Yeast Biocatalyst Research Articles

Engineering high production of fatty alcohols from methanol by constructing coordinated dual biosynthetic pathways

Microbial cell factories provide an efficient approach for the green manufacturing of chemicals. However, the excessive use of sugars increases the potential risk of food crisis. Methanol, an abundant feedstock, holds promise in facilitating low-carbon production processes. However, the current methanol bioconversion is hindered by limited regulatory strategies and relatively low conversion efficiency. Here, a yeast biocatalyst was extensively engineered for efficient biosynthesis of fatty alcohols through reinforcement of precursor supply and methanol assimilation in Pichia pastoris. Furthermore, the dual cytoplasmic and peroxisomal biosynthetic pathways were constructed by mating and exhibited robust production of 5.6 g/L fatty alcohols by using methanol as the sole carbon source. This study provides a heterozygous diploid P. pastoris strain with dual cytoplasmic and peroxisomal biosynthetic pathways, which achieved the highest fatty alcohol production from one-carbon feedstocks to date.

Clean Production of l-Alanyl-l-glutamine by an Efficient Yeast Biocatalyst Expressing α-Amino Acid Ester Acyltransferase without N-Glycosylation.

l-Alanyl-l-glutamine (Ala-Gln) is a widely used value-added dipeptide whose production relies heavily upon an efficient biocatalyst. The currently available yeast biocatalysts that express α-amino acid ester acyltransferase (SsAet) possess relatively low activity, which may be attributed to glycosylation. Here, to promote SsAet activity in yeast, we identified the N-glycosylation site as the Asn residue at position 442 and subsequently eliminated the negative effect of N-glycosylation on SsAet by removing artificial and native signal peptides to obtain K3A1, a novel yeast biocatalyst with significantly improved activity. Additionally, the optimal reaction conditions of strain K3A1 were determined (25 °C, pH 8.5, AlaOMe/Gln = 1:2), resulting in a maximum molar yield and productivity of approximately 80% and 1.74 g·(L·min)-1, respectively. Therefore, we developed a promising system to cleanly produce Ala-Gln in a safe, efficient, and sustainable manner, which may contribute to the future industrial production of Ala-Gln.

Synergistic of yeast Saccharomyces cerevisiae and glucose oxidase enzyme as co-biocatalyst of enzymatic microbial fuel cell (EMFC) in converting sugarcane bagasse extract into electricity

The microbial fuel cell (MFC) is an ecologically friendly alternative energy source. Due to the typically limited electron transfer in MFC systems, co-biocatalysts are necessary to enhance their performance. Enzymes are used as co-biocatalysts due to their superior ability to generate energy, and the system is known as an enzymatic microbial fuel cell (EMFC). One of the substrates that may be used is bagasse waste extracted from sugarcane. Saccharo­myces cerevisiae and the enzyme glucose oxidase (GOx) serve as co-biocatalysts in the breakdown of sugarcane bagasse waste in this study, which uses single-chamber EMFCs. In EMFC using sugarcane bagasse waste extract employing S. cerevisiae biocatalyst and glucose oxidase enzyme co-biocatalyst, the open circuit voltage was 0.56 V and the maximum power density was 146.65 mW m-2, an increase of 10.4 times to MFCs that solely employed only yeast biocatalyst. In addition, the chemical oxygen demand (COD) reduction achieved by this technology is 75 %. In addition, the pH of sugarcane bagasse waste extract samples treated with Saccharomyces cerevisiae yeast and GOx enzyme decreased from 4.6 to 4.2. This research demonstrates that adding the co-biocatalyst GOx enzyme may boost the performance of the traditional yeast MFC.

Recent advances on bio-based isobutanol separation

Bio-based isobutanol has attracted more and more attention due to its wide application and excellent fuel performance. Compared with ethanol, isobutanol has higher energy density, lower oxygen content, and hygroscopicity, making it an ideal gasoline additive or substitute. Bio-based isobutanol has been commercially produced from a variety of renewable feedstock, including corn, wheat, sorghum, barley, and sugar cane by using a yeast biocatalyst. The existing facility of ethanol plants is acquired to separate and purify isobutanol from the fermentation broth, meaning that the energy-intensive distillation process still cannot be replaced. There are still some challenges in the production of bio-based isobutanol, such as low product concentration, unexpected impurities, high separation cost, thus the development of an efficient and low-cost downstream separation process has become one of the keys to the industrial production of bio-based isobutanol. The separation methods for isobutanol recovery, including vacuum evaporation, adsorption, pervaporation, gas stripping, solvent extraction, salting-out, salting-out extraction, are summarized in this review. An outlook on the challenges and opportunities of isobutanol separation is also described in this review.

Candida zeylanoides as whole-cell biocatalyst to perform asymmetric bioreduction of benzophenone derivatives

Candida zeylanoides P1 was investigated as whole cell biocatalyst for the bioreduction of biaryl prochiral ketones into chiral carbinols, which can be used as pharmaceutical intermediate. Bioreduction of different biaryl ketones was carried out to their corresponding chiral biaryl carbinols such as (S)-(4-chlorophenyl) (phenyl) methanol (2a), which can be used in the synthesis of L-cloperastine drug, with antitussive, antiepidemic activity and bronchial musculature relaxant characteristics, in gram scale, enantiopure form (>99%) and excellent yields. The selectivity of C. zeylanoides P1 in enantioselective reduction of biaryl ketones was not affected by the steric and electronic effects of substrates. The current method demonstrates an encouraging green chemistry approach for the production of biaryl secondary chiral alcohols of pharmaceutical importance in mild, inexpensive and environmentally friendly process. The present study has many benefits since this yeast biocatalyst were successfully applied bioreduction of structurally bulky prochiral substrates, which cannot be reducted by chemical catalysis.

Early-stage performance evaluation of flowing microbial fuel cells using chemically treated carbon felt and yeast biocatalyst

The performance of closed-loop flowing-type microbial fuel cells using differently pretreated carbon felts is measured. Yeast cultivated from S. cerevisiae is used as biocatalyst, while glucose is the substrate. For the pretreatment of felt, acetone, nitric acid, and polyethyleneimine are employed. First the optimal conditions for yeast cultivation are quantitatively determined. As a result, a high yeast growth rate (1.083 h−1) and the optimal yeast growing time (48 h) for cell tests are obtained. The differently pretreated felts are analyzed by X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy and optical microscopy. Conductivity, charge transfer resistance, and CdbndO and CsbndN groups dangled on the felt are crucial parameters determining the performance of the microbial fuel cell. Particularly, the conjugation effects of pi-pi bonds and lone pairs facilitating the attachment of yeast to the CdbndO and CsbndN groups on the carbon felt promote (i) mutual adhesion between them and (ii) growth of yeast on CF-PEI. This correlation is confirmed by optical analysis of the felts after the cell tests. To evaluate the early-stage performance of the microbial fuel cells using the different felts, polarization curves are measured. In the measurements, the maximum power density of the cells depends on the superficial state of felts, while the performance of the cell using the PEI-treated felt is best, at 256.3 ± 11.5 mW·m−2. These data match other results attained by pretreatments.

Engineering of a high lipid producing Yarrowia lipolytica strain.

BackgroundMicrobial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways.ResultsWe describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.ConclusionsThe yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0492-3) contains supplementary material, which is available to authorized users.

Saccharomyces cerevisiae transcriptional reprograming due to bacterial contamination during industrial scale bioethanol production.

BackgroundThe bioethanol production system used in Brazil is based on the fermentation of sucrose from sugarcane feedstock by highly adapted strains of the yeast Saccharomyces cerevisiae. Bacterial contaminants present in the distillery environment often produce yeast-bacteria cellular co-aggregation particles that resemble yeast-yeast cell adhesion (flocculation). The formation of such particles is undesirable because it slows the fermentation kinetics and reduces the overall bioethanol yield.ResultsIn this study, we investigated the molecular physiology of one of the main S. cerevisiae strains used in Brazilian bioethanol production, PE-2, under two contrasting conditions: typical fermentation, when most yeast cells are in suspension, and co-aggregated fermentation. The transcriptional profile of PE-2 was assessed by RNA-seq during industrial scale fed-batch fermentation. Comparative analysis between the two conditions revealed transcriptional profiles that were differentiated primarily by a deep gene repression in the co-aggregated samples. The data also indicated that Lactobacillus fermentum was likely the main bacterial species responsible for cellular co-aggregation and for the high levels of organic acids detected in the samples.ConclusionsHere, we report the high-resolution gene expression profiling of strain PE-2 during industrial-scale fermentations and the transcriptional reprograming observed under co-aggregation conditions. This dataset constitutes an important resource that can provide support for further development of this key yeast biocatalyst.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0196-6) contains supplementary material, which is available to authorized users.

Exploiting cell metabolism for biocatalytic whole-cell transamination by recombinant Saccharomyces cerevisiae

The potential of Saccharomyces cerevisiae for biocatalytic whole-cell transamination was investigated using the kinetic resolution of racemic 1-phenylethylamine (1-PEA) to (R)-1-PEA as a model reaction. As native yeast do not possess any ω-transaminase activity for the reaction, a recombinant yeast biocatalyst was constructed by overexpressing the gene coding for vanillin aminotransferase from Capsicum chinense. The yeast-based biocatalyst could use glucose as the sole co-substrate for the supply of amine acceptor via cell metabolism. In addition, the biocatalyst was functional without addition of the co-factor pyridoxal-5′-phosphate (PLP), which can be explained by a high inherent cellular capacity to sustain PLP-dependent reactions in living cells. In contrast, external PLP supplementation was required when cell viability was low, as it was the case when using pyruvate as a co-substrate. Overall, the results indicate a potential for engineered S. cerevisiae as a biocatalyst for whole-cell transamination and with glucose as the only co-substrate for the supply of amine acceptor and PLP.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-5576-z) contains supplementary material, which is available to authorized users.

Production of ferulic acid and coniferyl alcohol by conversion of eugenol using a recombinant strain of Saccharomyces cerevisiae

ABSTRACTWe isolated an industrial strain of Saccharomyces cerevisiae 92411 able to convert coniferyl alcohol (2 g/l) into ferulic acid (1.8 g/l). To use eugenol as a substrate for the synthesis of natural ferulic acid, an optimized sequence of the Penicillium simplicissimum vaoA gene, encoding the flavoenzyme vanillyl‐alcohol oxidase (VAO), was expressed in the yeast 92411. To obtain a strong and stable expression of vaoA, an expression cassette flanked by Ty1 homologous sequences was constructed so the sequence encoding VAO could be integrated into various Ty1 loci of the host genome. The recombinant S. cerevisiae strain produced VAO. It synthesized ferulic acid directly from eugenol due to expression of the VAO activity and the natural ability of the strain to convert coniferyl alcohol into ferulic acid. In fermentation experiments, the strain produced up to 16.9 g/l of ferulic acid after 136 h. These results open new perspectives for the use of yeast biocatalysts in the biotechnological production of aromatic flavour from eugenol. Copyright © 2013 John Wiley & Sons, Ltd.

Enzyme-Mediated Bioremediation of Organophosphates Using Stable Yeast Biocatalysts

Organophosphates are extremely toxic compounds, which pose a threat to the environment and public due to their widespread use as common pesticides or due to their deliberate release as chemical weapons. The bacterial enzyme organophosphorus hydrolase (OPH, EC. 3.1.8.1) can hydrolyze, and thereby detoxify a broad range of organophosphate nerve agents. This enzyme therefore offers the opportunity for the development of naturally occurring newer bioremediation strategies. The aim of this research was to generate a stable yeast biocatalyst that was capable of hydrolyzing the poorly hydrolyzed P-S class of organophosphates. The genes encoding the wild-type OPH, or the enhanced variant enzyme S308L-OPH, were integrated into the ribosomal operon of the Saccharomyces cerevisiae genome to create a stable yeast biocatalyst.

Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3'-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94% CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64% CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170% hydrolytic activity and 155% synthetic activity compared with strain KCSe1 as well as 144% hydrolytic activity and 121% synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124% hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.

Meeting report: ACB‐2011 Shanghai – “Biotechnology for Better Life”

At the 9th APBioChEC (Nov. 24–28, 2009, Kobe, Japan), it was announced that the Asia Pacific Biochemical Engineering Conference (APBioChEC) would be transformed into the Asian Congress on Biotechnology (ACB) to facilitate an enlarged scope under the newly founded Asian Federation of Biotechnology (AFOB) [1]. “Biotechnology for Better Life” ... an avenue for young scientists/engineers to develop ... A total of 308 high-quality lectures, including 5 plenary lectures, 217 invited presentations from 10 sessions, 86 oral presentations from 3 special forums were delivered at this conference. In addition, 455 selected posters were presented from the submissions. In addition to this special issue of Biotechnology Journal, all the accepted full papers have been categorized according to their topics and will be published in five other international journals, including Biotechnology Advances, Advances in Biochemical Engineering/Biotechnology, Process Biochemistry, Frontiers of Chemical Science and Engineering and Interdisciplinary Sciences-Computational Life Sciences. Prof. Zi-xin Deng (Shanghai Jiaotong University, China), introduced the revival of the antibiotics legend and recent progress in novel antibiotic discovery. Three major paths for discovery of antibiotics were discussed: (i) natural screening; (ii) pathway engineering and combinatorial biosynthesis; and (iii) synthetic biology. Our knowledge of the natural products' biosynthetic mechanisms and advances in genomics, proteomics, metabolomics, metabolic engineering and synthetic biology are now providing better opportunities to generate novel compounds or optimal derivatives. Prof. Deng talked about his latest work on development of new antibiotics using tools from synthetic biology. Prof. Daniel I.C. Wang (Massachusetts Institute of Technology, USA), introduced glycoprotein glycosylation, which is a current challenge in the production of biopharmaceuticals. Prof. Wang also discussed glycoform heterogeneity and how to overcome this problem by process improvements and genetic manipulations for achieving greater glycoform quality. Prof. Jens Nielsen (Chalmers University of Technology, Sweden), discussed yeast as a platform cell factory in future biorefineries due to yeast's many advantages such as its general acceptance in industry, its robustness towards contaminations, its high alcohol and low pH tolerance, and a large number of available molecular biology techniques and omics tools for this organism. Prof. Nielsen also introduced a number of synthetic biology and systems biology tools for advancing the design and engineering of novel, efficient yeast biocatalysts to produce a range of different products (i.e., novel fuels and chemicals). See [2] for a review on yeast metabolic engineering by Prof. Nielsen. Prof. Jung-Keug Park (Dongguk University, Korea), presented a novel field of interdisciplinary science “wave biotechnology” and its related “industry wave bioindustry”. Prof. Park discussed the current status of research and development in wave biotechnology, and showed some on-going work in stem cell differentiation using electromagnetic and sonic wave to stimulate neural cells. From the second day to the fifth day of the conference, 308 oral presentations including 222 invited lectures took place in 10 scientific sessions on various topics: (i) Bioprocess Engineering, (ii) Systems Biology and Synthetic Biology, (iii) Enzyme and Protein Engineering, (iv) Nano- and Analytical Biotechnology, (v) Biorefinery, Biofuels and Bioproducts, (vi) Extremophiles and Marine Biotechnology, (vii) Agricultural and Food Biotechnology, (viii) Environmental Biotechnology, (ix) Pharmaceutical and Medical Biotechnology, (x) Stem Cells, Tissue Engineering, and Biomaterials. In addition, there were three special forums focused on current biotechnology R&D and business for biotechnologists and biochemical engineers in Asia, including: (i) Asian Bioindustry & Bioeconomy Forum, (ii) Young Scientists & Students (YSS) Forum, (iii) ISBBE Forum-The 5th International Symposium and Exhibition on Bioprocess & Bioproduct Engineering. sincere thanks to all for their support, participation and cooperation As we bid farewell to one successful event, we look forward to the next: the 2nd Asian Congress on Biotechnology will be held in December 2013 in New Delhi, India, as announced by Prof. Virendra S. Bisaria (Indian Institute of Technology, India) at the closing ceremony. Prior to that, the 15th International Biotechnology Symposium and Exhibition (IBS-2012) will be held in September 2012 in Daegu, Korea, as introduced by Prof. Tai Hyun Park (Seoul National University, Korea). We hope to see you all in Daegu and New Delhi! Congress website: www.acb-2011.org

Production and Characterization of Wine with Sugarcane Piece Immobilized Yeast Biocatalyst

In the present study, an economically cheap and sustainable food-grade yeast biocatalyst for wine production was successfully prepared. It was prepared through absorption technique by using fresh sugarcane (Saccharum officinarum L.) pieces as immobilizing support for yeast, Saccharomyces cerevisiae CFTRI 101. Immobilization was confirmed by scanning electron microscopy. Suitability of biocatalyst was investigated at low and room temperatures by using it in repeated batch. The fermentation rate and other parameters were compared with free yeast cells at different temperatures. In all cases, fermentation time was short (24 h at 30 °C and 98 h at 10 °C) and ethanol productivities were high as 4.2 g/l/h (3.33-fold at 30 °C and 2.31-fold at 10 °C). The volatile compounds methanol, ethyl acetate, propanol-1, isobutanol (2-methyl-1-propanol), and amyl alcohols (2-methyl-1-butanol and 3-methyl-1-butanol) that formed during fermentation were analyzed with the help of a gas chromatograph–flame ionization detector. The concentrations of ethyl acetate and methanol were not more than 100 mg/l in all cases, indicating an improvement in the product quality. Cell metabolism of immobilized yeast was not negatively affected by immobilization process. It was found that the immobilization of yeast cells on sugar pieces increased the fermentation rate and viability of yeast cells. Preliminary sensory tests recognized the fruity aroma, fine taste, and the overall improved quality of the produced wines.

Wine production by novel yeast biocatalyst prepared by immobilization on watermelon ( Citrullus vulgaris) rind pieces and characterization of volatile compounds

A novel yeast biocatalyst was prepared by using watermelon pieces as immobilizing support for yeast, Saccharomyces cerevisiae 101 strain for use in wine production. Immobilization was confirmed by electron microscopy. Suitability of biocatalyst was investigated by using it in repeated batch and continuous fermentations. The fermentation rate and other parameters were compared with free yeast cells at different temperatures. In all cases fermentation time was short (22 h at 30 °C and 80 h at 15 °C) and ethanol productivities were high (4 g/l/h). The volatile compounds, methanol, ethyl acetate, propanol-1, isobutanol and amyl alcohols that formed during fermentation were analyzed with the help of GC-FID. The concentrations of ethyl acetate and methanol were not more than 100 mg/l in all cases, indicating an improvement in the product. Cell metabolism of immobilized yeast was not much affected by immobilization. It was found that the immobilization of yeast on watermelon pieces increased the fermentation rate, vitality and viability of yeast cells. Preliminary sensory tests established the fruity aroma, fine taste and the overall improved quality of the produced wines.

Development, Production, and Application of Recombinant Yeast Biocatalysts in Organic Synthesis

The use of biocatalysts from yeast strains in organic synthesis is well established and covers a broad range of reaction classes. A particularly interesting reaction class is the regio- and stereospecific attachment of sugar moieties (i.e. glycosylation) to a variety of natural products, from small molecules up to oligosaccharides and proteins. Since the bioactivity of many therapeutics depends on the proper glycosylation, the improvement of glycosylation methodology by chemical synthesis, biocatalysis or in vivo approaches is of major interest. We have developed glycosyltransferase-toolkits for the straight-forward and quantitative transfer of a specific monosaccharide moiety to an acceptor substrate. The stable expression of the ?(1?4)-galactosyltransferase I in Saccharomyces cerevisiae and ?(1?3)-fucosyltransferase VI in Pichia pastoris has enabled these biocatalysts to be prepared in large-scale for use in organic synthesis. The application of galactosyltransferase using UDP-galactose and fucosyltransferase using GDP-fucose as NDP-sugar donor in the regio- and stereospecific galactosylation and fucosylation of small molecules is shown with a simplified reaction system. Optimisation of the reaction conditions allows for quantitative glycosylation reactions. The use of recombinant yeasts has been the key to performing this highly efficient glycosylation methodology and represents a building block of biocatalytic glycomics for the future.

Chemoenzymatic Route to Both Enantiomers of a 1‐Isopropyl‐3a‐methyloctahydroinden‐4‐one Derivative: A Synthetic Intermediate for Sesqui‐ and Diterpenoids

AbstractOn the way to a chemoenzymatic synthesis of a key intermediate for sesquiterpenoids and diterpenoids, 2‐methyl‐2‐(4‐methyl‐3‐oxopentyl)‐1,3‐cyclohexanedione was reduced with the whole cells of yeast biocatalysts. Torulaspora delbrueckii NBRC10921 reduced a cyclic ketone of three carbonyl groups in an enantiofacially selective manner (re‐face attack), but there was poor enantiotopic group selectivity between two carbonyl groups on the cyclohexane ring to yield a mixture of diastereomeric products. Candida floricola IAM13115 reduced mainly the pro‐(R) carbonyl group. In contrast, the reduction proceeded in an enantiofacially poorly selective manner to give another set of diastereomeric products. In both cases, another carbonyl group on the side chain worked as a ‘trapping arm’ of the resulting secondary alcohol. The diastereomeric products were effectively separated as the ‘syn’ or ‘cis’ isomer exclusively exist in the intramolecular hemiacetal structure, while ‘anti’ or ‘trans’ isomer being an equilibrated mixture of cyclic hemiacetal and open‐chain hydroxyketone (ca. 0.7 : 1). Starting separately from the enantiomerically enriched products as above, both enantiomers of the target compound, a key intermediate for terpenoids, were efficiently prepared via stereoselective ring closure under pinacol coupling reaction conditions. Furthermore, a daucane sesquiterpene intermediate, a hydroazulene derivative, was provided after one‐carbon homologation of the six‐membered ring.

Storage of immobilized yeast cells for use in wine-making at ambient temperature.

A comparative study of the storage and reuse of immobilized yeast cells on apple pieces, kissiris, and gamma-alumina was carried out. The immobilized biocatalysts were allowed to remain in the fermented alcoholic liquid after the end of each fermentation batch for extended periods at 30 degrees C before reactivation in batch fermentation for wine-making. The results showed that the biocatalysts were able to reactivate and ferment after successively increased periods of storage compared to free cell systems both on glucose medium and on grape must. In glucose medium, apple-, kissiris-, and gamma-alumina-supported biocatalysts reactivated after 120, 80, and 83 days, respectively. Possible storage periods for grape must were lower but remained high. Immobilized yeast biocatalyst on apple pieces produced wines with an improved volatiles composition compared to kissiris- and gamma-alumina-supported biocatalysts. There were no significant negative effects on the fermentation activity and volatile byproduct composition.

Use of glucose feeding to produce concentrated yeast cells.

A defined medium and fed-batch feeding process for the production of a yeast biocatalyst, developed at the 23-L scale, was scaled up to the 600-L pilot scale. Presterilized 100-L-vol plastic bags were implemented for the pilot-scale nutrient feeding. Medium of increased concentration Oqs implemented at the pilot scale, and equivalent dry cell weights were reached with a medium 80% more concentrated than that used at the laboratory scale. The higher medium concentration was believed to be necessary at the pilot scale owing to the additional heat stresses on key components (e.g., complexing of magnesium sulfate with phosphate), increased dilution during sterilization, lower evaporation rate owing to the lower vessel volume per minute air flow rate, and increased dilution owing to nutrient feeding or shot additions. Peak cell density was found to be somewhat insensitive to variations in residual glucose levels. These results suggest that defined medium developed at the laboratory scale may need to be further optimized at the pilot scale for equivalent performance.