Feed component inhibition in ethanolic fermentation by Saccharomyces cerevisiae

Inhibition by secondary feed components can limit productivity and restrict process options for the production of ethanol by fermentation. New fermentation processes (such as vacuum or extractive fermentation), while selectively removing ethanol, can concentrate nonmetabolized feed components in the remaining broth. Stillage recycle to reduce stillage waste treatment results in the buildup of nonmetabolized feed components. Continuous culture experiments are presented establishing an inhibition order: CaCl(2), (NH(4))(2)xSO(4) > NaCl, NH(4)Cl > KH(2)PO(4) > xylose, MgCl(2) > MgSO(4) > KCl. Reduction of the water activity alone is not an adequate predictor of the variation in inhibitory concentration among the different components tested. As a general trend, specific ethanol productivity increases and cell production decreases as inhibitors are added at higher concentration. We postulate that these results can be interpreted in terms of an increase in energy requirements for cell maintenance under hypertonic (stressed) conditions. Ion and carbohydrate transport and specific toxic effects are reviewed as they relate to the postulated inhibition mechanism. Glycerol production increases under hypertonic conditions and glycerol is postulated to function as a nontoxic osmoregulator. Calcium was the most inhibitory component tested, causing an 80%decline in cell mass production at 0.23 mol Ca(2+)/L and calcium is present at substantial concentration in many carbohydrate sources. For a typical final cane molasses feed, stillage recycle must be limited to less than onethird of the feed rate; otherwise inhibitory effects will be observed.     

Preparation of hydrolysates from tobacco stalks and ethanolic fermentation by Saccharomyces cerevisiae

Chipped tobacco stalks were subjected to steam pretreatment at 205 °C for either 5 or 10 min before enzymatic hydrolysis. Glucose (15.4–17.1 g/l) and xylose (4.5–5.0 g/l) were the most abundant monosaccharides in the hydrolysates. Mannose, galactose and arabinose were also detected. The hydrolysate produced by pretreatment for 10 min contained higher levels of all sugars than the 5 min-pretreated hydrolysate. The amounts of inhibitory compounds found in the hydrolysates were relatively low and increased with increasing pretreatment time. The hydrolysates were fermented with baker's yeast. Ethanol yield, maximum volumetric productivity and specific productivity were used as criteria of fermentability of the hydrolysates. The fermentation of the hydrolysates was only slightly inhibited compared to reference solutions having a similar composition of fermentable sugars. The ethanol yield in the hydrolysates was 0.38–0.39 g/g of initial fermentable sugars, whereas it was 0.42 g/g in the reference. The biomass yield was twofold lower in the hydrolysates than in the reference. The fermentation inhibition caused by the tobacco stalk hydrolysates was less than that caused by sugarcane bagasse hydrolysates obtained under the same hydrolysis conditions.     

Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae.

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With such supplementation, the viability of the yeast cells was maintained above 80% throughout the fermentation, while it dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effective than proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% with glycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested that glycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine bases increased the sugar uptake and ethanol production but did not allow the population to maintain the high level of cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence of particulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar utilization to 68, 75, 81, and 82%, respectively.     

Significance of the surface of immobilized Saccharomyces cerevisiae cells in ethanolic fermentation

S. cerevisiae cells immobilized in alginate beads show in many cases an increase of mean single cell volume during long-time fermentations (successive batch cycles). The biomass loading capacity of the gel beads is characterized by a maximum volume but not by a maximum number of cells occupying the gel volume. In our system this loading capacity, i.e. the maximum volume fraction of cells per volume of beads, amounted to about 0.54. As a more important result it must be stated that the specific product formation rate in the case of fermentations negligibly influenced by diffusion hindrance is related to the total surface of the viable cells but not to their total number, total volume or total dry weight.     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

Maltotriose fermentation by Saccharomyces cerevisiae

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l⁻¹ glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38. Received 13 January 2001/ Accepted in revised form 29 May 2001     

Continuous ethanolic fermentation by Saccharomyces cerevisiae immobilised in Ca-alginate beads hardened with Al3+

Summary The most suitable conditions for the hardening of alginate bioparticles by Al³⁺ obtained were: Al³⁺, 0.3 M; time of treatment 5 min and yeast concentration in the gel, 3 g dry weight/L. The hardened biocatalyst was used to carry out an ethanolic fermentation process in a semi-pilot packed-bed reactor and no breakage of the bioparticles was observed, although the lower part beads had a flat shape, which caused a 11% decrease in bed height. Ethanol productivities up to 31 g/L·h were obtained.     

Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

Background  

Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. The availability of NAD⁺ for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.     

Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

Mandarin essential oil as an antimicrobial in ethanolic fermentation: Effects on Limosilactobacillus fermentum and Saccharomyces cerevisiae

The antibacterial activity of citrus essential oils (EOs) in the context of combating Limosilactobacillus fermentum, one of the most important bacterial contaminants in the bioethanol production industry, has never been explored previously. Industrial processes usually utilize sulfuric acid for cell treatment to decrease bacterial contamination. However, due to the hazardous nature of sulfuric acid, an alternative to it is highly desirable. Therefore, in the present study, the efficacy of Fremont IAC 543 mandarin EO against a strain of L. fermentum (ATCC^® 9338™) was evaluated under proliferative/nonproliferative conditions, in both pure culture and co-culture with an industrial strain of Saccharomyces cerevisiae. The mandarin EO exhibited higher effectiveness against L. fermentum compared to that against S. cerevisiae under nonproliferative conditions (added to water rather than to culture medium). At the concentration of 0·05%, the EO was as effective as the acid solution with pH 2·0 in reducing the count of L. fermentum almost 5 log CFU ml^–1 cycles, while the concentration of 0·1% led to the complete loss of bacterial culturability. When L. fermentum was co-cultured with S. cerevisiae, the efficacy of the EO against the bacterial strain was reduced. However, despite this reduced efficacy in co-culture, mandarin EO may be considered effective in combating L. fermentum and could be applied in processes where this bacterium proves to be unfavourable and does not interact with S. cerevisiae.     

Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition

Summary The anaerobic glucose uptake (at 20°, pH 3.5) by resting cells of Saccharomyces cerevisiae followed unidirectional Michaelis-Menten kinetics and was competitively inhibited by l-sorbose; K _m and K _i were respectively 5.6×10^-4 m and 1.8×10^-1 m; V _max was 6.5×10^-8 moles mg^-1 min^-1. The aerobic uptake of glucose by resting yeast was also inhibited by l-sorbose but did not follow unidirectional Michaelis-Menten kinetics. Glucose-limited growth in the chemostat of a respiration-deficient mutant of S. cerevisiae was competitively inhibited by l-sorbose. As predicted by theory for transport-limited growth in the chemostat (van Uden, 1967) the steady state glucose concentrations were linear functions of the l-sorbose concentrations with different slopes at different dilution rates; K _m and K _i were respectively 7.2×10^-4 m and 1.8×10^-1 m. It is concluded that glucose transport was the rate-limiting step of anaerobic fermentation of S. cerevisiae and of growth of the mutant and that l-sorbose is a competitive inhibitor of active glucose transport in this yeast. The latter conclusion is accommodated in the transport model of van Steveninck and Rothstein (1965).     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Reaction kinetics of d-glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of d-glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of d-glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.     

NaCl stress inhibits maltose fermentation by Saccharomyces cerevisiae

While fermentation of 20 g glucose l^–1 by Saccharomyces cerevisiae was not impaired by high NaCl concentrations, fermentation of 20 g maltose l^–1 was significantly decreased by 0.7 M NaCl, and completely inhibited with 1.4 M NaCl. No glycerol was produced in response to the salt stress when yeast cells were fermenting maltose. Active maltose transport, and not intracellular hydrolysis, was the metabolic step severely impaired by the NaCl stress.     

Sucrose fermentation by Saccharomyces cerevisiae lacking hexose transport

Sucrose is the major carbon source used by Saccharomyces cerevisiae during production of baker's yeast, fuel ethanol and several distilled beverages. It is generally accepted that sucrose fermentation proceeds through extracellular hydrolysis of the sugar, mediated by the periplasmic invertase, producing glucose and fructose that are transported into the cells and metabolized. In the present work we analyzed the contribution to sucrose fermentation of a poorly characterized pathway of sucrose utilization by S. cerevisiae cells, the active transport of the sugar through the plasma membrane and its intracellular hydrolysis. A yeast strain that lacks the major hexose transporters (hxt1-hxt7 and gal2) is incapable of growing on or fermenting glucose or fructose. Our results show that this hxt-null strain is still able to ferment sucrose due to direct uptake of the sugar into the cells. Deletion of the AGT1 gene, which encodes a high-affinity sucrose-H(+) symporter, rendered cells incapable of sucrose fermentation. Since sucrose is not an inducer of the permease, expression of the AGT1 must be constitutive in order to allow growth of the hxt-null strain on sucrose. The molecular characterization of active sucrose transport and fermentation by S. cerevisiae cells opens new opportunities to optimize yeasts for sugarcane-based industrial processes.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

啤酒酵母胞外多糖发酵条件的研究

以啤酒酵母S－12为出发菌株,用紫外线＋氯化锂作为复合诱变剂,获得一株产胞外多糖量较高的变株S－12－4,比出发菌株提高33．3％,同时对变株进行了最佳培养条件的研究,结果表明：最适碳源和氮源分别为大米糖3％、酵母粉0．37％及NH4Cl0．32％,最适发酵条件为起始PH6．0、培养温度26℃,发酵周期为30h,在此基础上进行培养,变株S－12－4产胞外多糖最高可达38.2mg/100mL,比初始条件提高了54％。     

Static magnetic fields enhancement of Saccharomyces cerevisae ethanolic fermentation

Magnetic effects induced in ethanolic fermentation by Saccharomyces cerevisiae strain DAUFPE-1012 were studied during a 24 h exposure to 220 mT steady magnetic fields (SMF) at 23 +/- 1 degrees C, produced by NdFeB rod magnets. The magnets were attached diametrically opposed (N to S) to a cylindrical tube reactor. The biomass growth in the reactor culture media (yeast extract + glucose 2%) during 24 h was monitored by measurements of optical density, which was correlated to cell dry weight. Ethanol concentration and glucose level were measured every 2 h. The pH of the culture media was maintained between 4 and 5. As a result, biomass (g/L) increased 2.5-fold and ethanol concentration 3.4-fold in magnetized cultures (n = 8) as compared with SMF nonexposed cultures (n = 8). Glucose consumption was higher in magnetized cultures, which correlated to the ethanol yield.