Very High Gravity Research Articles

Truncation of CYR1 promoter in industrial ethanol yeasts for improved ethanol yield in high temperature condition

Abstract As the initiation of fermentation using the yeast Saccharomyces cerevisiae is always associated with a rapid drop in stress resistance, it is critical to develop yeast cells with elevated stress tolerances as well as normal growth and metabolite rates. In our study, the CYR1 promoter was serially truncated from the 3′end by 30 bp steps to finely modulating the activity of Adenylate cyclase, encoded by CYR1 and synthesizing cAMP from ATP. The constructed mutants AY15a-90 and AY15a-120, whose promoters carry 90- and 120-bp truncations, showed a 2- and 3-fold drop in adenylate cyclase activity, and improved heat and ethanol tolerances than AY15a. Interestingly, AY15a-90 and AY15a-120 also exhibited the same growth and fermentation rate as the strain AY15a. In very high gravity (VHG) fermentation at 40 °C, the yield of ethanol for AY15a-90 and AY15a-120 were 14% and 15% higher than that of AY15a, respectively. These results showed high stress tolerance and high fermentation activity could be compatible by finely modulating the expression of CYR1.

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

The bioethanol production from a novel non-grain feedstock, acorn starch, was studied in this work. The inhibition of tannin in strain growth was investigated, and the effect of tannin was negligible when the tannin concentration was lower than 1g/L in medium. Therefore, the extraction of tannin was performed using 40% (v/v) ethanol-water solution as the solvent for three times under the conditions of solid/liquid ratio 1:20, 60°C, 3h, by which more than 80% of tannin in acorn was extracted and the content of tannin in acorn decreased from 7.4% (w/w) to 1.5% (w/w). Very high gravity (VHG) fermentation technology was subsequently carried out to achieve a high ethanol concentration at 86.4g/L. A comprehensive process for bioethanol production from acorn starch was designed and a preliminary economic assessment was then performed revealing that this process appeared technically and economically justified.

Ethanol production by modified polyvinyl alcohol-immobilized Zymomonas mobilis and in situ membrane distillation under very high gravity condition

Concentration of ethanol from the fermentation broth is an energy-intensive process. Very high gravity (VHG) process with modified polyvinyl alcohol-immobilized Zymomonas mobilis cells integrated with in situ bioethanol removal via vacuum membrane distillation (VMD) was proposed to mitigate product inhibition effects in VHG fermentation. The proposed VHG+VMD system, at 300g/L glucose loading, produced 127.4g/L (16.1%v/v) ethanol, at 63.7g/L−h productivity and 84.9% glucose conversion rate, which is the highest in reported literature.

Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7

BackgroundClassical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes.ResultsError-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc.ConclusionsThis is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production.

Assessment of VHG (Very High Gravity) ethanol fermentation in continuous multistage with cell recovery, reactivation and recycling using a blend of juice and molasses from sugarcane as raw material

The very high gravity (VHG) process can improve the production through high ethanol titer up to 15% (v/v), nearly doubling the productivity [9.8kg/(m3·h)], reducing vinasse to 5.4 L/L and minimizing the energy demand. The concept of this new process of high productivity and efficiency is based on the yeast kinetic model aimed to preserve cell activity at high ethanol content under high recycling. The process design is for continuous mode in multistage reactors with decreasing fermentation temperature profile in each stage (34 to 28°C), standardization of mash in microbiological and physicochemical terms, feeding mash distributed between the first and second reactors, and intracellular detoxification through a second centrifugation followed by yeast treatment promoting membrane recovery and enzymatic restoration. The basic parameters were developed at bench scale in fed-batch with cell recycle. An oxidation-reduction potential sensor was successfully used to determine the micro-aeration requirement for final stage of sugar exhaustion, to promote a respiratory environment able to support high ethanol content, and as a tool to monitoring the reactivation stage. The optimized set of parameters obtained in bench scale were used for scaling-up the process to a continuous fermentation pilot unit set to operate uninterruptedly.

Effect of temperature on sugarcane ethanol fermentation: Kinetic modeling and validation under very-high-gravity fermentation conditions

In this work, a mechanistic model is developed to simulate the effect of temperature on Saccharomyces cerevisiae growth and ethanol production of batch fermentations. A wide temperature range is used to estimate the temperature-dependent kinetic parameters of the reaction kinetics. Because multi-parameter estimation problems are complex, an optimization-based procedure is used to determine the optimum parameter values. The calculated reaction rates are used to construct a mechanistic fed-batch model. Experimental data from several cycles of very-high-gravity (VHG) ethanol fermentation from sugarcane are used to validate the model. Acceptable predictions are achieved in terms of the residual standard deviation (RSD). In addition, a suitable fermentation temperature profile, nutrient supplementation and micro-aeration during cell treatment are essential factors to obtain a yield of up to 90%, with a productivity of 10.2g/Lh and an ethanol concentration of 120g/L.

Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch

Abstract Nitrogen sources, the critical media component, were optimized to enhance ethanol production by Saccharomyces cerevisiae in very high gravity (VHG) fermentation of corn starch (340 g/l). Screening experiments revealed yeast extract as an ideal nitrogen source for ethanol production. When yeast extract concentration was controlled at 2%, ethanol yield and fermentation efficiency reached approximately 20.3% and 84.5%, respectively, after 72 h of fermentation. To reduce ethanol production cost, yeast extract supplementation was partially replaced with less expensive nitrogen sources, namely urea and ammonium sulfate. Combined effects of the three nitrogen sources on ethanol production were determined through central composite design. The optimum combination of nitrogen sources (0.6% yeast extract, 69 mM urea, and 26 mM ammonium sulfate) enabled ethanol yield and fermentation efficiency comparable to those supplemented with 2% yeast extract, indicating that urea and ammonium sulfate synergistically enhanced ethanol production by S. cerevisiae in VHG fermentation.

Synergistic effects of cell wall degrading enzymes on rheology of cassava root mash

Abstract Control of rheological characteristics of substrates is important in ethanol fermentation, particularly in a very high gravity (VHG) process. In this study, the synergism of cell wall degrading enzymes on rheology of cassava root mash was studied under VHG condition. The root mash exhibited a shear-thinning behavior. Crude enzyme mixture from Aspergillus aculeatus showed a dose-dependent effect on reduction of complex viscosity and storage modulus of mash. The enzyme showed strong synergism with Trichoderma reesei cellulase and Aspergillus pectinase leading to marked reduction in mash viscosity compared with individual enzymes. The optimal mixture comprising the three enzymes at a 1:1:1 ratio at 0.05 mg protein/g dried solid led to a remarkable decrease of complex viscosity to 1.01 Pas at 15.79 rad/s compared with 832.40 Pas of the control after incubation at 45 °C for 2 h. Synergism could be related to complementary activities of glycosyl hydrolases from different microbial sources, according to proteomic analysis. Simultaneous saccharification and fermentation of the pretreated mash by Saccharomyces cerevisiae led to a final ethanol concentration of 15.47% (w/w) corresponding to 88.14% theoretical yield. The work showed for the first time the enzymatic modification of cassava root mash rheology related to degradation of cell wall polysaccharides.

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

Aeration is one of the significant factors affecting yeast physiology and impacts ethanol production. The aim of this study was to optimize the aeration rate and time for high levels of ethanol production under very high gravity (VHG) fermentation by Saccharomyces cerevisiae NP 01 using a factorial design based on response surface methodology (RSM). The changes in intracellular composition (trehalose and ergosterol) during the fermentation were also investigated. The ethanol production medium was sweet sorghum juice containing 280gl−1 of total sugar and 3.45gl−1 of urea. The fermentation was carried out at 30°C in a 2‐L fermenter. The results showed that the optimal aeration rate and aeration time for the VHG fermentation were 0.31vvm and 12.01h, respectively. Under the optimal conditions, the ethanol concentration (PE), productivity (Qp) and yield (Yp/s) were 127.80gl−1 (16.20%, v/v), 2.66gl−1h−1 and 0.49gg −1, respectively, whereas the PE values were 119.64 and 79.74gl−1 under no aeration with urea (positive control) and without urea (negative control) supplementation, respectively. The trehalose contents under optimal, positive and negative control conditions were 200, 192 and 154mgg DCW (dry cell weight)−1, respectively. The ergosterol contents under the optimal conditions were 3.35 and 5.74 fold higher than those of positive and negative controls, respectively. It can be conclude that ethanol fermentation efficiency is markedly related to the degree of aeration, trehalose accumulation and ergosterol synthesis in the plasma membranes of yeast cells.

Very high gravity ethanol and fatty acid production of Zymomonas mobilis without amino acid and vitamin.

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136g/L without amino acid and vitamin with a theoretical yield of 90%, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16% of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.

Redox potential driven aeration during very-high-gravity ethanol fermentation by using flocculating yeast.

Ethanol fermentation requires oxygen to maintain high biomass and cell viability, especially under very-high-gravity (VHG) condition. In this work, fermentation redox potential (ORP) was applied to drive the aeration process at low dissolved oxygen (DO) levels, which is infeasible to be regulated by a DO sensor. The performance and characteristics of flocculating yeast grown under 300 and 260 g glucose/L conditions were subjected to various aeration strategies including: no aeration; controlled aeration at −150, −100 and −50 mV levels; and constant aeration at 0.05 and 0.2 vvm. The results showed that anaerobic fermentation produced the least ethanol and had the highest residual glucose after 72 h of fermentation. Controlled aerations, depending on the real-time oxygen demand, led to higher cell viability than the no-aeration counterpart. Constant aeration triggered a quick biomass formation, and fast glucose utilization. However, over aeration at 0.2 vvm caused a reduction of final ethanol concentration. The controlled aeration driven by ORP under VHG conditions resulted in the best fermentation performance. Moreover, the controlled aeration could enhance yeast flocculating activity, promote an increase of flocs size, and accelerate yeast separation near the end of fermentation.

Energy evaluation of fuel bioethanol production from sweet sorghum using very high gravity (VHG) conditions

The bioethanol production from concentrated sweet sorghum juice (142–318 g/L sugar) was studied experimentally. The effects of the juice sugar content, juice concentration prior to fermentation (VHG conditions), fermentation efficiency, and fermentation time on the energy consumption of the industrial process were evaluated using Aspen Plus® software and experimental data. The use of sorghum varieties with high sugar content and of high performance industrial yeasts reduced significantly the energy consumption. However, the decrease in energy consumption in the product recovery by using VHG conditions was less than the increase in energy consumption necessary to get these conditions (juice concentration). The VHG technology can be justified if the concentration of the juice is needed for its storage to avoid spoilage. It can also be used when the sugar content in the raw juice is high and there is no need of juice concentration to work under VHG conditions.

Simultaneous liquefaction, saccharification and fermentation at very high gravity of rice at pilot scale for potable ethanol production and distillers dried grains composition

In this work, a simultaneous liquefaction, saccharification, and fermentation (SLSF) process at very high gravity (VHG) of broken rice for potable ethanol production was developed at pilot scale. The SLSF–VHG process was performed in a unique fermenter, at 30°C. Rice flour (RF) was dissolved in tap water to reach 311.5g/l dry matter and then the mixture was simultaneousely liquefied, saccharified, and fermented. Thanks to the addition of a raw starch hydrolyzing enzyme containing a mixture of alpha-amylase and gluco-amylase (Stargen 002 at 991.8 GAU/kg RF), gluco-amylase (Amigase Mega L at 0.035% w/w), protease (Fermgen at 600 SAPU/kg RF), active dry yeast Saccharomyces cerevisiae (Red Ethanol at 3.5×107cells/ml), KH2PO4 (4.8mM), and urea (16.0mM). Under these conditions, the SLSF–VHG process finished after 120h and achieved an ethanol content of 17.6% v/v corresponding to 86.3% of the theoretical ethanol yield. We scaled up this SLSF process at very high gravity at pilot scale (25l) and achieved an ethanol content of 17.0% v/v corresponding to a yield of 83.2% of the theoretical ethanol yield. Rice-based distillers dried grains (DDG) was produced from the whole stillage of SLSF process at very high gravity by being plate-filtered and dried. The obtained DDG had high contents of crude protein (47.5%) and fibers (15.8%). Our results suggest, the SLSF under VHG condition of broken rice as well as the recovery of protein-rich DDG could have a great potential for the ethanol and animal feeding industry in Vietnam.

Material flow management and cleaner production of cassava processing for future food, feed and fuel in Thailand

This research applied Material Flow Cost Accounting (MFCA) to identify the costs of material and energy loss, as well as the opportunity for technology improvement to increase productivity in starch and ethanol productivity in Thailand. The results showed that ethanol production incurred more loss cost than starch production because it entails several conversion processes. Scenario for technology improvement was evaluated and expanded to the broader scale of the whole country to assess the possibility of increasing cassava feedstock in order to meet the AEDP target. The result shows that the Very High Gravity (VHG) and Simultaneous Saccharification and Fermentation (SSF) processes can increase plant capacity and efficiency. Ethanol production from cassava pulp can also play an important role in meeting the Renewable and Alternative Energy Development Plan (AEDP) and utilizing by-product from starch processing, increasing its value and offsetting loss cost from starch production plant. Those improved options in the scenario can help to reduce cassava feedstock amount required to produce ethanol for the AEDP target. Yield improvement can satisfy feedstock requirement without the need of land for plantation expansion, reduce cassava export and allocation of more molasses for ethanol production.

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.

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

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.

Pilot scale simultaneous saccharification and fermentation at very high gravity of cassava flour for ethanol production

We developed a simultaneous saccharification and fermentation (SSF) process of cassava flour at very high gravity (VHG). Cassava flour (CF) was dissolved in water to reach 315.4g/l dry matter, and then the mixture was liquefied at 80°C for 90min by using alpha-amylase (3532AAU/kg CF) and beta-glucanase (2812U/kg CF). SSF of liquefied mash of cassava was performed at 30°C with the simultaneous addition of two glucoamylases (Distillase ASP at 540GAU/kg CF and Amigase Mega L at 0.035% w/w), active dry yeast (1.5×107 cells/l), urea (12mM) and KH2PO4 (4mM). Under these conditions, the SSF process finished after 72h. The ethanol content achieved 17.2% v/v corresponding to 86.1% of the theoretical ethanol yield at lab scale and decreased to 16.5% v/v corresponding to 83.6% of the theoretical ethanol yield at pilot scale. Therefore, the SSF of cassava flour under VHG condition could have a great potential for the ethanol industry in Vietnam and South East Asia.

Process Design for Very-high-gravity Ethanol Fermentation

Metabolic flux distribution may be altered by manipulating intracellular reducing equivalents. To favour ethanol synthesis by Saccharomyces cerevisiae, a reduced cytosolic environment is desired, otherwise biomass formation is favoured. Direct variation of intracellular NADH/NAD+ is difficult, however, indirect control through measurement of fermentation redox potential is applicable. To utilize fermentation redox potential into designing an ethanol fermentation process under very-high-gravity (VHG) conditions, correlations between yeast growth pattern and fermentation redox potential profile were established. Under VHG conditions, S. cerevisiae initially encounters osmotic stress resulting in a lengthy lag phase. As fermentation proceeds, the built-up of ethanol inhibits yeast propagation, resulting in sudden cell death and incomplete sugar conversion. Additionally, an operational scheduling for a continuous VHG ethanol fermentation, consisting of a chemostat device and an ageing vessel, was proposed and compared to the equivalent batch operation. Results show that the proposed operational scheduling is superior to the batch counterpart. Process design criteria for a chemostat device connecting to several equal-size ageing vessels were developed in an attempt to increase annual ethanol productivity.

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.