Very High Gravity Research Articles

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.

Ethanol fermentation of very high gravity (VHG) maize mashes by Saccharomyces cerevisiae with spent brewer's yeast supplementation

The subject of this research project was ethanol productivity from very high gravity maize mashes supplemented with spent brewer's yeast as a nutrient source. Batch fermentation of maize mashes (360 g of maize per kilogram of mash) supplemented with 30 g of spent brewer's yeast's wet biomass, dried spent brewer's yeast and yeast extract per kilogram of mash was conducted for 96 h at 30 °C. In all supplemented mashes higher ethanol production rate was observed in comparison to the control sample, especially in the initial stages of fermentation. After 96 h fermentation the ethanol concentration ranged from 120.95 to 142.34 g dm−3 in supplemented mashes (120.74 g dm−3 in the control sample) which corresponded with practical ethanol yields of 78.05–91.85% (77.91% in the control sample). It was also important that maximum ethanol yield in supplemented mashes was achieved after ca. 48 h and 72 h of fermentation while in the control sample ethanol production continued until the end of the process.

Evaluation of sweet potato for fuel bioethanol production: hydrolysis and fermentation

The enzymatic starch hydrolysis and bioethanol production from a variety of sweet potato developed for bioenergy purposes (K 9807.1) on the basis of its high starch yields, was studied. Drying at 55°C and 95°C of sweet potato neither affected the sugar content nor the starch enzymatic hydrolysis efficiency. Simultaneous saccharification and ethanol fermentations for dry matter ratio of sweet potato to water from 1:8 to 1:2 (w/v) were studied. Fresh sweet potato and dried at 55°C (flour) were assayed. At ratios of 1:8, similar results for fresh sweet potato and flour in terms of ethanol concentration (38–45 g/L), fermentation time (16 h) and sugar conversion (~ 100%) were found. At higher dry matter content, faster full conversion were observed using flour. A higher ratio than that for fresh sweet potato (1:2.2) did not improve the final ethanol concentration (100 g/L) and yields. High ethanol yields were found for VHG (very high gravity) conditions. The sweet potato used is an attractive raw matter for fuel ethanol, since up to 4800 L ethanol per hectare can be obtained.

Improvement of very-high-gravity ethanol fermentation from sweet sorghum juice by controlling fermentation redox potential

Effects of fermentation redox potential control (no control, −100mV and −150mV) on the improvement of ethanol productivity during very-high-gravity (VHG) fermentation from sweet sorghum juice (300g/l of total sugar) were investigated. Results showed that redox potential controlled at −150mV gave the highest ethanol production efficiencies (ethanol concentration or P value, 134.35±1.67g ethanol 1/l and ethanol productivity or Qp value, 2.80±0.03g ethanol 1/lh) at the fermentation time of 48h. Under the same condition without the redox potential control, the P and Qp values were 115.34±2.01g/l and 2.14±0.05g/lh, respectively at the fermentation time of 54h. To complete sugar utilization under redox potential controlled at −150mV, 16mM urea was supplemented into the VHG medium. Results showed that the P and Qp values were 140.23±1.88g/l and 2.92±0.01g/lh, respectively corresponding to sugar utilization of 96%. When the initial total sugar in the juice was reduced to 250g/l, no urea addition was required in order to complete sugar utilization, and the P and Qp values were 121.54±2.23g/l and 3.38±0.04g/lh, respectively at the fermentation time of 36h. These results clearly indicated that redox potential control coupling with supplementation of assimilable nitrogen levels significantly improved the ethanol production efficiency from the sweet sorghum juice under the VHG fermentation by Saccharomyces cerevisiae NP 01.

Global gene expression analysis of Saccharomyces cerevisiae grown under redox potential‐controlled very‐high‐gravity conditions

Redox potential (ORP) plays a pivotal role in yeast viability and ethanol production during very-high-gravity (VHG) ethanol fermentation. In order to identify the correlation between redox potential profiles and gene expression patterns, global gene expression of Saccharomyces cerevisiae was investigated. Results indicated that significant changes in gene expression occurred at the periods of 0 - 6 h and 30 - 36 h, respectively. Changes noted in the period of 0 - 6 h were mainly related to carbohydrate metabolism. In contrast, gene expression variation at 30 - 36 h could be attributed primarily to stress response. Although CDC19 was down-regulated, expression of PYK2, PDC6 and ADH2 correlated inversely with ORP. Meanwhile, expression of GPD1 decreased due to the depletion of dissolved oxygen in the fermentation broth, but expression of GPD2 correlated with ORP. Transcription of genes encoding heat shock proteins was characterized by uphill, downhill, valley and plateau expression profiles, accordingly to specific function in stress response. These results highlight the role of ORP in modulating yeast physiology and metabolism under VHG conditions.

Mutations of the TATA-binding protein confer enhanced tolerance to hyperosmotic stress in Saccharomyces cerevisiae

Previously, it was shown that overexpression of either of two SPT15 mutant alleles, SPT15-M2 and SPT15-M3, which encode mutant TATA-binding proteins, confer enhanced ethanol tolerance in Saccharomyces cerevisiae. In this study, we demonstrated that strains overexpressing SPT15-M2 or SPT15-M3 were tolerant to hyperosmotic stress caused by high concentrations of glucose, salt, and sorbitol. The enhanced tolerance to high glucose concentrations in particular improved ethanol production from very high gravity (VHG) ethanol fermentations. The strains displayed constitutive and sustained activation of Hog1, a central kinase in the high osmolarity glycerol (HOG) signal transduction pathway of S. cerevisiae. However, the cell growth defect known to be caused by constitutive and sustained activation of Hog1 was not observed. We also found that reactive oxygen species (ROS) were accumulated to a less extent upon exposure to high glucose concentration in our osmotolerant strains. We identified six new genes (GPH1, HSP12, AIM17, SSA4, USV1, and IGD1), the individual deletion of which renders cells sensitive to 50% glucose. In spite of the presence of multiple copies of stress response element in their promoters, it was apparent that those genes were not controlled at the transcriptional level by the HOG pathway under the high glucose conditions. Combined with previously published results, overexpression of SPT15-M2 or SPT15-M3 clearly provides a basis for improved tolerance to ethanol and osmotic stress, which enables construction of strains of any genetic background that need enhanced tolerance to high concentrations of ethanol and glucose, promoting the feasibility for VHG ethanol fermentation.

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.

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 > glycine > 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.

Dissolved carbon dioxide concentration profiles during very-high-gravity ethanol fermentation

The dissolved carbon dioxide (DCO2) evolved from Saccharomyces cerevisiae was measured for the duration of very-high-gravity (VHG) ethanol fermentation conducted at four glucose feeds (150, 200±0.21, 250±0.12, and 300±0.28g/L). The DCO2 concentration profiles depict evolution patterns that depend on the extent of glucose utilized. A mass balance for DCO2 was proposed by taking into account the physiochemical behavior of CO2 and biological processes occurring under VHG ethanol fermentation conditions. The proposed equations could interpret the underlying CO2 desorption, CO2 conversion, and CO2 evolution on the basis of yeast activity during VHG ethanol fermentation. Consequently for 150 and 200±0.21gglucose/L absence of yeast activity was interpreted in terms of zero CO2 evolution rate (CER(t)). In contrast, under 250±0.12 and 300±0.28gglucose/L conditions the presence of non-zero residual glucose resulted in non-zero CER(t). The mass balance equation could also interpret that the physical desorption of CO2 from the fermentation broth did not follow the equilibrium relation between dissolved and off-gas CO2 concentrations as defined by Henry's law under current experimental conditions.

A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions

High gravity (HG) or very high gravity (VHG) brewing has become popular in modern breweries due to its economic and product quality advantages. However, there are the negative impacts such as the fermentation performance of brewer’s yeast in HG or VHG wort, which are closely related to changes in cell physiological activity. In the present study, 3 kinds of worts, with different gravities, were used to examine the systematic effects on fermentation performance and physiological activity of lager yeast FBY009505 (Saccharomyces pastorianus) and ale yeast FBY0099 (Saccharomyces cerevisiae), as well as the resulting beer flavor. Results showed that the responses of FBY009505 and FBY0099 to the HG or VHG worts were similar. The specific fermentation rate and viability of cropped yeast of FBY009505 and FBY0099 were decreased with increasing wort gravity. The increased wort gravity resulted in the increase of energy charge and the decrease of α-glucosides transport rate and glycolytic enzyme activities. Moreover, the environmental stresses in the HG or VHG wort showed a higher inhibitory activity against α-glucoside transport than glycolytic enzymes. The content of intracellular trehalose and glycerol of FBY009505 and FBY0099 increased with the increase in wort gravity. The results from this study provided a potential means to systematically understand the physiology of brewer’s yeast under HG or VHG conditions.

English

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.

Transcriptome analysis of Saccharomyces cerevisiae at the late stage of very high gravity (VHG) fermentation

The effect of culture system on growth and economics performance of Orechromis niloticus (Nile tilapia) in concrete tanks was investigated. Four outdoor concrete tanks measuring 2.5 x 2 m was used for the study for 24 weeks culture period. The culture systems included the use of algae only at the stocking rates of 4 fishes/m2 (T1), algae plus commercial feed at the stocking rate of 10 fishes/m2 (T2), and commercial feed only at the stocking rate of 20 fishes/m2 (T3). The tanks were stocked with O. niloticusfingerlings with mean weight of 1.160, 1.030 and 0.255 g for T1, T2 and T3, respectively. Each treatment was replicated twice. Data collected were analyzed using analysis of variance and mean comparison was carried out using Duncan Multiple Range Test (DMRT). All tests were carried out at 5% probability level. The result on growth performance show that the intensive system (T1) was significantly different in some growth parameters (P < 0.05) such as yield (0.115, 0.830 and 1.260 kg/m3 for T1, T2 and T3, respectively), specific growth (1.975, 2.750 and 3.425% for T1, T2 and T3, respectively), survival rate (90, 80 and 91% for T1, T2 and T3, respectively), and feed conversion ratio (1.150 and1.030 for T2 and T3, respectively). Economically, the intensive system was more profitable with a gross margin of N 122, which was 40% more profitable than the extensive system (T1 with Gross margin of N80), and 269% than commercial feed plus algae (T2 with Gross margin of – N 194). The cost of production (incidence of cost) showed that it cost more (N 447) to produce O. niloticus using commercial feed plus algae (T2 ) in concrete tank than the use of algae only (T1) (N 268) and the use of commercial feed only (T3) (N380). Key words: Culture system, growth performance, economic performance,Oreochromic niloticus.

A Novel Strategy to Construct Yeast Saccharomyces cerevisiae Strains for Very High Gravity Fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.

The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae

A challenge associated with the ethanol productivity under very-high-gravity (VHG) conditions, optimizing multi-traits (i.e. byproduct formation and stress tolerance) of industrial yeast strains, is overcome by a combination of metabolic engineering and genome shuffling. First, industrial strain Y12 was deleted with a glycerol exporter Fps1p and hetero-expressed with glyceraldehydes-3-phosphate dehydrogenase, resulting in the modified strain YFG12 with lower glycerol yield. Second, YFG12 was subjected to three rounds of drug resistance marker-aided genome shuffling to increase its ethanol tolerance, and the best shuffled strain TS5 was obtained. Compared with wild strain Y12, shuffled strain TS5 not only decreased glycerol formation by 14.8%, but also increased fermentation rate and ethanol yield by 3.7% and 7.6%, respectively. Moreover, the system of genetic modification and Cre/loxP in aid of three different drug-resistance markers presented in the study significantly improved breeding efficiency and will facilitate the application of breeding technologies in prototrophic industrial microorganisms.

Medium Optimization for Improved Ethanol Production in Very High Gravity Fermentation

An optimal medium (300 g·L −1 initial glucose) comprising 6.3 mmol·L −1 Mg 2+ , 5.0 mmol·L −1 Ca 2+ , 15.0 g·L −1 peptone and 21.5 g·L −1 yeast extract was determined by uniform design to improve very high gravity (VHG) ethanol fermentation, showing over 30% increase in final ethanol (from 13.1% to 17.1%, by volume), 29% decrease in fermentation time (from 84 to 60 h), 80% increase in biomass formation and 26% increase in glucose utilization. Experiments also revealed physiological aspects linked to the fermentation enhancements. Compared to the control, trehalose in the cells grown in optimal fermentation medium increased 17.9-, 2.8-, 1.9-, 1.8- and 1.9-fold at the fermentation time of 12, 24, 36, 48 and 60 h, respectively. Its sharp rise at the early stage of fermentation when there was a considerable osmotic stress suggested that trehalose played an important role in promoting fermentation. Meanwhile, at the identical five fermentation time, the plasma membrane ATPase activity of the cells grown in optimal medium was 2.3, 1.8, 1.6, 1.5 and 1.3 times that of the control, respectively. Their disparities in enzymatic activity became wider when the glucose levels were dramatically changed for ethanol production, suggesting this enzyme also contributed to the fermentation improvements. Thus, medium optimization for VHG ethanol fermentation was found to trigger the increased yeast trehalose accumulation and plasma membrane ATPase activity.

Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

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.

The use of dried spent yeast as a low-cost nitrogen supplement in ethanol fermentation from sweet sorghum juice under very high gravity conditions

Dried spent yeast (DSY) was used as a low-cost nitrogen supplement for ethanol fermentation from sweet sorghum juice under very high gravity (VHG) conditions by Saccharomyces cerevisiae NP 01. The fermentation was carried out at 30oC in a 5-litre bioreactor. The results showed that DSY promoted ethanol production efficiencies. The ethanol concentration ( P ), productivity ( Q p ) and yield ( Y p/s ) of the sterile juice (total sugar of 280 g l -1 ) supplemented with 8 g l -1 of DSY were not different from those supplemented with yeast extract and/or peptone at the same amount. The initial yeast cell concentration of 5 x 10 7 cells ml -1 was found to be optimal for scale-up ethanol production. In addition, an increase in sugar concentration in inoculum preparation medium (from 10 to 100 g l -1 ) improved the ability of the inoculum to produce ethanol under the VHG conditions. When S. cerevisiae NP 01 grown in the juice containing 100 g l -1 of total sugar was used as the inoculum for ethanol fermentation, the P , Q p and Y p/s obtained were 108.98 ± 1.16 g l -1 , 2.27 ± 0.06 g l -1 h -1 and 0.47 ± 0.01 g g -1 , respectively. Similar results were also observed when the ethanol fermentation was scaled up to a 50-litre bioreactor under the same conditions. The cost of the sweet sorghum for ethanol production was US$ 0.63 per litre of ethanol. These results clearly indicate the high potential of using sweet sorghum juice supplemented with DSY under VHG fermentation for ethanol production in industrial applications.

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase-producing yeast Kluyveromyces marxianus Y179 and fed-batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch-based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l(-1) was achieved when 0·15 g l(-1) K(2) HPO(4) was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Jerusalem artichoke tubers are an alternative to grain-based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.

Ageing vessel design and optimization for continuous very-high-gravity ethanol fermentation processes

A continuous very-high-gravity (VHG) ethanol fermentation process design, consisting of a chemostat vessel connected to several equal-sized ageing vessels configured in parallel, was developed. The objective of the developed process is to have complete glucose utilization during fermentation stage. The process design integrates the conservation of mass principle and the experimental data of collected residual glucose profiles measured under VHG conditions. An ageing vessel involves three consecutive time periods: filling, ageing and operating. The ageing time is biological relevance, and is affected by the initial glucose concentration, the ethanol concentration, and the yeast viability in an ageing vessel. The operating time period is adjustable; a short operating time means a high discharge rate in order to empty an ageing vessel. The filling time links to the selection of the number of equal-sized ageing vessels that are installed downstream to a chemostat device. The developed process features the use of equal sized fermenters for all chemostat and ageing vessels so that the vessel exchangeability and the flexibility of fermentation operation are increased.