Improvement of Brazilian bioethanol production – Challenges and perspectives on the identification and genetic modification of new strains of Saccharomyces cerevisiae yeasts isolated during ethanol process

In Brazil, bioethanol is produced by sucrose fermentation from sugarcane by Saccharomyces cerevisiae in a fed-batch process that uses high density of yeast cells (15–25 % of wet weight/v) and high sugar concentration (18–22 % of total sugars). Several research efforts have been employed to improve the efficiency of this process through the isolation of yeasts better adapted to the Brazilian fermentation conditions. Two important wild strains named CAT-1 and PE-2 were isolated during the fermentation process and were responsible for almost 60 % of the total ethanol production in Brazil. However, in the last decade the fermentative substrate composition was much modified, since new sugar cane crops were developed, the use of molasses instead of sugar cane juice increase and with the prohibition of burning of sugarcane prior harvest. As consequence, these previously isolated strains are being replaced by new wild yeasts in most of ethanol plants. In this new scenario the isolation of novel better adapted yeasts with improved fermentative characteristics is still a big challenge. Here, we discuss the main aspects of Brazilian ethanol production and the efforts for the selection, characterization and genetic modifications of new strains with important phenotypic traits such as thermotolerance.     

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.     

Ethanol from Sugar Cane: Flask Experiments Using the EX-FERM Technique

Alcohol production at the laboratory scale from sugar cane pieces by the EX-FERM technique was studied with 37 strains of Saccharomyces spp. The EX-FERM process is novel in that it employs the simultaneous extraction and fermentation of the sucrose in a cane-water suspension. Two types of cane treatments were used: chips and shredded pith, either fresh or dried. A mother culture of the yeast was prepared in enriched cane juice and then added to the cane-water mixture. After static fermentation for 40 h at 30°C, the cane was removed, and fresh cane was added to the yeast-alcohol broth. After an additional 24 h, the cane was again removed and the liquor was analyzed. After the first 40-h cycle, sugar consumption was above 99% with 10 of the 37 yeast strains tested, and ethanol reached levels of 1.29 to 4.00 g per 100 ml, depending on the yeast strain. The final ethanol concentration reached 4.27 to 5.37 g per 100 ml, and sugar consumption was above 98% in three cases during a second EX-FERM cycle employing previously air-dried chips and pith. Product yields were within accepted values. Cane treatment did not appear to affect the results at this level.     

Production of Fructose and Ethanol from Cane Molasses Using Saccharomyces cerevisiae ATCC 36858

The purpose of this research was to study the possibility of the production of ethanol and enriched fructose syrups from sugar cane molasses using the yeast Saccharomyces cerevisiae ATCC 36858. In batch experiments with a total sugar concentration of between 96.7 g/l and 323.5 g/l, the fructose yield was above 90% of the theoretical value. The ethanol yield and volumetric productivity were in the range of 66% and 77% of the theoretical value, and between 0.53 g ethanol/l × h and 3.15 g ethanol/l × h, respectively. The fructose fraction in the carbohydrates content of the produced syrups was more than 95% when the total initial sugar concentration in the medium was below 273.8 g/l. Some oligosaccharides and glycerol were also produced in all tested media. The maximum amount of produced oligosaccharides including raffinose accounted for 13.4 g/l in the cane molasses medium with 323.5 g/l sugars in the initial phase of the fermentation process. The oligosaccharides produced and raffinose were completely consumed by the end of the fermentation process when the total initial sugar concentration was less than 191.3 g/l. The glycerol concentration was below 9.9 g/l. These findings are useful in the production of ethanol and high fructose syrups using sugar cane molasses.     

The production of ethanol from sucrose using Zymomonas mobilis

Summary A new single-batch fermentation process for the commercial production of ethanol from refined sucrose, raw sugar, sugar cane juice and sugar cane syrup has been developed using a highly adapted and efficient strain of Zymomonas mobilis. The process gives a 94–98% sucrose hydrolysis efficiency and a 95–98% ethanol conversion efficiency. Within 24–30 h, 200 g/l sucrose is converted to produce 95.5 g/l ethanol. Reinoculation is carried out from the fermented broth without the need for centrifugation or membrane filtration.     

Ethanol production by thermotolerant yeast and its UV resistant mutants.

Six thermotolerant yeasts were isolated at 37 degrees C from over-ripe grapes by serial dilution technique using glucose yeast extract medium. Purified yeast cultures were screened for ethanol production at 37 degrees C by batch fermentation, using cane molasses containing 20% sugars. Sugar conversion efficiency of these isolates varied from 66.0 to 88.5% and ethanol productivity from 1.11 to 1.73 ml/l/h. The highest ethanol producing isolate was exposed to UV radiations and 13 mutants were picked up from the UV treatment exhibiting 0.1 to 1.0%, survival. The UV mutants varied in cell size from parent as well as among themselves. Determination of ethanol produced by all the mutants revealed that only five mutants resulted in 4.5 to 6.2% increase in sugar conversion and 8.25 to 18.56% increase in ethanol concentration coupled with maximum ethanol productivity of 2.4 ml/l/h in 48 h of batch fermentation of cane molasses (20% sugars) at 37 degrees C temperature.     

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose‐accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter‐relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone‐like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.     

A comparative study of industrial and local yeast isolates

The biochemical properties of yeasts isolated from sugary substrates such as nectar, plam wine and sugar cane and identified as strains of Saccharomyces carlsbergensis and Saccharomyces cerevisiae were compared with those of imported industrial yeasts. The results presented here show that local yeasts better convert glucose, maltose and sucrose sugars at refrigeration temperature of 8°C than the imported ones. Significant differences existed in the amount of ethanol produced by both, the local and imported yeasts. Whereas the imported brewer's yeast exhibited copper sulphate resistance varying from 3.0mM to 7.0mM, the local isolates gave copper sulphate resistance values ranging from 2.0 to 15.0mM. The local yeast isolates also grew and flocculated faster than the industrial yeasts. The results are discussed in relation to the problems of the brewing industry in Nigeria, a third world's country.     

Ethanol production from sugar cane segments in a high solids drum fermentor

Summary A successful yeast fermentation for the production of relatively high concentration of ethanol (9% w/v) was carried out using sugar cane segments. Extraction of sugar from segments occurred simultaneously with ethanol formation. The beer produced was transferred to a fresh batch of sugar cane segments and the fermentation cycle was repeated successively three times with the same beer. A high cane to water ratio was obtained in a rotating drum fermentor which allowed for a minimal amount of liquid to be used during the fermentation process.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

Isolation of new ethanol-tolerant yeasts for fuel ethanol production from sucrose

Summary New ethanol-tolerant yeast strains were isolated from crude recycled yeasts used for fuel ethanol production in the 1983 sugar cane crop. The ethanol-tolerant isolates were able to produce and tolerate ethanol above 20% (v/v) in the fermentation of sugar cane syrup.     

EX-FERM ethanol production using chipped sugarcane in packed bed fermenters

Summary The operation of packed bed fermenters for ethanol production using the EX-FERM technique with two cycles of 24 h each is described. Twelve Saccharomyces strains were tested with sugarcane particles which had been previously dried and stored as the substrate. All the strains showed acceptable sugar consumption, in some cases above 97%, and ethanol yield coefficients. Sugar consumption values differed, significantly among yeast strains for both cycles. Specific initial rates of ethanol production for the strains ranged from 0.75 to 0.82 g/g· h. Sugar extraction from the particles influenced the first 4 h period of each cycle; final sugar extraction was about 96%. The initial yeast biomass figures were low, within 2–4 g/l, and the final distribution of yeast between solids and circulating liquid varied according to the yeast strain employed. Hydrolysis of sucrose into its components was demonstrated for selected yeast strains during the EX-FERM concurrent extraction and fermentation. The results of the present study support the validity of the operation of packed bed fermenters in cycles for the EX-FERM technique, and suggest employment of smaller cane particles.     

Technological options for biological fuel ethanol

The current paradigm to produce biotechnological ethanol is to use the yeast Saccharomyces cerevisiae to ferment sugars derived from starch or sugar crops such as maize, sugar cane or sugar beet. Despite its current success, the global impact of this manufacturing model is restricted on the one hand by limits on the availability of these primary raw materials, and on the other hand by the maturity of baker's yeast fermentation technologies. Revisiting the technical, economic, and value chain aspects of the biotechnological ethanol industry points to the need for radical innovation to complement the current manufacturing model. Implementation of lignocellulosic materials is clearly a key enabler to the billion-ton biofuel vision. However, realization of the full market potential of biofuels will be facilitated by the availability of an array of innovative technological options, as the flexibility generated by these alternative processes will not only enable the exploitation of heretofore untapped local market opportunities, but also it will confer to large biorefinery structures numerous opportunities for increased process integration as well as optimum reactivity to logistic and manufacturing challenges. In turn, all these factors will interplay in synergy to contribute in shifting the economic balance in favor of the global implementation of biotechnological ethanol.     

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Role of the maltose in the simultaneous-saccharification-fermentation process from raw wheat starch and Saccharomyces cerevisiae

During the simultaneous-saccharification-fermentation from raw wheat starch, amyloglucosidase and commercial yeast, Saccharomyces cerevisiae, the fermentescible sugars profile, at the beginning of the process, plays a great role in the process regulation. From a liquefied wort, fermentescible sugars were glucose, maltose and maltotriose at concentration of 2 g/l, 40 g/l and 7 g/l, respectively. A complete hydrolysis of starch leads to a potential glucose concentration of 150 g/l. The general mechanism of a simultaneous-saccharification-fermentation occurs into two steps: the production of fermentescible sugars and the consumption of these by the yeast. In our case, maltose, dominating sugar in the wort, is the most significant sugar in the process regulation because it was substrate not only for the amyloglucosidase but also for the yeast. The maltose consumption by the yeast is repressed by the glucose, itself produced by the saccharification. We demonstrated that the apparent drop of maltose concentration in the wort acts as an activator of the amyloglucosidase and this fact allows a rapid ethanol production. The process is regulated by different interactions between glucose, maltose and maltotriose, the three sugars that, on one hand, are produced by the enzyme and on the other hand are used by the yeast.     

Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration

With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factors-that needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.     

The use of tamarind waste to improve ethanol production from cane molasses

Tamarind wastes such as tamarind husk, pulp, seeds, fruit and the effluent generated during tartaric acid extraction were used as supplements to evaluate their effects on alcohol production from cane molasses using yeast cultures. Small amounts of these additives enhanced the rate of ethanol production in batch fermentations. Tamarind fruit increased ethanol production (9.7%, w/v) from 22.5% reducing sugars of molasses as compared to 6.5% (w/v) in control experiments lacking supplements after 72 h of fermentation. In general, the addition of tamarind supplements to the fermentation medium showed more than 40% improvement in ethanol production using higher cane molasses sugar concentrations. The direct fermentation of aqueous tamarind effluent also yielded 3.25% (w/v) ethanol, suggesting its possible use as a diluent in molasses fermentations. This is the first report, to our knowledge, in which tamarind-based waste products were used in ethanol production. Received 2 April 1998/ Accepted in revised form 13 November 1998     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Doubled sugar content in sugarcane plants modified to produce a sucrose isomer

Sucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades. Recently, genes have been cloned for bacterial isomerase enzymes that convert sucrose into sugars which are not metabolized by plants, but which are digested by humans, with health benefits over sucrose. We hypothesized that an appropriate sucrose isomerase (SI) expression pattern might simultaneously provide a valuable source of beneficial sugars and overcome the sugar yield ceiling in plants. The introduction of an SI gene tailored for vacuolar compartmentation resulted in sugarcane lines with remarkable increases in total stored sugar levels. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. The lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. This remarkable step above the former ceiling in stored sugar concentration provides a new perspective into plant source–sink relationships, and has substantial potential for enhanced food and biofuel production.