High concentration cultivation of Bifidobacterium longum in fermenter with cross-flow filtration

Summary High concentration cultivation of Bifidobacterium longum in a fermenter with cross-flow filtration using a ceramic filter is described. Continuous cross-flow filtration allowed complete recycling of the cells to the fermenter and also continuous separation of inhibitory metabolites. The final cell concentration attained in the cultivation was 54.4 g dry wt./l; this was seven times as high as that without cross-flow filtration. The time course of the cultivation with cross-flow filtration was predicted, based on the assumption that the specific growth rate can be expressed only as a function of concentrations of metabolites (acetate and lactate) in a culture broth.Nomenclature D dilution rate (h^-1) - m maintenance coefficient (h^-1) - OD ₅₇₀ optimal density at 570 nm - P _A acetate concentration (g/l) - P _A0 initial acetate concentration (g/l) - P _L lactate concentration (g/l) - P _L0 initial lactate concentration (g/l) - S lactose (substrate) concentration (g/l) - S ₀ initial lactose (substrate) concentration (g/l) - t cultivation time (h) - Y _x/s growth yield (g/g) - X dry cell concentration (g/l) - X ₀ initial dry cell concentration (g/l) - constant - constant     

Phenol degradation by a psychrotrophic strain of Pseudomonas putida

Summary Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, S_o, ranging from 14 to 1000 mg/1. The experimental data for 14<=S_o<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: _m=0.119 h^–1, K _S=5.27 mg/1 and K _I=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.Nomenclature K _S saturation constant (mg/l) - K _I substrate inhibition constant (mg/l) - specific growth rate (h^–1) - _m maximum specific growth rate without substrate inhibition (h^–1) - _max maximum achievable specific growth rate with substrate inhibition (h^–1) - S substrate (phenol) concentration (mg/l) - S_o initial substrate concentration (mg/l) - S_max substrate concentration corresponding to _max (mg/l) - t time (h) - X cell concentration, dry basis (mg DW/l) - X_f final cell concentration, dry basis (mg DW/l) - X_o initial cell concentration, dry basis (mg DW/l) - Y yield factor (mg DW cell produced/mg substrate consumed)     

Anaerobic digestion of palm oil mill effluent and condensation water waste: an overall kinetic model for methane production and substrate utilization

The process of anaerobic digestion is viewed as a series of reactions which can be described kinetically both in terms of substrate utilization and methane production. It is considered that the rate limiting factor in the digestion of complex wastewaters is hydrolysis and this cannot be adequately described using a Monod equation. In contrast readily assimilable wastewaters conform well to this approach. A generalized equation has thus been derived, based on both the Monod and Contois equations, which serves extreme cases. The model was verified experimentally using continuous feed anaerobic digesters treating palm oil mill effluent (POME) and condensation water from a thermal concentration process. POME represents a complex substrate comprising of unhydrolyzed materials whereas the condensation water is predominantly short chain volatile fatty acids. Substrate removal and methane production in both cases could be predicted accurately using the generalized equation presented.List of Symbols A (=K_skY/K_h) Kinetic parameter - B Specific methane yield, 1 of CH₄/g of substrate added B₀ Maximum specific methane yield, 1 of CH₄/g of substrate added at infinity - C Empirical constant in Contois equation - F Volumetric substrate removal rate, g/l day - k Hydrolysed substrate transport rate coefficient, 1/days - K (=YC) Kinetic parameter in Chen-Hashimoto equation - K _h Substrate hydrolysis rate coefficient, 1/days - K _s Half-saturation constant for hydrolysed substrate, g/l - M _v Volumetric methane production rate, 1 of CH₄/l day - MS Mineral solids, g/l - MSS Mineral suspended soilds, g/l - POME Palm oil mill effluent - R (=S_r/S_T0) Refractory coefficient - S _h Concentration of hydrolysed substrate, g/l - S _u Intracellular concentration of hydrolysed substrate, g/l - S ₀ Input biodegradable substrate concentration, g/l - S Biodegradable substrate concentration in the effluent or in the digester, g/l - S _r Refractory feed substrate concentration, g/l - S _T0 (=S₀+S_r) Total feed substrate concentration, g/l - S _T (S+S_r) Total substrate concentration in the effluent, g/l - TS Total solids, g/l - TSS Total suspended solids, g/l - VFA Total volatile fatty acids, g/l - VS Volatile solids, g/l - VSS Volatile suspended solids, g/l - X Biomass concentration, g/l - Y Biomass yield coefficient, biomass/substrate mass - Hydraulic retention time, days. - Specific growth rate of microorganisms, l/days - _m Maximum specific growth rate of microorganisms, l/days The authors wish to express their gratitude to the Departamento de Postgrado y Especialización del CSIC and to the Consejería de Educación y Ciencia de la Junta de Andalucia for their financial support of this work.     

Linear growth in microbial cultures limited by accumulated substrate

Summary The linear growth phase in cultures limited by intracellular (conservative) substrate is represented by a flat exponential curve. Within the range of experimental errors, the presented model fits well the data from both batch and continuous cultures ofEscherichia coli, whose growth is limited in that way.List of symbols D dilution rate, h^–1 - K_S saturation constant, g.L^–1 - S concentration of the limiting substrate, g.L^–1 - S_i concentration of the limiting substrate accumulated in the cells, g.g^–1 - S_o initial concentration of the limiting substrate, g.L^–1 - t time of cultivation, h - t₁ time of exhaustion of the limiting substrate from medium, h - t_o beginning of exponential phase, h - X biomass concentration, g.L^–1 - X₁ biomass concentration at the time of exhaustion of the limiting substrate from the medium, g.L^–1 - X_o biomass concn. at the beginning of exponential phase, g.L^–1 - biomass concn. at steady-state, g.L^–1 - Y growth yield coefficient (biomass/substrate) - specific growth rate, h^–1 - _m maximum specific growth rate, h^–1     

Mathematical modeling of production of microbial lipids

As a part of the investigations on the microbial lipid production using the yeast Rhodotorula gracilis, CFR-1, kinetics of the biomass synthesis has been studied using shake flask experiments. Using a medium containing a carbon to nitrogen ratio of 701, the rates of biomass production were followed at different initial substrate concentrations in the range of 20–100 kg/m³. A logistic model was found to be reasonably adequate to describe the kinetics of the growth of biomass; the maximum specific growth rate of 0.105 h^–1 was applicable for substrate concentrations less than 60 kg/m³, which gave reasonable agreement between predicted and actual biomass concentration values.List of Symbols S ₀, X ₀ kg/m³ Initial concentrations of sugar, non lipid biomass respectively - X, X(t) kg/m³ Concentrations of non lipid biomass at any time t - dX/dt kg/(m³ · h) Rate of biomass growth - h^–1 Specific growth rate - _max h^–1 Maximum specific growth rate - K _s mol/dm³ Monods constant - X _max kg/m³ Maximum biomass reached in a run     

Updated model of the batch acetone-butanol fermentation

Summary The recent models of the Acetone-Butanol fermentation did not adequately describe the culture inhibition by the accumulating metabolites and were unable to simulate the acidogenic culture dynamics at elevated pH levels. The present updated modification of the model features a generalised inhibition term and a pH dependent terms for intracellular conversion of undissociated acids into solvent products. The culture dynamics predictions by the developed model compared well with experimental results from an unconventional acidogenic fermentation ofC. acetobutylicum.Nomenclature A acetone concentration in the fermentation broth, [g/L] - AA total concentration of dissociated and undissociated acetic acid, [g/L] - AA _undiss concentration of undissociated acetic acid, [g/L] - APS Absolute Parameter Sensitivity - AT acetoin concentration in the fermentation broth, [g/L] - B butanol concentration in the fermentation broth, [g/L] - BA total concentration of dissociated and undissociated butyric acid, [g/L] - BA _undiss concentration of undissociated butyric acid, [g/L] - E ethanol concentration in the fermentation broth, [g/L] - f(T) inhibition function as defined in Equation (2) - k ₁ constant in Equation (4), [g substrate/g biomass] - k ₂ constant in Equation (4), [g substrate/(g biomass.h)] - k ₁ constant in Equation (5), [g substrate/(g biomass] - k ₂ constant in Equation (5), [g substrate/(g biomass.h)] - k ₃ constant in Equation (6), [g butyric acid/g substrate] - k ₄ constant in Equation (6), [g butyric acid/(g biomass.h)] - k ₅ constant in Equation (7), [g butanol/g substrate] - k ₆ constant in Equation (8), [g acetic acid/g substrate] - k ₇ constant in Equation (8), [g acetic acid/(g biomass.h)] - k ₈ constant in Equation (9), [g acetone/g substrate] - k ₉ constant in Equation (10), [g ethanol/g substrate] - k ₁₀ constant in Equation (11), [g acetoin/g substrate] - k ₁₁ constant in Equation (12), [g lactic acid/g substrate] - K _I Inhibition constant, [g inhibitory products/L] - ke maintenance energy requirement for the cell, [g substrate/(g biomass.h)] - K _AA acetic acid saturation constant, [g acetic acid/L] - K _BA butyric acid saturation constant, [g butyric acid/L] - K _S Monod's saturation constant, [g substrate/L] - LA lactic acid concentration in the fermentation broth, [g/L] - m _i ,n _i constants in Equation (14) - n empirical constant, dependent on degree of inhibition. - P concentration of inhibitory products (B+BA+AA), [g/L] - P _max maximum value of product concentration to inhibit the fermentation, [g/L] - pK_a equilibrium constant - r _A rate of acetone production, [g acetone/L.h] - r _AA rate of acetic acid production, [g acetic acid/L.h] - r _AT rate of acetoin production, [g acetoin/L.h] - r _B rate of butanol production, [g butanol/L.h] - r _BA rate of butyric acid production, [g butyric acid/L.h] - r _E rate of ethanol production, [g ethanol/L.h] - RPS Relative Parameter Sensitivity - r _LA rate of lactic acid production, [g lactic acid/L.h] - r _S dS/dt=total substrate consumption rate, [g substrate/L.h] - r _S substrate utilization rate, [g substrate/L.h] - S substrate concentration in the fermentation broth, [g substrate/L] - S ₀ initial substrate concentration, [substrate/L] - t time, [h] - X biomass concentration, [g/L] - Y _X yield of biomass with respect to substrate, [g biomass/g substrate] - Y _{P i} yield of metabolic product with respect to substrate, [g product/g substrate] Derivatives dX/dt rate of biomass production, [g biomass/L.h] - dP _i /dt rate of product formation, [g product/L.h] Greek letters specific growth rate of the culture, [h^–1] - I specific growth rate of the culture in the presence of the inhibitory products, [h^–1] - µ_max maximum specific growth rate of the culture, [h^–1]     

A heuristic approach to fed-batch optimisation of streptokinase fermentation

Previous studies have shown that the rate of formation of streptokinase, a secondary metabolite, in batch fermentation is proportional to the specific growth rate of the biomass, which in turn is inhibited by its substrate and the primary product (lactic acid). These kinetics suggest the suitability of fed-batch operation to increase the yield of streptokinase. A near-optimal feed policy has been calculated by the chemotaxis algorithm, and it shows a substrate feed rate decreasing nonlinearly and vanishing after 11 hours. This is followed by batch fermentation for a further 8 hours, at the end of which 12% more streptokinase is generated than by purely batch fermentation. Further improvements in productivity are possible.List of Symbols k _dh^–1 decay constant for active cells - k _ph^–1 decay constant for streptokinase - K _Igl^–1 inhibition constant for lactic acid - K_S gl^–1 inhibition constant for substrate - M gl^–1 lactic acid concentration - P gl^–1 streptokinase concentration - Q 1h^–1 substrate feed rate - S gl^–1 substrate concentration - S _ingl^–1 inlet concentration of substrate - t h time - t _bh end-point of batch fermentation - t _fh end-point of fed-batch fermentation - V l volume of broth in fermenter - V ₀ l initial value of V (at t=0) - V _ml maximum value of V - X gl^–1 total biomass concentration - X _agl^–1 concentration of active biomass - Y _MX yield coefficient for lactic acid from biomass - Y _PX yield coefficient for streptokinase from biomass - Y _XS yield coefficient for biomass from substrate Greek Letters h^–1 specific growth rate of biomass - _mh^–1 maximum specific growth rate     

Fluidized bed biomethanation of acetic acid

Summary The kinetics of acetate biomethanation was studied in a high recycle ratio biological fluidized bed reactor behaving in practice as a completely mixed reactor. The active biofilm consisted of bacteria from a methane fermenter that after spontaneous immobilization on the bed particles (sand) were adapted to acetate as the only carbon source. The effects of temperature (13°, 20°, 25° and 35°C), substrate concentration (500, 1000 and 1500 mg chemical oxygen demand (COD) l^-1) and hydraulic retention time (1 to 8 h) on substrate consumption were studied. Maximum substrate consumption (as % COD reduction) amounted from 25% (13°C, 1500 mg COD l^-1) to 93% (35°C, 500 mg COD l^-1). At 35°C the concentration of attached biomass presented a weakly increase with reactor substrate concentration (from 3.10 g VS l^-1 to 4.54 g VS l^-1 for 32 and 1150 mg COD l^-1 respectively). On the other hand when reducing , a sharp incrase in biomass loss coefficient was observed showing that excess biofilm growth was continuously removed by shearing forces. Thus in the assayed conditions the attached biomass concentration was basically determined by the bed superficial velocity. Result show that diffusional resistances are negligible. Data are fairly well correlated by a variable order kinetic model. The apparent reaction order is a function of temperature and increases from 0.27 to 0.7 when temperature decreases from 35° C to 13°C.Nomenclature b Total biomass loss coefficient (T^-1) - J Flux of substrate removal into the biofilm surface (ML^-2 T^-1) - J _d Flux of substrate removed into the biofilm surface in deep conditions (ML^-2 T^-1) - k Maximum specific rate of substrate utilization (T^-1) - K Variable order kinetic constant (T^-1 M^n-1 L^3n-3) - K _s9 Hall saturation constant (ML^-3) - n Reaction order - q Feed flow rate (L³ T^-1) - S Substrate concentration (ML^-3) - Se Effluent substrate concentration (ML^-3) - So Influent substrate concentration (ML^-3) - Se_min Minimum substrate concentration able to sustain a steady-state biofilm (ML^-3) - T Temperature - t Time(T) - V Bed volume (L³) - VS Volatile solids (M) - VSS Volatile suspended solids - X Attached biomass concentration (ML^-3) - X _c Effluent volatile suspended solids (ML^-3) - Y Yield coefficient - Hydraulic retention time (T) This work forms part of a Doctoral Thesis of senior author     

Ethanol fermentation by flocculating yeast: Performance and stability dependence on a critical fermentation rate

Summary A system coupling fermentor and decantor permitted strong accumulation of yeast flocs that were homogeneously suspended in the reactional volume. At 100–190 g/l glucose feed practically total substrate conversion was attained. At 130 g/l glucose feed the highest productivity (18.4 g.l.h) and the highest ethanol yield (90.6%) were reached with biomass levels of 80–90 g/l. We observed that the stability of this system is limited when a critical fermentation rate (D.S_o) close to 39–40 g/l.h (with corresponding ethanol productivities of 19–20 g/l.h) is reached. Higher fermentation rates provoked de-flocculation and lost of biomass.Symbols D dilution rate (h^–1) - E ethanol (g/l) - S_r residual substrate (g/l) - S_o substrate in the feed (g/l) - X biomass (g/l) - ethanol yield (%) - DS_o fermentation rate (g/l.h) (for S_r0) - P_E ethanol productivity (g/l.h)     

Adaptation of hybridoma cells to higher ammonia concentration

Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH₄Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH₄Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH₄Cl even under the ammonia serial change.List of symbols D_Feed d^-1 Dilution rate of fresh feed medium (=F_o/V) - D_Out d^-1 Dilution rate of cell suspension (=F₁/V) - F₁ ml·d^-1 Volumetric discharge rate of cell suspension - F₀ ml·d^-1 Volumetric flow rate of fresh feed medium - k_D h^-1 Specific death rate - P mmol·l^-1 Product concentration - S mmol·l^-1 Substrate concentration in culture broth - S₀ mmol·l^-1 Substrate concentration in feed medium - t d Cultivation time - V ml Working volume of reactor - X₀ cells·ml^-1 Total cell density - X_V cells·ml^-1 Viable cell density - Y_P/S mmol·mmol^-1 Yield of product from substrate - Y_X/S cells·mmol^-1 Yield of cells from substrate - mmol·cell^-1·h^-1 Specific production rate - h^-1 Specific growth rate - mmol·cell^-1·h^-1 Specific consumption rate of substrate     

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)     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.Nomenclature S glucose concentration (g/l) - X biomass concentration (g/l) - P ethanol concentration (g/l) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - YINX/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

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)     

Performance,kinetics, and substrate utilization in a continuous yeast fermentation with cell recycle by ultrafiltration membranes

Summary A continuous single stage yeast fermentation with cell recycle by ultrafiltration membranes was operated at various recycle ratios. Cell concentration was increased 10.6 times, and ethanol concentration and fermentor productivity both 5.3 times with 97% recycle as compared to no recycle. Both specific growth rate and specific ethanol productivity followed the exponential ethanol inhibition form (specific productivity was constant up to 37.5 g/l of ethanol before decreasing), similar to that obtained without recycle, but with greater inhibition constants most likely due to toxins retained in the system at hight recycle ratios.By analyzing steady state data, the fractions of substrate used for cell growth, ethanol formation, and what which were wasted were accounted for. Yeast metabolism varied from mostly aerobic at low recycle ratios to mostly anaerobic at high recycle ratios at a constant dissolved oxygen concentration of 0.8 mg/kg. By increasing the cell recycle ratio, wasted substrate was reduced. When applied to ethanol fermentation, the familiar terminology of substrate used for Maintenance must be used with caution: it is not the same as the wasted substrate reported here.A general method for determining the best recycle ratio is presented; a balance among fermentor productivity, specific productivity, and wasted substrate needs to be made in recycle systems to approach an optimal design.Nomenclature B Bleed flow rate, l/h - C _T Concentration of toxins, arbitrary units - D Dilution rate, h^-1 - F Filtrate or permeate flow rate, removed from system, l/h - F _o Total feed flow rate to system, l/h - K _s Monod form constant, g/l - P Product (ethanol) concentration, g/l - P _o Ethanol concentration in feed, g/l - PP} Adjusted product concentration, g/l - PD Fermentor productivity, g/l-h - R Recycle ratio, F/F _o - S Substrate concentration in fermentor, g/l - S _o Substrate concentration in feed, g/l - V Working volume of fermentor, l - V _MB Viability based on methylene blue test - X Cell concentration, g dry cell/l - X _o Cell concentration in feed, g/l - Y _ATP Cellular yield from ATP, g cells/mol ATP - Y _ATPS Yield of ATP from substrate, mole ATP/mole glucose - Y _G True growth yield or maximum yield of cells from substrate, g cell/g glucose - Y _P Maximum theoretical yield of ethanol from glucose, 0.511 g ethanol/g glucose - Y _P/S Experimental yield of product from substrate, g ethanol/g glucose - Y _x/s Experimental yield of cells from substrate, g cell/g glucose - S _NP/X Non-product associated substrate utilization, g glucose/g cell - k ₁, k₂, k₃, k₄ Constants - k ₁ ^APP , k ₂ ^APP Apparent k ₁, k₃ - k ₁ ^TRUE True k ₁ - m Maintenance coefficient, g glucose/g cell-h - m ^* Coefficient of substrate not used for growth nor for ethanol formation, g glucose/g cell-h - Specific growth rate, g cells/g cells-h, reported as h^-1 - _m Maximum specific growth rate, h^-1 - v Specific productivity, g ethanol/g cell-h, reported as h^-1 - v _m Maximum specific productivity, h^-1     

Lactose continuous fermentation with cells recycled by ultrafiltration and lactate separation by electrodialysis: model identification

Summary An off-line parameter estimation method has been developed to predict the dynamic behaviour of a continuous lactose fermentation system. The model used is an unstructured model taking into account cell growth, substrate consumption, and metabolite production (lactic acid). This method, based on the Hooke-Jeeves non-linear-programming technique, results in a good estimation of the biological parameters of the model, and so gives a better understanding of the different phenomena involved in lactose fermentation.Nomenclature Cp, Cs, Cz, Dp, Ds, Dz coefficients in system (A) - Fe bioreactor influent flow rate (1/h) - I current in the ED unit (A) - J lactate flux in the ED unit (g/h) - Kd mortality constant (h^-1) - Kp product inhibition constant (g/l) - Ks strbstrate saturation constant (g/l) - P ₀ product concentration in the bioreactor (g/l) - P ₁ product concentration in the D tank (g/l) - P _0r estimation of P ₀ (g/l) - Q ₀ retentate flow rate (UF influent) (1/h) - Q ₁ permeate flow rate (1/h) - Q ₂₂ cell bleed flow rate (1/h) - Q ₃ recycling flow rate in the ED (influent) (1/h) - Se substrate concentration in the influent (g/l) - S ₀ supstrate concentration in the bioreactor (g/l) - S ₁ substrate concentration in tank D (g/l) - S _0r estimation of S ₀ (g/l) - t time (h) - V ₀ fermentation broth volume (1) - V ₁ tank D volume (1) - X ₀ biomass concentration in the bioreactor (g/l) - Y _P/S (=1/Y _S/P) lactic acid yield coefficient (g lactic acid/g lactose consumed) - Y _X/S (=1/Y _S/X) cell yield coefficient (g cells produced/g lactose consumed) - Y _X/Z (=1/Y _Z/X) second cell yield coefficient (g cells produced/g nitrogen consumed) - Y _x, Y _m input mathematical parameters of the linear system (M ₂) - Ze nitrogen concentration in the influent (g/l) - Z ₀ nitrogen concentration in the bioreactor (g/l) - Z ₁ nitrogen concentration in tank D (g/l) - Z _0r estimation of Z ₀ (g/l) - , constants of the Luedeking and Piret's model - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1)     

On-line parameter and state estimation of continuous cultivation by extended Kalman filter

Summary The on-line estimation of biomass concentration and of three variable parameters of the non-linear model of continuous cultivation by an extended Kalman filter is demonstrated. Yeast growth in aerobic conditions on an ethanol substrate is represented by an unstructured non-linear stochastic t-variant dynamic model. The filter algorithm uses easily accessible data concerning the input substrate concentration, its concentration in the fermentor and dilution rate, and estimates the biomass concentration, maximum specific growth rate, saturation constant and substrate yield coefficient. The microorganismCandida utilis, strain Vratimov, was cultivated on the ethanol substrate. The filter results obtained with the real data from one cultivation experiment are presented. The practical possibility of using this method for on-line estimation of biomass concentration, which is difficult to measure, is discussed.Nomenclature D dilution rate (h^-1) - DO₂ dissolved oxygen concentration (%) - E identity matrix - F Jacobi matrix of the deterministic part of the system equations g - g continuousn-vector non-linear real function - h m-vector non-linear real function - K Kalman filter gain matrix - K _S saturation constant (kgm^-3) - K_S expectation of the saturation constant estimate - M Jacobi matrix of the deterministic part of the measurement equations h - P(t₀) co-variance matrix of the initial values of the state - P(t_k/t_k) c-variance matrix of the error in (t _k|t _k) - P(t_k+1/t_k) co-variance matrix of the error in (t _k+1|t _k - Q co-variance matrix of the state noise - R co-variance matrix of the output noise - S substrate concentration (kgm^-3) - S _i input substrate concentration - t time - t _k discrete time instant with indexk=0, 1, 2,... - u(t) input vector - v(t_k) measurement (output) noise sequence - w(t) n-vector white Gaussian random process - x(t₀) initial state of the system - (t₀) expectation of the initial state values - x(t) n-dimensional state vector - x(t_k) state vector at the time instantt _k - (t_k|t_k) expectation of the state estimate at timet _k when measurements are known to the timet _k - (t_k+1|t_k) expectation of the state prediction - X biomass concentration (kgm^-3) - expectation of the biomass concentration estimate - y(t_k) m-dimensional output vector at the time instantt _k - Y _XIS substrate yield coefficient - _X|S expectation of the substrate yield coefficient estimate - specific growth rate (h^-1) - _M maximum specific growth rate (h^-1) - expectation of the maximum specific growth rate estimate - state transition matrix     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

Fermentation ofd-xylose,d-glucose andl-arabinose mixture byPichia stipitis Y 7124: sugar tolerance

Summary The effect of substrate concentration (S ₀) on the fermentation parameters of a sugar mixture byPichia stipitis Y 7124 was investigated under anaerobic and microaerobic conditions. Under microaerobiosisP. stipitis maintained high ethanol yield and productivity when initial substrate concentration did not exceed 150 g/l; ethanol yield of about 0.40 g/g and volumetric productivity up to 0.39 g/l per hour were obtained. Optimal specific ethanol productivity (0.2 g/g per hour) was observed withS ₀=110 g/l. Under anaerobic conditionsP. stipitis exhibited the highest fermentative performances atS ₀=20 g/l; it produced ethanol with a yield of 0.42 g/g, with a specific rate of 1.1 g/g per day. When the initial substrate level increased, specific ethanol productivity declined gradually and ethanol yield was dependent on the degree of utilization of each sugar in the mixture.Abbreviations E _m maximum produced ethanol (g/l) - E ₀ initial ethanol (g/l) - E _v evaporated ethanol (g/l) - Q _p volumetric productivity of ethanol (g ethanol/l per hour or g/l per day) - q _p specific productivity of ethanol (g ethanol/g cells per hour) - q _pm maximum specific productivity of ethanol (g/l per hour) - S ₀ initial substrate concentration (g/l) - t _f time at which produced ethanol is maximum (h) - Y _p/s ethanol yield (g ethanol produced/g substrate utilized) - Y _x/s cell yeild (g cells produced/g substrate utilized) - Y _xo/xy xylitol yield (g xylitol produced/g xylose utilized) - probability coefficient - specific growth rate coefficient (h^-1 or d^-1)     

Long term shear effects on a hybridoma cell line by dynamic perfusion devices

The long term shear effects on a hybridoma cell line were studied by the simulation of a hollow fiber perfusion system. Various mechanical/environmental stress conditions were applied and steady state concentrations of live, dead and lysed cells were measured or calculated in a continuous culture. From mathematical modeling, it is shown that inclusion of a lysed cell index (LCI) renders a better fit to the material balance equation at steady state. The specific cell death rate increased with increasing shear force as expected only when the LCI was included. Without the inclusion of the LCI, the calculated specific cell growth rates are about 25–60% of the value when included. The results reported may lend some insight to design improvements since most perfusion devices add shear stresses to the cells in the reactor.List of Symbols b ml/hr continuous culture flow rate - D hr^–1 dilution rate (b/V) - m g glucose/10⁹ cells/hr specific maintenance coefficient - S ₀ g/l feed substrate concentration - S g/l reactor substrate concentration - t hr time - V ml reactor volume - X ⁺ cells/ml live cell concentration - X ^– cells/ml dead cell concentration - X ⁰ cells/ml lysed cell concentration - Y _x/s 10⁹ cells/g glucose cell/substrate yield coefficient - hr^–1 specific growth rate - hr^–1 specific death rate - hr^–1 specific lysis rate - hr^–1 specific lysis rate for simultaneous death and lysis