Effects of growth temperature on maximal specific growth rate,yield, maintenance,and death rate in glucose-limited continuous culture of the thermophilic Bacillus caldotenax

Summary The influence of temperature on the growth of the theromophilic Bacillus caldotenax was investigated using chemostat techniques and a chemically defined minimal medium. All determined growth constants, that is maximal specific growth rate, yield and maintenance, were temperature dependent. It was striking that the very large maintenance requirement was about 10 times higher than for mesophilic cells under equivalent conditions. A death rate, which was very substantial at optimal and supraoptimal growth temperatures, was estimated by comparing the maintenance for substrate and oxygen. There was no indication for a thermoadaptation as postulated by Haberstich and Zuber (1974).Symbols D Dilution rate (h^–1) - D_c=_max Critical dilution rate (h^–1) - E Temperature characteristic (J mol^–1) - k Organism constant - k_d Death rate coefficient (h^–1) - k_m Maintenance substrate coefficient estimated from M_O (h^–1) - M_O Maintenance respiration, mmol O₂ per g dry biomass and h (mmol g^–1h^–1) - M_O Maintenance respiration, taking k_d into account - m_S Maintenance substrate coefficient, g glucose per g dry biomass and h (h^–1) - OD Optical density at 546 nm - QO₂ Specific O₂-uptake rate (mmol g^–1h^–1) - Q _O2 ^V Specific O₂-uptake rate for viable portion of biomass (mmol g^–1 h^–1) - Q_S Specific glucose uptake rate (h^–1) - Q _S ^V Specific glucose uptake rate for viable portion of biomass (h^–1) - R Gas constant 8.28 J mol^–1K^–1 - S Substrate concentration in reactor (g l^–1) - S_O Influent substrate concentration (g l^–1) - T_max Maximal growth temperature (°C) - T_min Minimal growth temperature (°C) - X Dry biomass (g l^–1) - X_tOt=X Dry biomass containing dead and viable cells - X_v Viable portion of biomass - Y _O ^m Potential yield for O₂ corrected for maintenance respiration (g mol^–1) - Y _S ^m Potential yield for substrate corrected for maintenance requirement, g biomass per g glucose (–) - Specific growth rate (h^–1) - _max Maximal specific growth rate (h^–1)     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

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) (–)     

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)     

Limitations in substrate utilization efficiency by Zymomonas mobilis

Summary Optimal growth conditions for Zymomonas mobilis have been established using continuous cultivation methods. Optimal substrate utilization efficiency occurs with 2.5 g l^–1 yeast extract, 2.0 g l^–1 ammonium sulfate and 6.0 g l^–1 magnesium sulfate in the media. Catabolic activity is at its maximum with glucose uptake rates of 16–18 g l^–1 h^–1 and ethanol production rates of 8–9 g l^–1 h^–1, Q_g values of 22–26 and Q_p values between 11 and 13, which results in 40 g l^–1 h^–1 ethanol yields using a 100 g l^–1 substrate feed. Any increase in these parameters goes on cost of substrate utilization efficiency. Calcium pantothenate can not substitute yeast extract.Abbreviations G Glucose (%) - Pant Calcium pantothenate (mg l^–1) - D Dilution rate (h^–1) - NH₄ Ammonium sulfate (%) - M_g Magnesium sulfate (%) - S₁ Residual glucose in the fermenter (g l^–1) - S₀ Glucose feed (g l^–1) - Eth Ethanol concentration (g l^–1) - GUR Glucose uptake rate (g l^–1 h^–1) - Q_g Specific glucose uptake rate (g g^–1 h^–1) - Q_p Specific ethanol production rate (g g^–1 h^–1) - EPR Ethanol production rate (g l^–1 h^–1) - Y_g Yield coefficient for glucose (g g^–1) - Y_p Conversion efficiency (%) - C Biomass concentration (g l^–1) Present address: (Until June 1982) Institut für Mikrobiologie, TH Darmstadt, 6100 Darmstdt, Federal Republic of Germany     

Factors which influence the sedimentation characteristics of Torulopsis glabrata after growth in a recirculation system

Summary Some environmental affects on cell aggregation described in the literature are briefly summarized. By means of a biomass recirculation culture (Contact system), using the yeast Torulopsis glabrata, the aggregation behavior of cells in static and in dynamic test systems is described. Sedimentation times required to obtain 50 g · l^–1 yeast dry matter in static systems were always higher than in dynamic ones.In addition to, influencing the biomass yield, the specific growth rate of the yeast also affected cell aggregation. The specific growth rate and therefore the aggregation could be regulated by the biomass recirculation rate as well as by the sedimenter volume.Abbreviations f_o Overflow flow rate (l·h^–1) - f_R Recycle flow rate (l·h^–1) - f_t0t Total flow rate through the fermenter (l·h^–1) - g Gram - h Hour - D_R Fermenter dilution rate due to recycle (h^–1) - D_S Fermeter dilution rate due to substrate (h^–1) - D_tot Total fermenter dilution rate (h^–1) - l Liter - Specific growth rate (h^–1) - P_F Fermenter productivity (g·l^–1·h^–1) - P_FS Overall productivity (g·l^–1·h^–1) - R_pM Rates per minute - RS Residual sugar content in the effluent with respect to the substrate concentration (%) - Y Yield of biomass with respect to sugar concentration (%) - Sed 50 Sedimentation time to reach a YDM of 50 g·l^–1 (min) - V Volume (l) - V_F Fermenter volume (l) - V_Sed Sedimenter volume (l) - VVM Volumes per volume and minute - X_F YDM in the fermenter (g·l^–1) - X_F YDM in the recycle (g·l^–1) - X_S Yeast dry matter due to substrate concentration (g·l^–1) - YDM Yeast dry matter (g·l^–1)     

Studies on the variables and kinetics of SCP production from nopal fruit juice

Summary Submerged batch cultivation under controlled environmental conditions of pH 3.8, temperature 30°C, and K_La200 h^–1 (above 180 mMO₂ l ^–1 h^–1 oxygen supply rate) produced a maximum (12.0 g·l ^–1) SCP (Candida utilis) yield on the deseeded nopal fruit juice medium containing C/N ratio of 7.0 (initial sugar concentration 25 g·l ^–1) with a yield coefficient of 0.52 g cells/g sugar. In continuous cultivation, 19.9 g·l ^–1 cell mass could be obtained at a dilution rate (D) of 0.36 h^–1 under identical environmental conditions, showing a productivity of 7.2 g·l ^–1·h^–1. This corresponded to a gain of 9.0 in productivity in continuous culture over batch culture. Starting with steady state values of state variables, cell mass (C_X–19.9 g·l ^–1), limiting nutrient concentration (C_ln–2.5 g·l ^–1) and sugar concentration (C_S–1.5 g·l ^–1) at control variable conditions of pH 3.8, 30°C, and K_La 200 h^–1 keeping D=0.36 h^–1 as reference, transient response studies by step changes of these control variables also showed that this pH, temperature and K_La conditions are most suitable for SCP cultivation on nopal fruit juice. Kinetic equations obtained from experimental data were analysed and kinetic parameters determined graphically. Results of SCP production from nopal fruit juice are described.Nomenclature C_ln concentration of ammonium sulfate (g·l ^–1) - C_S concentration of total sugar (g·l ^–1) - C_X cell concentration (g·l ^–1) - D dilution rate (h^–1) - K_ln Monod's constant (g·l ^–1) - m maintenance coefficient (g ammonium sulfate cell^–1 h^–1) - m_(S) maintenance coefficient (g sugar g cell^–1 h^–1) - t time, h - Y yield coefficient (g cells/g ammonium sulfate) - Y_m maximum of Y - Y_S yield coefficient based on sugar consumed (g cells · g sugar^–1) - Y_S(m) maximum value of Y_S - ^µm maximum specific growth rate constant (h^–1)     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers

PVA-cryogels entrapping about 10⁹ cells of Acidithiobacillus ferrooxidans per ml of gel were prepared by freezing-thawing procedure, and the biooxidation of Fe²⁺ by immobilized cells was investigated in a 0.365 l packed-bed bioreactor. Fe²⁺ oxidation fits a plug-flow reaction model well. A maximum oxidation rate of 3.1 g Fe²⁺ l^–1 h^–1 was achieved at the dilution rate of 0.4 h^–1 or higher, while no obvious precipitate was determined at this time. In addition, cell-immobilized PVA-cryogels packed in bioreactor maintained their oxidative ability for more than two months under non-sterile conditions. Nomenclature: C _A0 – Concentration of Fe²⁺ in feed stream (g l^–1) C _A – Concentration of Fe^{2 +} in outlet stream (g l^{– 1}) D – Dilution rate of the packed-bed bioreactor (h^–1) F – Volumetric flow rate of iron solution (l h^–1) F _A0 – Mass flow rate of Fe²⁺ in the feed stream (g h^–1) K – Kinetic constant (l l^–1 h^–1) r _A – Oxidation rate of Fe²⁺ (g l^–1 h^–1) V – Volume of packed-bed bioreactor (l) X _A – Conversion ratio of Fe²⁺ (%)     

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.Abbreviations VNA Viable non-apoptotic cells - VA Viable apoptotic cells - NVNA Nonviable non-apoptotic or necrotic cells - NVA Nonviable apoptotic cells - CF Chromatin-free cells (late nonviable apoptotic cells) - AO Acridine orange - EB Ethidium Bromide - MAb Monoclocnal antibody - D.O. Dissolved oxygen - qMAb Specific MAb production rate (mg. (10⁹ cells)^–1.day^–1) - Specific growth rate (h^–1) - X_v Viable cell number (10⁵ cells.mL^–1) - X_t Total cell number (10⁵ cells.mL^–1) - Y_lac/glc Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)     

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l^-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: c_Glc – glucose concentration, mmol l^-1; c_Gln – glutamine concentration, mmol l^-1; c_Amm – ammonia concentration, mmol l^-1; c_Lac – lactate concentration, mmol l^-1; c_MAb – MAb concentration, mg l^-1; D – dilution rate, d^-1; D_i – dilution rate in the inner chamber of the membrane dialysis reactor, d^-1; D₀ – dilution rate in the outer chamber of the membrane dialysis reactor, d^-1; q*_FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1; q*_FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1.     

Lactate dehydrogenase isoenzymes of sperm cells and testes

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH_x from human sperm cells and rat testes were studied. 2. LDH_x shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H₄ and LDH-M₄ isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDH_x was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDH_x activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD⁺. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD⁺/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH_x. 9. The findings described are consistent with the idea that LDH_x is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.     

Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene

A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g⁻¹, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g⁻¹ (maximum theoretical yield, 1 g g⁻¹), and with high LDH activity.     

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     

Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations

Glucose repressed xylose utilization inCandida tropicalis pre-grown on xylose until glucose reached approximately 0–5 g l^–1. In fermentations consisting of xylose (93 g l^–1) and glucose (47 g l^–1), xylitol was produced with a yield of 0.65 g g^–1 and a specific rate of 0.09 g g^–1 h^–1, and high concentrations of ethanol were also produced (25 g l^–1). If the initial glucose was decreased to 8 g l^–1, the xylitol yield (0.79 g g^–1) and specific rate (0.24 g g^–1 h^–1) increased with little ethanol formation (<5 g l^–1). To minimize glucose repression, batch fermentations were performed using an aerobic, glucose growth phase followed by xylitol production. Xylitol was produced under O₂ limited and anaerobic conditions, but the specific production rate was higher under O₂ limited conditions (0.1–0.4 vs. 0.03 g g^–1 h^–1). On-line analysis of the respiratory quotient defined the time of xylose reductase induction.     

Microcalorimetric investigations of the metabolism of yeasts. Growth in chemostat cultures on glucose

Summary As part of a project on the production of penicillin, the penicillin production of two strains of Penicillium chrysogenum which have a different penicillin productivity was investigated in bubble column bioreactors and for comparison in stirred fermenters. The main interest of this study were the complicated interrelations between the stirrer speed, the stirrer type, the shear stress, the morphology of the mycelium and broth viscosity as well as the effect of the oxygen transfer behavior on antibiotic productivity.Stirred tank reactors with different turbine stirrers as well as with a draught tube and propeller were employed.The main variable investigated was the stirrer speed. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply of pulpy mycelia is improved and more cell mass is formed. This result is the same for both strains in all three reactors.If the oxygen partial pressure is near the lower cirtical value, a high percentage of the carbon source is converted into penicillin but the penicillin productivity is low due to a low percentage of penicillin producing cells. At oxygen partial pressures just above 8% saturation, the absolute penicillin productivity is maximal. At higher stirrer speeds and dissolved oxygen concentrations the penicillin production phase is shorter, cell growth is higher and a higher percentage of the carbon source is converted into CO₂.In reactors with a draught tube and propeller, a lower productivity is attained than in those with turbine stirrers.The behavior of the two strains is fairly similar. The higher producing strain, however, has a more distinct separation between its periods of growth and production than does the low producing one. At high stirrer speeds the increase in the cell growth rate is less significant and the substrate yield coefficients are higher for the high producing strain than for the low producing one.Symbols C Dissolved oxygen concentration (mg l^–1) - C^* C at saturation (mg l^–1) - k_La Volumetric mass transfer coefficient (h^–1) - OTR Oxygen transfer rate (mg l^–1 h^–1) - OUR Oxygen uptake rate (mg l^–1 h^–1) - rpm Impeller speed (min^–1) - X (Dry) biomass concentration (g kg^–1) - V_g Volumetric gas flow rate (Nl min^–1) - CMC Carboxymethyl cellulose     

Multiple growth inhibition of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in 1,3-propanediol fermentation

The inhibition of substrate and product on the growth of Klebsiella pneumoniae in anaerobic and aerobic batch fermentation for the production of 1,3-propanediol was studied. The cells under anaerobic conditions had a higher maximum specific growth rate of 0.19 h^–1 and lower tolerance to 110 g glycerol l^–1, compared to the maximum specific growth rate of 0.17 h^–1 and tolerance to 133 g glycerol l^–1 under aerobic conditions. Acetate was the main inhibitory metabolite during the fermentation under anaerobic conditions, with lactate and ethanol the next most inhibitory. The critical concentrations of acetate, lactate and ethanol were assessed to be 15, 19, 26 g l^–1, respectively. However, cells grown under aerobic conditions were more resistant to acetate and lactate but less resistant to ethanol. The critical concentrations of acetate, lactate and ethanol were assessed to be 24, 26, and 17 g l^–1, respectivelyRevisions requested 8 september; Revisions received 2 November 2004     

Influence of oxygen limitation on glucose metabolism in Hanseniaspora uvarum K5 grown in chemostat

Summary Growth and metabolite formation were studied as a function of oxygen feed rate, in glucose-limited chemostat cultures of Hanseniaspora uvarum K₅ at a dilution rate of 0.26 h^–1. Alcoholic fermentation occured at an oxygen feed rate of 80 mmol.l^–1.h^–1 . Below this value, pyruvate decarboxylase and alcohol dehydrogenase were present at high levels. In contrast, activities of oxidative metabolism enzymes, pyruvate dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase, decreased.     

Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX)

Summary The changes in cell wall strength of Hansenula polymorpha have been investigated in continuous cultures with respect to the recovery of methanol oxidase (MOX). Cultures grown on several substrate mixtures that enable induction of MOX have been compared with cultures grown on methanol as the sole inducer. The effects of dilution rate (D) on lysis properties have been studied. The cell wall strength was consistently influenced by growth media and D. Media containing glycerol/methanol showed the slowest lysis kinetics, with a large fraction of non-degradable cell wall material. In continuous cultures grown on a mixture of glucose and methanol both the resistance to zymolyase and the mean cell wall thickness increased at D<0.1 h^–1. The yield of MOX by zymolyase lysis is reproducible and up to 100% higher than that of the standard ultrasonic treatment. The lysis kinetics indicated that zymolyase punctures the cell wall; since the release rate of MOX is lower than that of protein, the cell contents will leak through. At D-values>0.2 h^–1, both protein and MOX release rates increase, reflecting a change in lysis mechanism due to the increased fraction of thin daughter cells. Kinetic analysis of zymolyase lysis using both physical and enzymatic methods provides information for achieving optimal recovery of MOX.Abbreviations and symbols C _MOX MOX activity [MOX units·g X^–1] - D dilution rate [h^–1] - MOX methanol oxidase - k_c decay rate constant of A 610 nm [min^–1] - k_d decay constant of MOX activity [min^–1] - k_dis dissociation rate constant [min^–1] - k_MOX release rate constant of MOX activity [min^–1] - k_p release rate constant of protein [min^–1] - R recovery efficiency of enzyme [-] - St stability of enzyme [-]     

An enzymatic route to produce pyruvate from lactate

A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l^–1 cell concentration in the broth was obtained with 20 g l^–1 (178 mM) sodium lactate and 4 g l^–1 NH₄Cl in the medium; and the biotransformation ability was 2.51 units ml^–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g^–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l^–1 h^–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.