Fermentation kinetics of Zymomonas mobilis near zero growth rate

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.     

Salt accumulation resulting from base added for pH control, and not ethanol, limits growth of Thermoanaerobacteriumthermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture

Thermoanaerobacter thermosaccharolyticum HG-8 was grown in continuous culture to characterize growth limitation at high feed substrate and product concentrations. Continuous fermentation of 50 and 73 g/L xylose at a dilution rate based on the feed flow, D(f), of 0.053 h(-)(1) and with the pH controlled at 7.0 by addition of KOH resulted in steady state utilization of >99% of the xylose fed and production of ethanol and acetic acid at a mass ratio of about 2:1. Continuous cultures of T. thermosaccharolyticum growing at D(f) = 0.053 h(-)(1) achieved complete utilization of 75 g/L xylose in the presence of 19.1 g/L K(+) (0.49 M) and an ethanol concentration of 22.4 g/L ethanol. When the feed to a culture initially at steady state with a 75 g/L xylose feed and D(f) = 0.053 h(-)(1) was increased to 87.5 g/L xylose, limitation of growth and xylose utilization was observed. This limitation was not relieved by repeating this feed upshift experiment in the presence of increased nutrient levels and was not reproduced by addition of ethanol to a steady-state culture fed with 75 g/L xylose. By contrast, addition of KCl to a steady-state culture fed with 75 g/L xylose reproduced the K(+) concentration, limitation of growth and xylose utilization, and product concentration profiles observed in the feed upshift experiment. The maximum concentration at which growth of batch cultures was observed was 0.43 M for KCl, NaCl, and equimolar mixtures of these salts, suggesting that the observed limitation is not ion-specific. These data support the interpretation that inhibition salt accumulation resulting from addition of KOH for pH control is the limiting factor manifested in the feed upshift experiment and that both nutrient limitation and ethanol inhibition played little or no role as limiting factors. More generally, salt inhibition would appear to be a possible explanation for the discrepancy between the tolerance to added ethanol and the maximum concentration of produced ethanol reported in the literature for fermentation studies involving thermophilic bacteria.     

Continuous ethanol production from Jerusalem artichoke tubers. I. Use of free cells of Kluyveromyces marxianus

The Continuous fermentation of Jerusalem artichoke juice to ethanol by free cells of Kluyveromyces marxianus UCD (FST) 55-82 has been studied in a continuous-stirred-tank bioreactor at 35 degrees C and pH 4.6. A maximum yield of 90% of the theoretical was obtained at a dilution rate of 0.05 h(-1). About 95% of the sugars were utilized at dilution rates lower than 0.15 h(-1). Volumetric ethanol productivity and volumetric biomass productivity reached maximum values of 7 g ETOH/L/h and 0.6 g dry wt/L/h, respectively, at a dilution rate of 0.2 h(-1). The maintenance energy coefficient for K. marxianus culture was found to be 0.46 g sugar/g biomass/h/ Oscillatory behavior was following a change in dilution rate from a previous steady state and from batch to continuous culture. Values of specific ethanol production rate and specific sugar uptake were found to increase almost linearly with the increase of the dilution rate. The maximum specific ethanol production rate and maximum specific sugar uptake rate were found to be 2.6 g ethanol/g/ cell/h and 7.9 sugars/g cell/h, respectively. Washout occurred at a dilution rate of 0.41 h(-1).     

The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol

The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol was studied in turbidostat cultures at constant and stepwise changed ethanol concentrations. Up to 50 g/L ethanol, the inhibition kinetics can be approximated by a linear relationship between the specific growth rate and the ethanol concentration. Above this level, deviations from this linearity are observed. The specific fermentation rates were less inhibited by ethanol than was the specific growth rate. The maximum ethanol concentration achieved was 72 g/L.The response time for the adaptation of a turbidstat culture to step changes in the ethanol concentration was markedly dependent on the concentration level, the response time being large at high ethanol concentrations.     

Growth characteristics of bakers' yeast in ethanol

The influence of temperature (15 degrees -40 degrees C) and pH (2.5-6.0) on the continuous growth of bakers' yeast (Saccharomyces cerevisiae) at steady state in 1% ethanol was investigated. Optimal temperature and pH were 30 degrees C and 4.5, respectively. The short-term effect of ethanol concentration (0.1-10.0%) on the yeast growth was assessed in batch culture. Up to 1% of ethanol, the yeast growth increased in function of the ethanol concentration in the medium. The biomass reached a maximum within the interval of 1-4% of ethanol (7.9 and 31.6 g/L, respectively) and decreased at higher concentrations. The residual ethanol concentration in the medium increased rapidly when the initial ethanol concentration exceeded 4%. The best-fit model obtained for growth inhibition as a function of ethanol concentrations was that of Tseng and Wayman: mu(m)S/)K + S( - i (S - S(theta)). With this model, the specific growth rate (mu) decreased linearly as the ethanol concentration increased between the threshold value (S(theta)) of 11.26 g/L to be fully inhibited at 70.00 g/L (S;) an inhibition constant (i) of 0.0048 g L(-1) h(-1), a maximum specific growth rate (mu(m)) of 0.284 h(-1), and a saturation constant (K) of 0.611 g/L were obtained.     

Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose

Pichia stipitis NRRL Y-7124 has potential application in the fermentation of xylose-rich waste streams, produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen-limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K(1)a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (C(ox)). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (micro) was a Monod function of C(ox). Specific oxygen uptake rate was proportional to mu by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady-state C(OX) depended only on D, while steady-state biomass concentration was controlled by both D and K(1)a. Given excess oxygen, cells grew subject to Monod limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong's model, and 64. 3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of micro. The dying portion increased with E and X.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Observed quasi-steady kinetics of yeast cell growth and ethanol formation under very high gravity fermentation condition

Using a generalSaccharomyces cerevisiae as a model strain, continuous ethanol fermentation was carried out in a stirred tank bioreactor with a working volume of 1,500 mL. Three different gravity media containing glucose of 120, 200 and 280 g/L, respectively, supplemented with 5 g/L yeast extract and 3 g/L peptone, were fed into the fermentor at different dilution rates. Although complete steady states developed for low gravity medium containing 120 g/L glucose, quasi-steady states and oscillations of the fermented parameters, including residual glucose, ethanol and biomass were observed when high gravity medium containing 200 g/L glucose and very high gravity medium containing 280 g/L glucose were fed at the designated dilution rate of 0.027 h⁻¹. The observed quasi-steady states that incorporated these steady states, quasi-steady states and oscillations were proposed as these oscillations were of relatively short periods of time and their averages fluctuated up and down almost symmetrically. The continuous kinetic models that combined both the substrate and product inhibitions were developed and correlated for these observed quasi-steady states.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

Continuous ethanol production from concentrated wood hydrolysates in an internal membrane-filtration bioreactor

Continuous culture for the production of ethanol from wood hydrolysate was carried out in an internal membrane-filtration bioreactor. The hydrolysate medium was sterilized at a relatively low temperature of 60 degrees C with the intention of reducing the formation of inhibitory compounds during the sterilization. The maximum ethanol concentration and productivity obtained in this study were 76.9 g/L and 16.9 g/L-h, respectively, which were much higher than those (57.2-67 g/L and 0.3-1.0 g/L-h) obtained in batch cultures using hydrolysate media sterilized at 60 degrees C. The productivity was also found to be much higher than that (6.7 g/L-h) obtained in a continuous cell retention culture using a wood hydrolysate sterilized at 121 degrees C. These results show that the internal membrane-filtration bioreactor in combination with low-temperature sterilization could be very effective for ethanol production from wood hydrolysate.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (µ) and the cell yield (Y_X/S). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down‐regulation of genes in the ethanol‐producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in‐phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y_P/S) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described. Biotechnol. Bioeng. 2010;106: 721–730. © 2010 Wiley Periodicals, Inc.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

Bioenergetics and end-product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation

Fermentation of xylose by Clostridium thermosaccharolyticum was studied in batch and continuous culture in which the limiting nutrient was either xylose, phosphate, or ammonia. Transient results obtained in continuous cultures with batch grown inoculum and progressively higher feed substrate concentrations exhibited ethanol selectivities (moles ethanol/moles other products) in excess of 11. The hypothesis that this high ethanol selectivity was a general response to mineral nutrient limitation was tested but could not be supported. Growth and substrate consumption were related by the equation q(s)(1 - Y(x) (c))G(ATP) = (mu/Y(ATP) (max)) + m, with q(s) the specific rate of xylose consumption (moles xylose/hour . g cells), Y(x) (c) the carbon based cell yield (g cell carbon/g substrate carbon), G(ATP) the ATP gain (moles ATP produces/mol substrate catabolized), mu the specific growth rate (1/h), Y(ATP) (max) the ATP-based cell yield (g cells/mol ATP), and m the maintenance coefficient (moles ATP/hour . g cells). Y(ATP) (max) was found to be 11.6 g cells/mol ATP, and m 9.3 mol ATP/hour . g cells for growth on defined medium. Different responses to nutrient limitation were observed depending on the mode of cultivation. Batch and immobilized cell continuous cultures decreased G(ATP) by initiating production of the secondary metabolites, propanediol, and in some cases, D-lactate; in addition, batch cultures increased the fractional allocation of ATP to maintenance and/or wastage. Nitrogen-limited continuous free-cell cultures maintained a constant cell yield, whereas phosphate-limited continuous free-cell cultures did not. In the case of phosphate limitation, the decreased ATP demand associated with the lowered cell yield was accompanied by an increased rate of ATP consumption for maintenance and/or wastage. Neither nitrogen or phosphorus-limited continuous free-cell cultures exhibited an altered G(ATP) in response to mineral nutrient limitation, and neither produced secondary metabolites. (c) 1993 John Wiley & Sons, Inc.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media

Aim of the present study was to evaluate the effect of exogenous additions of 1,3-propanediol (1,3-PDO) on microbial growth and metabolites production of Clostridium butyricum VPI 1718 strain, during crude glycerol fermentation. Preliminary batch cultures in anaerobic Duran bottles revealed that early addition of 1,3-PDO caused growth cessation in rather low quantities (15?g/L), while 1,3-PDO additions during the middle exponential growth phase up to 70?g/L resulted in an almost linear decrease of the specific growth rate (μ), accompanied by reduced glycerol assimilation, with substrate consumption being used mainly for energy of maintenance requirements. During batch trials in a 3-L bioreactor, the strain proved able to withstand more than 70?g/L of both biologically produced and externally added 1,3-PDO, whereas glycerol assimilation and metabolite production were carried on at a lower rate. Adaptation of the strain in high 1,3-PDO concentration environments was validated during its continuous cultivation with pulses of 1,3-PDO in concentrations of 31 and 46?g/L, where no washout phenomena were noticed. As far as C. butyricum cellular lipids were concerned, during batch bioreactor cultivations, 1,3-PDO addition was found to favor the biosynthesis of unsaturated fatty acids. Also, fatty acid composition was studied during continuous cultures, in which additions of 1,3-PDO were performed at steady states. Lipids were globally more saturated compared to batch cultures, while by monitoring of the transitory phases, it was noticed that the gradual diol washout had an evident impact in the alteration of the fatty acid composition, by rendering them more unsaturated.     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: II. Analysis of cell synchronization and metabolism

Sustained oscillations of biomass, ethanol, and ammonium concentrations, specific growth rate, and specific uptake rates of ethanol, ammonium, and oxygen were found in continuous cultures of Saccharomyces cerevisiae under controlled dissolved oxygen (DO), pH, and temperature conditions. The period of oscillations was approximately 2.5-3 h at a pH of 5.5 and 2-2.5 h at a pH of 6.5. Oscillations were observed only under conditions of low carbon (glucose below the minimum detectable level), nitrogen nutrient (ammonium concentration varied between 0.00001 and 0.0015M), and ethanol concentration (0.002-0.085 g/L) in the bioreactor.The oscillatory behavior at pH 5.5 was also characterized by partially synchronized cell growth and reproduction. Not only did the total percentage of budding cells oscillate with the same period as observed for the global biomass and nutrient concentrations, but the peaks in the individual subpopulations of initial budding, middle budding, and late budding cells appeared sequentially during the oscillation period. This provides strong evidence of the hypothesis that variations in metabolism during different periods in the cell cycle of a partially synchronized cell population are responsible for the observed oscillatory bioreactor behavior.The specific nutrient uptake rates for ammonium and oxygen as well as the net specific ethanol uptake rate oscillated with the same period as the biomass oscillations. These results show a dramatic increase in the ammonium and oxygen consumption rates prior to the initial budding of the synchronized subpopulation and a decrease in these rates during the late budding phase. At a pH of 5.5, the late budding phase is characterized by high specific ethanol productivity; however, the ethanol productivity lags the late budding phase at a pH pf 6.5. The observed time-varying metabolism in the oscillatory operating regime appears to be the result of the metabolic changes which occur during the cell cycle. Models which can predict the oscillatory biomass concentration and nutrient levels in this regime must be capable of predicting the concentrations and metabolic rates of the subpopulations as well.     

Continuous fermentation of feed streams containing D-glucose and D-xylose in a two-stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,(1,2,3) To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y-2460 and Saccharomyces cerevisiae NRRL Y-2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y-2235 fermented glucose but not xylose. Y-2460 consumed both sugars but fermented glucose inefficiently relative to Y-2235, and it suffered a diauxic lag lasting 10-20 h when given a sugar mixture. Immobilized Y-2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y-2460 exhibited an optimum productivity at 30-40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y-2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y-2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8-g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y-2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y-2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two-stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y-2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y-2235 plug flow reactor would enrich the hemicellulose-derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.