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.     

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).     

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.     

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.     

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 μA/mM in phosphate buffer), low detection limit (0.8 μM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).     

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     

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.     

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.     

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.     

Macrokinetic model for Gluconobacter oxydans in 2-keto-L-gulonic acid mixed culture

A set of kinetic models have been developed for the production of 2-keto-L-gulonic acid from L-sorbose by a mixed culture of Gluconobacter oxydans and Bacillus megaterium. A metabolic pathway is proposed for Gluconobacter oxydans, and a macrokinetic model has been developed for Gluconobacter oxydans, where the balances of some key metabolites, ATP and NADH are taken into account. An unstructured model is proposed for concomitant bacterium Bacillus megaterium. In the macrokinetic model and unstructured model, the mechanism of interaction between Gluconobacter oxydans and Bacillus megaterium is investigated and modeled. The specific substrate uptake rate and the specific growth rate obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between the substrate feeding rate and the main state variables, such as the medium volume, the biomass concentrations, the substrate, and the is set up. A closed loop regulator model is introduced to approximate the induction of enzyme pool during lag phase after inoculation. Experimental results demonstrate that the model is able to describe 2-keto-L-gulonic acid fermentation process with reasonable accuracy.     

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 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.     

混合培养中巨大芽孢杆菌对氧化葡萄糖酸杆菌的作用

为查明维生素Ｃ二步发酵混合培养中巨大芽孢杆菌与氧化葡萄糖酸杆菌间的关系,通过生长曲线测定、静息细胞实验及摇瓶发酵实验研究了巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产生２－酮基－Ｌ－古龙酸作用的影响;采用超滤分离、凝胶层析及聚丙烯酰胺凝胶电泳技术对巨大芽孢杆菌胞外液中具有促进氧化葡萄糖酸杆菌产酸作用的活性物质进行了分离和纯化。结果表明,大菌胞内液和胞外液均可促进小菌生长,大菌胞外液中具有该作用的组分分子     

Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains

Applied Microbiology and Biotechnology - The growing consumer demand for low-calorie, sugar-free foodstuff motivated us to search for alternative non-nutritive sweeteners. A promising sweet-tasting...     

Purification and properties of two different dihydroxyacetone reductases in Gluconobacter suboxydans grown on glycerol

It is well known that in oxidative fermentation microbial growth is improved by the addition of glycerol. In a wild strain, glycerol was converted rapidly to dihydroxyacetone (DHA) quantitatively in the early growth phase by the action of quinoprotein glycerol dehydrogenase (GLDH), and then DHA was incorporated into the cells by the early stationary phase. Two DHA reductases (DHARs), NADH-dependent (NADH-DHAR) (EC 1.1.1.6) and NADPH-dependent (NADPH-DHAR) (EC 1.1.1.156), were detected in the same cytoplasm of Gluconobacter suboxydans IFO 3255. The former appeared to be inducible and labile in nature while the latter was constitutive and stable. The two DHARs were separated each other and were finally purified to crystalline enzymes. This report might be the first one dealing with NADPH-DHAR that has been crystallized. The two DHARs were specific only to DHA reduction to glycerol and thus contributed to cytoplasmic DHA metabolism, resulting in an improved biomass yield with the addition of glycerol.     

Analysis of aldehyde reductases from Gluconobacter oxydans 621H

Two cytosolic nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductases, Gox1899 and Gox2253, from Gluconobacter oxydans 621H were overproduced and purified from Escherichia coli. The purified proteins exhibited subunit masses of 26.4 (Gox1899) and 36.7 kDa (Gox2253). Both proteins formed homo-octamers exhibiting native masses of 210 and 280 kDa, respectively. The substrate spectra, optimal reaction conditions, and kinetic constants were determined for Gox1899 and Gox2253. Both enzymes efficiently catalyzed the reduction of medium/long-chain aldehydes. However, Gox1899 had a wider substrate spectrum and was more catalytically efficient. The best activity with Gox1899 was found for aliphatic aldehydes of C6-C10. In contrast, Gox2253 had a limited substrate spectrum and reduced octanal, nonanal, and decanal. Both enzymes were unable to oxidize primary alcohols. Aldehyde removal may be of particular importance for Gluconobacter because the membrane-bound alcohol dehydrogenase rapidly oxidizes short to long-chain alcohols, and large quantities of aldehydes could enter the cell, making detoxification necessary.