Applications of yeast cell-surface display in bio-refinery

The dependency on depleting natural resources is a challenge for energy security that can be potentially answered by bioenergy. Bioenergy is derived from starchy and lignocellulosic biomass in the form of bioethanol or from vegetable oils in the form of biodiesel fuel. The acid and enzymatic methods have been developed for the hydrolysis of biomass and for transesterifiaction of plant oils. However, acid hydrolysis results in the production of unnatural compounds which has adverse effects on yeast fermentation. Recent advancements in the yeast cell surface engineering developed strategies to genetically immobilize amylolytic, cellulolytic and xylanolytic enzymes on yeast cell surface for the production of fuel ethanol from biomass. This review gives an insight in to the recent technological developments in the production of bioenergy, i.e, bioethanol using surface engineered yeast.     

The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes

Thermotolerant ethanol fermenting yeasts have been extensively used in industrial bioethanol production. However, little is known about yeast physiology under stress during bioethanol processing. This study investigated the physiological characteristics of the thermotolerant yeast Pichia kudriavzevii, strains NUNS-4, NUNS-5 and NUNS-6, under the multiple stresses of heat, ethanol and sodium chloride. Results showed that NUNS-4, NUNS-5 and NUNS-6 displayed higher growth rates under each stress condition than the reference strain, Saccharomyces cerevisiae TISTR5606. Maximum specific growth rates under stresses of heat (45°C), 15% v/v ethanol and 1·0 M sodium chloride were 0·23 ± 0·04 (NUNS-4), 0·11 ± 0·01 (NUNS-5) and 0·15 ± 0·01 h^–1 (NUNS-5), respectively. Morphological features of all yeast studied changed distinctly with the production of granules and vacuoles when exposed to ethanol, and cells were elongated under increased sodium chloride concentration. This study suggests that the three P. kudriavzevii strains are potential candidates to use in industrial–scale fermentation due to a high specific growth rate under multiple stress conditions. Multiple stress-tolerant P. kudriavzevii NUNS strains have received much attention not only for improving large-scale fuel ethanol production, but also for utilizing these strains in other biotechnological industries.     

<Emphasis Type="Italic">Cachaça</Emphasis> yeast strains: alternative starters to produce beer and bioethanol

This work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H₂S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.     

Large‐scale screening and characterisation of Lemna aequinoctialis and Spirodela polyrhiza strains for starch production

• Duckweed is considered a promising feedstock for bioethanol production due to its high biomass and starch production. Selection of duckweed strains with high starch accumulation is essential for application of duckweeds to bioethanol production. Geographic differentiation had a large influence on genetic diversity of duckweeds.
• Biomass production, starch content and starch amount in geographically isolated strains of 20 Lemna aequinoctialis and Spirodela polyrhiza were calculated to evaluate their potential for bioethanol production. The influence of different collection time, culture medium and NaCl concentration on starch accumulation of the best strains were analysed.
• The results showed that biomass production, starch content and starch production of duckweeds demonstrated clonal dependency. The best strain was L. aequinoctialis 6000, with biomass production of 15.38 ± 1.47 g m^?2, starch content of 28.68 ± 1.10% and starch production of 4.39 ± 0.25 g m^?2. Furthermore, starch content of L. aequinoctialis 6000 was highest after 8 h of light, tap water was the best medium for starch induction, and NaCl did not induce starch accumulation.
• This study suggests duckweed biomass production and starch production demonstrate clonal dependency, indicating that extensive clonal comparisons will be required to identify the most suitable isolates for duckweed selective breeding for bioethanol.

     

Bioethanol production from the hydrolysate of rape stem in a surface-aerated fermentor

In this study, we investigated the feasibility of producing bioethanol from the hydrolysate of rape stem. Specifically, the most ideal yeast strain was screened, and the microaeration was performed by surface aeration on a liquid medium surface. Among the yeast strains examined, Pichia stipitis CBS 7126 displayed the best performance in bioethanol production during the surface-aerated fermentor culture. Pichia stipitis CBS 7126 produced maximally 9.56 g/l of bioethanol from the initial total reducing sugars (about 28 g/l). The bioethanol yield was 0.397 (by the DNS method). Furthermore, this controlled surface aeration method holds promise for use in the bioethanol production from the xylose-containing lignocellulosic hydrolysate of biomass.     

Presence of proline has a protective effect on weak acid stressed Saccharomyces cerevisiae

Fermentation of sugars released from lignocellulosic biomass (LCMs) is a sustainable option for the production of bioethanol. LCMs release fermentable hexose sugars and the currently non-fermentable pentose sugars; ethanol yield from lignocellulosic residues is dependent on the efficient conversion of available sugars to ethanol, a side-product of the process is acetic acid production. Presence of acetic acid reduced metabolic output and growth when compared with controls; however, it was observed that incubation with proline had a protective effect, which was proline specific and concentration dependent; the protective effect did not extend to furan or phenolic stressed yeast cells. Proline accumulating strains displayed tolerance to acetic acid when compared with background strains, whereas, strains with a compromised proline metabolism displayed sensitivity. Sensitivity to weak acids appears to be reduced with the addition of proline; proline is an imino acid freely available as a nitrogen source in the aerobic phase of fermentations. Yeast strains with higher intracellular proline concentrations would be desirable for industrial bioethanol fermentations.     

Scientific challenges of bioethanol production in Brazil

Bioethanol (fuel alcohol) has been produced by industrial alcoholic fermentation processes in Brazil since the beginning of the twentieth century. Currently, 432 mills and distilleries crush about 625 million tons of sugarcane per crop, producing about 27 billion liters of ethanol and 38.7 million tons of sugar. The production of bioethanol from sugarcane represents a major large-scale technology capable of producing biofuel efficiently and economically, providing viable substitutes to gasoline. The combination of immobilization of CO₂ by sugarcane crops by photosynthesis into biomass together with alcoholic fermentation of this biomass has allowed production of a clean and high-quality liquid fuel that contains 93% of the original energy found in sugar. Over the last 30 years, several innovations have been introduced to Brazilian alcohol distilleries resulting in the improvement of plant efficiency and economic competitiveness. Currently, the main scientific challenges are to develop new technologies for bioethanol production from first and second generation feedstocks that exhibit positive energy balances and appropriately meet environmental sustainability criteria. This review focuses on these aspects and provides special emphasis on the selection of new yeast strains, genetic breeding, and recombinant DNA technology, as applied to bioethanol production processes.     

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.     

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.     

Mechanisms of weak acid-induced stress tolerance in yeasts: Prospects for improved bioethanol production from lignocellulosic biomass

Weak acids are known to have a negative impact on yeast performance, restraining production efficiency during the production of bioethanol and other fermentative yeast-derived products. These acids, which might be hydrophilic or lipophilic exert negative effects on yeasts when they diffuse into the cell in their unionized state as a result of their pH being lower than the pka of yeast growth medium. Consequently, the unionized acids dissociate into their respective cations and anions, as intracellular pH is typically neutral. Further, proton accumulation tends to reduce intracellular pH. As a result, the anions destabilize the internal cell machinery, thus affecting cellular metabolism on various levels. Overcoming this acid-mediated stress in budding yeast would in part, harness the potential of using lignocellulosic biomass hydrolysate – which is typically acetic acid-rich – as a cheaper feedstock for large-scale bioethanol production. Since organic acids are key intermediates in ethanol fermentation, this review focuses on the prospects of bioethanol production from lignocellulosic biomass using weak acid-tolerant strains of yeasts derived by metabolic engineering.     

酵母生物多样性开发及工业应用

酵母是一类包括酿酒酵母和非常规酵母在内的多种单细胞真菌的总称,其中酿酒酵母是应用较多的重要工业微生物,广泛应用于生物医药、食品、轻工和生物燃料生产等不同生物制造领域.近年来,研究者从不同生态环境中分离了大量的酵母菌株,鉴定了多个新种,也发现了抗逆性不同以及具有多种活性产物合成能力的菌株,证明天然酵母资源具有丰富的生物多...     

Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.     

Identification of glycolaldehyde as the key inhibitor of bioethanol fermentation by yeast and genome-wide analysis of its toxicity

Degradation of lignocellulose with pressurised hot water is an efficient method of bioethanol production. However, the resultant solution inhibits ethanol fermentation by Saccharomyces cerevisiae. Here, we first report that glycolaldehyde, which is formed when lignocellulose is treated with pressurised hot water, inhibits ethanol fermentation. The final concentration of glycolaldehyde formed by the treatment of lignocellulose with pressurised hot water ranges from 1 to 24 mM, and 1–10 mM glycolaldehyde was sufficient to inhibit fermentation. This result indicates that glycolaldehyde is one of the main substances responsible for inhibiting fermentation after pressurised hot water degradation of lignocellulose. Genome-wide screening of S. cerevisiae revealed that genes encoding alcohol dehydrogenase, methylglyoxal reductase, polysomes, and the ubiquitin ligase complex are required for glycolaldehyde tolerance. These novel findings will provide new perspectives on breeding yeast for bioethanol production from biomass treated with pressurised hot water.     

Autochthonous fermentation starters for the industrial production of Negroamaro wines

The aim of the present study was to establish a new procedure for the oenological selection of Saccharomyces cerevisiae strains isolated from natural must fermentations of an important Italian grape cultivar, denoted as “Negroamaro”. For this purpose, 108 S. cerevisiae strains were selected as they did not produce H₂S and then assayed by microfermentation tests. The adopted procedure made it possible to identify 10 strains that were low producers of acetic acid and hydrogen sulphide and showed that they completed sugar consumption during fermentation. These strains were characterized for their specific oenological and technological properties and, two of them, strains 6993 and 6920, are good candidates as industrial starter cultures. A novel protocol was set up for their biomass production and they were employed for industrial-scale fermentation in two industrial cellars. The two strains successfully dominated the fermentation process and contributed to increasing the wines’ organoleptic quality. The proposed procedure could be very effective for selecting “company-specific” yeast strains, ideal for the production of typical regional wines. “Winery” starter cultures could be produced on request in a small plant just before or during the vintage season and distributed as a fresh liquid concentrate culture.     

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Background  

Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials.     

Enhanced fermentative capacity of yeasts engineered in storage carbohydrate metabolism

During yeast biomass production, cells are grown through several batch and fed‐batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench‐top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic capacity of yeast which can be an interesting application for the food industry. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:20–24, 2015     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.