Observer design for differential-algebraic model of an aerobic culture of a recombinant yeast

The mathematical model of an aerobic culture of recombinant yeast presented in work by Zhang et al. (1997) is given by a differential-algebraic system. The classical nonlinear observer algorithms are generally based on ordinary differential equations. In this paper, first we extend the nonlinear observer synthesis to differential-algebraic dynamical systems. Next, we apply this observer theory to the mathematical model proposed in Zhang et al. (1997). More precisely, based on the total cell concentration and the recombinant protein concentration, the observer gives the online estimation of the glucose, the ethanol, the plasmid-bearing cell concentration and a parameter that represents the probability of plasmid loss of plasmid-bearing cells. Numerical simulations are given to show the good performances of the designed observer.Symbols C ₁ activity of pacing enzyme pool for glucose fermentation (dimensionless) - C ₂ activity of pacing enzyme pool for glucose oxidation (dimensionless) - C ₃ activity of pacing enzyme pool for ethanol oxidation (dimensionless) - E ethanol concentration (g/l) - G glucose concentration (g/l) - k _a regulation constant for (g glucose/g cell h^–1) - k _b regulation constant for (dimensionless) - k _c regulation constant for (g glucose/g cell h^–1) - k _d regulation constant for (dimensionless) - K _m1 saturation constant for glucose fermentation (g/l) - K _m2 saturation constant for glucose oxidation (g/l) - K _m3 saturation constant for ethanol oxidation (g/l) - L ( t) time lag function (dimensionless) - p probability of plasmid loss of plasmid-bearing cells (dimensionless) - P recombinant protein concentration (mg/g cell) - q _G total glucose flux culture time (g glucose/g cell h) - t culture time (h) - t _lag lag time (h) - X total cell concentration (g/l) - X ⁺ plasmid-bearing cell concentration (g/l) - Y ^F _{X / G} cell yield for glucose fermentation pathway (g cell/g glucose) - Y ^O _{X / G} cell yield for glucose oxidation pathway (g cell/g glucose) - Y _{X / E} cell yield for ethanol oxidation pathway (g cell/g ethanol) - Y _{E / X} ethanol yield for fermentation pathway based on cell mass (g ethanol·g cell) - ₂ glucoamylase yield for glucose oxidation (units/g cell) - ₃ glucoamylase yield for ethanol oxidation (units/g cell) - µ₁ specific growth rate for glucose fermentation (h^–1) - µ₂ specific growth rate for glucose oxidation (h^–1) - µ₃ specific growth rate for ethanol oxidation (h^–1) - µ_1max maximum specific growth rate for glucose fermentation (h^–1) - µ_2max maximum specific growth rate for glucose oxidation (h^–1) - µ_3max maximum specific growth rate for ethanol oxidation (h^–1)     

High productivity continuous ethanol fermentation with a flocculating mutant strain of Zymomonas mobilis

High fermenter (volumetric) ethanol productivities (80 g/lh^–1) were attained in a simple single-stage continuous-stirred-tank-reactor (CSTR) employing a flocculent mutant of Zymomonas mobilis with a feed containing 100g/l glucose. Under these conditions a final ethanol concentration of 47.6 g/l was obtained, representing a maximum conversion efficiency of 97% of theoretical.Nomenclature SR = Medium glucose concentration (g/l)X Biomass concentration (g/l) - P Ethanol concentration (g/l) - VP Volumetric productivity (g ethanol/l/h) - Yp/s Product yield coefficient (g ethanol/g glucose consumed) - Qp Specific rate of ethanol formation (g ethanol/g cells/h) - D Dilution rate (h^–1) - Dmax Maximum dilution rate: ie., highest dilution rate at which the effluent glucose concentration 4g/l (h^–1)     

Influence of exponentially decreasing feeding rates on fed-batch ethanol fermentation of sugar cane blackstrap molasses

Summary The ethanol yield was not affected and the ethanol productivity was increased when exponentially decreasing feeding rates were used instead of constant feeding rates in fed batch ethanol fermentations. The influences of the initial sugar feeding rate on the ethanol productivity, on the constant ethanol production rate during the feeding phase and on the initial ethanol production specific rate are represented by Monod-like equations.Nomenclature F reactor feeding rate (L.h^–1) - F_o initial reactor feeding rate (L.h^–1) - K time constant; see equation (l) (h^–1) - M_E mass of ethanol in the fermentor (g) - M_s mass of TRS in the fermentor (g) - M_x mass of yeast cells (dry matter) in the fermentor (g) - P ethanol productivity (g.L^–1.h^–1) - R ethanol constant production rate during the feeding phase (g.h^–1) - s standard deviation - S_o TRS concentration in the feeding mash (g.L^–1) - t time (h) - T fermentor filling-up-time (h) - T time necessary to complete the fermentation (h) - TRS total reducing sugars calculated as glucose (g.L^–1) - V_o volume of the inoculum (L) - V_f final volume of medium in the fermentor (L) - X_o yeast concentration of the inoculum (dry matter) (g.L^–1) - ethanol yield (% of the theoretical value) - initial specific rate of ethanol production (h^–1)     

The kinetics of ethanol production by Zymomonas mobilis on fructose and sucrose media

Summary Z.mobilis is strain ZM4 was grown on 250 g/l fructose and sucrose media in batch culture and on 100 and 150 g/l sucrose media in continuous culture. With fructose, a significant reduction in the growth rate and the cell yield was apparent although the other kinetic parameters were similar to those previously reported for fermentation of glucose. With sucrose the major differences were a reduction in ethanol yield, (due to levan formation) and a lower final ethanol concentration. Ethanol inhibition of sucrose metabolism occurred at relatively low ethanol concentrations compared to those inhibiting glucose metabolism.     

The production of ethanol plus fructose sweetener using fructose utilization negative mutants of Zymomonas mobilis

Summary Two mutants, unable to utilize fructose (Fru^–) as a sole source of carbon and energy, were isolated fromZymomonas mobilis following ethyl methane sulfonate (EMS) mutagenesis. The frequency of stable Fru^– mutants among survivors of mutagenesis was 1 in 10⁴. The two Fru^– mutants were able to cleave sucrose to glucose and fructose, and then ferment only the glucose to ethanol while accumulating fructose close to the theoretical value. Under controlled fermentation conditions, sucrose was converted to ethanol plus 80% or higher purity fructose syrup in a single-stage batch fermentation process, improving the Sucrotech Process significantly.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Effects of organism type and growth conditions on cell disruption by impingement

Data for disruption of C. utilis, S. cerevisiae and B. subtilis cells by impingement of a high velocity jet of suspended cells against a stationary surface are compared. Differences between organisms were observed, but there were no general differences found between yeast and bacteria. In addition, growth conditions were found to have an effect on disruption with cells grown at a high specific growth rate easier to disrupt than cells grown at a low rate.Nomenclature a exponent of pressure (dimensionless) - D dilution rate (h^\s-1) - K dimensional rate constant (Pa ^\s-) - N number of passes (dimensionless) - P operating pressure (Pa) - R fraction of cells disrupted (dimensionless) - u_m maximum specific growth rate (h^\s-1)     

Unmarked insertional inactivation in the gfo gene improves growth and ethanol production by Zymomonas mobilis ZM4 in sucrose without formation of sorbitol as a by-product,but yields opposite effects in high glucose

Sorbitol, one of the main by-products of growth on high sucrose concentrations, is catalyzed by glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28) in Zymomonas mobilis, which decreases the ethanol yield. In this study, an unmarked gfo mutant from Z. mobilis ZM4 was constructed using a site-specific FLP recombinase, and growth and ethanol production were evaluated with or without the addition of sorbitol to the media. The inactivation of gfo had contrasting effects in different substrates, especially at high concentrations. The maximum specific growth rate (μ_m) and theoretical ethanol yield value (Y_m) increased from 0.065 h⁻¹ and 60.56% to 0.094 h⁻¹ and 83.87% in 342 g/L sucrose, respectively. Conversely, in 200 g/L glucose, gfo inactivation decreased μ_m and Y_m from 0.15 h⁻¹ and 89.85% to 0.10 h⁻¹ and 67.59%, respectively, and prolonged the lag period from 16 h to 40 h. The addition of sorbitol slightly accelerated growth and sucrose hydrolysis by the gfo mutant in 342 g/L sucrose; however, addition of sorbitol restored the μ_m and Y_m of the gfo mutant in 200 g/L glucose to 0.14 h⁻¹ and 82.50%, respectively. Inactivation of gfo had a small effect on fructose utilization, and a positive one on mixture of glucose and fructose similar to that on sucrose. These results provide further understanding of the osmoregulation mechanisms in Z. mobilis and may help to exploit the biotechnological applications of this industrially important bacterium.     

Heterotrophic bacterial activity and primary production in a hypertrophic African lake

The relationship between heterotrophic bacteria and phytoplankton in the epilimnion (0–10 m) of hypertrophic Hartbeespoort Dam, South Africa, was examined by statistically analyzing three years of parallel measurements of heterotrophic bacterial activity (glucose uptake) and phytoplankton particulate and dissolved organic carbon production. Algal biomass ranged between 4.0 and 921.1 mg Chl a m^-3 at the surface. Primary production varied between 69.5 and 3010.0 mg C m^-2h^-1 while algal production of dissolved organic carbon (EDOC) ranged from 2.5 to 219.2 mg C m^-2h^-1. Bacterial numbers reached a summer peak of 44.23 × 10⁶ cells ml^-1 in the first year and showed no depth variation. The maximum rate of glucose uptake, V_max, reached a peak of 5.52 g C l^-1h^-1. V_max, maximum glucose concentration (K_t + S_n) and glucose turnover time (T_t) were usually highest at the surface and decreased with depth concomitant with algal production. At the surface, V_max was correlated to EDOC (r = 0.59, n = 67, p < 0.001) and primary production (r = 0.71, n = 70, p < 0.001). At 5 and 10 m, V_max was correlated to integral euphotic zone (~ 4 m) algal production and bacterial numbers. Glucose turnover time was inversely related to integral algal production (r = -0.72, n = 70, p < 0.001) and less strongly to bacterial numbers. The data indicated that although bacterial numbers and biomass were low relative to algal biomass in this hypertrophic lake, the heterotrophic bacteria attained high rates of metabolic activity as a result of enhanced algal production of available organic carbon.     

Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis

Summary Zymomonas mobilis is able to convert glucose and fructose to gluconic acid and sorbitol. The enzyme, glucose-fructose oxidoreductase, catalysing the intermolecular oxidation-reduction of glucose and fructose to gluconolactone and sorbitol, was formed in high amounts [1.4 units (U)·mg^-1] when Z. mobilis was grown in chemostats with glucose as the only carbon source under non-carbon-limiting conditions. The activity of a gluconolactone-hydrolysing lactonase was constant at 0.2 U·mg^-1. Using glucose-grown cells for the conversion of equimolar fructose and glucose mixtures up to 60% (w/v), a maximum product concentration of only 240 g·1^-1 of sorbitol was found. The gluconic acid accumulated was further metabolized to ethanol. After permeabilizing the cells using cationic detergents, maximum sorbitol and gluconic acid concentrations of 295 g·1^-1 each were reached; no ethanol production occurred. In a continuous process with -carrageenan-immobilized and polyethylenimin-hardened, permeabilized cells no significant decrease in the conversion yield was observed after 75 days. The specific production rates for a high yield conversion ( > 98%) in a continuous two-stage process were 0.19 g·g^-1·h^-1 for sorbitol and 0.21 g·g^-1·h^-1 for gluconic acid, respectively. For the sugar conversion of cetyltrimethylammonium bromide-treated -carrageenan-immobilized cells a V _max of 1.7 g·g^-1·h^-1 for sorbitol production and a K _m of 77.2 g·1^-1 were determinedOffprint requests to: B. Rehr     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

Combined alcoholic fermentation of D-xylose and D-glucose by four selected microbial strains: Process considerations in relation to ethanol tolerance

Summary As components of combined fermentation of both glucose and xylose to ethanol by separated or coculture processes, the effects of initial sugar concentrations on the fermentative performances ofPichia stipitis Y7124,Candida shehatae ATCC 22984,Saccharomyces cerevisiae CBS1200 andZymomonas mobilis ATCC10988 were investigated. From the characteristics of sugar and produced ethanol tolerances the most suitable microorganisms for the achievement of glucose and xylose fermentations have been selected with respect to different fermentation schemes.Nomenclature Tf fermentation time (hours) - Ef ethanol concentration (g/l) - Y_P/S ethanol yield (g of ethanol produced/g of sugar used) - qp average specific productivity of ethanol (g ethanol/g of cells per hour) - max maximum specific growth rate (h^–1)     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Simulation of the dynamics in the Baker's yeast process

Simulation of the dynamics in a fed batch process for production of Baker's yeast is discussed and applied. Experimental evidences are presented for a model of the energy metabolism. The model involves the concept of a maximum respiratory capacity of the cell. If the sugar concentration is increased above a critical value, corresponding to a critical rate of glycolysis and a maximum rate of respiration, then all additional sugar consumed at higher sugar concentrations is converted into ethanol.In a fed batch process with constant sugar feed the sugar concentration declines slowly. If ethanol is present when the sugar concentration declines below the critical value of 110 mg/dm³ fructose +glucose the metabolism switches rapidly into combined oxidation of sugar and ethanol. Thus, no diauxic growth is involved under process conditions. The rate of ethanol consumption is determined by the free capacity of respiration under these conditions. The invertase activity of the cells was found to be so high that mainly fructose and glucose were present in the medium, typically in the concentration range around 100 mg/dm³. These components are consumed at the same rate but with fructose at a higher concentration, indicating a higher K _s for fructose consumption.The model was used in simulation experiments to demonstrate the dynamics of the Baker's yeast process and the influence of different process conditions.List of Symbols DOT % air sat dissolved oxygen tension - F dm³/h rate of inlet medium flow - H kg/(dm³ % air sat.) oxygen solubility - K kg/m³ saturation constant specified by index - K _L a 1/h volumetric oxygen transfer coefficient - m g/(g · h) maintenance coefficient specified by index - P kg/(m³ · h) mean productivity of biomass in the process - q g/(g · h) specific consumption or production rate - S kg/m³ concentration of sugar in reactor - S ₀ kg/m³ concentration of inlet medium sugar medium t h process time - V dm³ medium volume - X kg/m³ concentration of biomass - Y g/g yield coefficient specified by index - 1/h specific growth rate Index aa anaerobic condition - c critical value - e ethanol - ec ethanol consumption - ep ethanol production - max maximum value - o oxygen - oe oxygen for growth on ethanol - os oxygen for growth on sugar - s sugar - x biomass     

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     

Ethanol fermentation using a fructose-negative mutant of Zymomonas mobilis

Summary The fermentation of an equimolar mixture of glucose and fructose into ethanol and sorbitol by a fructose negative mutant of Zymomonas mobilis is analysed using a recently described methodology (Ait-Abdelkader and Baratti, Biotechnol. Tech. 1993,329–334) based on polynomial fitting and calculation of instantaneous and overall parameters. These parameters are utilized to describe this mixed-substrate mixed-product fermentation.Nomenclature X biomass concentration, g/l - S total sugar concentration, g/l - Glu glucose concentration, g/l - Fru fructose concentration, g/l - Sor sorbitol concentration, g/l - P ethanol concentration, g/l - t fermentation time, h - specific growth rate, h^-1 - q_s specific sugar uptake rate, g/g.h - q_g specific glucose uptake rate, g/g.h - q_F specific fructose uptake rate, g/g.h - q_P specific ethanol productivity, g/g.h - q_Sor specific sorbitol productivity, g/g.h - Y_X/S biomass yield on total sugar, g/g - Y_P/S ethanol yield on total sugar, g/g - Y_Sor/S sorbitol yield on total sugar, g/g - Y_Sor/F sorbitol yield on fructose, (g/g) - Y_P/G ethanol yield on glucose, (g/g)     

Influence of linearly decreasing feeding rates on fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The ethanol yield was not affected and the ethanol productivity increased (10%) when linearly decreasing feeding rates were used instead of constant feeding rates in fed-batch ethanol fermentations.Nomenclature F reactor feeding rate (L.h^–1) - M_E mass of ethanol in the fermentor (g) - M_s mass of TRS in the fermentor (g) - M_x mass of yeast cells (dry matter) in the fermentor (g) - P ethanol productivity (g.L^–1.h^–1) - s standard deviation - S_o TRS concentration in the feeding mash (g.L^–1) - t time (h) - T fermentor filling-up time (h) - TRS total reducing sugars calculated as glucose (g.L^–1) - X_o yeast cells concentration (dry matter) in the inoculum (g.L^–1) - average ethanol yield (% of the theoretical value)     

Ethanol fermentation using a glucose negative mutant ofZymomonas mobilis

Summary The fermentation of an equimolar mixture of glucose and fructose into ethanol and sorbitol by a glucose negative mutant ofZymomonas mobilis was monitored. The results were analyzed using a recently described method based on polynomial fitting and calculation of intantaneous and overall parameters. These parameters described well the physiology of this mixed-substrate mixed-product fermentation. Growth of the mutant was greatly inhibited on this medium. Fructose was quantitatively converted into sorbitol while glucose was oxidized into gluconic acid .This latter product was utilized as substrate for cell growth and ethanol production.Nomenclature X biomass concentration, g/l - S total sugar concentration, g/l - Glu glucose concentration, g/l - Fru fructose concentration, g/l - Sor sorbitol concentration, g/l - P ethanol concentration, g/l - t fermentation time, h - specific growth rate, h^-1 - q_s specific sugar uptake rate, g/g.h - q_G specific glucose uptake rate, g/g.h - q_F specific fructose uptake rate, g/g.h - q_P specific ethanol productivity, g/g.h - q_Sor specific sorbitol productivity, g/g.h - Y_X/S biomass yield on total sugar, g/g - Y_P/S ethanol yield on total sugar, g/g - Y_Sor/S sorbitol yield on total sugar, g/g - y_Sor/f sorbitol yield on fructose, g/g - Y_P/G ethanol yield on glucose, g/g     

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4--d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca²⁺, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4--d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h^–1, biomass yield coefficient of 0.5 g g^–1 and feed substrate concentration of 200 g L^–1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L^–1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g^–1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.Nomenclature C _ox Dissolved oxygen concentration (mg L^–1) - CPR Carbon dioxide production rate (mmol h^–1) - C _s0 Glucose concentration in the feed (g L^–1) - C _s Substrate concentration in the fermenter (g L^–1) - C _s.crit Critical substrate concentration (g L^–1) - E Ethanol concentration (g L^–1) - F _s Substrate flow rate (g h^–1) - i Sample number (–) - K _e Constant in Equation 6 (g L^–1) - K _o Constant in Equation 7 (mg L^–1) - K _s Constant in Equation 5 (g L^–1) - m Specific maintenance term (h^–1) - OUR Oxygen up-take rate (mmol h^–1) - q _ox Specific oxygen up-take rate (h^–1) - q _ox.max Maximum specific oxygen up-take rate (h^–1) - q _p Specific product formation rate (h^–1) - q _s Specific substrate up-take rate (g g^–1 h^–1) - q _s.max Maximum specific substrate up-take rate (g g^–1 h^–1) - RQ Respiratory quotient (–) - S Total substrate in the fermenter at timet (g) - S ₀ Substrate mass fraction in the feed (g g^–1) - t Fermentation time (h) - V Instantaneous volume of the broth in the fermenter (L) - V ₀ Starting volume in the fermenter (L) - V _si Volume of samplei (L) - x Biomass concentration in the fermenter (g L^–1) - X ₀ Total amount of initial biomass (g) - X _t Total amount of biomass at timet (g) - Y _p/s Product yield coefficient on substrate (–) - Y _x/e Biomass yield coefficient on ethanol (–) - Y _x/s Biomass yield coefficient on substrate (–) Greek letters Moles of carbon per mole of yeast (–) - Moles of hydrogen atom per mole of yeast (–) - Moles of oxygen atom per mole of yeast (–) - Moles of nitrogen atom per mole of yeast (–) - Specific growth rate (h^–1) - _crit Critical specific growth rate (h^–1) - _E Specific ethanol up-take rate (h^–1) - _max.E Maximum specific ethanol up-take rate (h^–1)