Search

A stoichiometric model for Saccharomyces cerevisiae is reconstructed to analyze the continuous fermentation process of agave juice in Tequila production. The metabolic model contains 94 metabolites and 117 biochemical reactions. From the above set of reactions, 93 of them are linked to internal biochemical reactions and 24 are related to transport fluxes between the medium and the cell. The central metabolism of S. cerevisiae includes the synthesis for 20 amino-acids, carbohydrates, lipids, DNA and RNA. Using flux balance analysis (FBA), different physiological states of S. cerevisiae are shown during the fermentative process; these states are compared with experimental data under different dilution rates (0.04-0.12 h$ ^{-1} $). Moreover, the model performs anabolic and catabolic biochemical reactions for the production of higher alcohols. The importance of the Saccharomyces cerevisiae genomic model in the area of alcoholic beverage fermentation is due to the fact that it allows to estimate the metabolic fluxes during the beverage fermentation process and a physiology state of the microorganism.

In this study, a low-cost chemically defined (CD) culture medium was proposed and evaluated with the aim of replacing culture media such as yeast mold (YM) and yeast peptone dextrose (YPD), commonly used for growth and carotenoid production by Xanthophyllomyces dendrorhous. Initially, the CD culture medium was compared to the YM and YPD. The growth in optical density (OD) and carotenoid production (mg/L) of the cultures reached 4.88, 6.76, 5.79, and 0.67, 0.92, and 0.69, respectively. The CD culture served as the basis of an improved specific culture medium containing industrial glucose. Additionally, in this new formulation, vitamins, glutamate, and other compounds were evaluated. Industrial glucose more than doubled carotenoid production, however, the addition of vitamins was not essential for X. dendrorhous cultivation. Moreover, glutamate and Na2HPO4 proved to be highly significant factors (p-value &lt, 0.05), increasing carotenoid biosynthesis from 0.67 to 1.33 mg/L. The specific culture was successfully used in a bioreactor at 2 L and 110 L pilot-scale levels, increasing carotenoid production up to 2 mg/L. It was demonstrated that the CD-specific culture medium is an efficient alternative to conventional culture media to carry out carotenoid production at the laboratory and pilot levels, with promising potential for industrial scaling.

International audience; Enzymatic glycosylation is an efficient way to increase the water solubility and the bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs_SacB), Gluconacetobacter diazotrophicus (Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS) and Zymomonas mobilis (Zm_LevU) were screened for puerarin (daidzein-8-C-glucoside) fructosylation. Gd_LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of the acceptor forming beta-D-fructofuranosyl-(2 -> 6)-puerarin (P1a), while Bs_SacB, Lm_LevS and Zm_LevU synthesized puerarin-4'-O-beta-D-fructofuranoside (P1b) and traces of P1a. The Gd_LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin polyfructosides (PPFs). Bs_SacB elongated P1a more competently forming a linear series of water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS, HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_LsdA/Bs_SacB reactions yielded PPFs directly from puerarin with the acceptor conversion ranging 82-92 %. The bi-enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the intermediate product P1a and it is appropriate for use at industrial scale.

The isolation of autochthonous yeast species presents a good strategy to select new microorganisms for developing an adequate inoculum to carry out fermentations and generate representative products of the cider production zone. However, non-Saccharomyces yeasts have been considered to have low capacity to carry out a complete fermentation as Saccharomyces cerevisiae. In this work, five autochthonous yeasts from a cider fermentation process were isolated and identified as Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia membranaefaciens, P. kluyveri and Zygosaccharomyces rouxii. A series of fermentations were developed at laboratory level, using each species individually and it was observed that only S. cerevisiae was able to finish the process. K. marxianus consumed less than 50% of the sugars; P. kluyveri and Z. rouxii consumed less than 70% and P. membranaefaciens consumed more than 90% but the yield (ethanol produced for sugar consumed (YP/S)) was 0.39. Nevertheless, the addition of magnesium, zinc and nitrogen increased the fermentative capacity of almost all species: K. marxianus, Z. rouxii and P. kluyveri, showed an increase in ethanol production when nutrients were added, obtaining more than 80 g/L of ethanol, and showing that those nutrients are necessary to complete the fermentation. This work describes the potential use of different non-Saccharomyces species to carry out fermentation of apple juice and highlights the importance of certain nutrients to enable an efficient alcoholic fermentation and the generation of desirable volatile compounds for cider production.

The application of in situ near-infrared spectroscopy monitoring of xylose metabolizing yeast such as Pichia stipitis for ethanol production with semisynthetic media, applying chemometrics, was investigated. During the process in a bioreactor, biomass, glucose, xylose, ethanol, acetic acid, and glycerol determinations were performed by a transflection probe immersed in the culture broth and connected to a near-infrared process analyzer. Wavelength windows in near-infrared spectra recorded between 800 and 2200 nm were pretreated using Savitzky-Golay smoothing, second derivative and multiplicative scattering correction in order to perform a partial least squares regression and generate the calibration models. These calibration models were tested by external validation (78 samples). Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. Moreover, regressions coefficients (β) and variable influence in the projection plots were used to assess the results. A novelty is the use of β versus VIP dispersion plots to determine which vectors have more influence on the response in order to improve process comprehension and operability. Validated models were used in a real-time monitoring during P. stipitis NRRL Y7124 semisynthetic media fermentations.

Las levaduras utilizadas en la produccion de etanol de segunda generacion estan afectadas por compuestos inhibidores como el 5-hidroximetilfurfural (HMF) y el furfural que se liberan durante la etapa de hidrolisis; estos compuestos afectan la capacidad fermentativa de la levadura. Para encontrar nuevas levaduras con una capacidad excepcional para ser utilizadas en la produccion de etanol de segunda generacion; en este estudio se evaluo la respuesta fisiologica al estres por derivados de furano en la levadura nativa Kluyveromyces marxianus (SLP1), y se comparo con la levadura comercial Saccharomyces cerevisiae etanol red (ERD). Utilizamos un medio quimicamente definido anadido con HMF y furfural a diferentes concentraciones; una condicion de control sin inhibidores, y cuatro condiciones de estres, HMF 7 gL -1 , furfural 3gL -1 , HMF 3,5 gL -1 con furfural 1,5 gL -1 y HMF 7 gL -1 con furfural 3 gL -1 . K. marxianus exhibio una mayor capacidad para asimilar los compuestos inhibidores en menor tiempo que S. cerevisiae ERD; tambien, K. marxianus SLP1 mostro un mejor comportamiento para producir etanol en condiciones de inhibicion. A pesar de los efectos provocados por los compuestos inhibidores, las levaduras podrian producir etanol en un 80% de conversion. En conclusion, la levadura K. marxianus SLP1 puede ser una opcion para producir etanol de segunda generacion a nivel industrial.

The yeast Saccharomyces cerevisiae is commonly employed in industrial ethanol production, regardless of the capability of Kluyveromyces marxianus strains to produce ethanol at similar or higher levels and on inhibitory conditions. Therefore, in this work strains of S. cerevisiae (ethanol RED and AR5) and K. marxianus (SLP1 and OFF1) were compared for ethanol production from sugarcane bagasse (SCB) and wheat straw (WS) hydrolysates. As it is known, during the lignocellulosic hydrolysis not only free sugars were obtained (SCB, g L−1: glucose 7.64, xylose 8.38, arabinose 2.43; and WS, g L−1: glucose 6.07, xylose 6.36, arabinose 2.09) but also growth inhibitors of yeast such as hydroxymethylfurfural and furfural that could modify the fermentation capability. The volumetric ethanol productivity (Q p) was evaluated, and it was observed that the K. marxianus SLP1 was the most efficient for ethanol production reaching a Q p of 0.292 and 0.250 g L−1 h−1 on SCB and WS hydrolysates, respectively. In contrast, S. cerevisiae AR5 and ethanol RED exhibited a reduced Q p on SCB, but similar values of Q p to K. marxianus OFF1 on WS. The results obtained show that it is possible to select K. marxianus yeast strains for ethanol production using SCB and WS hydrolysates obtaining higher Q p than S. cerevisiae yeast strains. Considering the efficiency of ethanol production and the tolerance to inhibitors, K. marxianus strain SLP1 possesses a great potential as an industrial yeast for lignocellulosic ethanol production.

In the fermentation process of distilled spirits from Agave sugars, saponins inhibit the growth of yeasts during this procedure. This type of compound is present in the must, and the mechanisms of the cell growth inhibition are poorly understood. The fundamental objective of this work was to study the effect of saponins on the cell walls of the fermentative yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus. This study was performed in batch and continuous cultures, adding pulses of saponins extracted from the leaves of Agave durangensis and Agave salmiana ssp. crassispina. The saponin extract from A. durangensis exhibited a stronger growth inhibitory effect in both yeast strains than A. salmiana saponin extract due to the structure of the saponins. However, in K. marxianus growth recovery was observed, because these strain showed saponinase activity that performed the saponin hydrolysis. Finally, microscopic observations confirmed the changes in the wall cell of S. cerevisiae and K. marxianus .

Fructosylation can significantly improve the solubility, stability and bioactivity of phenolic compounds, increasing their health benefits. Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) was found to transfer the fructosyl unit of sucrose to different classes of phenolic compounds. Among the various acceptors tested, the isoflavone puerarin and the phenol coniferyl alcohol were the most efficiently fructosylated compounds, with conversion rates of 93% and 25.1%, respectively. In both cases, mono-, di-, and trifructosides were synthesized at a ratio of 37:14:1 and 32:8:1, respectively. Structural characterization of the puerarin mono-fructoside revealed that the enzyme transferred the fructosyl moiety of sucrose to the O6-position of the glucosyl unit of puerarin. The water solubility of fructosyl-β-(2→6)-puerarin was increased 23-fold, up to 16.2 g L−1, while its antioxidant capacity was only decreased 1.25-fold compared with that of puerarin.

Highlights • Yeast species influenced the de novo synthesis of 2-phenylethylacetate. • Inhibitory compounds showed a strong influence on cell growth and 2-phenylethylacetate production for the evaluated yeasts. • More than a 50 % reduction in the chemical and biochemical oxygen demand was achieved by yeast fermentation., Vinasses from the tequila industry are wastewaters with highly elevated organic loads. Therefore, to obtain value-added products by yeast fermentations, such as 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEA), could be interesting for industrial applications from tequila vinasses. In this study, four yeasts species (Wickerhamomyces anomalus, Candida glabrata, Candida utilis, and Candida parapsilosis) were evaluated with two different chemically defined media and tequila vinasses. Differences in the aroma compounds production were observed depending on the medium and yeast species used. In tequila vinasses, the highest concentration (65 mg/L) of 2-PEA was reached by C. glabrata, the inhibitory compounds decreased biomass production and synthesis of 2-PEA, and biochemical and chemical oxygen demands were reduced by more than 50 %. Tequila vinasses were suitable for the production of 2-phenylethylacetate by the shikimate pathway. A metabolic network was developed to obtain a guideline to improve 2-PE and 2-PEA production using flux balance analysis (FBA).

Two Pantoea anthophila bacterial strains were isolated from “tejuino”, a traditional Mexican beverage, and studied as β-galactosidase producers for galactooligosaccharides synthesis. Using 400 g/L of lactose, 50 °C, and 15 U/mL of β-galactosidase activity with ethanol-permeabilized cells, the maximum galactooligosaccharides (GOS) yield determined by High performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD) was 136 g/L (34% w/w of total sugars) at 96% of lactose conversion for Bac 55.2 and 145 g/L (36% w/w of total sugars) at 94% of lactose conversion for Bac 69.1. The main synthesized products were the disaccharides allolactose [Gal-β(1 → 6)-Glc] and 6-galactobiose [Gal-β(1 → 6)-Gal], as well as the trisaccharides 3′-galactosyl-lactose [Gal-β(1 → 3)-Gal-β(1 → 4)-Glc], 6-galactotriose [Gal-β(1 → 6)-Gal-β(1 → 6)-Gal], 3′-galactosyl-allolactose [Gal-β(1 → 3)-Gal-β(1 → 6)-Glc], and 6′-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc]. The β-galactosidases present in both strains showed a high transgalactosylation activity and formed principally β(1 → 3) and β(1 → 6) linkages. Considering the stability and bifidogenic properties of GOS containing such types of bonds, P. anthophila strains Bac 55.2 and Bac 69.1 possess a high potential as novel biocatalysts for prebiotic industrial production., This research was supported by the CONACYT project CB-2012-01/000000000181766, and by the Spanish Ministry of Economy and Competitiveness (projects BIO2013-48779-C4-1/3/4 and BIO2016-76601-C3-1-R/2-R/3-R).

Enzymatic fructosylation of organic acceptors other than sugar opens access to the production of new molecules that do not exist in nature. These new glycoconjugates may have improved physical-chemical and bioactive properties like solubility, stability, bioavailability, and bioactivity. This review focuses on different classes of acceptors including alkyl alcohols, aromatic alcohols, alkaloids, flavonoids, and xanthonoids, which were tested for the production of fructoderivatives using enzymes from the glycoside hydrolase (GH) families 32 and 68 that use sucrose as donor substrate. The enzymatic strategies and the reaction conditions required for the achievement of these complex reactions are discussed, in particular with regard to the type of acceptors. The solubility and pharmacokinetic and antioxidant activity of some of these new beta-D-fructofuranosides in comparison is reviewed and compared with their glucoside analogs to highlight the differences between these molecules for technological applications.

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

International audience; For tequila production, blue agave heads are cooked to hydrolyse their fructan content and release fermentable carbohydrates. However, this hydrolysis is not complete and the juice generated during cooking (cooking honey) contains fructans incompletely hydrolysed. Several distilleries perform an additional acidic hydrolysis on honey to improve fermentable carbohydrate recovery, but this treatment presents many disadvantages. As an alternative, we describe an enzymatic treatment with Fructozyme L, a preparation of inulinases. Optimal laboratory conditions for agave fructan hydrolysis using this preparation were 60 degrees C and a pH of 4.0-5.0. A concentration of 0.02% (V/V of honey) was needed to achieve more than 90% of fructan hydrolysis in the honey, after a 12 h treatment. These conditions were validated by two industrial trials during which enzymatic hydrolysates were successfully fermented and distilled, and volatile compound composition and sensory qualities of the tequila were assured. (C) 2010 Elsevier Ltd. All rights reserved.

The composition of water-soluble carbohydrates from Agave tequilana plants of 2, 4 and 6½ years were compared by HPLC, HPAEC-PAD, MALDI-TOF-MS and GC–MS. The plants of 2 years exhibited the highest levels of free monosaccharide and low molecular weight fructans (DP 3–DP 6) with potential application as prebiotics. A maximum of fructan polymerisation was achieved at 4 years with mean DP from 3 to 30, then it was decreased at 6½ years with mean DP from 4 to 24. The linkage analysis showed an increase and decrease in the branching degree from 2 to 6½ years with a maximum at 4 years, correlated to changes in t-β- d -Fruf linkages with increased and decreased synthesis of (2→1) and (2→6)-β- d -Fruf as well as 1,6-di-β- d -Fruf linkages.

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.

In traditional tequila production, the heads of Agave tequilana Weber var. azul are cooked in brick ovens to hydrolyze the fructan content and release fermentable sugars. The juice generated during cooking (known as “cooking honey”) was collected periodically in a tequila distillery and characterized to study the efficiency of fructan hydrolysis. The complex structure of fructans from A. tequilana was confirmed. The generation of 5-(hydroxymethyl)-furfural, an increase in absorbance and °Brix, and a decrease in pH and apparent average degree of polymerization of fructans during cooking were observed. The conversion of fructans in the flowing honey increased gradually from 20% at the onset of cooking to 98% after 25.5 h, where fructose represented more than 80% of the total carbohydrates. The proportion of non-hydrolyzed fructans in the cooking honey collected before this time resulted in a total ethanol loss of 6% in the tequila distillery investigated.

At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed.

Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application.

Alcoholic beverages are produced from a variety of raw materials, principally fruits and cereals. Various microorganisms are used to ferment the sugars present in the must. Fermentation can be making using either the addition of specific microorganisms or spontaneous fermentation from naturally occurring microorganisms. Both, the microorganisms and the raw material affect the volatile compounds that are produced, and these volatile compounds are responsible for the aroma and unique taste of these beverages (Nykanen. 1983, Moreno. 2009).

Fructanase and fructosyltransferase are interesting for the tequila process and prebiotics production (functional food industry). In this study, one hundred thirty non-Saccharomyces yeasts isolated from "Mezcal de Oaxaca" were screened for fructanase and fructosyltransferase activity. On solid medium, fifty isolates grew on Agave requilana fructans (ATF), inulin or levan. In liquid media, inulin and ATF induced fructanase activities of between 0.02 and 0.27 U/ml depending of yeast isolate. High fructanase activity on sucrose was observed for Kluyveromyces marxianus and Torulaspora delbrueckii, while the highest fructanase activity on inulin and ATF was observed for Issatchenkia orientalis, Cryptococcus albidus, and Candida apicola. Zygosaccharomyces bisporus and Candida boidinii had a high hydrolytic activity on levan. Sixteen yeasts belonging to K. marxianus, T. delbrueckii and C. apicola species were positive for fructosyltransferase activity. Mezcal microbiota proved to showed to be a source for new fructanase and fructosyltransferases with potential application in the tequila and food industry. (C) 2012 Elsevier Ltd. All rights reserved.

Aims: To study the effect of the addition of different nitrogen sources at high sugar concentration in the tequila fermentation process. Methods and Results: Fermentations were performed at high sugar concentration (170 g l−1) using Agave tequilana Weber blue variety with and without added nitrogen from different sources (ammonium sulfate; glutamic acid; a mixture of ammonium sulfate and amino acids) during the exponential phase of growth. All the additions increased the fermentation rate and alcohol efficiency. The level of synthesis of volatile compounds depended on the source added. The concentration of amyl alcohols and isobutanol were decreased while propanol and acetaldehyde concentration increased. Conclusions: The most efficient nitrogen sources for fermentation rate were ammonium sulfate and the mixture of ammonium sulfate and amino acids. The level of volatile compounds produced depended upon types of nitrogen. The synthesis of some volatile compounds increased while others decreased with nitrogen addition. Significance and Impact of the Study: The addition of nitrogen could be a strategy for improving the fermentation rate and efficiency in the tequila fermentation process at high sugar Agave tequilana concentration. Furthermore, the sensory quality of the final product may change because the synthesis of the volatile compounds is modified.

Few studies have been performed on the characterization of yeasts involved in the production of agave distilled beverages and their individual fermentation properties. In this study, a comparison and evaluation of yeasts of different origins in the tequila and wine industries were carried out for technological traits. Fermentations were carried out in high (300 g l(-1)) and low (30 g l(-1)) sugar concentrations of Agave tequilana juice, in musts obtained from Fiano (white) and Aglianico (red) grapes and in YPD medium (with 270 g l(-1) of glucose added) as a control. Grape yeasts exhibited a reduced performance in high-sugar agave fermentation, while both agave and grape yeasts showed similar fermentation behaviour in grape musts. Production levels of volatile compounds by grape and agave yeasts differed in both fermentations.

In the tequila industry, fermentation is traditionally achieved at sugar concentrations ranging from 50 to 100 g·L–1. In this work, the behaviour of the Saccharomyces cerevisiae yeast (isolated from the juices of the Agave tequilana Weber blue variety) during the agave juice fermentation is compared at different sugar concentrations to determine if it is feasible for the industry to run fermentation at higher sugar concentrations. Fermentation efficiency is shown to be higher (above 90%) at a high concentration of initial sugar (170 g·L–1) when an additional source of nitrogen (a mixture of amino acids and ammonium sulphate, different than a grape must nitrogen composition) is added during the exponential growth phase.Key words: Saccharomyces cerevisiae, fermentation efficiency, nitrogen source, tequila.

Sequential methodology based on the application of three types of experimental designs was used to optimize the astaxanthin production of the mutant strain 25-2 of Phaffia rhodozyma in shake flask cultures. The first design employed was a factorial design 2(5), where the factors studied were: pH, temperature, percent of inoculum, carbon and nitrogen concentrations, each one at two levels. This design was performed in two medium types: rich YM medium and minimal medium, based on date juice (Yucca medium). With this first design the most important factors were determined (carbon concentration and temperature) that were used in the second experimental strategy: the method of steepest ascent was applied in order to rapidly approach the optimum. Finally, a second-order response surface design was applied using temperature and carbon concentration as factors. The optimal conditions stimulating the highest astaxanthin production were: 19.7 degrees C temperature; 11.25 g l(-1) carbon concentration; 6.0 pH; 5% inoculum and 0.5 g l(-1) nitrogen concentration. Under these conditions the astaxanthin production was 8100 microg l(-1), 92% higher than the production under the initial conditions.

The present results concern the recombinant bacteriaEscherichia coli HB101(GAPDH) which produces glyceraldehyde 3-phosphate dehydrogenase. An unusual phenomenon was noticed concerning the plasmid stability of this strain growing in batch culture. The determination of sensitivity to ampicillin, whose resistance is carried by the plasmid, has been tested as a function of time on Petri dishes containing increasing concentrations of ampicillin in a batch culture in a complex medium without any selection pressure. A transitory decrease in the percentage of resistant cells has been noted during the exponential phase of growth. This phenomenon corresponds to a momentary plasmid instability probably due to a transitory gap between the growth rate of the cell and the duplication rate of the plasmid.

Analysis of amino acids on samples of a semi-synthetic medium containing casamino acids, on which an Escherichia coli strain (a genetically engineered one) has been cultivated, allowed the quantification of the amino acid utilization by this strain during batch and continuous cultures. In batch culture the main results were that the observed utilizations corroborated what is known about the transport of amino acids by E. coli . Indeed, amino acids are transported by common or specific carriers as described in the literature. In continuous cultures the amino acid utilization in terms of probable destination, either constitutive or energy source is studied. The following results were obtained. Amino acid utilizations (and corresponding rates) vary with glucose availability. Amino acids are differently distributed in the cell. Three classes exist: mainly protein amino acids, mainly energy amino acids, and slightly imported amino acids. Glucose availability has an influence on this distribution. When it increases, the energy utilization ratio decreases. An optimum for glucose availability conditions with regard to the recombinant protein produced by the E. coli strain used is confirmed. The results obtained during the continuous cultivations corroborate the hypothesis concerning the amino acid carriers made during the batch cultivations and confirm the fact that the mainly energy amino acids are the most rapidly utilized.

This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) by E. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L-1 of biomass and 6 g L-1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L-1, and in fed-batch experiments for glucose availability of 10 g h-1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.