Characterization of cellobiose fermentations to ethanol by yeasts

Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test cultures. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When higher carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of beta-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, beta-glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the beta-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When empolying these organisms in a simultaneous saccharification-fermentation of avicel, using Trichoderma reesei cellulase as the saccharifying agent, 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae.     

Studies on the use of a thermotolerant strain of Kluyveromyces marxianus in simultaneous saccharification and ethanol formation from cellulose

 The thermotolerant yeast strain, Kluyveromyces marxianus IMB3, was found to be capable of ethanol production during growth at 45°C on media containing milled paper and exogenously added commercial cellulase. At maximum achievable cellulose concentrations in shake-flask cultures, ethanol production increased to 6.6 g/l at 45°C, representing an overall level of conversion of 21% of the maximum theoretical yield. Subsequent studies involving variations in added cellulase concentrations to the batch systems demonstrated that ethanol yields could be increased to 10 g/l at 45°C, which represented 39% of the maximum theoretical yield. As a result of ethanol production at 45°C in the systems examined, we suggest that the thermotolerant ethanol-producing yeast strain K. marxianus represents a novel candidate for use in simultaneous saccharification and conversion of the resulting substrates to ethanol. Received: 9 June 1994/Received revision: 8 August 1994/Accepted: 12 August 1994     

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.     

Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

Evaluation of ethanol evaporation losses in acetic acid fermentations

Three different operation systems have been employed at laboratory, pilot plant and industrial scales. Developed experimentation has demonstrated that quantified ethanol losses minimize significantly when operating at low temperatures with low aerations and when mainly working with the closed system.     

Characteristics of cellulase preparations affecting the simultaneous saccharification and fermentation of cellulose to ethanol

The cellulase, Spezyme CP from Genencor, widely used for the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol, contained substances inhibitory to the growth of Klebsiella oxytoca P2, emphasising the need to check for inhibition effects in SSF experimentation. Also, the preparation contained enough -glucosidase activity to prevent cellobiose accumulation in SSF with a conventional non-cellobiose fermenting yeast: this finding is relevant to attempts to evaluate novel recombinant cellobiose-fermenting microbial strains.     

Evaluation of a recombinant Klebsiella oxytoca strain for ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with native cellobiose-utilising yeast strains and performance in co-culture with thermotolerant yeast and Zymomonas mobilis

In the simultaneous saccharification and fermentation to ethanol of 100 g l(-1) microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions. The time taken by K. oxytoca P2 to produce up to about 33 g l(-1) ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis. Ultimately, it produced slightly less ethanol (maximum 36 g l(-1)) than these organisms, reflecting its lower ethanol tolerance. Significant advantages were obtained by co-culturing K. oxytoca P2 with S. pastorianus, K. marxianus or Z. mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity. Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density. K. oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain. The usefulness of K. oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Classical and molecular analyses to characterize commercial dry yeasts used in wine fermentations

AIMS: The aim of the work was to apply PCR-temperature gradient gel electrophoresis (PCR-TGGE) and restriction enzyme analysis (RE) assays to identify commercially available starters of Saccharomyces cerevisiae sensu stricto complex. METHODS AND RESULTS: To characterize an analysed pool of 62 active dry yeasts of different brands used in wine fermentation practices, classical microbiological tests were also performed as well as evaluation of contamination with lactic acid bacteria and non-Saccharomyces yeasts. PCR-TGGE and RE were used in order to provide fast and reliable methods to identify and differentiate enological yeasts. Proposed molecular methods enabled to identify particular strains within 36 h after colony isolation and directly from dry yeast suspension. CONCLUSIONS: The methods are highly recommended to obtain reliable results on yeast strain differentiation in a significantly shorter time if compared to classical fermentation tests. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtaining of yeast strain differentiation in a short time and without plating is a good tool for a rapid discrimination among enological strains used as starters in enological practices.     

Role of water activity in ethanol fermentations

A separate role for water activity in the conversion of sugars to ethanol by two strains of yeast is identified. During fermentation of both single and mixed sugar substrates, the water activity was shown to remain constant during the logarithmic growth phase. This is despite the changes in concentration of substrates and product, the constancy reflecting the fact that the greater influence of ethanol on the solution activity is counterbalanced, in the early stages of the fermentation, by its low yield. The end of the log phase of growth coincides with the start of a period of gradually decreasing water activity. For the more ethanol-tolerant strain UQM66Y, growth was found to cease at a constant value of water activity while that for the less tolerant strain UQM70Y depended on both ethanol concentration and water activity. It is argued that water activity is a more appropriate variable than ethanol concentration for describing some of the nonspecific inhibitory effects apparent in ethanol fermentations. A straightforward method for the calculation of water activity during such fermentations based on the use of solution osmolality is presented.     

Microbial contamination of fuel ethanol fermentations

Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low pH and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species–species interactions between D. bruxellensis and S. cerevisiae are still poorly understood.     

Conversion of pentoses to ethanol by yeasts and fungi

Fermentation of D-xylose is of interest in enhancing the yield of ethanol obtainable from lignocellulosic hydrolysates. Such hydrolysates can contain both pentoses and hexoses, and while technology to convert hexoses to ethanol is well established, the fermentation of pentoses had been problematical. To overcome the difficulty, yeasts and fungi have been sought and identified in recent years that can convert D-xylose into ethanol. However, operation of their cultures in the presence of the pentose to obtain rapid and efficient ethanol production is somewhat more complex than in the archetype alcoholic fermentation, Saccharomyces cerevisiae on D-glucose. The complexity stems, in part, from the association of ethanol accumulation in cultures where D-xylose is the sole carbon source with conditions that limit growth, by oxygen in particular, although limitation by other nutrients might also be implicated. Aspects of screening for appropriate organisms and of the parameters that play a role in determining culture variables, especially those associated with ethanol productivity, are reviewed. Performance with D-xylose as sole carbon source, in sugar mixtures, and in lignocellulosic hydrolysates is discussed. A model that involves biochemical considerations of D-xylose metabolism is presented that rationalizes the effects of oxygen on cultures where D-xylose is the sole carbon source, notably effects of the specific rate of oxygen use on the rate and extent of ethanol accumulation. Alternate methods to direct fermentation of D-xylose have been developed that depend on its prior isomerization to D-xylose, followed by fermentation of the pentulose by certain yeasts and fungi. Factors involved in the biochemistry, use, and performance of these methods, which with some organisms involves sensitivity to oxygen, are reviewed.     

Evaluation of ethanol productivity from cellulose by clostridium thermocellum

Clostridium thermocellum, a thermophillic anaerobe, directly converts cellulose to ethanol. To estimate its ethanol production from cellulose, we used a new method based on material balance by which the efficiencies of the enzymes that convert cellulose to ethanol were calculated. Using this method, the maximum efficiency of ethanol production of two strains of C. thermocellum was estimated to be 0.05, with 0.67 as the theoretical maximum.     

Screening of thermotolerant yeasts as producers of superoxide dismutase

Abstract A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia . Higher yields at 45°C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.     

The role of indigenous yeasts in traditional Irish cider fermentations

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.     

Replicative inactivation and metabolic inhibition in yeast ethanol fermentations

Summary The effects of ethanol on the growth rates of twoSaccharomyces yeast strains were measured during normal batch fermentative growth and compared with those measured by initial rate studies. In the light of previous work, which has highlighted the loss of cell replicative ability caused by ethanol, the results imply that the observed reduction in growth rate reflects a mixture of true inhibition and replicative inactivation.     

Glycerol accumulation while recycling thin stillage in corn fermentations to ethanol

Summary By succesive recycling of the thin stillage in mashing and fermenting fresh corn, the glycerol content in each fermentation increased by about 0.4% and accumulated to a high of 2.1% in the beer of the fifth recycle. Glycerol concentration declined after the fifth recycle. The original fermentation contained 0.8% glycerol.Presented in part at the Society for Industrial Microbiology Annual Meeting, August 7–12, 1988, Chicago, IL.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Winemaking of musts at high osmotic strength by thermotolerant yeasts

Fermentation performance of 15 thermotolerant Saccharomyces cerevisiae in three musts from dried grapes at 25, 30, and 40° brix was studied. When the osmotic strength was increased, the volatile acidity and the SO₂ production in the wines also increased: in the must with 40° brix, yeasts produce from 1.63 to 3.65 g acetic acid l^–1 and from 40 to 73.6 mg SO₂ l^–1 due to osmotic stress. From 9.75 to 13.40 ethanol v/v production is observed in must at 30°brix, whereas at 40°brix there is a clear detrimental effect.