Continuous ethanol fermentation of lactose by a recombinant flocculating Saccharomyces cerevisiae strain.

Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L(-1) h(-1) (corresponding to a feed lactose concentration of 50 g L(-1) and a dilution rate of 0.55 h(-1)) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.     

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells.

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.     

Relationships between hydrodynamics and rheology of flocculating yeast suspensions in a high-cell-density airlift bioreactor

In this article a hydrodynamic and rheological analysis of a continuous airlift bioreactor with high-cell-density system is presented. A highly flocculating recombinant strain of Sacharomyces cerevisiae containing genes for lactose transport (lactose permease) and hydrolysis (beta-galactosidase) was exploited to ferment lactose from cheese whey to ethanol. The magnetic particle-tracer method was used to assess the effect of operational conditions (air-flow rate, biomass concentration) on hydrodynamic behavior of an airlift bioreactor during the fermentation process. Measurements of liquid circulation velocity showed the existence of a critical value of biomass concentration at which a dramatic deceleration of net liquid flow appeared with increasing biomass quantity. Rheological analysis revealed exponential increase of viscosity of the yeast floc suspension at the same biomass concentration of about 73 g/dm3 corresponding to 42.8% v/v of solid fraction. These facts have a particular importance for the successful processing of a high-cell-density airlift bioreactor as only a circulated flow regime will be favorable to keep the solid particles in suspension state and evenly distributed throughout the bioreactor.     

Application of Saccharomyces cerevisiae and Pichia stipitis karyoductants to the production of ethanol from xylose

Karyoductants of Saccharomyces cerevisiae V30 and Pichia stipitis CCY 39501 with the ability to ferment D-xylose to ethanol were isolated. The ability of these isolates to assimilate different sugars, ethanol tolerance and ethanol production from D-xylose was investigated. Karyoductants didn't grow on starch, lactose and cellobiose, like S. cerevisiae, but showed good growth on xylose and L-arabinose, like P. stipitis. All isolates fermented xylose to ethanol slower than P. stipitis and with lower yields, 0.09 - 0.16 g/g. They secreted also about 3.4 - 7.1 g/dm3 of xylitol to the culture medium (P. stipitis only 0.06 g/dm3). The karyoductants showed an average tolerance to ethanol when compared with the parent strains and fermented glucose in the presence of 6% alcohol whereas parent strain S. cerevisiae and P. stipitis showed exogenic ethanol tolerance of 9% and 3%, respectively.     

Continuous modeling of metabolic networks with gene regulation in yeast and in vivo determination of rate parameters

A continuous model of a metabolic network including gene regulation to simulate metabolic fluxes during batch cultivation of yeast Saccharomyces cerevisiae was developed. The metabolic network includes reactions of glycolysis, gluconeogenesis, glycerol and ethanol synthesis and consumption, the tricarboxylic acid cycle, and protein synthesis. Carbon sources considered were glucose and then ethanol synthesized during growth on glucose. The metabolic network has 39 fluxes, which represent the action of 50 enzymes and 64 genes and it is coupled with a gene regulation network which defines enzyme synthesis (activities) and incorporates regulation by glucose (enzyme induction and repression), modeled using ordinary differential equations. The model includes enzyme kinetics, equations that follow both mass-action law and transport as well as inducible, repressible, and constitutive enzymes of metabolism. The model was able to simulate a fermentation of S. cerevisiae during the exponential growth phase on glucose and the exponential growth phase on ethanol using only one set of kinetic parameters. All fluxes in the continuous model followed the behavior shown by the metabolic flux analysis (MFA) obtained from experimental results. The differences obtained between the fluxes given by the model and the fluxes determined by the MFA do not exceed 25% in 75% of the cases during exponential growth on glucose, and 20% in 90% of the cases during exponential growth on ethanol. Furthermore, the adjustment of the fermentation profiles of biomass, glucose, and ethanol were 95%, 95%, and 79%, respectively. With these results the simulation was considered successful. A comparison between the simulation of the continuous model and the experimental data of the diauxic yeast fermentation for glucose, biomass, and ethanol, shows an extremely good match using the parameters found. The small discrepancies between the fluxes obtained through MFA and those predicted by the differential equations, as well as the good match between the profiles of glucose, biomass, and ethanol, and our simulation, show that this simple model, that does not rely on complex kinetic expressions, is able to capture the global behavior of the experimental data. Also, the determination of parameters using a straightforward minimization technique using data at only two points in time was sufficient to produce a relatively accurate model. Thus, even with a small amount of experimental data (rates and not concentrations) it was possible to estimate the parameters minimizing a simple objective function. The method proposed allows the obtention of reasonable parameters and concentrations in a system with a much larger number of unknowns than equations. Hence a contribution of this study is to present a convenient way to find in vivo rate parameters to model metabolic and genetic networks under different conditions.     

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.     

A biochemically structured model for ethanol fermentation by Kluyveromyces marxianus: A batch fermentation and kinetic study

Anaerobic batch fermentations of ricotta cheese whey (i.e. containing lactose) were performed under different operating conditions. Ethanol concentrations of ca. 22 g L⁻¹ were found from whey containing ca. 44 g L⁻¹ lactose, which corresponded to up to 95% of the theoretical ethanol yield within 15 h. The experimental data could be explained by means of a simple knowledge-driven biochemically structured model that was built on bioenergetics principles applied to the metabolic pathways through which lactose is converted into major products. Use of the model showed that the observed concentrations of ethanol, lactose, biomass and glycerol during batch fermentation could be described within a ca. 6% deviation, as could the yield coefficients for biomass and ethanol produced on lactose. The model structure confirmed that the thermodynamics considerations on the stoichiometry of the system constrain the metabolic coefficients within a physically meaningful range thereby providing valuable and reliable insight into fermentation processes.     

Kinetics of batch ethanol fermentation of cheese-whey powder (CWP) solution as function of substrate and yeast concentrations

A lactose utilizing yeast strain, Kluyveromyces marxianus DSMZ-7239 was used for ethanol formation from cheese-whey powder (CWP) solution in batch experiments. Effects of initial substrate (CWP) and yeast concentrations on the rate and extent of ethanol formation were investigated. The initial pH and oxidation-reduction potential (ORP) was kept at 5 and -250 mV, respectively. The rate and extent of ethanol formation increased with increasing CWP concentration up to 156 g l(-1) (75 g l(-1) sugar) and then decreased for larger CWP concentrations due to substrate inhibition at high sugar concentrations. The ethanol yield coefficient was also maximum (0.54 g EtOH/g sugar) and equal to the theoretical yield at CWP concentration of 156 g l(-1). The growth yield coefficient was found to be Y(x/s)=1.2+/-0.1g biomass g sugar(-1). The rate of sugar utilization and ethanol formation also increased linearly with increasing initial biomass concentrations. A kinetic model describing the rate of sugar utilization and substrate inhibition as function of the initial substrate and the biomass concentrations was developed. The kinetic constants were determined using the experimental data. Model predictions of sugar utilization rates were in good agreement with the experimental data. The results indicated that the initial sugar concentration should be below 75 g l(-1) (CWP<156 g l(-1)) and the initial biomass should be above 850 mg l(-1) to obtain high rates and yields of ethanol formation and to avoid substrate inhibition.     

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.     

Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation

In ethanol fermentation, instantaneous biomass yield of the yeast Saccharmoyces cerevisiae was found to decrease (from 0.156 to 0.026) with increase in ethanol concentration (from 0 to 107 g/L), indicating a definite relationship between biomass yield and product inhibition. A suitable model was proposed to describe this decrease which incorporates the kinetic parameters of product inhibition rather than pure empirical constants. Substrate inhibition was found to occur when substrate concentration is above 150 g/L. A similar definite relationship was observed between substrate inhibition and instantaneous biomass yield. A simple empirical model is proposed to describe the declines in specfic growth rate and biomass yield due to substrate inhibition. It is observed that product inhibition does not have any effect on product yield whereas substrate inhibition significantly affects the product yield, reflecting a drop in overall product yield from 0.45 to 0.30 as the initial substrate concentration increases from 150 to 280 g/L. These results are expected to have a significant influence in formulating optimum fermentor design variables and in developing an effective control strategy for optimizing ethanol producitivity.     

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

Overexpression of GLT1 in fps1DeltagpdDelta mutant for optimum ethanol formation by Saccharomyces cerevisiae

Glycerol is the main byproduct produced under anaerobic ethanol fermentations by Saccharomyces cerevisiae and consumes a considerable amount of substrate. To verify the metabolic phenotype predications for increasing ethanol formation, two engineered S. cerevisiae KAM-14, KAM-15 strains were constructed for possible redirection of glycerol carbon flux into ethanol by overexpression of GLT1 in the fps1DeltagpdDelta mutant. The engineered strains KAM-14 and KAM-15 compared to the control strain KAM-2, produced 12.24% and 10.42% higher ethanol, 39.72% and 31.03% lower glycerol yield during anaerobic batch fermentations, respectively. The maximum specific growth rates of KAM-14 and KAM-15 were found to be relatively lower than that of KAM-2 during the exponential growth phase. In the meantime, the biomass concentrations of both KAM-14 and KAM-15 were similar to KAM-2. Acetate and pyruvate concentrations of KAM-14 and KAM-15 were greatly decreased comparing to those of KAM-2, respectively. These experimental results approved the metabolic pathway strategies to improve ethanol formation.     

Sensitivity analysis of a biofilm model describing a one-stage completely autotrophic nitrogen removal (CANON) process.

A mathematical model for nitrification and anaerobic ammonium oxidation (ANAMMOX) processes in a single biofilm reactor (CANON) was developed. This model describes completely autotrophic conversion of ammonium to dinitrogen gas. Aerobic ammonium and nitrite oxidation were modeled together with ANAMMOX. The sensitivity of kinetic constants and biofilm and process parameters to the process performance was evaluated, and the total effluent concentrations were, in general, found to be insensitive to affinity constants. Increasing the amount of biomass by either increasing biofilm thickness and density or decreasing porosity had no significant influence on the total effluent concentrations, provided that a minimum total biomass was present in the reactor. The ANAMMOX process always occurred in the depth of the biofilm provided that the oxygen concentration was limiting. The optimal dissolved oxygen concentration level at which the maximum nitrogen removal occurred is related to a certain ammonium surface load on the biofilm. An ammonium surface load of 2 g N/m2. d, associated with a dissolved oxygen concentration level of 1.3 g O2/m3 in the bulk liquid and with a minimum biofilm depth of 1 mm seems a proper design condition for the one-stage ammonium removal process. Under this condition, the ammonium removal efficiency is 94% (82% for the total nitrogen removal efficiency) (30 degrees C). Better ammonium removal could be achieved with an increase in the dissolved oxygen concentration level, but this would strongly limit the ANAMMOX process and decrease total nitrogen removal. It can be concluded that a one-stage process is probably not optimal if a good nitrogen effluent is required. A two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.     

Lactose utilization by Saccharomyces cerevisiae strains expressing Kluyveromyces lactis LAC genes

Whey generated in cheese manufacture continues being an industrial problem without a satisfactory solution. Genetic modification of the yeast S. cerevisiae to obtain strains able to utilize lactose, is a prerequisite for the utilization of this yeast to convert cheese whey into useful fermentation products (i.e. biomass, heterologous protein and other recombinant products). Although the construction of S. cerevisiae Lac(+) strains has been achieved by different strategies, most of these strains have unsuitable characteristics, such as genetic instability of the Lac phenotype or diauxic growth. In previous communications we have described the construction of genetically stable strains of S. cerevisiae that assimilate lactose with a high efficiency. These strains carry multiple copies of Kluyveromyces lactis LAC4 and LAC12 genes, which code for a beta-galactosidase and a lactose permease, respectively. In this work we report additional results about the effect of gene dosage, and analyze the performance of a selected strain in the bioconversion of cheese whey. Additionally, we describe the construction of a new strain, which combines the Lac(+) phenotype with additional properties of biotechnological interest: flocculence, and the ability to hydrolyze starch.     

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.     

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae

Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

Selection of yeast strain and fermentation conditions for high-yield ethanol production from lactose and concentrated whey

A total of 65 yeast strains were screened for their ability to grow and ferment lactose in a standard DURHAM tube test at 30 °C. Based on the kinetic parameters for lactose and whey lactose fermentations in shake flask cultures, the strain Candida psedotropicalis 65 was chosen for further studies. Some of the cultural parameters affecting ethanolic fermentations on lactose were standardized. At an initial lactose concentration of 100–120 g/l in the medium containing concentrated whey or lactose, at 40 °C and within 48 h, the selected strain reached an ethanol concentration of 41–59 g/l, an ethanol productivity of 1.3–3.0 g/l/h, a lactose consumption of 99%, an ethanol yield 0.4–0.49 g/g and a biomass yield of 0.027 g/g.     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h(-1), feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30 degrees C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.