Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol

Investigations into physiological aspects of glycerol conversion to dihydroxyacetone (DHA) by Gluconobacter oxydans ATCC 621 were made. The activity levels of the enzymes involved in the three catabolic pathways previously known and the effects of specific inhibitors and uncoupling agents on cellular development, DHA synthesis, and cellular respiratory activity were determined. It was established that only two catabolic pathways are involved in glycerol dissimilation by this micro-organism. The only enzyme responsible for DHA production is membrane-bound glycerol dehydrogenase, which employs oxygen as the final acceptor of reduced equivalents without NADH mediation. The ketone is directly released into the culture broth. As the glycolytic and carboxylic acid pathways are absent, the pathway provided by the membrane-bound enzyme is indispensable for the energy requirements of G. oxydans. The cytoplasmic pathway, which begins by phosphorylation of glycerol followed by a dehydrogenation to dihydroxyacetone phosphate, allows growth of the bacterium. At the same time, the substrate transport mode was characterized as facilitated diffusion using radioactive [1(3)-³H]-glycerol. Concerning the DHA inhibition of microbial activity, the enzymatic study of the membrane-bound glycerol dehydrogenase showed the enzymatic origin of this phenomenon: a 50% decrease of the enzyme activity was observed in the presence of 576 mm DHA. The decrease in the rate of penetration of glycerol into cells in the presence of DHA indicates that growth inhibition is essentially due to the high inhibition exerted by the ketone on the substrate transport system.     

Repeated use of immobilized Gluconobacter oxydans cells for conversion of glycerol to dihydroxyacetone

Dihydroxyacetone (DHA) is of great interest in the fine chemical and pharmaceutical industry; therefore, the discovery of suitable biocatalysts for the efficient production of it is very necessary. In the experiment, Gluconobacter oxydans was immobilized in polyvinyl alcohol (PVA). Various parameters of the immobilized cells were investigated. The results have shown that the optimal conversion conditions by the immobilized cells were at 30 degrees C and pH 6.0. The immobilized cells remained very active over the period of 14 days for storage and only lost 10% of its original activity. Repeated use of immobilized cells for conversion of glycerol to DHA was carried out in a 1.5 L stirred tank reactor, the average conversion rate was about 86%. Despite the high shear stress, bead shape was not affected, even after five consecutive conversion cycles. The regenerated biocatalyst could recover 90% of its initial activity.     

Hydrogen peroxide as an oxygen source for immobilized Gluconobacter oxydans converting glycerol to dihydroxyacetone

Summary A flow injection analysis (FIA) system with amperometric detection was developed for measuring hydrogen peroxide which was used as an oxygen source for immobilized cells. A constant concentration of peroxide in the reactor was maintained by processing the analytical signal in a computer programmed as a PI-regulator. The concentration of dissolved oxygen was followed using a commercial Clark-electrode. The simultaneous measurements of hydrogen peroxide and dissolved oxygen are discussed with respect to process control.Conversion of glycerol to dihydroxyacetone by Gluconobacter oxydans immobilized in calcium alginate was used as a model system.Initial specific productivity increased with increasing hydrogen peroxide concentration. However, decreases in viable counts, enzymatic activities and overall productivities were noted. Various techniques for improving operational stability are discussed.     

Aldehyde production by alcohol oxidation with Gluconobacter oxydans

The microbial oxidation of various primary alcohols to the corresponding aldehydes has been investigated. A focused screening performed amongst some acetic acid bacteria showed that a newly isolated strain of Gluconobacter oxydans oxodizes various short-chain aliphatic alcohols to the corresponding aldehydes with negligible acid production. 3-Methyl-1-butanol (isoamyl alcohol) proved to be the better substrate with high yields (more than 90%) without by-product formation. This biotransformation also occurs with continuous or semicontinuous addition of substrate since the volatile product is removed from the medium under vigorous aeration conditions. Product recovery is attained either by the use of cold traps or by reversible complex formation.     

Semisynthetic culture medium for growth and dihydroxyacetone production by Gluconobacter oxydans

Only three vitamins (pantothenate, p-amino benzoic acid, nicotinic acid) and two amino acids (serine, glutamine) were required in the growth medium for Gluconobacter oxydans which allowed the concentration of yeast extract to be reduced to 5–10% of the previous concentration. When compared with data from cultivations with complex media, the new medium gave a lower yield (about 0.02 g biomass per g glycerol) and comparable growth rate (0.24 to 0.38 h–1) but a higher productivity (10.3 g dihydroxyacetone/gh).     

Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone

Gluconobacter oxydans could be immobilized as a biocatalyst for the conversion of glycerol to dihydroxyacetone. To reduce the production cost, the cells were produced from agricultural byproducts. Corn meal hydrolysate and corn steep liquor were employed to replace of sorbitol and yeast extract as medium for G. oxydans cell production. The optimal medium contained 80 g/L reducing sugar, 25 g/L corn steep liquor, and 10 g/L glycerol. The cell mass was about 4.22 g/L and the glycerol dehydrogenase activity was about 5.23 U/mL. For comparison, the cell mass was about 4.0 g/L and the glycerol dehydrogenase activity was about 5.35 U/mL cultured in sorbitol and yeast extract medium. These studies shown the corn meal hydrolysate and corn steep liquor medium was similar in performance to a nutrient-rich medium, but the cost of production was only 15% of that cultured in sorbitol and yeast extract medium. It was an economical process for the production of G. oxydans cells as biocatalyst for the conversion of glycerol to dihydroxyacetone in industry.     

Kinetic aspects of glucose oxidation by Gluconobacter oxydans cells immobilized in calcium alginate

Summary Gluconobacter oxydans subspecies suboxydans (ATCC 621 H), when growing at high glucose concentrations, oxidizes this substrate incompletely and gluconic acid accumulates in the medium in almost stoichiometric amounts. Such cells were harvested and entrapped in various alginate gels. The preparation with the highest retention of glucose oxidizing activity was used in further studies with the aim of developing an efficient process for continuous gluconic acid production.The retention of activity increases (up to 95%) as the alginate concentration in the gel decreases or the cell/alginate weight ratio is enhanced. In the latter case, however, transport of oxygen to and inside the biocatalyst beads rapidly becomes rate-limiting and thus lowers the efficiency of the biocatalyst. Similarly, the efficiency decreases as the size of the biocatalyst beads increases. In no case rate-limitation by transport of glucose was found. Thus, biocatalyst activity per unit volume of support, diameter of the biocatalyst beads, and aeration efficiency are important parameters for reactor design.     

Asymmetric oxidation by Gluconobacter oxydans

Asymmetric oxidation is of great value and a major interest in both research and application. This review focuses on asymmetric oxidation of organic compounds by Gluconobacter oxydans. The microbe can be used for bioproduction of several kinds of important chiral compounds, such as vitamin C, 6-(2-hydroxyethyl)amino-6-deoxy-α-l-sorbofuranose, (S)-2-methylbutanoic acid, (R)-2-hydroxy-propionic acid and 5-keto-d-gluconic acid. Characteristics of the bacteria and research progress on the enantioselective biotransformation process are introduced.     

Continuous production of gluconic acid by immobilized Gluconobacter oxydans cell bioreactor

Summary Living Gluconobacter oxydans cells were attached on fibrous nylon carrier. Free gluconic acid was directly continuously produced in an aerated tubular immobilized-cell bioreactor for at least 6 months, with a volumetric productivity of at least 5 g/lh at 100 g/l substrate glucose and about 80 g/l product gluconic acid concentrations. The highest volumetric productivity in respect to glucose concentration was obtained with 175 g/l glucose, with about 120 g/l product gluconic acid level. With self-directing optimization procedure in respect to maximum product gluconic acid level, productivities as high as about 12–15 g/lh were obtained at relatively high substrate feed rate of 0.166 l/lh and relatively low aeration rate of 0.5 l/lmin. The highest glucose conversion of about 96% was obtained with a long residence time, at the lowest substrate feed rate used at a relatively low aeration rate, resulting however in a significant increase in ketogluconic acid production.     

Inhibitory effects of glycerol on Gluconobacter oxydans

Summary The inhibitory effects of glycerol on Gluconobacter oxydans were measured separately. The kinetics of oxygen uptake rate representing the DHA production, the CO₂ evolution rate representing the assimilation of the product, and the specific growth rate were mathematically modelled. Glycerol does not inhibit DHA formation and CO₂-evolution.now: Institut für Biotechnologie, TU Graz, Petersgasse 12, 8010 Graz, Austria     

Oxygen supply to immobilized cells: 5. Theoretical calculations and experimental data for the oxidation of glycerol by immobilized Gluconobacter oxydans cells with oxygen or p-benzoquinone as electron acceptor

Theoretical calculations of reaction kinetics were done for one-step reactions catalyzed by cells immobilized in spherical beads. The reactions catalyzed by free cells were assumed to obey Michaelis-Menten kinetics for a one-substrate reaction. Both external (outside the beads) and internal (inside the beads) mass transfer of the substrate were considered for the immobilized preparations. The theoretical calculations were compared with experimental data for the oxidation of glycerol to dihydroxyacetone by Gluconobacter oxydans cells immobilized in calcium alginate gel. Glycerol was present in excess so that the reaction rate was limited by oxygen. The correlation between experimental data and theoretical calculations was quite good. The calculations showed how the overall effectiveness factor was influenced by, for example, the particle size and the cell density in the beads. In most cases the reaction rate was mainly limited by internal mass transfer of the substrate (oxygen). As shown previously, p-benzoquinone can replace oxygen as the electron acceptor in this reaction. The same equations for reaction kinetics and mass transfer were used with p-benzoquinone as the rate-limiting substrate. Parameters such as diffusivity, maximal reaction rate, and K were, of course, different. In this case also, the correlation between the model and the experimental results was quite good. Much higher production rates were obtained with p-benzoquinone as the electron acceptor compared to when oxygen was used. The reasons for this fact were that p-benzoquinone gave a higher maximal reaction rate for free cells and the solubility of p-benzoquinone was higher than for oxygen. Different methods of increasing the rate of microbial oxidation reactions are discussed.     

Kinetics of polyol oxidation with Gluconobacter oxydans

Summary The influence of culture pH on the metabolism of Gluconobacter oxydans was determined. An acidic milieu during growth of the organism enhances the oxidation rate. The CO₂ evolution rate representing the assimilation of the product is inhibited by a low pH value. Growth of the bacteria is possible both on glycerol and DHA in separate phases, which is not a controlled as diauxic growth. Product formation follows Luedeking-Piret kinetics.now: Institut für Biotechnologie, TU Graz, Petersgasse 12, 8010 Graz, Austria     

Intracytoplasmic membrane formation and increased oxidation of glycerol growth of Gluconobacter oxydans.

Gluconobacter oxydans is well known for the limited oxidation of compounds and rapid excretion of industrially important oxidation products. The dehydrogenases responsible for these oxidations are reportedly bound to the cell's plasma membrane. This report demonstrates that fully viable G. oxydans differentiates at the end of exponential growth by forming dense regions at the end of each cell observed with the light microscope. When these cells were thin sectioned, their polar regions contained accumulations of intracytoplasmic membranes and ribosomes not found in undifferentiated exponentially growing cells. Both freeze-fracture-etched whole cells and thin sections through broken-cell envelopes of differentiated cells demonstrate that intracytoplasmic membranes occur as a polar accumulation of vesicles that are attached to the plasma membrane. When cells were tested for the activity of the plasma membrane-associated glycerol dehydrogenase, those containing intracytoplasmic membranes were 100% more active than cells lacking these membranes. These results suggest that intracytoplasmic membranes are formed by continued plasma membrane synthesis at the end of active cell division.     

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.     

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 5.6 g/l yeast extract, 4.7 g/l glycerol, 42.1 g/l mannitol, 0.5 g/l K₂HPO₄, 0.5 g/l KH₂PO₄, 0.1 g/l MgSO₄·7H₂O, and 2.0 g/l CaCO₃ 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 72 h of biotransformation using 10 g/l resting cells at 30 °C, pH 5.0 and 1.5 vvm of aeration rate.     

Molecular simulation of pyrroloquinoline quinine-dependent glycerol dehydrogenase in Gluconobacter oxydans

During the fermentation process from glycerol to 1,3-dihydroxyacetone (DHA) by Gluconobacter oxydans, the increase in the concentration of glycerol shows obvious inhibition on the cell growth and DHA production. Researches on the interaction mechanism between glycerol and glycerol dehydrogenase (sldha) are important to improve the conversion rate from glycerol to DHA and to enhance the strains tolerance to glycerol. At present, the 3D structure of sldha is still unknown. So we analysed the 3D structure and then found the binding sites of glycerol with sldha. In the present study, we constructed the 3D structure of sldha by the homology modelling method based on Modeller 9v6 software. Four proteins, 1yiqA, 1kb0A, 1kv9A and 1lrwA, from Protein Data Bank were chosen as templates, since they have the highest similarities with sldha in Protein Data Bank which is 38%, 37%, 39% and 38%, respectively. The molecular dynamics simulation of constructed 3D structure of sldha by Gromacs 4.0.5 was carried out. Finally, the binding sites of Ala715 and H719 were found through the molecular docking simulation between glycerol and sldha by using Autodock 4.2.     

Optimization of sorbose production from sorbitol by Gluconobacter oxydans

The basic parameters were studied influencing the conversion of orbitol to sorbose by Gluconobacter oxydans(industrial strain from FARMAKON Co., Czechoslovakia). The most effective conversion in the stationary phase was reached at pH 5.0, no inhibitory effect of sorbitol in a concentration ranging from 20 to 200 g/l and a minimum inhibitory effect of the sorbose concentration up to 200 g/l were observed. According to the optimum conditions mentioned above the optimized course of the fed-batch cultivation was proposed. The final concentration of sorbose of 410 g/l was reached after 36 hours.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

Electron transport system associated with direct glucose oxidation in Gluconobacter oxydans

Summary The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic membrane inGluconobacter oxydans (ATCC 9937) has been postulated. High turnover of dehydrogenases under oxygen enrichment conditions is explained on the basis of a simplistic electron transport chain comprising cytochrome c553 (MW 23000) as a subunit of dehydrogenase and a cytochrome b562. The electron transport chain under low dissolved oxygen tension (DOT) is shown to comprise a number of cytochrome c species with very low midpoint potential difference.