Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Summary Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation.With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:Nomenclature D dilution rate (1/h) - DOT dissolved oxygen tension (%) - m_o2 maintenance coefficient (mmol O₂/g cell mass h) - q_o2 specific oxygen uptake rate (mmol O₂/g cell mass h) - q_s specific xylose uptake rate (g xylose/g cell mass h) or (mmol xylose/g cell mass h) - q_x specific xylitol production rate (g xylitol/ g cell mass h) or (mmol xylitol/ g cell mass h) - q_e specific ethanol production rate (g ethanol/ g cell mass h) or (mmol ethanol/ g cell mass h) - q_CO2 specific carbon dioxide production rate (mmol CO₂/g cell mass h) - S xylose concentration (g/1) - Y_cm/s cell mass yield coefficient, (g cell mass/mmol xylose) or (g cell mass/ g xylose consumed) - Y_cm/O2 cell mass yield coefficient, (g cell mass/mmol O₂) - Y_X/S xylitol yield coefficient (g xylitol/g xylose consumed) - Y_x/O2 xylitol yield coefficient (g xylitol/mmol O₂) - Y_e/s ethanol yield coefficient (g ethanol/g xylose consumed) - OUR oxygen uptake rate (mmol O₂/1h) - specific growth rate (1/h)     

Measurement of oxygen uptake and carbon dioxide production rates ofmammalian cells using membrane mass spectrometry

A method for the measurement of oxygen uptake and carbon dioxide production rates in mammalian cell cultures using membrane mass spectrometry is described. The small stirred reactor with a volume of 15 ml and integrated pH-control permits the economical application of isotopically labelled substrates and ¹³C-labelled bicarbonate buffer. Repetitive experiments showed the reproducibility of the method. In one case bicarbonate-free HEPES buffer was used and carbon dioxide production was measured using the intensity of the peak at m/z = 44(¹²CO₂). In all other cases H¹³CO₃ ⁻ -buffer was applied and also¹²CO₂ was measured. The minimum cell density required was only 2 × 10⁴ cells ml⁻¹. In the hybridoma T-flask cultivation studied here the measured specific oxygen uptake and carbon dioxide production rates were reasonably constant during the exponential growth phase and decreased significantly afterwards. Estimated respiratory quotients were always between0.90 and 0.92 except in HEPES-buffer, where a value of 0.67 was found. In the latter case specific oxygen uptake rate was higher than in bicarbonate buffered culture, however, carbon dioxide production rate was lower, and viable cell density was lowest. The addition of phenazine methosulfate, an artificial electron acceptor, increased both rates resulting in highest viable cell density but also highest lactate production rate. Glucose and glutamine pulse-feeding increased final cell density. The method described is directly applicable for samples from batch, fed-batch and continuous cultivations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Conversion of cellulose into fungal cell mass

Summary Chaetomium cellulolyticum (ATCC 32319) was cultivated on glucose, Avicel and/or Sigmacell in a 20-1 stirred tank batch reactor. The substrate (cellulose) concentration, the cell mass concentration (through protein and/or nitrogen content), reducing sugar concentration, the enzyme activity, the alkali consumption rate, the dissolved O₂ and CO₂ concentrations in the outlet gas were measured. The specific growth rate, the substrate yield coefficient, cell productivity, the oxygen consumption rate, the CO₂ production rate and the volumetric mass transfer coefficient were determined. At the beginning of the growth phase the oxygen utilization rate exhibits a sharp maximum. This maximum could be used to start process control. Because of the long lag phase periodic batch operation is recommended.Symbols CP cell protein concentration (g l^–1) - FPA FP enzyme activity (IU l^–1) - GP dissolved protein concentration (g l^–1) - IU international unit of enzyme activity - k_La volumetric mass tranfer coefficient (h^–1) - LG alkali (1 n NaOH) consumption (ml) - LGX specific alkali consumption rate per cell mass (ml g^–1 h^–1) - P cell mass productivity (g l^–1 h^–1) - specific oxygen consumption rate per cell mass (g g^–1 h^–1) - Q aeration rate (volumetric gas flow rate per volume of medium, vvm) (min^–1) - N impeller speed (revolution per minute, rpm) (min^–1) - S substrate concentration (g l^–1) - S⁰ S at t_F=0 (g l^–1) - S₀ S in feed (g l^–1) - SR acid consumption (ml) - TDW total dry weight (g l^–1) - T temperature (° C) - t_F cultivation time (h) - U substrate conversion - X cell mass concentration (g l^–1) - Y_X/S vield coefficient - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1)     

Über den Einfluß niedriger und hoher O2-Partialdrucke auf den Sauerstoff- und Kohlendioxidumsatz von Amaranthus und Phaseolus während der Lichtphase

Summary Carbon dioxide and oxygen gas exchange of illuminated Amaranthus and Phaseolus leaves was measured from 0–600 ppm of CO₂ in an open system.At low oxygen concentration (2% O₂) the ratio of CO₂ uptake to O₂ evolution came close to 1.At high oxygen partial pressure (42% O₂) the O₂ compensation point of an Amaranthus leaf was increased and oxygen evolution was depressed. Accordingly the CO₂/O₂ quotients were variable; the lowest value of 1,9 differed significantly from 1,0.The oxygen and carbon dioxide compensation points of a Phaseolus leaf were increased at high oxygen concentration (42% O₂) and oxygen evolution as well as carbon dioxide uptake were reduced. Therefore the ratios CO₂ over O₂ varied and differed greatly from 1,0.It was concluded that the nature of photosynthates is regulated by the gas composition around the leaves.     

Physiologically motivated strategies for control of the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production

The efficiency of the fed-batch cultivation of recombinant Escherichia coli AT2471 for phenylalanine production is highly dependent on the distribution of the carbon flow between the main process products — biomass, phenylalanine, acetic acid and carbon dioxide. In order to enhance the process performance, the effects of several factors, namely glucose feeding, tyrosine feeding and oxygen supply, were investigated experimentally. As a result, a set of control strategies was developed, designed to tolerate phenylalanine synthesis at the expense of the remaining products. The DO was controlled to prevent acetic acid excretion due to oxygen limitation. The total amount of tyrosine fed was used to provide an optimal balance between biomass synthesis and that of phenylalanine. Special algorithms for control of the glucose feed rate were applied to eliminate the threat of acetic acid excretion due to overfeeding, and at the same time, to reduce excessive CO₂ evolution caused by unnecessarily severe glucose limitation. The joint application of these strategies resulted in greatly improved efficiency in the phenylalanine production process: the final phenylalanine concentration reached 46 g/l, the yield was above 17%, and the productivity-0.85 g/l·h. In combination, these data exceed the results reported by others, and are much higher than those obtained by use before the implementation of the proposed complex of techniques.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Continuous cultivation of Saccharomyces cerevisiae. II. Growth on ethanol under transient-state conditions

Growth of Saccharomyces cerevisiae LBG H 1022 on ethanol under transient-state conditions was studied. As a cultivation device, an aerated Chemap fermentor combined with continuously working gas analyzers for oxygen and carbon dioxide was used. Yeast cell dry matter, substrate concentration, specific oxygen uptake, specific carbon dioxide release, and respiration quotient were measured during the different transient states. Depending on which range of the dilution rate the initial steady state was found, we obtain different responses to the shift experiment. For the lower range, up to D = 0.07, we deal with damped oscillations ranging above and below the steady-state values. For the higher specific growth rates, the rate of damping is strongly enhanced and the shape of the curves becomes an asymptotic approach to the final steady states.     

An adaptive on-line control feed rate system in a laboratory scale bakers yeast process

Summary A new control policy for the on-line optimization of the nutrient supply in bakers yeast process is proposed. A feed rate corresponding to minimal substrate uptake time was shown to be optimal for cell yield and specific growth rate. Cultivation results of baker's yeast are presented.Nomenclature c glucose concentration in wort (mol.l^–1) - C total glucose used (mol) - c_e ethanol concentration in wort (mg.l^–1) - c_p glucose concentration in fresh medium (mol.l^–1) - dt/dc glucose consumption time (sec.mol^–1) - F substrate feed rate (litre.hr^–1) - q_c glucose uptake rate (mol.hr^–1) - Q_c specific glucose uptake rate (moll.g^–1.hr^–1) - qO₂ oxygen uptake rate (mol.hr^–1) - QO₂ specific oxygen uptake rate (mol.g^–1.hr^–1) - r_x productivity (g.l^–1.hr^–1) - t time (hr) - x biomass concentration (g.l^–1) - X total biomass (g) - Y_x/c cell yield (g.g^–1): (g.mol^–1) - Y_o/c consumed oxygen to glucose ratio (mol.mol^–1)     

Data consistency,yield, maintenance,and hysteresis in batch cultures of Candida lipolytica cultured on n-hexadecane

Candida lipolytica was cultured batchwise using n-hexadecane as the main carbon source. Biomass production, n-hexadecane consumption, oxygen consumption, and carbon dioxide evolution were measured to follow the fermentation. The consistency of the measured data was examined using integrated and instantaneous available electron and carbon balances. Values of the “true” growth yield, η_max, and maintenance coefficient, m_e were estimated using three different sets of data (biomass and n-hexadecane, oxygen and biomass, and CO₂ and biomass), and the results were compared with estimates obtained from literature data. Hysteresis patterns were observed in plots of specific rates of oxygen consumption and carbon dioxide evolution versus specific growth rate.     

Respiratory activity and growth kinetics of Candida yeasts related to carbon sources and available energy

Summary Three yeasts of the genus Candida (Candida intermedia, candida lipolytica and Candida tropicalis) were cultivated batchwise on three different carbon sources: glucose, acetate, and hexadecane. Growth curves, oxygen uptake rates, CO₂ evolution rates and the amount of oxygen required for biomass production were determined. The data were compared and discussed from the point of maximum specific growth rate, maximum oxygen uptake rate, carbon conversion into CO₂ and biomass, consumption of oxygen and available energy for cell synthesis. The results indicated a relationship between _m m, Y_s, Y_O, and for different carbon sources. Y_O and were in the same order of magnitude for acetate (0.58 and 0.38 respectively) and hexadecane (0.45 and 0.40 respectively). These values were remarkably lower than those for glucose (1.26 and 0.54 respectively).Symbols av e^– Available electrons per mol of substrate (dimensionless) - E_av Energy available per mol of substrate (dimensionless) - C_d Dissimilated carbon (%) - m Maximum specific rate of oxygen uptake (mMO₂ h^–1 g^–1) - RQ CO₂ evolved per O₂ consumed - mol. wt. Molecular weight - Y_ATP Biomass mass yield based on mol of ATP generated (g) - Biomass mass yield based on available energy (g) - Y_M Biomass mass yield based on mol of organic substrate (g) - Y_O Biomass mass yield based on oxygen consumed (gg^–1) - 1/Y_O Oxygen consumed for one gram of biomass produced (gg^–1) - Y_s Biomass mass yield based on organic substrate (dimensionless) - b Reductance degree of biomass (equiv. available electrons/g atom carbon) - s Reductance degree of organic substrate (equiv. available electrons/g atom carbon) - Fraction of energy in organic substrate which is converted to biomass - b Weight fraction carbon in biomass (dimensionless) - s Weight fraction carbon in organic substrate (dimensionless) - _m Maximum specific growth rate (h^–1)     

Control of continuous fed-batch fermentation process using neural network based model predictive controller

Cell growth and metabolite production greatly depend on the feeding of the nutrients in fed-batch fermentations. A strategy for controlling the glucose feed rate in fed-batch baker’s yeast fermentation and a novel controller was studied. The difference between the specific carbon dioxide evolution rate and oxygen uptake rate (Q _c − Q _o) was used as controller variable. The controller evaluated was neural network based model predictive controller and optimizer. The performance of the controller was evaluated by the set point tracking. Results showed good performance of the controller.     

Control of carbon source supply and dissolved oxygen by use of carbon dioxide concentration of exhaust gas in fed-batch culture

Accurate and automatic control strategies for a feedback-control system of volatile carbon source feeding and dissolved oxygen (DO) level were investigated. To maintain the optimal ethanol concentration for microbial growth, carbon dioxide concentration in exhaust gas was used as a stepwise control parameter of ethanol feeding. A proportional-differential (PD) control program was used to correct the errors. The coefficient of stepwise control was calculated stoichiometrically, and parameters of PD were experimentally preset and were not changed during cultivation. DO was also controlled by the PD control and the stepwise program based on carbon dioxide concentration of the exhaust gas. Agitation speed and partial pressure of oxygen of the inlet gas were changed stepwise in accordance with the oxygen consumption rate. The stepwise coefficients were estimated from stoichiometry and material balance of molecular oxygen. The PD control program was only used for the agitation speed control to correct the fluctuations of DO level. The parameters did not need to be changed during cultivation. By use of these sophisticated control programs for fed-batch culture of Candida brassicae, ethanol concentration and DO level were accurately controlled at 3.4–3.7 g/l and 2.2–2.8 ppm, respectively, while cell mass concentration reached about 80 g/l. No manual operation was needed.     

Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor

A computer-aided on-line real-time monitoring system for plant cell bioprocesses was established and applied to the cultivation of Perilla frutescens plant cells in a bioreactor. This system calculated several informative process variables which were useful for the identification of the physiological states of the plant cells during cultivation. Some variables, such as the respiratory quotient (RQ), pH, and specific carbon dioxide evolution rate (SCER), could be used for the identification of the growing phase of cell cultures. The results also suggest that the oxygen uptake rate (OUR) and the specific OUR (SOUR) may depend on the accumulation of anthocyanin (a secondary metabolite) in P. frutescens cell cultures.     

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

HETEROTROPHY OF FOUR MARINE PHYTOPLANKTERS AT LOW SUBSTRATE CONCENTRATIONS1

Three pelagic marine phytoplankters, Coccolithus huxleyi, Skeletonema costatum, and Thalassiosira rotula, and a facultative heterotroph, Cyclotella cryptica, have been exposed to three organic substrates, viz, glucose, acetate, and glutamate, at low concentrations (organic carbon 0.25 mg/liter). Experiments were performed in the dark and light and the net assimilation of substrate was measured by using radiocarbon. The dark uptake of carbon dioxide was also determined, together with photosynthesis at near optimum light intensity. The expected heterotrophy was detected with Cyclotella cryptica. Thalassiosira rotula was found to assimilate glutamate at an appreciable rate. In all cases, however, the short-term uptake of carbon dioxide in the dark was the greatest assimilation rate measured. Values are discussed in relation to their ecological significance and it is concluded that heterotrophic survival of these and probably most other algae in the open ocean would be impossible unless they were in contact with a high concentration of substrate in the form of particulate matter.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H₂-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H₂, carbon dioxide (CO₂), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.     

Studies on the kinetics and mechanism of BOD exertion in dilute systems

The mechanisms and kinetic course of BOD exertion were compared in both open and closed systems. Two open reactors, a simulated stream device, and an open stirred reactor were employed, and the closed systems consisted of standard BOD bottles and 2.4-liter vessels. In the closed systems, both quiescent and stirred conditions of incubation were examined. Biological solids concentration, bacteria and protozoa concentration, substrate analysis, and chemical oxygen demand as well as biochemical oxygen utilization were employed to assess the performance of these systems. Oxygen uptake rate constants were observed to increase with increasing concentration o carbon source, thus militating against irect use of the usual dilution technique for predicting rate of deoxygenation in receiving streams. The relationship between specific O₂ uptake rate and substrate concentration approximated a hyperbolic function similar to the Mono relationship for specific growth rate and substrate concentration. A technique using an open stirred reactor than the standard BOD bottle dilution technique is recommended.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Production of extracellular lipase by Candida cylindracea CBS 6330

In this study we investigated the influences of aeration, substrate type and concentration on extracellular lipase production in a batch fermentor. The use of air enriched with pure oxygen is the most suitable for the lipase production. Additionally, we found that the presence of fats in the culture broth did not affect the value of the volumetric mass transfer coefficient of oxygen in our system. Olive oil or oleic acid was used as carbon sources. In both cases, the maximal specific rate of growth, μ_max, was the same but the highest activity was obtained when 10?g/dm³ of olive oil were used as an initial substrate concentration.