Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

Isolation and Screening of Yeasts That Ferment d-Xylose Directly to Ethanol

Natural habitats of yeasts were examined for the presence of strains able to produce ethanol from d-xylose. Black knots, insect frass, and tree exudates were screened by enrichment in liquid d-xylose-yeast extract medium. These and each d-xylose-assimilating yeast in a collection from cactus fruits and Drosophila spp. were tested for alcohol production from this sugar. Among the 412 isolates examined, 36 produced more than 1 g of ethanol liter from 20 g of d-xylose liter, all under aerated conditions. Closer examination of the strains indicated that their time courses of d-xylose fermentation followed different patterns. Some strains produced more biomass than ethanol, and among these, ethanol may or may not be assimilated rapidly after depletion of d-xylose. Others produced more ethanol than biomass, but all catabolized ethanol after carbohydrate exhaustion. Ethanol production appeared best at low pH values and under mild aeration. Possible correlations between the nutritional profiles of the yeasts and their ability to produce ethanol from d-xylose were explored by multivariate analysis. d-Xylose appeared slightly better utilized by yeasts which rate poorly in terms of fermentation. The fermentation of d-glucose had no bearing on d-xylose fermentation. No specific nutritional trait could discriminate well between better d-xylose fermentors and other yeasts.     

Ethanol production from d-galactose and glycerol by Pachysolen tannophilus

Pachysolen tannophilus has recently been shown to be able to convert d-xylose, a pentose, to ethanol. Previously, d-xylose had been considered to be nonfermentable by yeasts. The present study shows that the organism can be used to obtain ethanol from other carbohydrates previously considered as nonfermentable, either by P. tannophilus in particular, d-galactose, or by yeasts in general, glycerol. Such identification for d-galactose allows P. tannophilus to be considered for fermentation of four of the five major plant monosaccharides: d-glucose, d-mannose, d-galactose and d-xylose. The ability to ferment glycerol is of potential use, in part, for the conversion of glycerol derived from algae into ethanol.     

Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce extracellular cellulase [see 1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

d-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae

We used commercial bakers' yeast (Saccharomyces cerevisiae) to study the conversion of d-xylulose to ethanol in the presence of d-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for d-xylulose fermentation was 35 degrees C, and the optimal pH range was 4 to 6. The fermentation of d-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of d-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of d-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from d-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield.     

Alcoholic Fermentation of d-Xylose by Yeasts

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.     

Fermentation of d-Xylose and l-Arabinose to Ethanol by Erwinia chrysanthemi

Erwinia spp. are gram-negative facultative anaerobes within the family Enterobacteriacae which possess several desirable traits for the conversion of pentose sugars to ethanol, such as the ability to ferment a broad range of carbohydrates and the ease with which they can be genetically modified. Twenty-eight strains of Erwinia carotovora and E. chrysanthemi were screened for the ability to ferment d-xylose to ethanol. E. chrysanthemi B374 was chosen for further study on the basis of its superior (4%) ethanol tolerance. We have characterized the fermentation of d-xylose and l-arabinose by the wild type and mutants which bear plasmids containing the pyruvate decarboxylase gene from Zymomonas mobilis. Expression of the gene markedly increased the yields of ethanol (from 0.7 up to 1.45 mol/mol of xylose) and decreased the yields of formate, acetate, and lactate. However, the cells with pyruvate decarboxylase grew only one-fourth as fast as the wild type and tolerated only 2% ethanol. Alcohol tolerance was stimulated by the addition of yeast extract to the growth medium. Xylose catabolism was characterized by a high saturation constant K(s) (4.5 mM).     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I – Yeasts in general

There are a number of process advantages which could be exploited through the use of thermophilic microorganisms for ethanol production. Energy savings through reduced cooling costs, higher saccharification and fermentation rates, continuous ethanol removal and reduced contamination have stimulated a search for routes to thermophilic or thermotolerant yeasts. These routes have included screening existing culture collections, temperature adaptation, mutagenesis and molecular techniques and finally isolating new strains. Varying success has been achieved, however, the most thermotolerant yeasts have come from fresh isolations from environments which experience high temperatures. Thermotolerant yeasts have been investigated for the following potential applications: simultaneous saccharification and fermentation of cellulose, where the high fermentation temperature allows more rapid and efficient enzymatic cellulose hydrolysis; whey fermentation, where high salt and low fermentable substrate concentrations make conditions difficult; and fermentation of D-xylose and cellobiose, which is essential for efficient conversion of woody biomass to ethanol. Ethanol and temperature tolerance are important characteristics for commercial yeast strains. Both characteristics are interactive and generally decrease with increasing temperature and ethanol concentration. Considerable research has been directed towards investigation of fatty acid composition changes in response to these stresses and the role of heat shock proteins in tolerance mechanisms. If thermotolerant yeasts are to be used in commercial processes, bioreactor configuration will play an important part in the design of production processes. Batch and fed-batch systems have been shown to be useful in some circumstances as have continuous flow systems, however, some of the newly isolated thermotolerant yeasts such as Kluyveromyces marxianus do not show the high growth rate under anaerobic conditions that is characteristic of Saccharomyces cerevisiae. Various immobilization techniques appear to offer a means of presenting and maintaining high biomass in anaerobic continuous flow reactors.     

Effects of borate on isomerization and yeast fermentation of high xylulose solution and acid hydrolysate of hemicellulose

d-Xylose has been isomerized by immobilized d-glucose isomerase (EC nomenclature is now d-xylose isomerase, d-xylose ketol-isomerase, EC 5.3.1.5; EC 5.3.1.18 is a deleted EC entry). Temperature has a profound influence on the equilibrium concentration of d-xylulose. When 1 md-xylose was isomerized in the presence of various concentrations of borate, maximum conversion (80%) was observed at 0.2 m sodium tetraborate. Temperature (40–69°C) and pH (6.0–7.5) had an insignificant effect on the equilibrium when borate was present. d-Xylose (0.5 m) was isomerized by d-glucose isomerase in the presence of various concentrations of sodium tetraborate (0.0125–0.25 m). Based on the initial rate of ethanol production and the fraction of total sugar converted into ethanol after 24 h of yeast fermentation, an optimum tetraborate concentration of 0.05 m was determined for both isomerization and fermentation. At an acidic pH, the rate of fermentation was faster than at neutral pH when borate was included in the d-xylose—d-xylulose system. Acid hydrolysate of bagasse hemicellulose could not be fermented at a pH lower than 5. Therefore, a compromise condition, pH 6.0, was chosen for fermentation.     

Purification and characterization of the d-xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene and the d-xylulokinase (ATP: d-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The d-xylose isomerase gene was separated from the d-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The d-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the d-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first d-xylose isomerase gene that has been isolated and extensively purified from any organism. d-Xylose isomerase, the enzyme product of the d-xylose isomerase gene, is responsible for the conversion of d-xylose to d-xylulose, as well as d-glucose to d-fructose. It is widely believed that yeast cannot ferment d-xylose to ethanol primarily because of the lack of d-xylose isomerase in yeast. d-Xylose isomerase (also known as d-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the d-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting d-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Potentialities and limitations of direct alcoholic fermentation of starchy material with amylolytic yeasts

Summary The fermentation characteristics of a large number of starch-degrading yeasts were compared. None of the amylolytic yeasts currently recognized, appear to be entirely suitable for direct alcoholic fermentation of starchy biomass. The species capable of extensive starch hydrolysis produce only low amounts of ethanol from glucose and dextrin, one of the major limitations being their low ethanol tolerances. Some of the less-active yeasts have much better glucosefermentation characteristics, but dextrin conversion is limited probably due to the nature of their enzyme systems. Using an -amylase dextrin (22.5% w/v), ethanol yields of about 70% were obtained with Saccharomyces diastaticus strains. Through associative fermentation of S. diastaticus and other selected amylolytic yeasts slightly better yields, however not exceeding 80%, were obtained.     

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

Fermentation of d-Xylose to Ethanol by Genetically Modified Klebsiella planticola

d-Xylose is a plentiful pentose sugar derived from agricultural or forest residues. Enteric bacteria such as Klebsiella spp. ferment d-xylose to form mixed acids and butanediol in addition to ethanol. Thus the ethanol yield is normally low. Zymomonas spp. and most yeasts are unable to ferment xylose, but they do ferment hexose sugars to ethanol in high yield because they contain pyruvate decarboxylase (EC 4.1.1.1), a key enzyme that is absent from enteric bacteria. This report describes the fermentation of d-xylose by Klebsiella planticola ATCC 33531 bearing multicopy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene markedly increased the yield of ethanol to 1.3 mol/mol of xylose, or 25.1 g/liter. Concurrently, there were significant decreases in the yields of formate, acetate, lactate, and butanediol. Transconjugant Klebsiella spp. grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even in the absence of selective pressure by antibiotics to maintain the plasmid. Ethanol production was 31.6 g/liter from 79.6 g of mixed substrate per liter chosen to simulate hydrolyzed hemicellulose. The physiology of the wild-type of K. planticola is described in more detail than in the original report of its isolation.     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.     

Kinetic studies of the enzymatic isomerization of xylose

d-Xylose isomerase catalyses the conversion of the common pentose, d-xylose, to its keto-isomer, d-xylose. This reaction is of interest because many microorganisms that are unable to metabolize d-xylose can utilize d-xylulose. The kinetics of a commonly used immobilized whole-cell isomerase, Sweetzyme Q, have been determined from initial rate studies on the forward and reverse reactions. The effect of pH, temperature, and substrate and product concentration on enzyme activity have all been examined. Reaction rates were modelled with the Michaelis-Menten equation. Using constants determined from Lineweaver-Burk plots, the rate equation accurately simulated experimental conversion data.     

Changes in microflora and biochemical composition during the Baceman stage of traditional indonesian Kecap (soy sauce) production

Indonesian soy sauce (kecap) is made from black soybeans in a traditional way which involves two microbiological stages: a solid-state fermentation and a brine fermentation. This study is concerned with the brine fermentation, called baceman. Samples from different kecap producers were analyzed for (bio)chemical content and micro-organisms. It was found that the final composition of the baceman differed from manufacturer to manufacturer, and even within companies large differences were found in microflora and the amounts of fermentation products, formol nitrogen and salt concentration. The main fermentation products were lactate, acetate, glycerol and ethanol. Pediococcus halophilus, staphylococci, a coryneform bacterium and yeasts belonging to Candida, Debaromyces and Sterigmatomyces were isolated from the brines. Compared to Japanese soy sauce production, fermentation by yeasts does not play an important role in Indonesian kecap production. This is due to the fact that kecap is made from whole soybeans only, which are poor in sugars. After fermentation by P. halophilus no substrates are left for growth and ethanol production by yeasts. The presence of film forming yeasts can even lead to spoilage of the product.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

Pervaporation behavior and integrated process for concentrating lignocellulosic ethanol through polydimethylsiloxane (PDMS) membrane

The effects of by-products from ethanol fermentation and hydrolysates of lignocelluloses on ethanol diffusion through polydimethylsiloxane (PDMS) membranes with/without silicalite-1 were investigated. A pervaporation process was integrated with lignocellulosic fermentation to concentrate bioethanol using bare PDMS membranes. Results showed that yeasts, solid particles, and salts increased ethanol flux and selectivity through the membranes (PDMS with/without silicalite-1), whereas glucose exerted negative effects on the performance. On bare PDMS membrane, the performance was not obviously affected by the existence of aliphatic acids. However, on PDMS-silicalite-1 membrane, a remarkable decrease in ethanol selectivity and a rapid growth of total flux in the presence of aliphatic acids were observed. These phenomena were due to the interaction of acids with silanol (Si–OH) groups to break the dense membrane surface. On the PDMS membranes with/without silicalite-1, degradation products of lignocellulosic hydrolysates such as furfural and hydroxyacetone slightly influenced separation performance. These results revealed that an integrated process can effectively eliminate product inhibition, improve ethanol productivity, and enhance the glucose conversion rate.     

Ethanol production from pentoses by immobilized microorganisms

The capabilities of immobilized Fusarium oxysporum f. sp. lini, Mucor sp., and Saccharomyces cerevisiae in fermenting pentose to ethanol have been compared. S. cerevisiae was found to have the best fermentation rate on d-xylulose of 0.3 g l^?1 h^?1. By using a separate isomerase column for converting d-xylose to d-xylulose and a yeast column for converting d-xylulose to ethanol, an ethanol concentration of 32 g l^?1 was obtained from 10% d-xylose. The ethanol yield was calculated to be 64% of the theoretical yield.