Challenges and opportunities in improving the production of bio-ethanol

Optimizing bioethanol production from sweet sorghum stem juice under very high gravity fermentation and temperature stress conditions

Background: The energy crisis and climate change necessitate studying and discovering of new processes involved in the production of alternative and renewable energy sources. Very high gravity (VHG) fermentation is one such process improvement aimed at increasing both the rate of fermentation and ethanol concentration. The technology involves preparation and fermentation of media containing 300 g or more of dissolved solids per liter to get a high amount of ethanol. Findings: Saccharomyces cerevisiae was inoculated to the very high gravity medium containing 30% to 40% w/v glucose with and without supplementation of three selected fruit pulps (mango, banana, and sapota). The fermentation experiments were carried out in batch mode. The effect of supplementation of 4% fruit pulp/puree on the metabolic behavior and viability of yeast was studied. Significant increase in ethanol yields up to 83.1% and dramatic decrease in glycerol up to 35% and trehalose production up to 100% were observed in the presence of fruit pulp. The fermentation rate was increased, and time to produce maximum ethanol was decreased from 5 to 3 days with increased viable cell count. The physical and chemical factors of fruit pulps may aid in reducing the osmotic stress of high gravity fermentation as well as enhanced ethanol yield. Conclusions: It was found that fruit pulp supplementation not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. Production of high ethanol concentration by the supplementation of cheap materials in VHG sugar fermentation will eliminate the expensive steps in the conventional process and save time.

Tannin extraction pretreatment and very high gravity fermentation of acorn starch for bioethanol production

DNA microarray analysis was used to investigate the expression profile ofSaccharomyces cerevisiae genes in glycolysis pathway, trehalose and steroid biosynthesis and heat shock proteins (HSP) in response to harsh environment under the late stage of very high gravity (VHG) fermentation. The data show that only a few genes (GPM2, PGM1, GAL10 and PGM1) involved in glycolysis pathway and trehalose biosynthesis were up-regulated. Five genes that encode heat shock proteins (HSP26, HSP10, HSP42, HSP78 and HSP82) were up-regulated. Among these five genes, there was a strong expression increase of about 84-fold for HSP26.The results of this study revealed adverse VHG fermentation conditions stress response pattern and suggested interesting information about the mechanisms involved in adaptation of cells to the complex VHG fermentation environment. This identification of genes provides information that will help to genetically modify yeast to further maintain the fermentation fitness and improve its fermentation capacity and process. Key words: DNA microarray, gene expression, yeast, stress, very high gravity (VHG) fermentation.

High ethanol production under optimal aeration conditions and yeast composition in a very high gravity fermentation from sweet sorghum juice by Saccharomyces cerevisiae

During fermentation, yeast cells encounter a number of stresses, including hyperosmolarity, high ethanol concentration, and high temperature. Previous deletome analysis in the yeast Saccharomyces cerevisiae has revealed that SOD1 gene encoding cytosolic Cu/Zn-superoxide dismutase (SOD), a major antioxidant enzyme, was required for tolerances to not only oxidative stress but also other stresses present during fermentation such as osmotic, ethanol, and heat stresses. It is therefore possible that these fermentation-associated stresses may also induce endogenous oxidative stress. In this study, we show that osmotic, ethanol, and heat stresses promoted generation of intracellular reactive oxygen species (ROS) such as superoxide anion in the cytosol through a mitochondria-independent mechanism. Consistent with this finding, cytosolic Cu/Zn-SOD, but not mitochondrial Mn-SOD, was required for protection against oxidative stress induced by these fermentation-associated stresses. Furthermore, supplementation of ROS scavengers such as N-acetyl-L-cysteine (NAC) alleviated oxidative stress induced during very high gravity (VHG) fermentation and enhanced fermentation performance at both normal and high temperatures. In addition, NAC also plays an important role in maintaining the Cu/Zn-SOD activity during VHG fermentation. These findings suggest the potential role of ROS scavengers for application in industrial-scale VHG ethanol fermentation.

In very high gravity (VHG) fermentation, yeast cells are subjected to a multitude of challenging conditions, including the osmotic pressure exerted by the high sugar content of the wort and the stress factors associated with the high ethanol concentrations present at the end of the fermentation cycle. The response of this biological system to abiotic stresses may be enhanced through biochemical and physiological routes. Silica may play a significant role in regulating the cellular homeostasis of yeast. Alternatively, it is expected that this outcome may be achieved through biochemical responses from the effects of vitamins on yeast cells and the physiological yeast route changing by the culture medium aeration. The objective of this study was to investigate the effects of adding 500 mg L−1 of silica on corn ethanol wort medium and the possibility of supplementing the same wort with vitamins alongside aeration (0.2 v v−1 min−1) as an alternative resource to sustain the fermentation yield rather than adding silica in a fed-batch fermentation cycle with yeast recycling. Upon completion of the five fermentation cycles, yeast samples subjected to the treatment with the addition of silica exhibited a 3.1% higher fermentation yield in comparison to the results observed in the vitamins plus aeration medium bath. Even though greater biomass production (19.1 g L−1) was observed through aerobic yeast behavior in vitaminized supplemented corn medium, the provided silica had a more beneficial effect on yeast stress relief for very high gravity fermentation in a corn hydrolyzed wort with cell recycling.

Ethanol production from mixtures of sweet sorghum juice and sorghum starch using very high gravity fermentation with urea supplementation

There have been numerous developments in ethanol fermentation technology since the beginning of the new millennium as ethanol has become an immediate viable alternative to fast-depleting crude reserves as well as increasing concerns over environmental pollution. Nowadays, although most research efforts are focused on the conversion of cheap cellulosic substrates to ethanol, methods that are cost-competitive with gasoline production are still lacking. At the same time, the ethanol industry has engaged in implementing potential energy-saving, productivity and efficiency-maximizing technologies in existing production methods to become more viable. Very high gravity (VHG) fermentation is an emerging, versatile one among such technologies offering great savings in process water and energy requirements through fermentation of higher concentrations of sugar substrate and, therefore, increased final ethanol concentration in the medium. The technology also allows increased fermentation efficiency, without major alterations to existing facilities, by efficient utilization of fermentor space and elimination of known losses. This comprehensive research update on VHG technology is presented in two main sections, namely VHG brewing, wherein the effects of nutrients supplementation, yeast pitching rate, flavour compound synthesis and foam stability under increased wort gravities are discussed; and VHG bioethanol fermentation studies. In the latter section, aspects related to the role of osmoprotectants and nutrients in yeast stress reduction, substrates utilized/tested so far, including saccharide (glucose, sucrose, molasses, etc.) and starchy materials (wheat, corn, barley, oats, etc.), and mash viscosity issues in VHG bioethanol production are detailed. Thereafter, topics common to both areas such as process optimization studies, mutants and gene level studies, immobilized yeast applications, temperature effect, reserve carbohydrates profile in yeast, and economic aspects are discussed and future prospects are summarized.

Viscosity reduction of cassava for very high gravity ethanol fermentation using cell wall degrading enzymes from Aspergillus aculeatus

High gravity (HG) and very high gravity (VHG) fermentations are increasingly attractive within the brewing industry as a means of optimizing process efficiency and energy-saving. However, the use of highly concentrated worts is concomitant with a number of biological stress factors that can impact on yeast quality and fermentation performance. In order to eliminate or reduce potentially detrimental effects, brewing yeast respond to their environment by shunting carbon into different metabolic end products, which assist in the protection of cells, but also impact on final ethanol yield. The purpose of this research was to investigate the impact of substrate sugar concentration on carbon partitioning in brewing fermentations. This was conducted using a series of laboratory-scale fermentations with worts of 13°P, 18°P and 24°P, pitched using lager and ale yeast strains. Fermentation performance was assessed with respect to the uptake of wort sugars and the production of key carbon-based metabolites, leading to a calculation of yeast central carbon flux. Analysis of carbon assimilation and dissimilation revealed that changes in intracellular trehalose, glycogen, higher alcohols and esters were observed, however the production of yeast biomass acted as the major trade-off with ethanol production. The data presented here shows for the first time the requirements of yeast populations during HG and VHG conditions and the factors that have a major impact on key performance indicators. This data has major significance for fermentation-based industries globally and is especially important for those sectors seeking to maximize yield from existing resources through high gravity fermentations.

The changes in the proton efflux rate (PER) during fermentation of normal gravity (NG), high gravity (HG) and very high gravity (VHG) wort by a lager yeast (Saccharomyces pastorianus) were monitored using an optimized PER test method. The values of the proton efflux rate in S. pastorianus decreased with increasing initial wort gravity. Moreover, the difference in the proton efflux rate values at the beginning of the fermentation was lower than at the end of fermentation from normal gravity to very high gravity brewing. These results demonstrated that the proton efflux rate in S. pastorianus was inhibited in the later stages of high gravity and very high gravity brewing. Furthermore, the changes of the proton efflux rate in S. pastorianus under the high ethanol concentration conditions appeared to depend on the concentration of ethanol in the fermentation liquid. A better negative correlation (P > 0.001, r = −0.95) between the ethanol concentration at >4% (w/v) and the proton efflux rate was found. The changes of the proton efflux rate in the cells treated with exogenous ethanol confirmed that higher concentrations of ethanol could significantly inhibit proton efflux in S. pastorianus. This study offers a possible way to monitor and explain the performance of yeast in the complex environment of high gravity and very high gravity brewing.

Grain pearling and very high gravity (VHG) fermentation technologies for fuel alcohol production from rye and triticale

BackgroundThe optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions.ResultsChemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of Bud31 and Hpr1 was found to lead to the increase of both ethanol yield and fermentation rate, while Pho85, Vrp1 and Ygl024w expression is required for maximal ethanol production in VHG fermentations. Five genes, Erg2, Prs3, Rav1, Rpb4 and Vma8, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate.ConclusionsThe identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers.

Bioethanol fermentation under very high gravity (VHG) conditions by using sago hampas hydrolysate (SHH) supplemented with yeast extract as a fermentation medium has resulted in an incomplete metabolism of glucose, leading to relatively low bioethanol production in comparison to the theoretical titre. Therefore, additional supplementation of the fermentation medium is necessary to increase the yeast tolerance towards inhibitors and high concentration of glucose and bioethanol. This work investigates the effect of supplementing SHH media with various nutrients on bioethanol fermentation under VHG conditions. The nutrients included magnesium sulphate (0.12 g/L), urea (3 g/L), glutamic acid (5 g/L), and peptone (5 g/L). Our results showed that culture supplemented with peptone has significantly improved the yeast growth by 0.9-fold and glucose consumption efficiency by 10% compared to the control cultures. Besides that, the media formulation has also increased bioethanol production by 13%, with a maximum concentration of 126.20 ± 3.0 g/L. In general, the results suggest an improved formulation of fermentation medium consisting of SHH for bioethanol production under VHG conditions. These results will provide useful insights into the development of bioethanol production from sago-based feedstock in the future.