Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris

Pichia pastoris is widely used for the production of recombinant proteins in industrial biotechnology. In general, industrial production processes describe fed-batch processes based on the specific growth rate. Recently, we introduced the specific substrate uptake rate (q _s) as a novel parameter to design fed-batch strategies for P. pastoris. We showed that a dynamic feeding strategy where the feed was adjusted in steps to the maximum specific substrate uptake rate was superior to more traditional strategies in terms of specific productivity. In the present study, we compare three different dynamic feeding strategies based on q _s for a recombinant P. pastoris strain with respect to cell physiology, methanol accumulation, productivity and product quality. By comparing (A) a feeding profile at constant high q _s, (B) a periodically adjusted feeding profile for a stepwise q _s ramp, and (C) a feeding profile at linear increasing q _s, we evaluated potential effects of the mode of feeding. Although a dynamic feeding strategy with stepwise increases of q _s to q _{s max} resulted in the highest specific productivity, a feeding profile where the feeding rate was stepwise increased to a constant high q _s value was superior in terms of the amount of active enzyme produced and in the amount of accumulated methanol. Furthermore, this feeding strategy could be run automatically by integrating an online calculator tool, thus rendering manual interventions by the operator unnecessary.     

Soft-sensor assisted dynamic investigation of mixed feed bioprocesses

Recombinant mixed feed bioprocesses are characterized by the controlled feeding of multiple defined carbon sources aiming at increased productivities. However, mixed feed process design is challenging due to physiological constraints such as adaptation times and catabolite repression.A novel soft-sensor assisted dynamic method that allows the science-based process design with respect to co-utilization of primary and secondary substrate was developed. The method is based on the control of the specific uptake rates of primary and secondary substrate via a combination of a rate-based soft sensor and in-line infrared spectroscopy. Maximum secondary substrate specific uptake rates and adaptation times are determined by a combination of dynamic pulse and ramp experimentation.The power of the presented method was demonstrated on a recombinant Escherichia coli pBAD mixed feed process with d-glucose as primary and l-arabinose as secondary carbon source. Onset of catabolite repression was observed once a total specific substrate uptake rate of 1.0 g/gh was exceeded. Adaptation times to l-arabinose were determined as ～10 min.The presented method can be considered generically applicable for the physiological investigation of mixed feed systems. Furthermore, metabolic capabilities of the promising but yet unexplored E. coli pBAD mixed feed system were explored for the first time.     

A new process for red pigment production by submerged culture of Monascus purpureus

Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH?of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specific consumption rate of glucose (q_G) was higher than the specific consumption rate of ethanol (q_E), whereas the specific growth rate was greatest with ethanol. The specific production rate of red pigment (p_OD) and pigment yield (Y_OD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H₂SO₄-pretreated black strap molasses (containing 150 g sugars/l) in Vogel's medium. On the basis of comparison of kinetic parameters, namely the volumetric substrate uptake rate (Q _s), and specific substrate uptake rate (q _s), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and had the ability to overproduce citric acid.     

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)     

Variation of ADM1 by using temperature-phased anaerobic digestion (TPAD) operation

The objective of the study was to examine the application of the Anaerobic Digestion Model No. 1 (ADM1) developed by the IWA task group for mathematical modelling of anaerobic process. Lab-scale temperature-phased anaerobic digestion (TPAD) process were operated continuously, and were fed with co-substrate composed of dog food and flour. The model platform implemented in the simulation was a derivative of the ADM1. Sensitivity analysis showed that k_m.process (maximum specific uptake rate) and K_S.process (half saturation value) had high sensitivities to model components. Important parameters including maximum uptake rate for propionate utilisers (k_m.pro) and half saturation constant for acetate utilisers (K_S.ac) in the thermophilic digester and maximum uptake rate for acetate utilisers (k_m.ac) in the mesophilic digester were estimated using iterative methods, which optimized the parameters with experimental results. Simulation with estimated parameters showed good agreement with experimental results in the case of methane production, uptake of acetate, soluble chemical oxygen demand (SCOD) and total chemical oxygen demand (TCOD). Under these conditions, the model predicted reasonably well the dynamic behavior of the TPAD process for verifying the model.     

Physiological description of multivariate interdependencies between process parameters,morphology and physiology during fed‐batch penicillin production

Optimization of productivity and economics of industrial bioprocesses requires characterization of interdependencies between process parameters and process performance. In the case of penicillin production, as in other processes, process performance is often closely interlinked with the physiology and morphology of the organism used for production. This study presents a systematic approach to efficiently characterize the physiological effects of multivariate interdependencies between bioprocess design parameters (spore inoculum concentration, pO₂ control level and substrate feed rate), morphology, and physiology. Method development and application was performed using the industrial model process of penicillin production. Applying traditional, statistical bioprocess analysis, multivariate correlations of raw bioprocess design parameters (high spore inoculum concentration, low pO₂ control as well as reduced glucose feeding) and pellet morphology were identified. A major drawback of raw design parameter correlation models; however, is the lack of transferability across different process scales and regimes. In this context, morphological and physiological bioprocess modeling based on scalable physiological parameters is introduced. In this study, raw parameter effects on pellet morphology were efficiently summarized by the physiological parameter of the biomass yield per substrate. Finally, for the first time to our knowledge, the specific growth rate per spore was described as time‐independent determinant for switching from pellet to disperse growth during penicillin production and thus introduced as a novel, scalable key process parameter for pellet morphology and process performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:689–699, 2014     

Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves

Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO₂ uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de-epoxidation), as well as nonphotochemical quenching (q_NP), and the rate constant of radiationless energy dissipation (k_D). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO₂ uptake differed widely and light saturation of CO₂ uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO₂ uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q_NP, increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO₂ uptake was approached, q_NP showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k_D, which opens the possibility that zeaxanthin is related to the rapidly relaxing “high-energy-state quenching” in leaves.     

Specific light uptake rates can enhance astaxanthin productivity in <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

Lumostatic operation was applied for efficient astaxanthin production in autotrophic Haematococcus lacustris cultures using 0.4-L bubble column photobioreactors. The lumostatic operation in this study was performed with three different specific light uptake rates (q _e) based on cell concentration, cell projection area, and fresh weight as one-, two- and three-dimensional characteristics values, respectively. The q _e value from the cell concentration (q _e1D) obtained was 13.5 × 10^?8 μE cell^?1 s^?1, and the maximum astaxanthin concentration was increased to 150 % compared to that of a control with constant light intensity. The other optimum q _e values by cell projection area (q _e2D) and fresh weight (q _e3D) were determined to be 195 μE m^?2 s^?1 and 10.5 μE g^?1 s^?1 for astaxanthin production, respectively. The maximum astaxanthin production from the lumostatic cultures using the parameters controlled by cell projection area (2D) and fresh weight (3D) also increased by 36 and 22 % over that of the controls, respectively. When comparing the optimal q _e values among the three different types, the lumostatic cultures using q _e based on fresh weight showed the highest astaxanthin productivity (22.8 mg L^?1 day^?1), which was a higher level than previously reported. The lumostatic operations reported here demonstrated that more efficient and effective astaxanthin production was obtained by H. lacustris than providing a constant light intensity, regardless of which parameter is used to calculate the specific light uptake rate.     

Effects of combining biological treatment and activated carbon on hexavalent chromium reduction

The objectives of the present work were: (a) to analyze the Cr(VI) removal by combining activated sludge (AS) with powdered activated carbon (PAC), (b) to analyze the effect of PAC and Cr(VI) on the growth kinetics of activated sludge, and (c) to determine if the combined method (AS-PAC) for Cr(VI) removal can be considered additive or synergistic with respect to the individual processes. Chromate removal was improved by increasing PAC concentrations in both PAC and AS-PAC systems. Cr(VI) removal using the AS-PAC system was higher than using AS or PAC. The increase of Cr(VI) caused longer lag phase and lower observed specific growth rate (μ_obs), biomass yield (Y_X/S), and specific growth substrate consumption rate (q_S) of activated sludge; additionally, PAC did not enhance the growth kinetic parameters (μ_obs, Y_X/S, q_S). Cr(VI) reduction in AS-PAC system was the result of the additive effect of each individual Cr(VI) removal process.     

Inhibition of the Fermentation of Propionate to Methane by Hydrogen, Acetate, and Propionate

Inhibition of the fermentation of propionate to methane and carbon dioxide by hydrogen, acetate, and propionate was analyzed with a mesophilic propionate-acclimatized sludge that consisted of numerous flocs (size, 150 to 300 μm). The acclimatized sludge could convert propionate to methane and carbon dioxide stoichiometrically without accumulating hydrogen and acetate in a propionate-minimal medium. Inhibition of propionate utilization by propionate could be analyzed by a second-order substrate inhibition model (shown below) given that the substrate saturation constant, K_s, was 15.9 μM; the substrate inhibition constant, K_i, was 0.79 mM; and the maximum specific rate of propionate utilization, q_m, was 2.15 mmol/g of mixed-liquor volatile suspended solids (MLVSS) per day: q_s = q_mS/[K_s + S + (S²/K_i)], where q_s is the specific rate of propionate utilization and S is the initial concentration of undissociated propionic acid. For inhibition by hydrogen and acetate to propionate utilization, a noncompetitive product inhibition model was used: q_s = q_m/[1 + (P/K_p)ⁿ], where P is the initial concentration of hydrogen or undissociated acetic acid and K_p is the inhibition constant. Kinetic analysis gave, for hydrogen inhibition, K_p(H2) = 0.11 atm (= 11.1 kPa, 71.5 μM), q_m = 2.40 mmol/g of MLVSS per day, and n = 1.51 and, for acetate inhibition, K_p(HAc) = 48.6 μM, q_m = 1.85 mmol/g of MLVSS per day, and n = 0.96. It could be concluded that the increase in undissociated propionic acid concentration was a key factor in inhibition of propionate utilization and that hydrogen and acetate cooperatively inhibited propionate degradation, suggesting that hydrogenotrophic and acetoclastic methanogens might play an important role in enhancing propionate degradation to methane and carbon dioxide.     

Effect of glucose and oxygen on β-lactam biosynthesis by Cephalosporium acremonium

The effects of glucose consumption rate (q_s) and oxygen limitation on the control of cephalosporin C (Ceph C) biosynthesis and the activities of deacetoxycephalosporin C synthetase/hydroxylase (DAOC-SH) and acetyl coenzyme A: deacetylcephalosporin C o-acetyltransferase (DAC-AT) were investigated in cultivations of the highly productive Cephalosporium acremonium strain TR87 under conditions similar to those used in industrial production. A carefully optimised time course of q_s during the first part of fed batch cultivations was essential for maximal Ceph C production. The actual glucose concentration in the medium was of secondary importance. A decrease of q_s between 20 and 35 h of cultivation was found to induce the early onset of antibiotic synthesis. By subsequently maintaining q_s at a relatively low level using a controlled feed of glucose and a limiting amount of phosphate, maximal production rates were obtained. Oxygen starvation after the onset of Ceph C production led to a pronounced increase in penicillin N formation, a reduced Ceph C yield (−30%) and a strongly reduced activity of the two enzymes tested. In general, neither the time course nor the absolute levels of the two enzyme activities directly correlated with the actual production rates of Ceph C. This is the first time where an independent parameter (q_s) has been demonstrated to be responsible for triggering the synthesis of an antibiotic.     

Kinetic behavior of heterogeneous populations in completely mixed reactors

The kinetic behavior of heterogeneous microbial populations was studied in a continuous flow completely mixed reactor operated at various dilution rates. Glucose was used as the growth-limiting nutrient. The physiological growth parameters for cells harvested from continuous flow reactors were determined using batch experiments. It, was found that the growth parameters, maximum growth rate (μ_m), saturation constant (k_s), and cell yield (Y) vary for each dilution rate, and cannot be considered as precise constants in depicting the kinetic behavior of heterogeneous populations. In addition, it was found that the yield coefficients obtained from batch experiments were always lower than those obtained from continuous flow experiments. Levels of substrate and biological solids calculated for different dilution rates using growth constants from batch experiments did not agree with the experimental values observed in steady-state experiments. However, when the yield values from, the continuous flow experiments were used in conjunction with batch values for μ_m and k_s the theoretical and experimental dilute-out curves agreed fairly closely (within the range needed for engineering prediction) until the culture began to wash out of the unit. In general, the data substantiated the use of the single phase relationship between growth rate and substrate concentration described by the Monod equation, μ = μ_mS/(k_s + s).     

The <Emphasis Type="Italic">E. coli</Emphasis> pET expression system revisited—mechanistic correlation between glucose and lactose uptake

Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q _s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q _s,lac) and q _s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q _s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q _s,lac and q _s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.     

Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor

The influence of dissolved oxygen concentration on the nitrification kinetics was studied in the circulating bed reactor (CBR). The study was partly performed at laboratory scale with synthetic water, and partly at pilot scale with secondary effluent as feed water. The nitrification kinetics of the laboratory CBR as a function of the oxygen concentration can be described according to the half order and zero order rate equations of the diffusion-reaction model applied to porous catalysts. When oxygen was the rate limiting substrate, the nitrification rate was close to a half order function of the oxygen concentration. The average oxygen diffusion coefficient estimated by fitting the diffusion-reaction model to the experimental results was around 66% of the respective value in water. The experimental results showed that either the ammonia or the oxygen concentration could be limiting for the nitrification kinetics. The latter occurred for an oxygen to ammonia concentration ratio below 1.5–2 gO₂/gN-NH₄ ⁺ for both laboratory and pilot scale reactors. The volumetric oxygen mass transfer coefficient (k _L a) determined in the laboratory scale reactor was 0.017?s^?1 for a superficial air velocity of 0.02?m s^?1, and the one determined in the pilot scale reactor was 0.040?s^?1 for a superficial air velocity of 0.031?m?s^?1. The k _L a for the pilot scale reactor did not change significantly after biofilm development, compared to the value measured without biofilm.     

Effects of salinity on rates of protein synthesis and oxygen uptake in the post-larvae and juveniles of the tropical prawn Macrobrachium rosenbergii (de Man)

Protein synthesis is a major determinant of growth and yet little is known about the environmental factors that influence protein synthesis rates in farmed freshwater prawns. To this end, post-larvae and juveniles of Macrobrachium rosenbergii were exposed to various salinities (0, 14, 30‰) to determine whole-animal rates of fractional protein synthesis (k_s) and oxygen uptake. In the post-larvae that migrate upstream from brackish to freshwater areas, whole-animal k_s was unaffected by salinity, but rates of oxygen uptake were significantly lower at 14‰. In the freshwater juveniles, a different response was observed, as mean k_s was significantly higher at 14‰ compared with 0‰, but rates of oxygen uptake remained unchanged. Such differences are thought to be related to the energetic costs of osmoregulation and to the ability to maintain osmotic gradients in freshwater. In an additional experiment, acclimation temperature (20, 26, 30 °C) had a direct effect on k_s in juveniles held at 0‰. In all cases, changes in k_s resulted from alterations in RNA activity at constant RNA capacity. In juveniles at least, whole-animal rates of protein synthesis were highest at 14‰ and 30 °C which corresponds to the optimal salinity and temperature recommended for the growth and culture of M. rosenbergii.     

Improvement of a mammalian cell culture process by adaptive,model-based dialysis fed-batch cultivation and suppression of apoptosis

Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products. In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented. On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis. Maximum cell density could be increased by a factor of ~5–7 by dialysis fed-batch cultivation. On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3. These factors resulted in more product formation (by a factor ~2). Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.Abbreviations A membrane area (dm²) - c _Glc,F glucose concentration in nutrient feed (mmol L^–1) - c _Glc,FD glucose concentration in dialysis feed (mmol L^–1) - c _Glc,i glucose concentration in inner reactor chamber (mmol L^–1) - c _Glc,o glucose concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Lac,FD lactate concentration in dialysis feed (mmol L^–1) - c _Lac,i lactate concentration in inner reactor chamber (mmol L^–1) - c _Lac,o lactate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _LS,FD limiting substrate concentration in dialysis feed (mmol L^–1) - c _LS,i limiting substrate concentration in inner reactor chamber (mmol L^–1) - c _LS,o limiting substrate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Mab monoclonal antibody concentration (mg L^–1) - F _D feed rate of dialysis feed (L h^–1) - F _Glc feed rate of nutrient concentrate feed (L h^–1) - K _d maximum death constant (h^–1) - k _d,LS death rate constant for limiting substrate (mmol L^–1) - k _Glc monod kinetic constant for glucose uptake (mmol L^–1) - k _Lac monod kinetic constant for lactate uptake (mmol L^–1) - k _LS monod kinetic constant for limiting substrate uptake (mmol L^–1) - K _Lys cell lysis constant (h^–1) - K _S,Glc monod kinetic constant for glucose (mmol L^–1) - K _S,LS monod kinetic constant for limiting substrate (mmol L^–1) - µ cell-specific growth rate (h^–1) - µ _d cell-specific death rate (h^–1) - µ _d,min minimum cell-specific death rate (h^–1) - µ _max maximum cell-specific growth rate (h^–1) - P _Glc membrane permeation coefficient for glucose (dm h^–1) - P _Lac membrane permeation coefficient for lactate (dm h^–1) - P _LS membrane permeation coefficient for limiting substrate (dm h^–1) - q _Glc cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Glc,max maximum cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Lac cell-specific lactate uptake/production rate (mmol cell^–1 h^–1) - q _Lac,max maximum cell-specific lactate uptake rate (mmol cell^–1 h^–1) - q _LS cell-specific limiting substrate uptake rate (mmol cell^–1 h^–1) - q _LS,max maximum cell-specific limiting substrate uptake rate (mmol cell ^–1 h^–1) - q _Mab cell-specific antibody production rate (mg cell^–1 h^–1) - q _MAb,max maximum cell-specific antibody production rate (mg cell^–1 h^–1) - t time (h) - V _i volume of inner reactor chamber (culture chamber) (L) - V _o volume of outer reactor chamber (dialysis chamber) (L) - X _t total cell concentration (cells L^–1) - X viable cell concentration (cells L^–1) - Y _Lac/Glc kinetic production constant (stoichiometric ratio of lactate production and glucose uptake) (–)     

Phenylalanine production from a rec Escherichia coli-strain in fed-batch culture

The production of phenylalanine from a plasmid-harboring auxotrophic Escherichia coli mutant (E. coli W3110 Δtyr, Δtrp, Δphe/pJN6) was studied in two types of constantly-fed-batch cultures. The plasmid contains genes essential for phenylalanine production. In tyrosine fed-batch cultures the cell mass was increased and the strong inhibition and repression of phenylalanine synthesis by tyrosine was avoided. In this way r_p can be increased since production also occurred during the growth phase. Experiments with different feed rates of tyrosine, corresponding to different growth rates, showed that a high μ during the feeding period was necessary for obtaining a high q_p in the non-growth period.Glucose fed-batch cultures were employed to reduce the byproduct formation that occurred if excess of glucose was present in the culture liquid. By choosing a proper feed rate q_s in the non-growing cells could be controlled at a reduced level suitable for obtaining a high Y_p/s. The byproduct formation was thereby reduced and an average Y_p/s of 0.20 was obtained from the non-growing cells.