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     

Glycerol inhibition of growth and dihydroxyacetone production byGluconobacter oxydans

The evidence, kinetic aspects, and modelization of the inhibitory effect of glycerol on dihydroxyacetone (DHA) production byGluconobacter oxydans have been studied. The comparison of the maximal productivities and specific rates evaluated for initial concentrations of 31, 51, 76, 95, and 129 g L^–1 of substrate showed that glycerol exerts an inhibitory effect both on growth and DHA production: decrease of the growth-specific rate and of the specific rate of DHA production with increase of the initial glycerol content. The inhibition phenomenon was attributed to an immediate effect of glycerol on the biological activity. It was also established that the presence of glycerol at high concentration induces an increase in the time necessary for the cells to reach their maximal level of specific rates. This result tends to show that glycerol brings into play on the biological system the capacity to reach its optimal range of activity. The main models found in the literature dealing with substrate inhibition phenomena were then tested on experimental data. The exponential model describes at best the glycerol inhibition on growth (=0.53e^(–S/93.6)) and on DHA production (q_P=7e^(–S/76.7)). The kinetic study and modelization of the inhibition effect of glycerol on DHA production allows one, therefore, to fill the gap in the fundamental knowledge of this industrial fermentation, to show the maladjustment of the classical fermentation process used (batch), and to reconsider the conception for the optimization of the production (proposition of more adapted process like fed-batch and/or biphasic systems).     

Inhibitory effect of dihydroxyacetone onGluconobacter oxydans: Kinetic aspects and expression by mathematical equations

Summary Microbial conversion of glycerol into dihydroxyacetone (DHA) byGluconobacter oxydans was subjected to inhibition by excess substrate. Comparison of cultures containing increasing initial DHA contents (0 to 100 g l^–1) demonstrated that DHA also inhibited this fermentation process. The first effect was on bacterial growth (cellular development stopped when DHA concentration reached 67 gl^–1), and then on oxidation of glycerol (DHA synthesis only occurred when the DHA concentration in the culture medium was lower than 85 g l^–1). Productivity, specific rates and, to a lesser extent, conversion yields decreased as initial concentrations of DHA increased. The changes in the specific parameters according to increasing initial DHA contents were described by general equations. These formulae satisfactorily express the concave aspect of the curves and the reduction in biological activity when the cells were in contact with DHA concentrations of up to 96 g l^–1.Abbreviations X, S, P biomass, substrate, product concentrations - r _x,r _s,r _p rates of growth, consumption and production - ,q _s,q _p specific rates of growth, glycerol consumption and DHA production - Y _x/s, Y_p/s conversion yields of substrate into biomass and product - K _s constant of affinity of cells to the substrate - K _ip product inhibition constant - P _m threshold concentration of DHA in substrate     

Study of the inhibitory effect of the product dihydroxyacetone on Gluconobacter oxydans in a semi-continuous two-stage repeated-fed-batch process

The influence of the product inhibition by dihydroxyacetone (DHA) on Gluconobacter oxydans for a novel semi-continuous two-stage repeated-fed-batch process was examined quantitatively. It was shown that the culture was able to grow up to a DHA concentration of 80 kg m⁻³ without any influence of product inhibition. The regeneration capability of the reversibly product inhibited culture from a laboratory-scale bioreactor system was observed up to a DHA concentration of about 160 kg m⁻³. At higher DHA concentrations, the culture was irreversibly product inhibited. However, due to the robust membrane-bound glycerol dehydrogenase of G. oxydans, product formation was still active for a prolonged period of time. The reachable maximum final DHA concentration was as high as 220 kg m⁻³. The lag phases for growth increased exponentially with increasing DHA threshold values of the first reactor stage. These results correlated well with fluorescence in situ hybridization (FISH) measurements confirming that the number of active cells decreased exponentially with increasing DHA concentrations.     

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     

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.     

Process development of oxygen-demanding reactions utilizing a simple design with parallel glass tube reactors – Evaluated using Gluconobacter oxydans (DSM 24525)

Abstract

To investigate the possibility of using simple glass tubes as reactors for oxygen-demanding reactions, a setup was assembled to study the initial rate of conversion of glycerol to dihydroxyacetone (DHA) using Gluconobacter oxydans. Several parallel 10 mL glass tubes were incubated in a temperature-controlled shaker. The concentration of DHA was determined using a fast spectrophotometric HPLC-based method that could process 3 samples/min. It was shown that the obtained results were reproducible and the reaction rates remained constant throughout the reaction. Further, the system reached a high volumetric activity of 15.48 g DHA L^{? 1} h^{? 1} consuming 86 mmol L^{? 1} h^{? 1} oxygen before the system became mass-transfer limited, indicating a high diffusion of oxygen. It was concluded that the reactor system is well suited for process development where the requirement for oxygen is high and that the assay developed can be used to determine the initial rate of DHA production.     

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.     

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.     

REVERSIBLE KINETIC MODEL FOR THE SHORT-TERM REGULATION OF METHYLAMMONIUM UPTAKE IN TWO PHYTOPLANKTON SPECIES,DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Methylammonium, an ammonium analog, was used to study the short-term kinetics of ammonium uptake in a diatom, Phaeodactylum tricornutum Bohlin, and a green alga, Dunaliella tertiolecta Butcher. Time courses of methylammonium disappearance were measured over a wide range of initial substrate concentrations for the two species. It was shown that feedback inhibition, described mathematically by a reversible enzyme kinetic model, can be used to explain the data. For the two species, there was good agreement between the kinetic parameters obtained from the analysis of the uptake versus substrate curve and those from the fit of the reversible kinetic model to the time-course data. All time courses of CH₃NH₃⁺ disappearance could be described by constants V_m and k_s. Ammonium time-course data show some similarities to its analog, methylammonium. Our study suggests that the apparent change in V_m and k_s with time measured after the addition of saturating ammonium concentrations reflects an uncoupling between transport and assimilation of the substrate rather than a real change in the kinetic parameters of the transport mechanism.     

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.     

Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293

Gluconobacter melanogenus 3293 converts glycerol to dihydroxyacetone(DHA) during exponential growth on a yeast extract-phosphate medium at pH 7. The efficiency of this conversion in 25-liter batch fermentations has been found to increase over threefold, when oxygen tension is controlled by increasing the partial pressure of oxygen in the aeration. Conversion of glycerol to DHA does not occur under oxygen-limited fermentation conditions. When the dissolved oxygen tension was maintained at 0.05 atmospheres (using oxygen-enriched air), quantitative conversion of up to 100 g of glycerol/liter to DHA was obtained in 33 h. The amount of glycerol converted can be increased without increasing impeller speed or aeration rate. This increase is not the result of increased production of cell mass. The specific conversion of glycerol to DHA increased from 12.2 g of DHA/g of cell mass at the point of maximum conversion to 35.8 with oxygen enrichment. This increased specific production occurred even though the specific growth rate during the period of oxygen enrichment decreased from 0.23 to 0.06/h.     

Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient

Aims: To improve the yield and productivity of docosahexaenoic acid (DHA) by Schizochytrium sp. in terms of the analysis of microbial physiology. Methods and Results: A two‐stage oxygen supply control strategy, aimed at achieving high concentration and high productivity of DHA, was proposed. At the first 40 h, K_La was controlled at 150·1 h^?1 to obtain high μ for cell growth, subsequently K_La was controlled at 88·5 h^?1 to maintain high q_p for high DHA accumulation. Finally, the maximum lipid, DHA content and DHA productivity reached 46·6, 17·7 g l^?1 and 111 mg l^?1 h^?1, which were 43·83%, 63·88% and 32·14% over the best results controlled by constant K_La. Conclusions: This paper described a two‐stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp. Significance and Impact of the study: This study showed the advantage of two‐stage control strategy in terms of microbial physiology. As K_La is a scaling‐up parameter, the idea developed in this paper could be scaled‐up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in continuous cultures

The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O₂ and CO₂ were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO₂,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO₂,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO₂, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO₂,max measured in the BOM. Accordingly, only the kinetic constant Ks/K _i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K _s/K _i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999     

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.     

Reduction of extracellular dehydroascorbic acid by K562 cells

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent) K_m 7 μM , V_max 3·25 pmol min^?1 (10⁶ cells)^?1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0·1 pmol min^?1 (10⁶ cells)^?1 (1 μM of DHA^?1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and thenoyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min^?1 (10⁶ cells^?1). At a concentration of DHA of 100 μM this is 25 per cent of the observed reduction.     

Optimization of the microbial synthesis of dihydroxyacetone from glycerol with <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis>

An optimized repeated-fed-batch fermentation process for the synthesis of dihydroxyacetone (DHA) from glycerol utilizing Gluconobacter oxydans is presented. Cleaning, sterilization, and inoculation procedures could be reduced significantly compared to the conventional fed-batch process. A stringent requirement was that the product concentration was kept below a critical threshold level at all times in order to avoid irreversible product inhibition of the cells. On the basis of experimentally validated model calculations, a threshold value of about 60 kg m^-3 DHA was obtained. The innovative bioreactor system consisted of a stirred tank reactor combined with a packed trickle-bed column. In the packed column, active cells could be retained by in situ immobilization on a hydrophilized Ralu-ring carrier material. Within 17 days, the productivity of the process could be increased by 75% to about 2.8 kg m^-3 h^-1. However, it was observed that the maximum achievable productivity had not been reached yet.Abbreviations K _O Monod half saturation constant of dissolved oxygen (kg m^-3) - K _S Monod half saturation constant of substrate glycerol (kg m^-3) - O Dissolved oxygen concentration (kg m^-3) - P Product concentration (kg m^-3) - P _crit Critical product concentration constant (kg m^-3) - S Substrate concentration (kg m^-3) - t Time (s) - X Biomass concentration (dry weight) (kg m^-3) - Y _P/S Yield coefficient of product from substrate - Y _X/S Yield coefficient of biomass from substrate - Growth dependent specific production rate constant (kg m^-3) - Growth independent specific production rate constant (s^-1) - Specific growth rate (s^-1) - _max Maximum specific growth rate constant (s^-1)     

Kinetics of Mercury Reduction by Serratia marcescens Mercuric Reductase Expressed by Pseudomonas putida Strains

Mercury (Hg) resistance is widespread among microorganisms and is based on the intracellular transformation of Hg(II) to less toxic elemental Hg(0). The use of microbial consortia to demercurize polluted wastewater streams and environments has been demonstrated. To develop efficient and versatile microbial cleanup strategies requires detailed knowledge of transport and reaction rates. This study focuses on the kinetics of the key enzyme of the microbial transformation, e.g., the mercuric reductase (MerA) under conditions closely resembling the cell interior. To this end, previously constructed and characterized Pseudomonas putida strains expressing MerA from Serratia marcescens were applied. Of the P. putida strains considered in this study P. putida KT2442::mer73 constitutively expressing broad spectrum mercury resistance (merTPAB) yielded the highest mercuric reductase (MerA) activity directly after cell disruption. MerA in the raw extract was further purified (about 100 fold). Reduction rates were measured for various substrates (HgCl₂, Hg₂SO₄, Hg(NO₃)₂ and phenyl mercury acetate) up to high concentrations dependent on the purification grade. In all cases, a pronounced substrate inhibition was found. The kinetic constants determined for the cell raw extract are in agreement with those measured for intact cells. However, the rate data exhibit reduced affinity and inhibition with rising purification grade (specific activity). Therefore, the findings seemingly point to reactions preceding the catalytic reduction. Based on simplified assumptions, a kinetic model is suggested which reasonably describes the experimental findings and can advantageously be applied to the bioreactor design.     

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K _m and V _max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K _m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V _max,app>V _max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k _L a positively. The k _L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k _L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r _DHA (9.75 mg l⁻¹ h⁻¹) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.