2-ketogluconic Acid Research Articles

Directing cell catalysis of glucose to 2-keto-d-gluconic acid using Gluconobacter oxydans NL71

2-Keto-d-gluconic acid (2-KGA) is a high-value-added product from lignocellulosic straw-based biorefining process obtained via cell catalysis using Gluconobacter oxydans (G. oxydans); nonetheless, the economical production has some limitations because of the formation of undesirable mixture products of keto-gluconic acid from glucose. In this study, it was demonstrated that pulse addition of hydrogen peroxide (H2O2) to synthetic media was beneficial to improve the conversion rate of glucose to 2-KGA by 1.47-fold in G. oxydans. Moreover, some lignocellulosic degradation compounds served as substitute and played the role of H2O2 on corn stover hydrolysate for the production of 2-KGA. By conducting qRT-PCR analysis, eight dehydrogenase genes were identified in G. oxydans, which were responsible for regulating 2-KGA production. Moreover, most of the identified genes were found to produce membrane-bound glucose dehydrogenase and gluconate 2-dehydrogenase, and were up-regulated by adding H2O2 or degradation compounds. These up-regulated genes could be responsible for directing cell catalysis of glucose to 2-KGA in G. oxydans.

Improved Production of Pyrroloquinoline Quinone by Simultaneous Augmentation of Its Synthesis Gene Expression and Glucose Metabolism in Klebsiella pneumoniae.

Klebsiella pneumoniae can naturally synthesize pyrroloquinoline quinone (PQQ), but current low yield restricts its commercialization. Here, we reported that PQQ production can be improved by simultaneously intensifying PQQ gene expression and glucose metabolism. Firstly, tandem repetitive tac promoters were constructed to overexpress PQQ synthesis genes. Results showed that when three repeats of tac promoter were recruited to overexpress PQQ synthesis genes, the recombinant strain generated 1.5-fold PQQ relative to the strain recruiting only one tac promoter. Quantitative real-time PCR (qRT-PCR) revealed the increased transcription levels of PQQ synthesis genes. Next, fermentation parameters were optimized to augment the glucose direct oxidation pathway (GDOP) mediated by PQQ-dependent glucose dehydrogenase (PQQ-GDH). Results demonstrated that the cultivation conditions of sufficient glucose (≥ 32g/L), low pH (5.8), and limited potassium (0.7nmol/L) significantly promoted the biosynthesis of gluconic acid, 2-ketogluconic acid, and PQQ. In optimum shake flask fermentation conditions, the K. pneumoniae strain overexpressing PQQ synthesis genes under three repeats of tac promoter generated 363.3nmol/L of PQQ, which was 2.6-fold of that in original culture conditions. In bioreactor cultivation, this strain produced 2371.7nmol/L of PQQ. To our knowledge, this is the highest PQQ titer reported so far using K. pneumoniae as a host strain. Overall, simultaneous intensification of pqq gene expression and glucose metabolism is effective to improve PQQ production.

Rhizosphere-Associated Pseudomonas Suppress Local Root Immune Responses by Gluconic Acid-Mediated Lowering of Environmental pH

The root microbiome consists of commensal, pathogenic, and plant-beneficial microbes [1]. Most members of the root microbiome possess microbe-associated molecular patterns (MAMPs) similar to those of plant pathogens [2]. Their recognition can lead to the activation of host immunity and suppression of plant growth due to growth-defensetradeoffs [3, 4]. We found that 42% of the tested root microbiota, including the plant growth-promoting rhizobacteria Pseudomonas capeferrum WCS358 [5, 6] and Pseudomonas simiae WCS417 [6, 7], are able to quench local Arabidopsis thaliana root immune responses that aretriggered by flg22 [8], an immunogenic epitope of the MAMP flagellin [9], suggesting that this is animportant function of the root microbiome. Ina screen for WCS358 mutants that lost their capacity to suppress flg22-induced CYP71A12pro:GUS MAMP-reporter gene expression, we identified the bacterial genes pqqF and cyoB in WCS358, which are required for the production of gluconic acid and its derivative 2-keto gluconic acid. Both WCS358 mutants are impaired in the production of these organic acids and consequently lowered their extracellular pH to a lesser extent thanwild-type WCS358. Acidification of the plant growth medium similarly suppressed flg22-induced CYP71A12pro:GUS and MYB51pro:GUS expression, and the flg22-mediated oxidative burst, suggesting a role for rhizobacterial gluconic acid-mediated modulation of the extracellular pH in the suppression of root immunity. Rhizosphere population densities of the mutants were significantly reduced compared to wild-type. Collectively, these findings show thatsuppression of immune responses is an important function of the root microbiome, as it facilitates colonization by beneficial root microbiota.

The maize mycorrhizosphere as a source for isolation of arbuscular mycorrhizae-compatible phosphate rock-solubilizing bacteria

Phosphate chemical fertilizers are costly and raise concerns about environmental pollution through industrial production. The use of phosphate rock (PRs) emerges as a more sustainable alternative for agriculture. The aim of this work was to isolate phosphate rock-solubilizing bacteria (PRSB) from maize mycorrhizosphere, having growth promoting traits and that will be arbuscular mycorrhizae fungi (AMF) compatible. Bacteria were isolated from the mycorrhizosphere of maize and tested for rock phosphate solubilization, production of organic acids, phosphatases and phytase activities, and growth promotion traits. The capacity of some strains to enhance the dry weight of maize plants was determined in a growth chamber experiment. The compatibility of the selected strains with Rhizophagus irregularis under in vitro conditions was also tested. Out of 118 isolates from maize, eight belonging to Asaia lannaensis, Rahnella sp., Pantoea sp., and Pseudomonas sp. were found to be the best PRSB. On solid media, all strains mobilized P from tricalcium phosphate, hydroxyapatite, and PRs. A. lannaensis was the only PRSB showing visible solubilization of AlPO4 and FePO4. All the PRSBs solubilized PR by producing D-gluconic acid and 2-ketogluconic acid and by lowering the pH. Most strains presented IAA and siderophore production and different biofilm formation and motility capacities. All strains improved the dry weight of maize seedlings compared with non-inoculated plants. The results proved that PRSBs were able to grow on R. irregularis hyphae as the sole in vitro C source. Bacteria isolated from the mycorrhizosphere of maize shows effective solubilization of phosphorus from PR with different reactivity levels. The traits of these bacteria as growth promoters and their biocompatibility with AMF show their potential as inoculants. Improvement of the agronomic effectiveness of PRs is relevant for developing countries that use PRs directly as P-fertilizers (less expensive than soluble P-fertilizers) for sustainable agriculture.

Plant-Beneficial <i>Pseudomonas</i> Spp. Suppress Local Root Immune Responses by Gluconic Acid-Mediated Lowering of Environmental pH

The plant root microbiome consists of commensal, pathogenic and plant-beneficial microbes. Most members of the root microbiome possess microbe-associated molecular patterns (MAMPs) similar to those of plant pathogens. Their recognition by the cell surface-localized pattern recognition receptors can lead to the initiation of host immune signaling and suppression of plant growth due to growth-defense tradeoffs. We found that 42% of the tested root microbiota, including the plant growth-promoting rhizobacteria Pseudomonas capeferrum WCS358 (WCS358) and Pseudomonas simiae WCS417, are able to quench local Arabidopsis thaliana root immune responses that were triggered by the synthetic MAMP flg22, suggesting that this is an important function of the root microbiome. In a screen for WCS358 mutants that lost their capacity to suppress flg22-induced CYP71A12pro:GUS MAMP-reporter gene expression, we identified the bacterial genes pqqF and cyoB in WCS358. CyoB and pqqF are required for the production of gluconic acid and its derivative 2-keto gluconic acid. Both WCS358 mutants are impaired in the production of these organic acids and consequently lowered their extracellular pH to a lesser extent than wild-type WCS358. Acidification of the plant growth medium similarly suppressed flg22-induced CYP71A12pro:GUS and MYB51pro:GUS expression as well as the flg22-mediated oxidative burst, suggesting a role for rhizobacterial gluconic acid-mediated modulation of the extracellular pH in the suppression of local root immunity. Rhizosphere population densities of the mutants were significantly reduced compared to wild-type. Collectively, these findings show that suppression of local root immune responses is an important function of the root microbiome, as it facilitates colonization by beneficial root microbiota.

Characterization of mineral phosphate solubilizing and plant growth promoting bacteria from termite soil of arid region.

Five highly efficient phosphate solubilizing bacteria, viz., Pantoea sp. A3, Pantoea sp. A34, Kosakonia sp. A37, Kosakonia sp. B7 and Bacillus sp. AH9 were isolated from termitorial soils of Sanjivani island of southern Maharashtra, India. These isolates were characterized and explored for phosphate solubilization and plant growth promotion. Among these, Bacillus sp. AH9 showed highest phosphate solubilization index (3.5) and solubilization efficiency (250%) on Pikovskaya agar. Interestingly, Pantoea sp. A34 displayed maximum mineral phosphate solubilization (1072.35mg/L) in liquid medium and during this period the pH dropped to 3.13. All five isolates had highest P solubilization at 48h after inoculation. During mineral phosphate solubilization, both gluconic acid and 2-keto gluconic acid were produced by Kosakonia and Bacillus isolates, while only 2-keto gluconic acid was detected in Pantoea isolates. Highest organic acid (39.07 ± 0.04g/L) production was envisaged in Bacillus sp. AH9, while Pantoea sp. A34 produced the least amount (13.00 ± 0.01g/L) of organic acid. Seed bacterization with Pantoea sp. A3 and Kosakonia sp. A37 resulted in ~ 37% and ~ 53% increase in root length of tomato seedlings, respectively, while Pantoea sp. A34 and Kosakonia sp. B7 had deleterious effects on root length as well as overall growth of the seedlings. To our knowledge, this is the first report of plant growth promoting potential of microorganisms isolated from termitorial soil of Sanjivani island, which is a drought-prone area. Therefore, such efficient growth promoting P solubilizers can offer an effective solution for sustainable agriculture in arid, dryland farming and drought-prone regions.

Non-capsulated mutants of a chemical-producing Klebsiella pneumoniae strain.

To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae. The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4×105 CFUμg-1 DNA, which is 10 times higher than that of the wild strain. 52.2g 1,3-propanediol L-1, 30.7g 2,3-butanediol L-1, and 175.9g 2-ketogluconic acid L-1 were produced by non-capsulated mutants, which were 10-40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8-2.2 cP. Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

2-Ketogluconic acid and pyrroloquinoline quinone secreting probiotic Escherichia coli Nissle 1917 as a dietary strategy against heavy metal induced damage in rats

ObjectiveThe objective of this study was to develop genetically modified probiotic E. coli Nissle 1917 (EcN-23) slowly releasing 2-ketogluconic acid (2-KG) asa chelator and pyrroloquinoline quinone (PQQ) as an antioxidant against heavy metal induced damage. MethodsMale rats were exposed separately to cadmium (Cd), mercury (Hg), lead (Pb) for amonth and Cd-Pb together for 4months. ResultsEcN-23 significantly decreased oxidative stress induced by Cd and Hgas compared to orally given PQQ and 2-KG and also prevented LPS induced damage in Pb treated rats along with enhanced gastric emptying and survival. Moreover, oxidative damage, histopathological damage and dislipidemia markers were maintained at near normal in rats on long term coexposure of Cd and Pb without any loss in essential metal ions. ConclusionTreatment of EcN-23 is effective against Cd, Pb and Hg toxicity as well asCd and Pb coexposure.

Efficient coproduction of gluconic acid and xylonic acid from lignocellulosic hydrolysate by Zn(II)-selective inhibition on whole-cell catalysis by Gluconobacter oxydans

With Zn(II)-selective inhibition on the whole-cell catalysis of Gluconobacter oxydans NL71, gluconic acid and xylonic acid were coproduced efficiently from the hydrolysate of corn stover. Further metabolism of gluconic acid to the by-product 2-ketogluconic acid was prevented by addition of 10g/L ZnCl2. Remarkably, yields of 93.91% of gluconic acid and 93.36% of xylonic acid were obtained with the supplement of ZnCl2 in the synthetic medium, without by-product production. After optimization of the concentrations of ZnCl2 and inocula of the strain, maximum amounts of gluconic acid and xylonic acid were coproduced at titers of 63.01g/L and 33.81g/L, with an overall utilization of 100% of the sugars in the enzymatic hydrolysate of corn stover. The results showed execution of our objective to prove this novel bioconversion method for simultaneously producing gluconic acid and xylonic acid, which would benefit subsequent studies on the comprehensive utilization of lignocellulosic materials.

Production of Chemicals by <em>Klebsiella pneumoniae</em> Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Erratum to: Identification and characterization of the rhizosphere phosphate-solubilizing bacterium Pseudomonas frederiksbergensis JW-SD2, and its plant growth-promoting effects on poplar seedlings

The rhizospheric bacterium JW-SD2 was identified as Pseudomonas frederiksbergensis based on phenotypic features, the Biolog Identification System and 16S rRNA sequence analysis. The phosphate-solubilizing activity, acidification in culture media, growth rate and organic acid secretion of JW-SD2 were investigated during 192 h of cultivation. The phosphate solubilized by JW-SD2 reached 7.75 mM. The decrease of pH and increase of titratable acidity were closely correlated (Pearson’s r = −0.953 and 0.969, respectively) with the phosphate-solubilizing activity. High concentrations of gluconic, 2-ketogluconic, pyruvic, maleic and malic acids were detected before 96 h of culture, when the strain displayed a high level of phosphate-solubilizing activity, indicating that these organic acids were efficient components in phosphate solubilization. However, acetic acid did not affect phosphate solubilization as shown by a remarkable increase at 144 h of culture when the phosphate-solubilizing activity decreased. The phosphate-solubilizing ability of JW-SD2 was significantly (P < 0.01) affected by environmental factors. Over a broad ranges of temperature (20−35 °C), pH (4−9), salinity (0−3.0 %), and volume of medium (1/5−3/5 of flask volume), the phosphate solubilized by JW-SD2 remained above 4.00 mM, demonstrating good potential in adapting to a changing environment. The inoculation experiments indicated that JW-SD2 could significantly (P < 0.05) promote growth of poplar (Populus euramericana cv. NL-895) in both sterilized and non-sterilized soils. The effects of plant growth promotion were greater in non-sterilized than in sterilized soil. During the 150 days of the trial, the effects of plant growth promotion by JW-SD2 first increased then decreased over time, suggesting that, in field applications, the periodic supplementation of the strain into the rhizosphere should be considered.

The Life History of Pyrroloquinoline Quinone (PQQ): A Versatile Molecule with Novel Impacts on Living Systems

Pyrroloquinoline quinone (PQQ) acting as redox cofactor of Glucose dehydrogenase is orthocyclic antioxidant acting under multifarious environmental stress and rich conditions in prokaryotes as well as eukaryotes. Microbes are the exclusive source of PQQ biosynthesis and for biocatalysis of glucose into gluconic acid and 2-ketogluconic acid by gram positive and negative bacteria. This study focus to describe PQQ biography (1979-2016) highlighted its applications acts as biocatalyst by enhanced production of NADH from pqqC, involved in several important mechanisms like bacterial energy transduction by m-ATPase, production of biocontrol substances, growth stimulating activities and DNA repair. PQQ acting as anti-neurological, anti-degenerative, anti-melanogenic and anti-cancer agent due to its antioxidant nature by scavenging free radicals. PQQ is modulator of immunity by CD4 cells count and IL-2, sleep maintenance by PGC-1 alpha pathway and inflammation due to ROS. The mineral phosphate solubilization results in plant growth promotional, biocontrol, antifungal and ISR activities. PQQ nanoparticles used for production of Biofuels cells and sulfonated polymers. It acts as a modulator of diverse signaling pathways like STAT, MAPK, JAK, JNK, P13K/Akt, mTOR, EGFR and Raps for cell proliferation, differentiation, apoptosis, Box translocation and metabolic pathways by phosphorylation of ADP and suppression of reactive oxygen species. It protects from oxidative stress by acting as Alpha AA modulator of lysine metabolism, TrxR1 in selenium metabolism, low density lipoprotein in lipid metabolism, ATP production in Energy, Glucose and carbohydrate metabolism. It has been structurally characterized with bioinformatics tools and future perspectives being molecular modeling and docking for analytical drug design.

Production of xylonic acid by Klebsiella pneumoniae.

The glucose oxidation pathway is important for glucose catabolism in Klebsiella pneumoniae. Gluconic acid and 2-ketogluconic acid are intermediates of this pathway, and the production of these two chemicals has been developed in K. pneumoniae mutants. Catalysis characteristic research in this study has shown that xylose is a suitable substrate of the glucose dehydrogenase of this pathway. Here, using xylose as substrate, xylonic acid was accumulated in the broth of K. pneumoniae culture, and this process was dependent upon acidic conditions. Using a mixture of glucose and xylose as substrates, a mixture of xylonic acid and gluconic acid was produced by the Δgad mutant of K. pneumoniae; gluconic acid was synthesized early, and xylonic acid synthesis began after most glucose was consumed. Using the hydrolysate of bamboo as substrate, mixture of 33g/L gluconic acid and 14g/L xylonic acid were produced by K. pneumoniae Δgad. In fed-batch fermentation, 103g/L xylonic acid was produced after 79h culture, with a conversion ratio of 1.11g/g. This is the first report of xylonic acid produced by K. pneumoniae. Production of xylonic acid and gluconic acid using bamboo hydrolysate is a novel approach for biomass utilization.

Inoculation of genetically modified endophytic Herbaspirillum seropedicae Z67 endowed with gluconic and 2-ketogluconic acid secretion, confers beneficial effects on rice (Oriza sativa) plants

Background and aims Herbaspirillum seropedicae (Hs) Z67 a diazotrophic endophyte was genetically engineered for secretion of 2-keto-D-gluconic acid by heterologous expression of genes for pqq synthesis and gluconate dehydrogenase to study its beneficial effect on plants.

Trapping of phosphate solubilizing bacteria on hyphae of the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM 197198

A simple method is described for trapping phosphate solubilizing bacteria (PSB) strongly attached to the hyphae of the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis (Ri). Bacteria were isolated from the hyphosphere of mycorrhizal leek plants growing on Turface previously inoculated with soil suspensions, obtained from the mycorrhizosphere of mycorrhizal plants growing in agricultural settings or maple forests in Quebec, Canada. Among the best PSB strongly attached to the hyphae of Ri, 26 isolates belonged to Burkholderia spp. and one was identified as Rhizobium miluonense. Four hyphobacteria exhibiting high potential of inorganic and organic P mobilization were further compared with four equivalent mycorrhizobacteria directly isolated from mycorrhizospheric soils sampled. In general, hyphobacteria were superior in mobilizing P from hydroxyapatite and from a low reactivity igneous phosphate rock from Quebec. Release of gluconic acid or the product of its oxidation 2-ketogluconic acid, are the main mechanisms involved in P solubilization. In a two compartments Petri plate system, Ri extraradical hyphal exudates, supported PSB growth and activity. In the absence of PSB Ri showed a negligible P solubilization activity. In the presence of PSB a substantial increase in P mobilization was observed, and the superiority of hyphobacterial activity was also observed under this system. Our results suggest that in developing a bioinoculant based on selected PSB, their interaction with AMF hyphae should not be overlooked.

Gac-mediated changes in pyrroloquinoline quinone biosynthesis enhance the antimicrobial activity of Pseudomonas fluorescens SBW25.

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In Pseudomonas fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors.

2-Keto-D-gluconate-yielding membrane-bound D-glucose dehydrogenase from Arthrobacter globiformis C224: purification and characterization.

Glucose dehydrogenase (GlcDH) is the rate-limiting catalyst for microbial conversion of glucose to the important organic acid 2-ketogluconic acid (2KGlcA). In this study, a d-glucose dehydrogenase was purified from the industrial 2KGlcA producer Arthrobacter globiformis C224. After four purification steps, the GlcDH was successfully purified over 180 folds and specific activity of 88.1 U/mg. A single protein band of 87 kDa was detected by SDS-PAGE. The purified GlcDH had the broad substrate specificity with the Km values for d-glucose, d-xylose, d-galactose and maltose of 0.21 mM, 0.34 mM, 0.46 mM and 0.59 mM, respectively. The kinetic studies proved that A. globiformis GlcDH followed the ping-pong kinetic mechanism. The GlcDH showed an optimum catalytic activity at pH 5.0 and 45 °C with the stable activity at temperature of 20–40 °C and pH of 6.0–7.0. Organic solvents, metal ions or EDTA could significantly influence the GlcDH activity to different degrees.

Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

2-Ketogluconic acid production by Klebsiella pneumoniae is a pH-dependent process, strictly proceeding under acidic conditions. Unfortunately, cell growth is inhibited by acidic conditions, resulting in low productivity of 2-ketogluconic acid. To overcome this deficiency, a two-stage fermentation strategy was exploited in the current study. During the first stage, the culture was maintained at neutral pH, favoring cell growth. During the second stage, the culture pH was switched to acidic conditions favoring 2-ketogluconic acid accumulation. Culture parameters, including switching time, dissolved oxygen levels, pH, and temperature were optimized for the fed-batch fermentation. Characteristics of glucose dehydrogenase and gluconate dehydrogenase were revealed in vitro, and the optimal pHs of the two enzymes coincided with the optimum culture pH. Under optimum conditions, a total of 186 g/l 2- ketogluconic acid was produced at 26 h, and the conversion ratio was 0.98 mol/mol. This fermentation strategy has successfully overcome the mismatch between optimum parameters required for cell growth and 2-ketogluconic acid accumulation, and this result has the highest productivity and conversion ratio of 2-ketogluconic and produced by microorganism.

Effect of Carbon, Nitrogen Sources and Abiotic Stress on Phosphate Solubilization by Bacterial Strains Isolated from a Moroccan Rock Phosphate Deposit

Experiments were conducted to evaluate the solubilization of tri-calcium phosphate (TCP) by phosphatesolubilizing bacterial isolates PSB 4, 5 and 6 identified as Enterobacter sp., Bacterium DR172, and Enterobacter hormaechei, respectively, in addition to Acinetobacter sp. used as a positive control strain. The study was carried out in culture media using different carbon and nitrogen sources, and under different abiotic stress conditions such as high salt, pH, and temperature. The effect of EDTA on growth was also tested. Organic acids produced by the PSB isolates were also determined by reverse phase HPLC. The isolates behave differently with different types of N- and C-sources and seem to adapt to different conditions, with glucose significantly promoting TCP solubilization in all cases. The highest production of orthophosphate (866 mg.L-1, pH 3.2) was showed by Enterobacter hormaechei using glucose as C-source, (NH 4 ) 2 SO 4 as N-source, at 37°C and pH 7, and without any addition of EDTA. The solubilized phosphate also did attaint 110.71% in comparison with control when sorbitol was used as a C-source by Bacterium DR172. We also noticed a decrease in pH due to the organic acids secreted to the medium. Newly carboxylic acids were identified to be produced by these strains in addition to others previously identified, 2 ketogluconic acid was found when galactose was used as a C-source for all isolates except for Acinetobacter sp. which secreted lactic, glutaric and glucuronic acids. Analysis of the Solubilization Index (SI) of the bacterial strains at different temperatures revealed different effectiveness to degrade and assimilate TCP, the SI being maximal at 25°C for the three isolates (4.17, 3.83 and 4.44, for PSB4, 5 and 6), whereas for Acinetobacter sp. the highest SI (3.83) was achieved at 30°C.