High Gravity Fermentation Research Articles

Substrate and Product Inhibition on Yeast Performance in Ethanol Fermentation

A batch fermentation utilizing Saccharomyces cerevisiae BY4742 was conducted to determine the inhibitory effects of highly concentrated substrate and product levels on yeast. Experiments were performed to determine the largest dosage of substrate and the largest product concentration that the yeast could tolerate in a very high gravity fermentation process. The yeast’s growth and fermentation activities were characterized by changes in the biomass and ethanol yield under different substrate and product concentrations during fermentation. All of the experiments were performed at a pH of 5.0 and a temperature of 35 °C with a stirring rate of 180 r/min and a fermentation time of 96 h. Furthermore, five cycles of acclimatization were conducted to improve the yeast’s tolerance to ethanol. Ethanol yield was maximized at 95% with a product concentration of 39 g/L and substrate dosage of 80 g/L. The system exhibited an obvious increase in cell growth and ethanol production with increasing substrate dosage up to a...

Assessing the potential of wild yeasts for bioethanol production.

Bioethanol fermentations expose yeasts to a new, complex and challenging fermentation medium with specific inhibitors and sugar mixtures depending on the type of carbon source. It is, therefore, suggested that the natural diversity of yeasts should be further exploited in order to find yeasts with good ethanol yield in stressed fermentation media. In this study, we screened more than 50 yeast isolates of which we selected five isolates with promising features. The species Candida bombi, Wickerhamomyces anomalus and Torulaspora delbrueckii showed better osmo- and hydroxymethylfurfural tolerance than Saccharomyces cerevisiae. However, S. cerevisiae isolates had the highest ethanol yield in fermentation experiments mimicking high gravity fermentations (25 % glucose) and artificial lignocellulose hydrolysates (with a myriad of inhibitors). Interestingly, among two tested S. cerevisiae strains, a wild strain isolated from an oak tree performed better than Ethanol Red, a S. cerevisiae strain which is currently commonly used in industrial bioethanol fermentations. Additionally, a W. anomalus strain isolated from sugar beet thick juice was found to have a comparable ethanol yield, but needed longer fermentation time. Other non-Saccharomyces yeasts yielded lower ethanol amounts.

Effect of fruit pulp supplementation on rapid and enhanced ethanol production in very high gravity (VHG) fermentation

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.

Production of ethanol through very high gravity fermentation by Saccharomyces cerevisiae wild strains

Production of ethanol through very high gravity fermentation by Saccharomyces cerevisiae wild strains

Effects of magnesium ions on both VHG batch and continuous fruit wine fermentations

In Poland, fruit wines or aromatized fruit wines are semi-sweet or sweet and contain approximately 15–16% (v/v) ethanol. Their production can be classified as a very high-gravity (VHG) fermentation. Magnesium has a beneficial effect on VHG fermentations as it protects the yeast cells against ethanol, osmotic and temperature stress. The effect of the magnesium concentration in an apple must, containing 32% sugars, on the fermentative parameters of batch and continuous fermentations was assessed. In the batch process, a magnesium concentration of ~8.5 mg/L resulted in decreased the ethanol production in comparison to a magnesium concentration of ~250, 490 and 970 mg Mg2+/L. The highest amount of Mg also caused a metallic taste. A continuous fermentation was carried out for 2.5 months in a four-column packed-bed fermentor. The medium contained ~50, 250 and 490 mg Mg2+/L and the yeast was immobilized on foam glass. During the continuous fermentation, no differences at p ≤ 0.05 in terms of fermentative parameters were seen with magnesium additions. The same beginning amount of magnesium ions in the medium led to a similar use of this element, both in batch and in continuous fermentation. The more Mg2+ that was present in the medium, the more Mg2+ was used by the yeast. The results suggest that the minimal dose of magnesium, under the described conditions, is 50 mg/L, corresponding to the amount of Mg in the medium prepared using concentrated apple juice and tap water. This finding has industrial significance, as Polish wine companies prepare their fruit musts using tap water. Copyright © 2014 The Institute of Brewing & Distilling

Optimization of very high gravity fermentation process for ethanol production from industrial sugar beet syrup

In order to reduce production costs and environmental impact of bioethanol from sugar beet low purity syrup 2, an intensification of the industrial alcoholic fermentation carried out by Saccharomyces cerevisiae is necessary. Two fermentation processes were tested: multi-stage batch and fed-batch fermentations with different operating conditions. It was established that the fed-batch process was the most efficient to reach the highest ethanol concentration. This process allowed to minimize both growth and ethanol production inhibitions by high sugar concentrations or ethanol. Thus, a good management of the operating conditions (initial volume and feeding rate) could produce 15.2% (v/v) ethanol in 53 h without residual sucrose and with an ethanol productivity of 2.3 g L h−1.

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

Cassava is an important feedstock for bioethanol production; however, its use in very high gravity (VHG) fermentation is limited by the high viscosity of mash due to the presence of plant cell wall derived polysaccharides. In this study, viscosity reduction of cassava root mash, chips, and pulp was achieved using a mixture of cell wall degrading enzymes prepared from solid state fermentation of Aspergillus aculeatus BCC17849. Proteomic analysis showed the mixture contained endo- and exo-acting cellulases, hemicellulases, and pectinases from various glycosyl hydrolase families. Enzymatic pretreatment of cassava substrates with the enzyme mixture containing endoglucanase, FPase, xylanase, polygalacturonase, β-glucanase, and mannanase activities at 45°C, pH 5.0 for 2h reduced viscosity to the operating level of <500mPas, equivalent to the final viscosity of 3.0–51.3% of the initial levels. Simultaneous saccharification and fermentation of the pretreated root mash (32% initial solid) by Saccharomyces cerevisiae with a conventional high-temperature liquefaction process and an uncooked process using a raw starch degrading amylase at 32°C for 96h led to a final ethanol concentration of 19.65 and 17.54% (v/v), respectively. The results demonstrated potential of the enzyme for improving process efficiency and economics in VHG bioethanol production.

Comparison of strategies to overcome the inhibitory effects in high-gravity fermentation of lignocellulosic hydrolysates

High-gravity (HG) technology aims at generating final ethanol concentrations above 50 kg m−3 in order to reduce the cost of the distillation step. The generation of higher amounts of inhibitors during the pretreatment step is one of the challenges that accompany the increase in initial dry matter. Detoxification of spruce hydrolysate, adaptation of the cells before fermentation, supplementation with nutrients, and washing of solids were the strategies compared in this study. They represent different approaches to cope with the inhibitory effects, and we compared their efficiencies using a thermotolerant strain of Saccharomyces cerevisiae at temperatures from 30 °C up to 40 °C.The dilute acid-pretreated spruce used as substrate in this study was not fermentable under HG conditions (200 g kg−1 water-insoluble solids) when no improvement method was applied. In HG simultaneous saccharification and fermentation at 30 °C combined with a 24 h pre-hydrolysis step, the detoxification of pretreated spruce with reducing agent (Na2S2O4) gave the best result with an ethanol yield of 57% (on total sugars) of the maximum theoretical and a volumetric productivity of 1.58 g dm−3 h−1. In HG separate hydrolysis and fermentation, nutrients supplementation gave better final ethanol yields than detoxification of the material, reaching an ethanol yield of about 60% of the theoretical (on total sugars). The results obtained, showed an increase in severity of inhibitory effects with temperature increase. Improved cell viability was observed when detoxified material was used and also when yeast extract addition was coupled with adaptation of the cells to the hydrolysate.

Influence of Some Process Parameters on Defined Characteristics of Sorghum Beer during Very High Gravity Fermentation

This study was carried out to investigate the influence of some process parameters on defined characteristics of sorghum beer during very high gravity fermentation. The yeast (Saccharomyces uvarum)was pitched in very High Gravity Wort from two varieties (red and white) of sorghum (Sorghum bicolor). Changes in some process parameters like pH, specific gravity, percentage alcohol by volume, free amino nitrogen, total reducing sugar, total nitrogen and total protein were investigated over a period of 5 days of fermentation. Results showed that pH values decreased as fermentation proceeded with increased total acidity while alcohol contents of the fermenting wort increased from the first day to the fifth day of fermentation. There were observed decrease in specific gravity, total reducing sugar, free amino nitrogen (FAN), Percentage total nitrogen and Percentage total protein from first day to the fifth day of fermentation. The red sorghum had a lower pH, higher specific gravity and alcohol content than the white sorghum. This showed that Saccharomyces uvarum can grow well in very high gravity sorghum wort and pH, specific gravity, free alpha amino nitrogen is an important aspect of the wort for yeast fermentation.

Energy-Efficient Production of Cassava-Based Bio-Ethanol

Fuel ethanol is an important renewable and sustainable fuel, produced in China by fermentation of mostly corn, wheat and cassava feedstock. Fermentation produces an ethanol-lean broth (10 to 12 vol%). Ethanol is recovered by distillation, followed by a molecular sieve drying beyond the azeo-tropic point. The distillation and molecular sieve operations consume most of the total energy used, with the steam consumption currently being ~1.8 kg/kg ethanol, including 0.5 kg/kg ethanol in the final molecular sieve stage during regeneration. The objectives of the paper are fourfold: 1) firstly to study the distillation process of a large-scale cassava-based fuel ethanol production (200,000 tons per year), by field measurements and by using an Aspen Plus V8.2 simulation, with and without energy integration of condensers and reboilers, resulting in a distillation steam consumption of ~1.3 kg/kg ethanol; 2) secondly, to examine the effects of using Very High Gravity (VHG) fer-mentation of cassava meal mash. By using VHG fermentation, the ethanol concentration in the fermenter broth is significantly increased, to about 19 vol% (15.4 wt%). The steam consumption is then reduced to ~0.94 kg/kg, representing a considerable saving in comparison with the current operation. Applying VHG fermentation needs minor additional investment, rapidly recovered through the energy savings and the smaller size of equipment; 3) thirdly, to assess the application of a hybrid operation, where pervaporation will be used to selectively and continuously remove ethanol from the fermenter broth, thus slightly increasing the fermentation yield by reducing the risk of ethanol inhibition, whilst producing an ehtanol-rich permeate (about 30 wt%); and finally 4) to demonstrate that the production cost of cassava-based ethanol can substantially be reduced by applying the proposed improvements.

Relationship of trehalose accumulation with ethanol fermentation in industrial Saccharomyces cerevisiae yeast strains

The protective effect and the mechanisms of trehalose accumulation in industrial Saccharomyces cerevisiae strains were investigated during ethanol fermentation. The engineered strains with more intercellular trehalose achieved significantly higher fermentation rates and ethanol yields than their wild strain ZS during very high gravity (VHG) fermentation, while their performances were not different during regular fermentation. The VHG fermentation performances of these strains were consistent with their growth capacity under osmotic stress and ethanol stress, the key stress factors during VHG fermentation. These results suggest that trehalose accumulation is more important for VHG fermentation of industrial yeast strains than regular one. The differences in membrane integrity and antioxidative capacity of these strains indicated the possible mechanisms of trehalose as a protectant under VHG condition. Therefore, trehalose metabolic engineering may be a useful strategy for improving the VHG fermentation performance of industrial yeast strains.

English

&nbsp; The advent of highly efficient, environmentally friendly and cost effective fermentation technology has given impetus to research in the field of optimizing nutritional parameters for optimum yeast fermentative performance. Very high gravity fermentation is a novel fermentation technology that provides an increased production capacity from same size fermentation facilities, with outstanding benefits that includes: high ethanol yield per fermentable mash, considerable savings in energy and process water usage, and effluents with low biological oxygen demand amongst others. Limitations to full commercialization of the technology have been attributed to deleterious effects of the fermentation condition on yeast physiology which include high osmotic stress and ethanol toxicity amongst others. The impact of these physiological stresses on yeast cells during high substrate fermentation manifest as sluggish and incomplete fermentation with high residual sugars in beer, reduced ethanol yield, disproportionate synthesis of esters and generation of respiratory deficient yeast crop. However, compelling evidence has implicated magnesium ions with numerous biological processes and more importantly, with the role of curtailing the impact of these stress conditions. Hence, this review highlights two potential stress conditions of very high gravity fermentation; their mechanism of inhibition versus yeast stress response mechanism, role of magnesium ions in yeast physiology and its impact on fermentation processes. The knowledge emphasized herein will be of practical importance to industrial fermentation processes, as it provides feasible clues to improving yeast fermentative performance under high substrate conditions - with perspectives to precise magnesium regulation in yeast. &nbsp; Key words:&nbsp;Very high gravity fermentation, osmotic stress, ethanol stress, yeast stress tolerance, magnesium ion.

Impacts of Premature Yeast Flocculation Factors on Yeast Physiological Characteristics and Metabolite Profiles during Stirred and Unstirred High-Gravity Fermentations

Premature yeast flocculation (PYF) is a sporadic condition of brewery fermentations in which yeast settles out from the fermenting wort earlier or more heavily than usual and before full attenuation has been reached. Factors originating from malt (i.e., high molecular weight polysaccharides and antimicrobial peptides) are believed to cause this accelerated flocculation. The impacts of PYF factors on yeast physiological characteristics and metabolite profiles were investigated during high-gravity (15°P) stirred and unstirred laboratory-scale fermentations. The PYF positive (PYF+) malt was known to have caused PYF in brewery fermentations and was confirmed as PYF+ using our in-house PYF test. However, comparing a range of yeast health, growth, and metabolic aspects of fermentation (gravity drop, ethanol yield, and CO2 evolution), only minor differences were observed between PYF+ and PYF negative (PYF–) fermentations. In particular, sugar uptake was not significantly affected by the PYF status of the wort. Hence, it is unlikely that significant amounts of membrane-disruptive antimicrobial peptides were present in this PYF+ sample; rather, it is more likely that it was inducing PYF primarily through the “bridging” polysaccharide mechanism. The main differences observed in these experiments were between the stirred and unstirred fermentations. Keeping yeast in suspension by mechanical agitation enabled more rapid yeast growth and fermentation progression.

Effects of Lys and His supplementations on the regulation of nitrogen metabolism in lager yeast

Significant positive correlations between wort fermentability and the assimilation of Lys and His under normal-gravity and high-gravity conditions indicated that Lys and His were the key amino acids for lager yeast during beer brewing. In order to obtain insight into the roles of Lys and His in nitrogen regulation, the influences of Lys, His and their mixture supplementations on the fermentation performance and nitrogen metabolism in lager yeast during high-gravity fermentation were further investigated in the present study. Results showed that Lys and His supplementations improved yeast growth, wort fermentability, ethanol yield and the formation of flavor volatiles. Lys supplementation up-regulated Ssy1p-Ptr3p-Ssy5p (SPS)-regulated genes (LYP1, HIP1, BAP2 and AGP1) dramatically compared to nitrogen catabolite repression (NCR)-sensitive genes (GAP1 and MEP2), whereas His supplementation activated SPS-regulated genes slightly in exponential phase, and repressed NCR-sensitive genes significantly throughout the fermentation. Lys and His supplementations increased the consumption of Glu and Phe, and decreased the consumption of Ser, Trp and Arg. Moreover, Lys and His supplementations exhibited similar effects on the fermentation performance, and were more effective than their mixture supplementation when the same dose was kept. These results demonstrate that both Lys and His are important amino acids for yeast nitrogen metabolism and fermentation performance.

Production of a high concentration of ethanol from potato tuber by high gravity fermentation

To produce a high concentration of ethanol from viscous potato tuber mash, potato tuber mash containing high contents of solids (28%) was prepared by grinding the potato tuber without the addition of water. The viscosity of the potato mash was reduced by using Viscozyme (0.1%) at 50°C for 30 min. The potato mash was then liquefied using Liquozyme (0.1%) at 90°C for 30 min and optimal conditions for the simultaneous saccharification and fermentation (SSF) of the potato mash for ethanol production were investigated using statistical methods. Using 24 factorial design, saccharifying-enzyme and incubation temperature were found to be important factors. Using response surface methodology, the optimal saccharifying-enzyme dosage and incubation temperature were determined to be 1.45 AGU/g dry matter and 31.3°C, respectively. Under these optimal conditions for SSF, 14.92%(v/v) ethanol with 91.0% of theoretical yield was produced after 60 h, and all the starch was completely used up.

3′ Truncation of the GPD1 Promoter in Saccharomyces cerevisiae for Improved Ethanol Yield and Productivity

Glycerol is a major by-product in bioethanol fermentation by the yeast Saccharomyces cerevisiae, and decreasing glycerol formation for increased ethanol yield has been a major research effort in the bioethanol field. A new strategy has been used in the present study for reduced glycerol formation and improved ethanol fermentation performance by finely modulating the expression of GPD1 in the KAM15 strain (fps1Δ pPGK1-GLT1 gpd2Δ). The GPD1 promoter was serially truncated from the 3' end by 20 bp to result in a different expression strength of GPD1. The two engineered promoters carrying 60- and 80-bp truncations exhibited reduced promoter strength but unaffected osmostress response. These two promoters were integrated into the KAM15 strain, generating strains LE34U and LE35U, respectively. The transcription levels of LE34U and LE35U were 37.77 to 45.12% and 21.34 to 24.15% of that of KAM15U, respectively, depending on osmotic stress imposed by various glucose concentrations. In very high gravity (VHG) fermentation, the levels of glycerol for LE34U and LE35U were reduced by 15.81% and 30.66%, respectively, compared to KAM15U. The yield and final concentration of ethanol for LE35U were 3.46% and 0.33% higher, respectively, than those of KAM15U. However, fermentation rate and ethanol productivity for LE35U were reduced. On the other hand, the ethanol yield and final concentration for LE34U were enhanced by 2.28% and 2.32%, respectively, compared to those of KAM15U. In addition, a 2.31% increase in ethanol productivity was observed for LE34U compared to KAM15U. These results verified the feasibility of our strategy for yeast strain development.

Dried Spent Yeast and Its Hydrolysate as Nitrogen Supplements for Single Batch and Repeated-Batch Ethanol Fermentation from Sweet Sorghum Juice

Dried spent yeast (DSY) and its hydrolysate (DSYH) were used as low-cost nitrogen supplements to improve ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 under very high gravity (VHG) fermentation (280 g·L−1 of total sugar) conditions. The supplemented DSY and DSYH concentrations were 11, 16 and 21 g·L−1, corresponding to a yeast extract nitrogen content of 6, 9 and 12 g·L−1, respectively. The initial yeast cell concentration for ethanol fermentation was approximately 5 × 107 cells·mL−1. The fermentation was carried out in single batch mode at 30 °C in 1-L air-locked bottles with an agitation rate of 100 rpm. Ethanol production from the juice with and without yeast extract (9 g·L−1) was also performed as control treatments. The results showed that DSY at 21 g·L−1gave the highest ethanol concentration (PE, 107 g·L−1) and yield (Yp/s, 0.47 g·g−1). The use of DSYH at the same DSY concentration improved ethanol productivity (Qp), but not PE and Yp/s. The ethanol production efficiencies of the juice under DSY and DSYH supplementations were markedly higher than those without nutrient supplementation. However, the PE and Qp values of the juice containing 21 g·L−1 of DSY was approximately 7 g·L−1 and 0.62 g·L−1·h−1 lower than those under the presence of yeast extract (9 g·L−1), respectively. At the end of the single batch fermentation under the optimum DSY concentration, the sugar consumption was approximately 80%. Therefore in the repeated-batch fermentation, the initial total sugar was reduced to 240 g·L−1. The results showed that the system could be carried out at least 20 successive batches with the average PE, Yp/s and Qp of 95 g·L−1, 0.46 g·g−1 and 1.45 g·L−1·h−1, respectively.

Impact of an acid fungal protease in high gravity fermentation for ethanol production using indian sorghum as a feedstock

This study evaluated the conventional jet cooking liquefaction process followed by simultaneous saccharification and fermentation (SSF) at 30% and 35% dry solids (DS) concentration of Indian sorghum feedstock for ethanol production, with addition of acid fungal protease or urea. To evaluate the efficacy of thermostable α-amylase in liquefaction at 30% and 35% DS concentration of Indian sorghum, liquefact solubility, higher dextrins, and fermentable sugars were analyzed at the end of the process. The liquefact was further subjected to SSF using yeast. In comparison with urea, addition of an acid fungal protease during SSF process was observed to accelerate yeast growth (μ), substrate consumption (Q(s)), ultimately ethanol yield based on substrate (Y(p/s)) and ethanol productivity based on fermentation time (Q(p)). The fermentation efficiency and ethanol recovery were determined for both concentrations of Indian sorghum and found to be increased with use of acid fungal protease in SSF process.

High Level Ethanol Production by Nitrogen and Osmoprotectant Supplementation under Very High Gravity Fermentation Conditions

Optimization of nutrient supplements i.e., yeast extract (1, 3 and 5 g·L−1), dried spent yeast (DSY: 4, 12 and 20 g·L−1) and osmoprotectant (glycine: 1, 3 and 5 g·L−1) to improve the efficiency of ethanol production from a synthetic medium under very high gravity (VHG) fermentation by Saccharomyces cerevisiae NP 01 was performed using a statistical method, an L9 (34) orthogonal array design. The synthetic medium contained 280 g·L−1 of sucrose as a sole carbon source. When the fermentation was carried out at 30 °C, the ethanol concentration (P), yield (Yp/s) and productivity (Qp) without supplementation were 95.3 g·L−1, 0.49 g·g−1 and 1.70 g·L−1·h−1, respectively. According to the orthogonal results, the order of influence on the P and Qp values were yeast extract &gt; glycine &gt; DSY, and the optimum nutrient concentrations were yeast extract, 3; DSY, 4 and glycine, 5 g·L−1, respectively. The verification experiment using these parameters found that the P, Yp/s and Qp values were 119.9 g·L−1, 0.49 g g−1 and 2.14 g·L−1·h−1, respectively. These values were not different from those of the synthetic medium supplemented with 9 g·L−1 of yeast extract, indicating that DSY could be used to replace some amount of yeast extract. When sweet sorghum juice cv. KKU40 containing 280 g·L−1 of total sugar supplemented with the three nutrients at the optimum concentrations was used as the ethanol production medium, the P value (120.0 g·L−1) was not changed, but the Qp value was increased to 2.50 g·L−1·h−1.

Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast

The objective of this study was to investigate the changes in free amino acids (FAA) composition by supplementing three commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of wort mashing, and monitoring the effects on the assimilation pattern of FAA and fermentation performance of lager yeast (Saccharomyces pastorianus) during normal and high gravity fermentations. Proteases supplementation significantly improved the extract yield and FAA level of mashed worts. Normal gravity worts treated with Flavorzyme and Neutrase exhibited higher fermentability, ethanol production and flavor volatiles concentration compared to the control worts, while these beneficial effects were observed in high gravity worts treated with Protamex and Neutrase. The reason for the above results is proposed to be the change in the assimilation pattern of FAA in lager yeast with increased wort gravity, especially for the improved assimilation ratios of Leu, Arg, Phe, His, Asp and Val. In normal gravity fermentations, there were strong correlations between the assimilation amounts of Lys, Leu, Arg and His and fermentability, while in high gravity fermentations, these good correlations were found with only Lys and His. The present study suggested that optimizing the composition of FAA by supplementing proteases during wort mashing was beneficial to beer brewing for improving fermentation performance of lager yeast and flavor volatiles formation.