rhizosphere soil samples (pH ? 5.50) with rice – pulses (green gram/black gram) cropping system were collected and phosphate solubilising bacteria were isolated. In vitro characterization of the PSB isolates were conducted with calcium, aluminium and iron phosphates to recover soluble P. All the five strains i.e. Bacillus cereus BLS18 (KT582541), Bacillus amyloliquefaciens CTC12 (KT633845), Burkholderia cepacia KHD08 (KT717633), Burkholderia cepacia KJR03 (KT717634), Burkholderia cepacia K1 (KM030037) could solubilize Ca3(PO4)2, AlPO4, FePO4, and Fe3(PO4)2. Higher recovery of soluble P was with Ca3(PO4)2 while the least was with AlPO4. All the strains exibited a trend similar with respect to P recovery i.e. Ca3(PO4)2 > FePO4 > AlPO4 > Fe3(PO4)2. B. amyloliquefaciens CTC12 was most efficient in solubilizing calcium and iron phosphates whereas B. cepacia KHD08 recovered maximum P with aluminium phosphate. All the inorganic salt fortified mediums showed a significant decline in pH which necessitated the identification of compounds present in the mediums. Organic acids viz; acetic, citric, gluconic, lactic, malic, succinic, tartaric acids in the mediums were identified by HPLC. Tartaric acid was only found in the mediums supplemented with AlPO4. B. amyloliquefaciens CTC12 and B. cepacia KHD08 showed promising results in in vitro analysis of P solubilization. The present study is focused on problematic acid soils where phosphorous is unavailable and mostly fixed with aluminium and iron ultimately making it unavailable for the crops to take up. This leads to unbalanced and frequent use of chemical fertilizer. Hence the study is a significant attempt to characterize native PSBs with capacity to solubilize Al-P and Fe-P

Progress in vitamin C biosynthesis related dehydrogenases / 生物工程学报

Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.

Whole-cell biotransformation for simultaneous synthesis of L-2-aminobutyric acid and D-gluconic acid in recombinant Escherichia coli / 生物工程学报

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, expressing L- threonine dehydratase from Escherichia coli, and co-expressing leucine dehydrogenase from Bacillus cereus and glucose dehydrogenase from Bacillus subtilis for cofactor regeneration, was constructed and used for one-pot production of L-2-aminobutyric acid (L-ABA) and D- gluconic acid from L-threonine and D-glucose. We used shake-flask culture to study the whole-cell catalytic condition including temperature, pH, proper permeabilization of cells and optimal wet cells amount. Moreover, the whole-cell catalyst was cultured in 5-L fermentor by fed-batch fermentation, and 164 g/L L-threonine and 248 g/L D-glucose were converted to 141.6 g/L L-ABA and 269.4 g/L D-gluconic acid. The whole-cell catalyst is promising to fulfill industrial requirements for L-ABA and D-gluconic acid.

Chiral separation of fourteen amino alcohols by nonaqueous capillary electrophoresis / 药学学报

We developed a new method for chiral separation of fourteen amino alcohols by nonaqueous capillary electrophoresis (NACE) with the D-(+)-gluconic acid δ-lactone-boric acid complex as chiral selector. In order to achieve good enantioseparation, the effects of D-(+)-gluconic acid δ-lactone and boric acid concentrations, triethylamine concentration, as well as capillary temperature were systematically investigated. The optimized conditions were identified as follows:an uncoated fused silica capillary of 50 μm ID with a total length (Ltot) of 55 cm and an effective length (Leff) of 45 cm; 200 mmol·L-1 D-(+)-gluconic acid δ-lactone, 80 mmol·L-1 boric acid, and 57.4 mmol·L-1 triethylamine in methanol; positive pressure injection at 2.9 psi for 2 s; capillary temperature, 25 ±0.2℃; applied voltage, +15 kV; detection wavelength, 214 nm. Under the optimized conditions, a good chiral resolution was achieved in most of the tested drugs. This method provides a foundation for the development and application of new chiral selectors of polyhydroxy compound-boric acid complexes in chiral drugs analysis by NACE.

Initial pH of medium affects organic acids production but do not affect phosphate solubilization

The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

Levans production by Gluconacetobacter diazotrophicus

Background: Growth of Gluconacetobacter diazotrophicus with glucose as carbon an energy source has been extensively studied. However, there are no reports in the literature describing growth of G. diazotrophicus in cultures containing sucrose as carbon source. The first step in sucrose pathway and production of levans was investigated. Biomass, levans, gluconic acid and keto gluconic acids production and levansucrase activity were determined in cultures with different sucrose concentration and nitrogen sources. Results: The biomass production was maximal in cultures containing 100 g x L-1 sucrose and inorganic nitrogen. Gluconic acid production was observed under all conditions tested, at levels up to 9 g x L-1 in cultures with sucrose excess and biological N2-fixation (BNF). Keto gluconic acids were detectable only in cultures with sucrose excess and supplemented with organic nitrogen sources. Levans production, although observed in all cultures, was maximal in batch culture with 100 g x L-1 of sucrose and BNF, concomitant with a significant expression of extracellular levansucrase. Conclusions: Ours results not only describe some unknown aspects of G. diazotrophicus physiology, but open up the possibility of developing a technology of levans production by this organism using culture media with sucrose (or some cheaper substitute, like molasses) and without the addition of any N-source because of its ability of fixing atmospheric N2.

Invertase, glucose oxidase and catalase for converting sucrose to fructose and gluconic acid through batch and membrane-continuous reactors / Invertase de glicose oxidase e catalase para a conversão de sacarose para ácido glucónico e frutose através de lote e de membrana-contínuas reactores

Conversion of sucrose into fructose and gluconic acid using invertase, glucose oxidase and catalase was studied by discontinuous (sequential or simultaneous addition of the enzymes) and continuous (simultaneous addition of the enzymes in a 100 kDa-ultrafiltration membrane reactor) processes. The following parameters were varied: concentration of enzymes, initial concentration of substrates (sucrose and glucose), pH, temperature and feeding rate (for continuous process). The highest yield of conversion (100 percent) was attained through the discontinuous (batch) process carried out at pH 4.5 and 37 ºC by the sequential addition of invertase (14.3 U), glucose oxidase (10,000 U) and catalase (59,000 U).

Neste trabalho estudou-se a conversão da sacarose em frutose e ácido glicônico, usando as enzimas invertase, glicose oxidase e catalase, através do emprego de processo descontínuo (com adição sequencial ou simultânea das enzimas) e contínuo (adição simultânea das enzimas em reator com membrana acoplado à membrana de ultrafiltração de 100 kDa). Os parâmetros variados foram: a concentração das enzimas, a concentração inicial dos substratos (sacarose e glicose), o pH, a temperatura e a vazão específica de alimentação (processo contínuo). Obteve-se rendimento de 100 por cento, quando a conversão foi conduzida por processo descontínuo em pH 4,5 e a 37 ºC com adição seqüencial das enzimas invertase (14,3 U), glicose oxidase (10.000 U) e catalase (59.000 U).

Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil

A mineral phosphate solubilizing bacterium, Burkholderia cepacia DA23 has been isolated from cultivated soils. Phosphate-solubilizing activities of the strain against three types of insoluble phosphate were quantitatively determined. When 3 percent of glucose concentration was used for carbon source, the strain had a marked mineral phosphate-solubilizing activity. Mineral phosphate solubilization was directly related to the pH drop by the strain. Analysis of the culture medium by high pressure liquid chromatography identified gluconic acid as the main organic acid released by Burkholderia cepacia DA23. Gluconic acid production was apparently the result of the glucose dehydrogenase activity and glucose dehydrogenase was affected by phosphate regulation.

Uma bactéria capaz de solubilizar fosfato mineral, Burkholderia cepacea DA23, foi isolada de solo cultivado. A capacidade dessa bactéria solubilizar o fosfato de três tipos de fosfato insolúvel foi quantificada. Quando foi utilizada glicose a 3 por cento como fonte de carbono, a bactéria apresentou uma intensa atividade solubilizante de fosfato, sendo a solubilização diretamente relacionada com a queda de pH causada pela bactéria. A análise do meio de cultura por cromatografia líquida de alta pressão indicou o ácido glicônico como principal ácido produzido por Burkholderia cepacea DA23. Aparentemente, a produção de ácido glicônico foi causada pela atividade da glicose desidrogenase. A enzima foi afetada pela regulação do fosfato.

Emprego de reator com membrana na obtenção da frutose e ácido glicônicoa partir da sacarose / Fructose and gluconic acid production by sucrose convesion in a membrane bioreactor

Frutose e Ácido Glicônico são produtos importados empregados em diferentes setores nas áreas química, farmacêutica e alimentícia, representando um mercado de dois milhões de dólares (US$ 2,0 milhões) por ano. Por sua vez, a sacarose pode ser empregada como matéria-prima para a obtenção destes produtos através de conversão enzimátiva empregando invertase e glicose-oxidase. O uso de biorreatores com membrana (MBR) mostra-se interessante em processos enzimáticos, pois, ao serem empregados em processos contínuos permitem, simultaneamente, produção e separação dos produtos, reduzindo a formação de subprodutos e, eventual, inibição da enzima por excesso de substrato ou produtos. A sacarose é convertida em xarope de açúcar invertido (solução equimolar de frutose e glicose) pela invertase (Bioinvert`marca registrada’, enzima comercial), seguido pela oxidação da glicose em ácido glicônico pela ação da glicose oxidase (GO). O processo de conversão multi-enzimático da sacarose foi obtido através da alimentação de sacarose (50 mM) em reator com membrana (MBR) contendo invertase (24 U/mL), glicose-oxidase (0,5 U/mL) e catalase (470 U/mL) e operando com vazão específica de 6,0 ‘h POT. -1’, 35`graus’C e pH 5,5. As condições operacionais otimizadas possibilitaram a conversão completa da sacarose (X = 100 %) e da glicose resultante (Y = 100%) com velocidades específicas de reação de 4,2 mmol/U.h, 0,60 mmol/U.h e 0,00062 mmol/U.h, respectivamente, para a invertase, glicose oxidase e catalase. A respeito da oxidação da glicose, a adição de catalase no meio reacional se fez necessária para minimizar os efeitos inibitórios sobre a GO através do peróxido de hidrogênio formado

The fructose and gluconic acid are products of great application in chemical, pharmaceutical and food industry. The actual Brazilian market for these compounds is about US$ 2 millions, here as the sucrose, the raw-material used for their production, represents about 2.4% of the Brazil's GNP. This conversion increases the value added to the sugarcane, usually marketed as a commodity, because the fructose and gluconic acid are more valuable products than sucrose. The use of membrane bioreactor (MBR), which operates under mild conditions regarding internal pressure, temperature and pH, has been growing along the years for enzyme catalyzed processes. Moreover, in the MBR the reaction and separation of the products occur simultaneously, avoiding the formation of by-products and the eventual inhibition of the enzyme caused by excess of substrate or products. The sucrose is converted to the inverted syrup (an equimolar solution of fructose and glucose) by invertase (in this work was employed Bioinvert®, a commercial invertase) followed by the oxidation of glucose in gluconic acid by the glucose oxidase (GO). The multi-enzymatic conversion of sucrose was attained when carried out under initial substrate of 50mM and invertase, glucose oxidase and catalase concentrations, respectively, of 24.0 U/mL, 0.5 U/mL and 470 U/mL in a membrane reactor utilizing a dilution rate of 6.0 h-1, 35ºC and pH 5.5. The optimized operational conditions led to a conversion yield of 100% for sucrose hydrolysis and glucose oxidation steps resulting in enzyme productivity of 4.2 mmol/U.h, 0.60 mmol/U.h and 0.00062 mmol/U.h, respectively, to invertase, glucose oxidase and catalase. In regard to the glucose oxidation, the addition of catalase in the reaction medium was necessary, in order to minimize the inhibition of the GO by the hydrogen peroxide formed.

STUDY ON THE FERMENTATION CONDITION PRODUCING 2-KETO-LGLUCONIC ACID BY USING MIXED CULTURE OF MICROORGANISM / 微生物学通报

Vitamin C precusor-2-keto-L-gulonic acid can be prepared directly by mixed culture of Ghiconobacter oxy-dans SCB329 and Guconobacter subaxydans SCB110. To obtained its high yield, firstly, the proportion of the two micro- organisms, the ingredients of medium and the initial pH were optimized in shake flaskd, then L9 (34) orthogonal experiment confirmed that urea, C. S. L had high degree statistical meaning. Based on these data, an optimized fermentation media was obta ined: D-Sorbitol 9g, C. S.L1.5g, Urea1.5g, KH2PO40.1g, CaCO30.2g. By-product can be inhabited to the greatest extent and the yield increases by 20%.