Kinetic Characterization of Apical <Emphasis Type="SmallCaps">D</Emphasis>-Fructose Transport in Chicken Jejunum

In mammals, D-fructose transport takes place across the brush-border membrane of the small intestine through GLUT5, a member of the facilitative glucose transporter family. In the present paper, we describe and characterize for the first time the apical transport of D-fructose in chicken intestine. Brush-border membrane vesicles (BBMV) were obtained from jejunum of 5- to 6-wk-old chickens. D-Fructose uptake by BBMV from chicken jejunum comprises a saturable component and a simple diffusion process. The maximal rate of transport (V_max) for D-fructose was 2.49 nmol·(mg prot)^–1·s^–1, the Michaelis constant (K_m) was 29 mM, and the diffusion constant (K_d) was 25 nl·(mg prot)^–1·s^–1. The apical transport of D-fructose was Na⁺-independent, phlorizin-, phloretin-, and cytochalasin B-insensitive, and did not show cis-inhibition by D-glucose or D-galactose. These properties, together with the detection of specific GLUT5 mRNA, indicate the presence of a low-affinity high-capacity GLUT5-type carrier in the chicken jejunum, responsible for the entry of D-fructose across the brush-border membrane of enterocytes.     

Development of a continuous system for the degradation of a cyanuric acid by adsorbed Pseudomonas sp. NRRL B-12228

Cyanuric acid in high concentrations (15.5 mm) was degraded completely by Pseudomonas sp. NRRL B-12228 independently of glucose concentration. In the batch fermentations there was a relation between the glucose concentration, on the one hand, and the liberation of ammonia or production of protein, on the other. The greater the supply of carbon, the more biomass was produced, and fewer NH _inf4 ^sup+ ions were released. Continuous fermentations using adsorbed cells could be performed to degrade cyanuric acid. In spite of different glucose feeding there was only a negligible difference in residues of s-triazine. In a one-step continuous system with dilution rates between 0.021 h^–1 and 0.035 h^–1, even a ratio of 0.65 between glucose and cyanuric acid was not sufficient to degrade the cyanuric acid supplied (320–540 mol l^–1 h^–1) completely. When a continuous two-step system was applied with dilution rates between 0.035 h^–1 and 0.056 h^–1, the consumption of carbon source could be minimized while s-triazine degradation up to 860 mol l^–1 h^–1 was complete. In this way the ratio between glucose and cyanuric acid could be increased to 0.25 (molar C:N ratio = 0.33:1). Thereby the process was made considerably more economic.     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture

Summary The effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes from glucose was investigated in a microaerobic continuous culture. At a dilution rate of 0.20 h^–1 and a fixed oxygen uptake rate (OUR) of 31.5 mmol l^–1 h^–1 the biomass concentration increased with pH ranging from 5.0 to 7.0, while the specific ATP requirement of the cells decreased. In the pH range 5.5–6.5 the product concentration (butanediol + acetoin) was maximal and nearly constant. However, the specific production continuously declined with increasing pH. Experiments with addition of acetic acid showed that the various effects of pH are due to inhibition of the by-product acetic acid on cell growth. The strength of the acetic and inhibition depended only on the concentration of its undissociated form [HAc]. The biomass concentration and the specific OUR were also only functions of [HAc], irrespective of the pH. Although the specific ATP requirement (q ATP) strongly depended on the pH, [HAc] at constant pH. Offprint requests to: W.-D. Deckwer     

Characterisation of kinetic parameters and metabolic transition of Corynebacterium glutamicum on l-lysine production in continuous culture

Kinetic parameters and physiological states of Corynebacterium glutamicum at the growing and l-lysine-overproducing phase were characterised in continuous culture on threonine-limited complex and minimal media. High l-lysine productivity occurred at dilution rates ranging from 0.1 h^–1 to 0.3 h^–1 on threonine-limited complex medium, and at dilution rates ranging from 0.1 h^–1 to 0.15 h^–1 in minimal medium. l-Lysine yields of 0.25 g/g (0.31 g/g as l-lysine hydrochloride) in complex medium, and of 0.17 g/g (0.21 g/g as l-lysine hydrochloride) in minimal medium, corresponding respectively to intrinsic yields of 0.533 g/g and 0.572 g/g were obtained. These intrinsic yield factors are closed to the theoretical ones (0.608 g/g, 0.75 mol/mol). Intrinsic biomass yields were calculated as 0.658 g/g in complex medium and 0.283 g/g in minimal medium. CO₂ production has been clearly related to l-lysine production. According to our results on specific uptake rates and specific productivities in complex medium, metabolic rearrangement should occur during the transition from the growing phase to the l-lysine-overproducing phase. This phenomenon was further investigated.     

Lactic acid productivity of a continuous culture of Lactobacillus delbreuckii

Summary Maximum volumetric productivities of biomass (1.40 gl^–1h^–1) and lactic acid (8.93 gl^–1h^–1) for a continuous culture ofLactobacillus delbreuckii occurred between dilution rates 0.35h^–1 and 0.40h^–1. All major nutrients were in excess in these cultures. Glucose utilisation was complete at dilution rates of 0.1h^–1 and lower. Product and biomass yields were constant in the dilution rate range studied (0.05h^–1 to 0.50h^–1).     

Expression of glf Z.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum

A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)⁻¹ h⁻¹. Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)⁻¹ h⁻¹, yielding 87 g l⁻¹ D-mannitol from 93.7 g l⁻¹ D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD⁺ ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l⁻¹ D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)⁻¹ h⁻¹ was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells. Dedicated to Prof. Hermann Sahm on the occasion of his 65th birthday.     

Physiological aspects of l-lysine production: effect of nutritional limitations on a producing strain of Corynebacterium glutamicum

The effect of nutritional limitations, such as phosphorus and carbon, on the production of l-lysine by Corynebacterium glutamicum was studied in continuous culture. We observed that phosphate-limited cultures at low growth rates were favourable to l-lysine production. l-Lysine was produced when a culture at low dilution rate (0.03 h^–1) was established. A dilution rate of about 0.04 h^–1 should be maintained in order to assure good productivity and an l-lysine yield of 0.53 g/g. Under carbon-limiting conditions the maintenance energy and growth yield of 0.03 g/g·g^–1·h^–1 and 0.41 g/g, respectively, have been obtained. Under these limiting conditions the l-lysine production was not favoured even at lower dilution rates.Correspondence to: N. Coello     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Metabolic and kinetic studies of hybridomas in exponentially fed-batch cultures using T-flasks

Exponentially fed-batch cultures (EFBC) of a murine hybridoma in T-flasks were explored as a simple alternative experimental tool to chemostats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode using an exponentially increasing and predetermined stepwise feeding profile of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution rates below the maximum growth rate. In particular, steady-state for growth rate and concentration of viable cells, glucose, and lactate was attained at different dilution rates between 0.005 and 0.05 h^–1. For such a range, the glucose and lactate metabolic quotients and the steady-state glucose concentration increased, whereas total MAb, volumetric, and specific MAb production rates decreased 65-, 6-, and 3-fold, respectively, with increasing dilution rates. The lactate from glucose yield remained relatively constant for dilution rates up to 0.03 h^–1, where it started to decrease. In contrast, viability remained above 80% at high dilution rates but rapidly decreased at dilution rates below 0.02 h^–1. No true washout occurred during operation above the maximum growth, as concluded from the constant viable cell number. However, growth rate decreased to as low as 0.01 h^–1, suggesting the requirement of a minimum cell density, and concomitant autocrine growth factors, for growth. Chemostat operation drawbacks were avoided by EFBC in T-flasks. Namely, simple and stable operation was obtained at dilution rates ranging from very low to above the maximum growth rate. Furthermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.Abbreviations EFBC exponentially-fed batch culture - CSC continuous suspended culture - MAb monoclonal antibody - D dilution rate - q _i metabolic quotient or specific rate of consumption or production of i     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Production of thermostable xylanases in batch and continuous culture by Thermomonospora fusca KW 3

Summary The actinomycete Thermomonospora fusca KW 3 produced novel thermostable xylanases in batch and continuous cultures in media containing insoluble xylan. The production of xylanases could be induced with oat spelt or beech xylan. Very low activities were detected when the strain was grown on glucose or xylose. In continuous cultivations, mycelial wall growth could be prevented using a stirrer speed controller. Homogeneous mixing of the insoluble substrate was obtained by vibrating the flexible tubes. T. fusca KW 3 could be grown on insoluble xylan at growth rates as high as 0.23 h^–1, equivalent to a doubling time of 3 h. Xylanase activity decreased from maximum levels of 2.5 units (U) ml^–1 with increasing dilution rate and was nearly constant at a level of 0.5 U ml^–1 with dilution rates greater than 0.1 h^–1. Correspondence to: P. Röthlisberger     

The influence of cyclic glucose feeding on a continuous bakers' yeast culture

Conclusions Except for the pronounced adaptation-hysteresis effect, the pulse experiments exhibited the expected trend: deviation from the steady feed reference curve was greatest at lowest dilution rates. Under conditions of lowest glucose level the effect of pulsing would be expected to cause the largest fluctuations in glucose, causing a certain fraction of the cells to ferment. Generally over the entire dilution rate range the biomass production was decreased and the ethanol was increased by pulsing the feed stream. There is, however, some evidence that pulse feeding can trigger quite unexpected results. Point (6) at D=0.3 h^–1 in Fig. 1 exhibit a biomass productivity which was about 20% greater than the continuous feeding reference value (DX=3.6 kg m^–3 h^–1 as compared with 3.0 kg m^–3 h^–1). Such performance would be of significant commercial value, but the poor reproducibility due to adaptation, as seen here, certainly would preclude its application.The results obtained should also be applicable to fed batch operation at the corresponding glucose level. Further experiments including the variation of the glucose feeding period would be necessary to obtain a conclusive picture. The observed phenomena are likely to occur in other fermentations and could eventually explain some of the problems existing with scale up of fermentation processes.Symbols D dilution rate h^–1 - P product (ethanol) concentration kg m^–3 - Q_O2 specific oxygen uptake rate mol kg^–1 s^–1 - Q_CO2 specific CO₂ production rate mol kg^–1 s^–1 - S substrate (glucose) concentration kg m^–3 - X biomass concentration kg m^–3 - Y_P/S yield of ethanol from glucose kg kg^–1 - Y_X/S yield of biomass from glucose kg kg^–1     

Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose

In order to improve the production rate of l-lysine, a mutant of Corynebacterium glutamicum ATCC 21513 was cultivated in complex medium with gluconate and glucose as mixed carbon sources. In a batch culture, this strain was found to consume gluconate and glucose simultaneously. In continuous culture at dilution rates ranging from 0.2 h⁻¹ to 0.25 h⁻¹, the specific l-lysine production rate increased to 0.12 g g⁻¹ h⁻¹ from 0.1 g g⁻¹ h⁻¹, the rate obtained with glucose as the sole carbon source [Lee et al. (1995) Appl Microbiol Biotechnol 43:1019–1027]. It is notable that l-lysine production was observed at higher dilution rates than 0.4 h⁻¹, which was not observed when glucose was the sole carbon source. The positive effect of gluconate was confirmed in the shift of the carbon source from glucose to gluconate. The metabolic transition, which has been characterized by decreased l-lysine production at the higher glucose uptake rates, was not observed when gluconate was added. These results demonstrate that the utilization of gluconate as a secondary carbon source improves the maximum l-lysine production rate in the threonine-limited continuous culture, probably by relieving the limiting factors in the lysine synthesis rate such as NADPH supply and/or phosphoenolpyruvate availability. Received: 16 May 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Two-stage continuous fermentation of Clostridium acetobutylicum: effects of pH and dilution rate

Summary The kinetics of a two-stage continuous fermentation of Clostridium acetobutylicum have been studied. The pH and the dilution rate have been shown to be two essential factors for process optimization. An increase in pH or dilution rate in the first stage decreased solvent production in the second fermentor. To achieve optimal solvent production, the pH had to be maintained at 4.5 in the first stage and between 4.5 and 5.0 in the second stage. Dilution rates of 0.08 h^–1 and 0.04 h^–1,respectively, in the first and second fermentors allowed a high solvent concentration. When the pH was maintained at 4.5 in each stage and when the dilution rates were 0.08 h^–1 and 0.04 h^–1 in the first and second fermentors respectively, 21 g/l solvent concentration was achieved. A conversion yield of 0.36 g solvents/g glucose consumed was obtained with total consumption of glucose. Biomass was only produced in the first stage together with 40% of the solvents, indicating that solvent production had to be induced in the first fermentor. Offprint requests to: J. M. Engasser     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.