Deciphering styrene oxide tolerance mechanisms in Gluconobacter oxydans mutant strain.

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L-1 styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses. Physiological changes are one of the main factors responsible for the high OS tolerance in mutant strains. Moreover, the P-type ATPase GOX_RS04415 and the LysR family transcriptional regulator GOX_RS04700 were also verified as critical genes for styrene oxide tolerance. The tolerance mechanisms of OSs can be used in biocatalytic chassis cell factories to synthesize compounds and degrade environmental pollutants. This study provides new insights into the mechanisms underlying the toxicological response to OS stress and offers potential targets for enhancing the solvent tolerance of G. oxydans.

Antibacterial activity of Ricinus communis plant extract against antibiotic resistant Helicobacter pylori and Gluconobacter oxydans isolated from fresh apple juices samples.

Bacteria were isolated from samples of Fresh Apple juices from shops of three different localities of Lahore. Analysis of samples from Liberty, Anarkali and Yateem khana Markets show different levels of contamination. There were pathogenic and non-pathogenic bacteria in all samples and were identified by the morphological and biochemical tests. Most of the plasmids of pathogenic bacteria were 4kb in their molecular size. Ribotyping of 16S ribosomal RNA gene sequencing was done to confirm Helicobacter pylori strain and Gluconobacter oxydans. The highest sensitivity of 210mm was shown by Enterobacter sp. against Aztheromysine disk (15µg) while Micrococcus sp. was highly resistant against all of the Antibiotics applied. The antibiotic resistance of pathogenic bacteria was also checked against Ricinus communis plant's extracts, all isolated bacterial pathogens were resistant but only, E.coli was inhibited at 300µl of the extracts. Presence of pathogenic bacteria in Apple juice samples was due to contamination of sewage water in drinking water while some of these pathogenic bacteria came from Apple's tree and other from store houses of fruits.

Electrodialytic bioproduction of xylonic acid in a bioreactor of supplied-oxygen intensification by using immobilized whole-cell Gluconobacter oxydans as biocatalyst.

Immobilized whole-cell fermentation has been proven to be an effective method to improve the performance and cost-effectiveness of Gluconobacter oxydans ATCC 621. In the bio-oxidation of xylose to xylonic acid, the oxygen supply through the immobilized beads is a well-known factor that limits the biocatalytic performance of Gluconobacter oxydans as obligate aerobic bacteria. The activity of immobilized cells could be efficiently improved by execution of pressurized pure oxygen supply strategy. Subsequently, in order to further enhance the production efficiency of xylonic acid and reduce end-product inhibition, online-electrodialysis was employed. Finally, a design of pressurized oxygen supply bioreactor combining with online-electrodialysis was put forward for implementing successive production of xylonic acid. The central features of this a highly integrated design are feasible and thus might enable cost-competitive bacterial xylonic acid production.

Enhancement of 5-keto-d-gluconate production by a recombinant Gluconobacter oxydans using a dissolved oxygen control strategy.

The rapid and incomplete oxidation of sugars, alcohols, and polyols by the gram-negative bacterium Gluconobacter oxydans facilitates a wide variety of biological applications. For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a promising precursor of the industrial substance L-(+)-tartaric acid, G. oxydans DSM2343 was genetically engineered to strain ZJU2, in which the GOX1231 and GOX1081 genes were knocked out in a markerless fashion. Then, a secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 was heterologously expressed in G. oxydans ZJU2. The 5-KGA production and cell yield were increased by 10% and 24.5%, respectively. The specific activity of GCD towards gluconate was 1.75±0.02 U/mg protein, which was 7-fold higher than that of the sldAB in G. oxydans. Based on the analysis of kinetic parameters including specific cell growth rate (µ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp), a dissolved oxygen (DO) control strategy was proposed. Finally, batch fermentation was carried out in a 15-L bioreactor using an initial agitation speed of 600 rpm to obtain a high µ for cell growth. Subsequently, DO was continuously maintained above 20% to achieve a high qp to ensure a high accumulation of 5-KGA. Under these conditions, the maximum concentration of 5-KGA reached 117.75 g/L with a productivity of 2.10 g/(L·h).

Identification of mannitol as compatible solute in Gluconobacter oxydans.

Gluconobacter oxydans is an industrially important bacterium owing to its regio- and enantio-selective incomplete oxidation of various sugars, alcohols, and polyols. The complete genome sequence is available, but it is still unknown how the organism adapts to highly osmotic sugar-rich environments. Therefore, the mechanisms of osmoprotection in G. oxydans were investigated. The accumulation and transport of solutes are hallmarks of osmoadaptation. To identify potential osmoprotectants, G. oxydans was grown on a yeast glucose medium in the presence of 100 mM potassium phosphate (pH 7.0) along with various concentrations of sucrose (0-600 mM final concentration), which was not metabolized. Intracellular metabolites were analyzed by HPLC and (13)C NMR spectroscopy under stress conditions. Both of these analytical techniques highlighted the accumulation of mannitol as a potent osmoprotectant inside the stressed cells. This intracellular mannitol accumulation correlated with increased extracellular osmolarity of the medium. For further confirmation, the growth behavior of G. oxydans was analyzed in the presence of small amounts of mannitol (2.5-10 mM) and 300 mM sucrose. Growth under sucrose-induced osmotic stress conditions was almost identical to control growth when exogenous mannitol was added in low amounts. Thus, mannitol alleviates the osmotic stress of sucrose on cellular growth. Moreover, the positive effect of exogenous mannitol on the rate of glucose consumption and gluconate formation was also monitored. These results may be helpful to optimize the processes of industrial product formation in highly concentrated sugar solutions.

Quantifying the sensitivity of G. oxydans ATCC 621H and DSM 3504 to osmotic stress triggered by soluble buffers.

In Gluconobacter oxydans cultivations on glucose, CaCO3 is typically used as pH-buffer. This buffer, however, has disadvantages: suspended CaCO3 particles make the medium turbid, thereby, obstructing analysis of microbial growth via optical density and scattered light. Upon searching for alternative soluble pH-buffers, bacterial growth and productivity was inhibited most probably due to osmotic stress. Thus, this study investigates in detail the osmotic sensitivity of G. oxydans ATCC 621H and DSM 3504 using the Respiratory Activity MOnitoring System. The tested soluble pH-buffers and other salts attained osmolalities of 0.32-1.19 osmol kg(-1). This study shows that G. oxydans ATCC 621H and DSM 3504 respond quite sensitively to increased osmolality in comparison to other microbial strains of industrial interest. Osmolality values of >0.5 osmol kg(-1) should not be exceeded to avoid inhibition of growth and product formation. This osmolality threshold needs to be considered when working with soluble pH-buffers.

Improving Gluconobacter oxydans performance in the in situ removal of the inhibitor for asymmetric resolution of racemic 1-phenyl-1,2-ethanediol.

Gluconobacter oxydans DSM2003 was used to catalyze the oxidation of racemic 1-phenyl-1,2-ethanediol (PED) for the production of (S)-enantiomer. The oxidative product mandelic acid produced strong inhibition to this reaction and largely reduced the activity of biocatalyst, which was the key problem in the reaction. In order to overcome this bottleneck, an anion exchange resin was selected and introduced as adsorbent for the in situ removal of the inhibitor from the reaction system. This method increased the substrate concentration from 12 to 60 g/L and the yield of (S)-PED by approximately five times from 4.9 g/L, on the premise that the enantiomeric excess (ee) value of (S)-PED remained above 96% and the reaction time was no more than 20 h. Moreover, the final space-time yield was over 1.2g/L/h, which was higher than that reported from previous studies.

Draft genome sequence of Gluconobacter oxydans WSH-003, a strain that is extremely tolerant of saccharides and alditols.

Gluconobacter oxydans is known for its incomplete oxidation of a wide range of alcohols, sugars, and acids in a bioprocess. The corresponding oxidation products are secreted almost completely into the medium. Here, we present the high-quality draft genome sequence of G. oxydans WSH-003, an industrial strain with both high l-sorbose productivity and extreme tolerance to saccharides and alditols.

A novel technique for in situ aggregation of Gluconobacter oxydans using bio-adhesive magnetic nanoparticles.

Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension.

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor.

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6g/l yeast extract, 4.7 g/l glycerol, 42.1g/l mannitol, 0.5 g/l K(2)HPO(4), 0.5 g/l KH(2)PO(4), 0.1g/l MgSO(4)·7H(2)O, and 2.0 g/l CaCO(3) with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8±2.4% of conversion rate of glycerol to DHA was achieved after 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate.

Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans.

Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant DeltaadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. DeltaadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting DeltaadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on DeltaadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

Capillary electrophoresis for the monitoring of carboxylic acid production by Gluconobacter oxydans.

Determination of carboxylic acids in Gluconobacter oxydans fermentations of wheat straw hydrolyzate was carried out. This matrix is of complex composition containing carbohydrates, organic compounds (e.g., amino acids, toxins), and inorganic salts making the analysis challenging even with separation techniques. A method based on capillary electrophoresis with indirect UV detection was developed for the simultaneous quantification of 18 carboxylic acids. The background electrolyte solution of ammonia, 2,3-pyridinedicarboxylic acid, and Ca2+ and Mg2+ salts, containing myristyltrimethylammonium hydroxide as a dynamic capillary coating reagent, was validated for the robust and repeatable separation of the carboxylic acids. Intraday relative standard deviations in the optimized method were less than 1.6% for migration times and between 1.0% and 5.9% for peak area. Interday relative standard deviations were less than 5.0% for migration times and between 5.7% and 9.3% for peak area. With 11 nl injected, detection limits for the analytes were between 10 and 43 micromol/l. Detection limits ranged from 0.1 to 0.5 pmol at signal-to-noise ratio of 3. The results demonstrated that wheat straw hydrolyzate was a suitable substrate for G. oxydans with a product yield of 45% for the formation of xylonic acid from xylose and 96% for the formation of gluconic acid from glucose.

Biochemical and spectroscopic properties of cyanide-insensitive quinol oxidase from Gluconobacter oxydans.

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.

Modelling bacterial spoilage in cold-filled ready to drink beverages by Acinetobacter calcoaceticus and Gluconobacter oxydans.

AIMS: Mathematical models were created which predict the growth of spoilage bacteria in response to various preservation systems. METHODS AND RESULTS: A Box-Behnken design included five variables: pH (2.8, 3.3, 3.8), titratable acidity (0.20%, 0.40%, 0.60%), sugar (8.0, 12.0, 16.0 * Brix), sodium benzoate concentration (100, 225, 350 ppm), and potassium sorbate concentration (100, 225, 350 ppm). Duplicate samples were inoculated with a bacterial cocktail (100 microl 50 ml(-1)) consisting of equal proportions of Acinetobacter calcoaceticus and Gluconobacter oxydans (5 x 10(5) cfu ml(-1) each). Bacteria from the inoculated samples were enumerated on malt extract agar at zero, one, two, four, six, and eight weeks. CONCLUSION: The pH, titratable acidity, sugar content, sodium benzoate, and potassium sorbate levels were all significant factors in predicting the growth of spoilage bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This beverage spoilage model can be used to predict microbial stability in new beverage product development and potentially reduce the cost and time involved in microbial challenge testing.

Optimized synthesis of L-sorbose by C(5)-dehydrogenation of D-sorbitol with Gluconobacter oxydans.

The optimization of L-sorbose synthesis by regiospecific dehydrogenation of D-sorbitol using Gluconobacter oxydans is reported. The current L-sorbose production processes that are based on G. oxydans and other bacterial strains are suboptimal as to yield and rate of L-sorbose synthesis. One reason for these problems is the toxicity that is induced by the substrate D-sorbitol when used in concentrations of >10% (w/v). This phenomenon significantly limits the potentials of L-sorbose production from an industrial point of view. The goal of this study was to develop a fast production process that yields L-sorbose in stoichiometric amounts starting from D-sorbitol concentrations that exceed 10% (w/v). A gradual improvement of the inoculum build-up procedure, culture medium composition, and process parameters ultimately led to a theoretically maximal L-sorbose productivity (200 g L(-1) of L-sorbose from 200 g L(-1) of D-sorbitol in 28 h of fermentation) using a Gluconobacter oxydans mutant strain that was selected under conditions of substrate inhibition. Because the D-sorbitol/L‐sorbose bioconversion is used to mass-produce vitamin C, the procedure reported here will contribute to a more efficient and more economic synthesis of vitamin C.