Showing papers by "Bärbel Hahn-Hägerdal published in 1999"

Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts

Abstract: The influence of the factors acetic acid, furfural, and p-hydroxybenzoic acid on the ethanol yield (YEtOH) of Saccharomyces cerevisiae, bakers' yeast, S. cerevisiae ATCC 96581, and Candida shehatae NJ 23 was investigated using a 2(3)-full factorial design with 3 centrepoints. The results indicated that acetic acid inhibited the fermentation by C. shehatae NJ 23 markedly more than by bakers' yeast, whereas no significant difference in tolerance towards the compounds was detected between the S. cerevisiae strains. Furfural (2 g L-1) and the lignin derived compound p-hydroxybenzoic acid (2 g L-1) did not affect any of the yeasts at the cell mass concentration used. The results indicated that the linear model was not adequate to describe the experimental data (the p-values of curvatures were 0.048 for NJ 23 and 0.091 for bakers' yeast). Based on the results from the 2(3)-full factorial experiment, an extended experiment was designed based on a central composite design to investigate the influence of the factors on the specific growth rate (mu), biomass yield (Yx), volumetric ethanol productivity (QEtOH), and YEtOH. Bakers' yeast was chosen in the extended experiment due to its better tolerance towards acetic acid, which makes it a more interesting organism for use in industrial fermentations of lignocellulosic hydrolysates. The inoculum size was reduced in the extended experiment to reduce any increase in inhibitor tolerance that might be due to a large cell inoculum. By dividing the experiment in blocks containing fermentations performed with the same inoculum preparation on the same day, much of the anticipated systematic variation between the experiments was separated from the experimental error. The results of the fitted model can be summarised as follows: mu was decreased by furfural (0-3 g L-1). Furfural and acetic acid (0-10 g L-1) also interacted negatively on mu. Furfural concentrations up to 2 g L-1 stimulated Yx in the absence of acetic acid whereas higher concentrations decreased Yx. The two compounds interacted negatively on Yx and YEtOH. Acetic acid concentrations up to 9 g L-1 stimulated QEtOH, whereas furfural (0-3 g L-1) decreased QEtOH. Acetic acid in concentrations up to 10 g L-1 stimulated YEtOH in the absence of furfural, and furfural (0-2 g L-1) slightly increased YEtOH in the absence of acetic acid whereas higher concentrations caused inhibition. Acetic acid and furfural interacted negatively on YEtOH.

Influence of Furfural on anaerobic glycolytic kinetics of Saccharomyces cerevisiae in batch culture

Abstract: Furfural was reduced to furfuryl alcohol by Saccaromyces cerevisiae with a yield of 0.97 +/- 0.01 mmol/ mmol causing a lag phase in cell growth. In the presence of 29 mM, furfural the cell-mass yield on glucose decreased from 11.0 +/- 0.1 mg/mmol (0.06 +/- 0.0006 g/g) in a reference fermentation without furfural to 9.7 +/- 0.07 mg/ mmol (0.05 +/- 0.0004 g/g), whereas the ethanol yield on glucose increased from 1.6 +/- 0.03 to 1.7 +/- 0.03 mmol/mmol. No glycerol was excreted during furfural reduction, and the lag phase in acetate production was extended from 1 h in the reference fermentation to 5 h in the presence of furfural. Acetaldehyde and pyruvate were excreted during the furfural reduction phase. Cell growth and cell maintenance were proportional to glucose consumption during the entire fermentation, whereas the cell-mass yield on ATP produced was low during furfural reduction. These observations indicate that furfural addition to a batch culture decreased cell replication without inhibiting cell activity (designated as replicative inactivation). The absence of glycerol production during furfural reduction suggests that furfural acted as an alternative redox sink oxidizing excess NADH formed in biosynthesis. A mechanistic mathematical model was developed that described accurately the fermentation in the absence and presence of furfural. The model was based on the assumptions that: (i) furfural reduction to furfuryl alcohol by NADH-dependent dehydrogenases had a higher priority than reduction of dihydroxyacetone phosphate to glycerol; and (ii) furfural caused inactivation of cell replication. The effect of furfural on cell replication is discussed in relation to acetaldehyde formation. Inactivation of cell replication was modeled by considering two populations within the cell culture, both metabolically active, but only one replicating. The kinetic description was developed as a tool to estimate transient fluxes of carbon, NADH/NAD+ and ATP/ADP.

Nutrient requirements of lactococci in defined growth media

Abstract: Many attempts have been made for the last six decades to design defined media for species of the lactococcus group. The general outcome of the studies suggests that this group is heterogeneous with respect to specific requirements for nutrients. Lactococcal species are limited in various metabolic pathways. Early attempts to trace the required nutrients were not always successful because of the poor quality of analysis and the presence of impurities in the medium components.

Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

Abstract: We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.

Simultaneous enzymatic wheat starch saccharification and fermentation to lactic acid by Lactococcus lactis

Abstract: Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For saccharification the commercial enzyme mixture SAN Super 240 L, having α-amylase, amyloglucosidase and protease activity, was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations of 90 g/l and 180 g/l and SAN Super concentrations between 3 μl/g and 8 μl/g starch. Kinetic models, developed for the saccharification and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat flour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme activity.

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Abstract: Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase.

Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources.

Abstract: Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l−1d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l−1d-glucose or raffinose was used as co-substrate together with 50 g l−1d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation.

Production of ethanol from xylose

Abstract: This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant yeast strains transformed with xylose reductase and/or xylitol dehydrogenase enzyme genes. A yeast strain transformed with the xylose reductase gene is capable of reducing xylose to xylitol and consequently of producing xylitol in vivo. If both of these genes are transformed into a yeast strain, the resultant strain is capable of producing ethanol on xylose containing medium during fermentation. Further, the said new yeast strains are capable of expressing the said two enzymes. Xylose reductase produced by these strains can be used in an enzymatic process for the production of xylitol in vitro.

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction

Abstract: A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35degreesC at 5% phosphate, and increased linearly to 63degreesC at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 211-218, 1999. (Less)

Genetically engineered yeast and mutants thereof for the efficient fermentation of lignocellulose hydrolysates

Abstract: The present invention provides genetically engineered expression vectors, and recombinant cells comprising those vectors, or portions of those vectors. The vectors comprise a mutant form of a gene encoding an aldose reductase (AR) enzyme in which only a portion of the gene is present on the vector. The mutated aldose reductase sequence serves as a site for homologous crossing over of vector-encoded sequences and a host cell genome. Recombinant cells made using the vector of the invention lack an aldose reductase gene and are capable of fermenting lignocellulose hydrolysates to ethanol in high quantities. The invention also provides recombinant vectors and cells with multiple copies of genes encoding enzymes involved in the conversion of lignocellulose or lignocellulose hydrolysates to ethanol. Accordingly, the invention provides methods of making recombinant cells and methods of efficiently producing ethanol from lignocellulose-containing compositions.

Oxygen-dependent xylitol metabolism in Pichia stipitis

Abstract: Pichia stipitis CBS 6054 was cultivated in chemostat cultures under aerobic and oxygen-limited conditions with xylitol alone, a mixture of xylitol and glucose and a mixture of xylitol and xylose. Xylitol metabolism was strictly respiratory and no ethanol was formed. Simultaneous feeding of xylitol and glucose and xylitol and xylose to oxygen-limited xylitol-pregrown cells resulted in ethanol formation. In vitro both pyruvate decarboxylase activity and alcohol dehydrogenase activity were present in cells metabolising xylitol under oxygen-limited conditions; however, this did not result in ethanol formation. Glucose, xylose and xylitol utilisation, respectively, were compared under anaerobic conditions with regard to growth rate, carbon source and oxygenation level during pre-cultivation. Irrespective of pre-growth conditions, xylitol was not metabolised under anaerobic conditions, whereas ethanol was formed from both xylose and glucose. Anaerobic xylose utilisation required induction of a xylose-utilising metabolic pathway during pre-cultivation.

Polymer conjugates of polyethylene glycols or oxides with polyethyleneimine or polypropyleneimine for extracting carboxylic acids from solutions

Abstract: The invention provides novel polymer conjugates that are especially useful in the separation of carboxylic acids from complex fermentation compositions. The polymer conjugates comprise polyethylene glycols or oxides and polyethyleneimine or polypropyleneimine in various ratios. The novel polymer conjugates permit enhanced extraction of carboxylic acids from fermentation systems not only through their use in partitioning the carboxylic acids from various polymeric components of the fermentation system, but also through their use in adjusting the pH of the system for optimal performance and separation.