Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems

Summary Direct alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l^–1 of dextrin S. diastaticus produced 77 g·l^–1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l^–1 was obtained with a medium containing 400 g·l^–1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l^–1 of ethanol was produced from 400 g·l^–1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l^–1 of ethanol from 200 g·l^–1 of dextrin, corresponding to an ethanol productivity of 7.6g·l^–1·h^–1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l^–1 and 80 g·l^–1 of ethanol using initial dextrin concentrations of 200 and 250 g·l^–1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l^–1·h^–1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation.     

Enhanced production of extracellular α-amylase and glucoamylase by amylolytic yeasts using β-cyclodextrin as carbon source

Summary Several amylolytic yeasts from the genera Candida, Cryptococcus, Filobasidium, Lipomyces, Saccharomycopsis, Schwanniomyces, and Trichosporon can utilize -cyclodextrin as a sole carbon source. For most species significantly higher yields of both -amylase and glucoamylase are obtained as compared to with starch. This novel inducer of yeast amylases should therefore be useful in the characterization of these amylolytic enzymes and their regulation.     

Fructose syrups and ethanol production by selective fermentation of inulin

Jerusalem artichoke is a favorable substrate for inulin or fructose syrup production. The sugar content and the fructose ratio of inulin depend on various factors, particularly on the date of harvest. Incomplete fermentation of extracts by selected yeasts allows the production of inulin with increased fructose content. The yeast strains (Saccharomyces cerevisiae, S. diastaticus...) are chosen for their ability to ferment sucrose and inulin small polymers, but not easily inulin large polymers. A good increase in the fructose ratio and a good yield in residual sugars can be obtained with the better strains. After fermentation and acid or enzymatic hydrolysis, extracts from early and late harvested tubers lead to syrups of good quality containing up to 95% and 90% of fructose respectively. This fermentative enrichment process is competitive with others (for example, chromatographic enrichment), is appropriate to raw extracts, simplifies the purification steps, and also permits the simultaneous benefit of production of by-products in the form of ethanol and yeast (in addition to the pulps). Unhydrolyzed inulin polymers with high fructose content can be recovered by this selective fermentation.     

Development of Rapidly Fermenting Strains of Saccharomyces diastaticus for Direct Conversion of Starch and Dextrins to Ethanol

Alcoholic fermentation, growth, and glucoamylase production by 12 strains of Saccharomyces diastaticus were compared by using starch and dextrins as substrates. Haploid progeny produced from a rapidly fermenting strain, SD2, were used for hybridization with other S. diastaticus and Saccharomyces cerevisiae haploids. Alcoholic fermentation and enzyme production by hybrid diploids and their haploid parents were evaluated. Although the dosage of the STA or DEX (starch or dextrin fermentation) genes may enhance ethanol production, epistatic effects in certain strain combinations caused decreases in starch-fermenting activity. Both the nature of the starch or dextrin used and the fermentation medium pH had substantial effects on alcohol production. Commercial dextrin was not as good a substrate as dextrins prepared by digesting starch with α-amylase. Crude manioc starch digested by α-amylase was fermented directly by selected hybrids with almost 100% conversion efficiency. The manioc preparation contained adequate minerals and growth factors. This procedure should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere. A rapidly fermenting haploid strain, SD2-A8, descended from strain SD2, contains two unlinked genes controlling formation of extracellular amylase. A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed.     

Fermentation characteristics as criteria for selection of cachaça yeast

The fermentation characteristics of 24 strains of Saccharomyces cerevisiae and one strain of Candida apicola, C. famata, C. guilliermondii, Hanseniospora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe were evaluated for the production of cachaça. They were isolated from small cachaça distilleries (27), industrial cachaça distilleries (2) and one sugarcane alcohol distillery. The yeasts showed significant differences in ethanol yield, substrate conversion, efficiency, conversion factors of substrate into ethanol (Y _p/s), cells (Y _x/s), organic acids (Y _ac/s) and glycerol (Y _g/s), and maximum specific growth rate ( _max). In general the S. cerevisiae strains showed better fermentation potential, with yields between 83 and 91% and _max between 0.450 and 0.640 h^–1, several of them being comparable with the high performance yeast used in the industrial production of ethanol, which was adopted as a reference. The non-Saccharomyces strains showed high efficiency, very low ethanol yield and very high Y _ac/s and Y _g/s values, except Pichia subpelliculosa, which behaved very similarly to the S. cerevisiae strains. Hierarchical Cluster Analysis and Principal Component Analysis showed the fermentation yield (or substrate conversion) as being the variable which contributed most to the separation of the strains into different groups.     

Alcoholic fermentation of starch containing media using yeast protoplast fusion products

Summary Fermentation of starch based industrial media was tested with yeast fusion products previously described, from a Baker's yeastSaccharomyces cerevisiae and Saccharomyces diastaticus and from a highly flocculentSaccharomyces cerevisiae andSaccharomyces diastaticus. The (somatic) fusion products were capable to produce more ethanol than parental strains after 96 h of batch fermentation. The aim of this work was to reduce the amount of enzyme used in saccharification by using good fermenting amylolytic yeast strains.     

Evaluation of the cellobiose-fermenting yeastBrettanomyces custersii in the simultaneous saccharification and fermentation of cellulose

Summary Brettanomyces custersii (CBS 5512) was identified as a promising glucose- and cellobiose-fermenting yeast for the simultaneous saccharification and fermentation (SSF) of cellulose for ethanol production. In SSF studies with 75 g/L of cellulose,B. custersii produced 32 g/L of ethanol in just 3 days (75% of theoretical yield). This yield represents an increase of more than 16% over the yields of other fementative yeasts and the time to achieve it is less than that with other organisms. In addition, the ethanol tolerance ofB. custersii seems to be greater than that of other cellobiose-fermenting yeasts considered to date. Overall, the combination of higher yields, rates, and ethanol concentrations obtained withB. custersii improves the economics of ethanol production.     

Simultaneous production of sugars and ethanol from inulin rich-extracts in a chemostat

Summary Incomplete fermentation of inulin-containing extracts by Saccharomyces diastaticus allows the simultaneous production of ethanol and syrups with increased fructose content. The yeast strain used ferments sucrose and inulin small polymers but does not easily ferment inulin large polymers. After batch fermentation a production of 62.5 g/L ethanol and 75 g/L of sugars containing up to 94 % fructose can be obtained. A continuous fermentation was performed in a chemostat permitting the adjustment of both productions according to the dilution rate with a maximal ethanol productivity of 3.9 g/L.h.     

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.     

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae.

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.     

Spheroplast fusion of a brewing yeast strain with Saccharomyces diastaticus

Summary One haploid and one diploid strain of Saccharomyces diastaticus carrying genes responsible for glucoamylase synthesis were fused with a brewing polyploid Saccharomyces uvarum lager strain. With the spheroplast fusion technique, the ability to use dextrin and starch was introduced in the brewing yeast. Spheroplasts of the strains to be used were obtained by enzymatic digestion of the cell walls. Fusion took place in polyethylene glycol; complete cells were then regenerated in hypertonic medium containing 3% agar at 37°C. In the first fusion experiment melibiose was used as carbon source; in the second fusion experiment glycerol was employed as carbon source, for the parental Saccharomyces diastaticus diploid strain was a petite mutant. Fusion products were capable of utilizing melibiose and dextrin as carbon sources.     

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Summary An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l^-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l^-1 glucose.Similar experiments with batch cultures of certain non-fermentative yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l^-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells^-1·h^-1.     

The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts

Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of ≥20 ml air l⁻¹ mash min⁻¹ showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.     

Simultaneous saccharification and fermentation of lime-treated biomass

Simultaneous saccharification and fermentation (SSF) was performed on lime-treated switchgrass and corn stover, and oxidatively lime-treated poplar wood to determine their compatibility with Saccharomyces cerevisiae. Cellulose-derived glucose was extensively utilized by the yeast during SSF. The ethanol yields from pretreated switchgrass, pretreated corn stover, and pretreated-and-washed poplar wood were 72%, 62% and 73% of theoretical, respectively, whereas those from -cellulose were 67 to 91% of theoretical. The lower ethanol yields from treated biomass resulted from lower cellulose digestibilities rather than inhibitors produced by the pretreatment. Oxidative lime pretreatment of poplar wood increased the ethanol yield by a factor of 5.6, from 13% (untreated) to 73% (pretreated-and-washed).     

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for second-generation bioethanol production

Background

Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.

Results

Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse.

Conclusions

This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.

     

Simultaneous saccharification and fermentation of cellulose: Effect of ethanol and cellulases on particular stages

The effects of ethanol and Trichoderma reesei cellulase on the saccharification and fermentation processes as well as the tolerance of the cellulase complex for ethanol have been investigated. The studies were conducted with respect to their usefulness in the process of simulataneous saccharification and fermentation of cellulose to ethanol. The following results were obtained. (1) Fermentative activity of Kluyveromyces fragilis yeasts was gradually depressed with increasing intial ethanol concentrations and temperature of fermentation between 35–46°C. (2) Crude cellulase preparation introduced to the culture broth to a final enzyme activity of 0.5 to 2.0 FPU/ml had not distinct effect on the biomass production, ethanol yield, and glucose uptake by yeasts in 48 h fermentation at 43°C. On the other hand, only a negligible decrease in the cellulase complex activity was observed during fermentation process. (3) Saccharification of wheat straw was inhibited by at least 1% w/v ethanol. (4) The enzymes of the cellulase system showed a high stability to exposure to ethanol for 48 h at 43°C.     

Schwanniomyces: SCP and ethanol from starch

Summary Application of Schwanniomyces yeasts to single cell protein or alcohol production is feasible based on cell yields of 60% aerobically, and ethanol yields of 86% of theoretical in associative fermentation.     

Identification and characterization of yeast isolated from indonesian fermented food

Yeast strains with amylolytic activity were isolated from cassavatapé and its precursor,ragi. they were divided into two groups based on their characteristics: group 1, possessing high amylolytic activity and low ethanol yield; and group 2, possessing low amylolytic activity and high ethanol yield. The major strains of the group 1 were identified asEndomyces fibuliger, and those of group 2 were identified asPichia anomala. Based on 18S rDNA analysis, an isolate fromragi that had a high amylolytic activity was thought to be an undescribed species that was related to the basidiomycetous genera.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

Comparison of industrial yeast strains for fermentation of spent sulphite pulping liquor fortified with wood hydrolysate

Ethanol production from spent sulphite pulping liquor (SSL) was compared for four different yeasts. A second strain of S. cerevisiae as well as a 2-deoxyglucose-resistant strain formed through protoplast fusions between S. uvarum and S. diastaticus produced up to 27% more ethanol from SSL fortified with hydrolysis sugars than was produced by S. cerevisiae. The incremental improvement in ethanol yield appeared to vary with the degree of fortification, ranging from 5.8% for unfortified SSL, to 27% for the highest level of fortification tested. Decreasing fermentation rates were observed for SSL fortified with glucose, mannose and galactose, respectively. Sugar uptake rates in SSL fortified with glucose, galactose and mannose were 6.8, 2.8 and 2.0 g L⁻¹ h⁻¹, respectively. However, when these sugars were fermented along with a glucose cosubstrate, the rate at which the combined glucose/mannose medium was fermented was nearly identical to that of the glucose control. Received 18 April 1996/ Accepted in revised form 27 August 1996