Tryptophan Production by a Lipoic Acid Auxotroph of Enterobacter aerogenes Having Both Pyruvic Acid Productivity and High Tryptophanase Activity

A new process for tryptophan production was established using a lipoic acid auxotrophic mutant, Enterobacter aerogenes l-12, which has both pyruvic acid productivity and tryptophanase activity. The process consists of the production of pyruvic acid from glucose by the washed cells and the subsequent conversion of the acid to tryptophan by the tryptophanase itself in the presence of indole and NH₄C1.

To prepare washed cells of which the tryptophanase activity and the pyruvic acid productivity were both high, it was best to culture the strain in a medium containing 1 % Polypepton, 0.2 % glucose, 3 μg/1 dl-lipoic acid, 0.05 % l-tryptophan, and mineral salts. The optimum composition of the reaction mixture for the pyruvic acid production by the washed cells was established. Under these conditions, 17 g/1 of pyruvic acid was accumulated from 5 % glucose after 36 hr of incubation. Thus, the conversion of the pyruvic acid to tryptophan was done by adding indole, NH₄C1, pyridoxal-5′-phosphate, Triton X-100, and KOH to adjust the pH to 9.0 to the above reaction mixture. As a result, the pyruvic acid was rapidly converted to tryptophan, and the concentration of 14 g/1 was obtained after 36 hr (total 72 hr).     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Control of gene expression from the SUC2 promoter of Saccharomyces cerevisiae with the aid of a glucose analyser

Summary A Saccharomyces cerevisiae strain harbouring the recombinant plasmid pSMF38TMA was cultured in a jar fermentor under the control of glucose concentration. In the recombinant plasmid, the mouse -amylase gene was fused to the S. cerevisiae SUC2 promoter. When glucose concentration in the medium was controlled at 10 g/l, the gene expression was completely repressed. On the other hand, the -amylase was produced and secreted in the medium at a very high level, around 200 mg/l as evaluated from the specific activity of commercially available human salivary amylase, when the glucose was kept at 0.15 g/l. This amount was almost 20-fold that obtained at 10 g/l glucose. The specific growth rate of the yeast in this culture was almost 60% of that attained with 10 g/l glucose. To obtain higher cell growth and productivity, the yeast was at first cultured at 2 g/l glucose and the concentration was then lowered to 0.15 g/l. By this control of the glucose concentration, on-off regulation of gene expression from the SUC promoter could be attained.     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells     

Transformation of protoplasts ofCellulomonas flavigena

Summary Protoplasts ofCellulomonas flavigena (Cm^s) were transformed with plasmid pC194. Transformation frequency was 2.72×10^–3 in MR-1 regeneration medium with 2 g/ml chloramphenicol. Transformation conditions are described.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Increase of hybridoma productivity using an original dialysis culture system

Hybridoma cell growth and monoclonal antibody production were investigated with a laboratory-made system in which cells were grown in dialysis tubing (MW cut-off 25 kD). The dialysis system contained 10 ml of cell suspension and was immersed in 200 ml of culture medium which when replaced or was at 4-day intervals. With this system, monoclonal antibody concentrations similar to those observed in ascites (concentrations in the order of one gramme per liter) were obtained. With no medium replacement, the antibody production was 3.3 g/l and the cell productivity 3.2×10^–8 g of IgM produced per cell in one minute. With medium replacement the antibody production was higher, 4.4 g/l but the cell productivity was lower, 1.49×10^–8 g per cell in one minute. Cells cultivated in non-optimized conditions were better producers than cells growing in a good environment.Abbreviations FCS fetal calf serum - Ig immunoglobulin - MAb monoclonal antibody - MW molecular weight - MWCO molecular weight cut off - RM replaced medium - NRM non replaced medium     

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

Summary RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp^– phenotype to Trp⁺. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

Xylitol production from D-xylose byCandida guillermondii: Fermentation behaviour

Summary The ability ofCandida guillermondii to produce xylitol from xylose and to ferment individual non xylose hemicellulosic derived sugars was investigated in microaerobic conditions. Xylose was converted into xylitol with a yield of 0,63 g/g and ethanol was produced in negligible amounts. The strain did not convert glucose, mannose and galactose into their corresponding polyols but only into ethanol and cell mass. By contrast, fermentation of arabinose lead to the formation of arabitol. On D-xylose medium,Candida guillermondii exhibited high yield and rate of xylitol production when the initial sugar concentration exceeded 110 g/l. A final xylitol concentration of 221 g/l was obtained from 300 g/l D-xylose with a yield of 82,6% of theoretical and an average specific rate of 0,19 g/g.h.Nomenclature Q_p average volumetric productivity of xylitol (g xylitol/l per hour) - q_p average specific productivity of xylitol (g xylitol/g of cells per hour) - So initial xylose concentration (g/l) - tf incubation time (hours) - Y_P/S xylitol yield (g of xylitol produced/g of xylose utilized) - Y_E/S ethanol yield (g of ethanol produced/g of substrate utilized) - Y_X/S cells yield (g of cells/g of substrate utilized) - specific growth rate coefficient (h^–1) - _max maximum specific growth rate coefficient (h^–1)     

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

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

l-threonine production by l-aspartate- and l-homoserine-resistant mutant of Escherichia coli

Summary Growth and l-threonine productivity of l-threonine producer Escherichia coli H-4290 were inhibited by precursor amino acids, l-homoserine and l-aspartate. l-Threonine hyper-producers were isolated among the mutants resistant to l-homoserine and l-aspartate. Mutants H-4351 (Hom^r) and H-4578 (Hom^r, Asp^r) accumulated 22.2 g/l and 24.3 g/l of l-threonine in test tube cultures, while the parental strain H-4290 accumulated 18.2 g/l. The enzyme level of aspartokinase I (first enzyme of the threonine operon) was enhanced 2.3 times (H-4351) and 3 times (H-4578) that of H-4290. Mutant H-4578 accumulated 76 g/l of l-threonine in a 2-l jar fermentor after 75 h cultivation.Abbreviations DAP diaminopimeric acid - Met ^l poor growth in methionine-free medium - AHV -amino--hydroxyvaleric acid - Thr-N^- lack of ability to utilize l-threonine as a nitrogen source - Rif rifampicin - Lys+Met^r resistant to l-lysine and dl-methionine     

Transformation of Salmonella typhimurium TA1538 by optimized electroporation

Summary Standard electroporation of Salmonella typhimurium TA1538 by the plasmid pAMH70 yielded 1.3 × 10² transformants/g of plasmid DNA. Three parameters: resistance (U₁), voltage (U₂) and dose of plasmid DNA (U₃) were optimized with two Doehlert designs. Transformation was increased 75 fold. Optimal values for U₁, U₂ and U₃ were 761.0 , 1.6 kV and 96.1 ng respectively. The most important parameters were U₂ and the couple U₁/U₂.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Genetic transformation of Clostridium botulinum hall a by electroporation

Summary A method was developed for the introduction of plasmids into Clostridium botulinum by electroporation. A 4.4 kb plasmid vector, pGK12, which contains genes for resistance to erythromycin (Em^r) and chloramphenicol (Cm^r) was electroporated into C. botulinum type A (Hall A). The highest transformation efficiency was obtained using midlog phase cells, 10% PEG 8000 as the electroporation solution, and 2.5 kV field strength. The transformation efficiency was highest (10³ transformants/g of DNA) when 1 g of plasmid DNA and 4 × 10⁸ CFU/ml of recipient cells were used. Plasmid DNA recovered from the transformants was indistinguishable from that introduced on the basis of restriction enzyme digestion and agarose gel electrophoresis.     

Localized cutaneous nocardiosis in Japan

A turbidometric method was used to measure Candida albicans yeast cell growth and to quantitate the postantifungal effect (PAFE) after exposure to various concentrations of flucytosine and amphotericin B, alone and in combination, for 2 hr at 30°C. The drug concentrations used in the PAFE assays were determined by initial MIC and FIC (fractional inhibitory concentration) evaluations. The PAFE was calculated by the difference in time (hr) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. A synergistic PAFE was evidenced with combinations of the two drugs at concentrations below their individual MICs. Combinations of flucytosine (0.012 to 0.049 g ml^–1) and amphotericin B (0.195 to 0.39 g ml^–1) produced PAFEs ranging from 6.3 to 21.8 hr. These PAFEs persisted from 0.3 to 14.7 hr longer than those achieved when each of the two agents was assayed separately.     

An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

In this work, metabolite and antibody production kinetics of hybridoma cultures were investigated as a function of cell density and growth rate in a homogeneous perfusion reactor. Hydrophilized hollow fiber polypropylene membranes with a pore size of 0.2 m were used for medium perfusion. Oxygen was supplied to the cells through thin walled silicone tubing. The mouse-mouse hybridoma cells were grown in three identical bioreactors at perfusion rates of 1.1, 2.0, and 3.2/day for a period of eight days during which the viable cell concentrations reached stable values of 2.6×10⁶, 3.5×10⁶, and 5.2×10⁶ cells/ml, respectively. Total cell densities reached values ranging from 8×10⁶ to 1×10⁶ cells/ml. Specific substrate consumption and product formation rates responded differently to changes in cell density and apparent specific growth rate, which were not varied independently. Using multiple regression analysis, the specific glucose consumption rate was found to vary with viable cell density while the specific glutamine uptake and lactate production rates varied with both viable cell density and apparent specific growth rate. These results suggest that cell density dictates the rate of glucose consumption while the cell growth rate influences how glucose is metabolized, i.e., through glycolysis or the TCA cycle. The specific antibody production rate was found to be a strong function of cell density, increasing as cell density increased, but was essentially independent of the specific growth rate for the cell line under study.List of Symbols MAb monoclonal antibody - X _v viable cell density (cells/ml) - X _d nonviable cell density (cells/ml) - specific growth rate (1/day) - k _d specific death rate (1/day) - D dilution rate (1/day) - S _f substrate concentration in feed (g/l or mM) - S substrate concentration (g/l or mM) - P _f product concentration in feed (g/l or g/ml) - P product concentration (g/l or ug/ml) - q _s specific consumption rate of substrate (g/hr/cell or mmol/hr/cell) - q _p specific production rate of product (g/hr/cell) - q _MAb specific production rate of monoclonal antibody (g/hr/cell) This work was supported in part by a grant for the National Science Foundation (BCS-9157851) and by matching funds from Merck and Monsanto. We sincerely thank Mr. Roland Buchele of Akzo Inc. (Germany) for donation of the polypropylene membranes, Dr. Michael Fanger (Dartmouth Medical School) for the hybridoma cell line, Dr. Sadettin Ozturk (Verax Corp., Lebanon, NH) for technical discussions regarding reactor design, and Dr. Derrick Rollins (Iowa State University) for advice on statistical methods.     

The viscoelasticity of entangled actin networks: the influence of defects and modulation by talin and vinculin

Rheological measurements of the frequency-dependent complex elastic module G^*() of entangled F-actin solutions in the frequency range 10^–5 – 1 Hz were carried out in three dynamic regimes: 1.) A terminal relaxation from gel-like to liquid-like behaviour measured at frequencies < _d ^–1 2.) a rubber-type plateau and 3.) a regime determined by chain conformational transitions at frequencies > _i ^–1. A major point of interest was to clarify whether rheological, high precision measurements can yield quantitative information about the influence of talin and vinculin on the structure, chain dynamics, elasticity and viscoelasticity of actin filaments with time. We show that in the regime reflecting internal chain dynamics (10^–2 to 1 s time domain), F-actin behaves as a random coil of the Rouse type. This contrasts with dynamic light scattering and correlation spectroscopic studies of actin filament flickering, which indicate that filaments behave as semiflexible rods. The internal chain dynamics, which are determined by thermically excited bending undulations, exhibit a persistence length of 0.3–1 m Evidence is provided that this discrepancy is due to a cross-over of semiflexible rod behaviour at excitation wavelengths () below approximately 1 gm to random-coil behaviour at 1 µ (expected at a frequency 1 Hz). The random coil behaviour is largely determined by defects in actin filaments leading to sharp bends of the chain which act as semiflexible hinges. Talin produces drastic effects on the time course of viscoelasticity during actin polymerization. It promotes the rapid formation of short filament fragments ( 1 gmm, within time scales of min) which anneal slowly into long filaments (within several hours), most probably by fusion. The viscoelasticity depends on the coexistence of short and very long filaments indicated by the elongation of the rubber plateau. The most dramatic effect is a reduction of the ratio of the terminal ('Ed) to the Rouse relaxation time of _i by more than one order of magnitude (_d/_i = 100 compared to ratio _d/_i = 2000 for pure actin). From this it is concluded that talin causes a remarkable decrease in the effective segment length of the macromolecule and, thus induces an increase in chain stiffness. Vinculin on the other hand shows no such effect. Correspondence to: E. Sackmann     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

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

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