High Gravity Ethanol Fermentation Research Articles

Gaining insights into the responses of individual yeast cells to ethanol fermentation using Raman tweezers and chemometrics

Saccharomyces cerevisiae is the most common microbe used for the industrial production of bioethanol, and it encounters various stresses that inhibit cell growth and metabolism during fermentation. However, little is currently known about the physiological changes that occur in individual yeast cells during ethanol fermentation. Therefore, in this work, Raman spectroscopy and chemometric techniques were employed to monitor the metabolic changes of individual yeast cells at distinct stages during high gravity ethanol fermentation. Raman tweezers was used to acquire the Raman spectra of individual yeast cells. Multivariate curve resolution-alternating least squares (MCR-ALS) and principal component analysis were employed to analyze the Raman spectra dataset. MCR-ALS extracted the spectra of proteins, phospholipids, and triacylglycerols and their relative contents in individual cells. Changes in intracellular biomolecules showed that yeast cells undergo three distinct physiological stages during fermentation. In addition, heterogeneity among yeast cells significantly increased in the late fermentation period, and different yeast cells may respond to ethanol stress via different mechanisms. Our findings suggest that the combination of Raman tweezers and chemometrics approaches allows for characterizing the dynamics of molecular components within individual cells. This approach can serve as a valuable tool in investigating the resistance mechanism and metabolic heterogeneity of yeast cells during ethanol fermentation.

Rheological characteristics and genotype correlation of cassava root for very high gravity ethanol production: The influence of cassava varieties and harvest times.

In very high gravity (VHG) ethanol fermentation, the rheological properties of native cassava significantly influence heat and mass transfer, mixing energy, and, thus, the yield of all steps. This study investigated the effect of cassava varieties and their harvest times on starch liquefaction, saccharification, and fermentation. The genotype correlation of the starch properties was revealed for the most suitable cassava varieties. First, the starch content, amylose content, total reducing sugar from liquefaction and saccharification, pasting properties, and ethanol yields of six cassava varieties (Huay Bong 60, Hanatee, Kasetsart 50, Pirun 1, Pirun 2, and Rayong 11) at 6-, 9-, 12-, and 15-month harvest times were evaluated. The amylose content increased significantly from the 6th to the 12th month but slightly decreased at the 15th month. It was observed that the starch content contributed to a more substantial influence on the change in peak viscosity than on the amylose content. Ethanol fermentation using Saccharomyces cerevisiae TISTR5606 showed that the Rayong 11 variety at the 15-month harvest time provided the highest ethanol concentration of 104.7±4.1gL-1 and an ethanol yield of 0.4±0.1gethanolg-1 reducing sugar, which corresponded to 74.5% of the theoretical yield.

Co-production of ethanol and biodiesel from sweet sorghum juice in two consecutive fermentation steps

Sugars from sweet sorghum stalks can be used to produce ethanol and also to grow oleaginous yeasts. Instead of two separate processes, in this paper we propose a different route producing ethanol and microbial oil in two consecutive fermentation steps. Three yeasts were compared in the first ethanol producing step. In the second step four different oleaginous yeasts were tested. Sweet sorghum juice was first clarified and concentrated. High gravity ethanol fermentation was carried out with concentrated juice with 23.7 g/100 mL of total sugars and without added nutrients. Total sugars were 2.5 times more than the original clarified juice. One yeast gave the best overall response over the two other tested; relative high ethanol productivity, 1.44 g ethanol/L·h−1, and 90% of sugar consumption. Aeration by flask agitation produced superior results than static flasks for all yeasts. Microbial oil production was done employing the residual liquid left after ethanol separation. The pooled residual liquid from the ethanol distillation contained 7.08 g/mL of total carbohydrates, rich in reducing sugars. Trichosporon oleaginosus and Lipomyces starkeyi produced higher dry biomass, total sugar consumption and oil productivity than the other two oleaginous yeasts tested; with values around 25 g/L, 80%, and 0.55 g oil/L·h−1 respectively. However, the biomass oil content in all yeasts was relatively low in the range of 14 to 16%. The two step process is viable and could be considered an integral part of a consolidated biorefinery from sweet sorghum. How to cite: Rolz C, de León R, Mendizábal de Montenegro AL. Co-production of ethanol and biodiesel from sweet sorghum juice in two consecutive fermentation steps. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.05.002.

Comparison of yeast extract prepared by autolysis or steam explosion as a cheap nutrient supplement for very high gravity ethanol fermentation of cassava starch

Comparison of yeast extract prepared by autolysis or steam explosion as a cheap nutrient supplement for very high gravity ethanol fermentation of cassava starch

A review of recent advances in high gravity ethanol fermentation

Abstract The technology of ethanol fermentation under high gravity (HG) and very high gravity (VHG) conditions has witnessed significant progress over the last three decades owing to economic and environmental benefits. Research efforts have been directed toward comprehensive development of the technology. As a result, final ethanol concentrations in excess of 15% v/v are being achieved using saccharine and starchy substrates. Moreover, during the recent past, researchers have explored the possibilities of using unconventional and cost-effective substrates as well as nitrogen supplements in HG and VHG fermentations. On the other hand, considerable emphasis has been placed on selecting industrial strains, flocculating yeasts, and construction of novel strains that exhibit both osmotolerance and high ethanol yielding capabilities under VHG conditions. And, to further increase the cost-effectiveness, priority has been given for fermentation process control aspects such as control of redox potential and dissolved CO2 profiling, process optimization and modelling and simulation strategies. HG lignocellulosic ethanol production is an emerging area for research.

Sequential high gravity ethanol fermentation and anaerobic digestion of steam explosion and organosolv pretreated corn stover

The present work investigates the suitability of pretreated corn stover (CS) to serve as feedstock for high gravity (HG) ethanol production at solids-content of 24wt%. Steam explosion, with and without the addition of H2SO4, and organosolv pretreated CS samples underwent a liquefaction/saccharification step followed by simultaneous saccharification and fermentation (SSF). Maximum ethanol concentration of ca. 76g/L (78.3% ethanol yield) was obtained from steam exploded CS (SECS) with 0.2% H2SO4. Organosolv pretreated CS (OCS) also resulted in high ethanol concentration of ca. 65g/L (62.3% ethanol yield). Moreover, methane production through anaerobic digestion (AD) was conducted from fermentation residues and resulted in maximum methane yields of ca. 120 and 69mL/g volatile solids (VS) for SECS and OCS samples, respectively. The results indicated that the implementation of a liquefaction/saccharification step before SSF employing a liquefaction reactor seemed to handle HG conditions adequately.

Osmotic stress Response of Saccharomyces cerevisiae under HG and Elevated Temperature Environment

The High Gravity (HG) ethanol fermentation at high temperature is very attractive and promising technology for fuel ethanol production. This study was designed to improve the osmotic as well as thermal behavior of the Saccharomyces cerevisiae strain isolated from distillery waste. Therefore, initial pH and substrate concentrations were optimized for this strain. The S. cerevisiae was subjected to thermal treatment to improve its fermentation ability without significant yield losses. At pHo 5.0, 95g/L ethanol was produced with the productivity (Qp) value of 1.02. The activation energy Ea value calculated at 30-40oC was 16.48kcal/mol indicating the thermal tolerance of the strain SC36. The results of glucose optimization revealed that at 250g/L glucose concentration, Qp, Yp/s and Yp/x value of 1.53g/Lh, 0.443g/g substrate and 41.4g/g biomass were obtained. The strain’s potential to be able to ferment very high gravity medium is very promising for fuel ethanol production

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.

S533.099Using Viscosity Reducing Enzyme as Annexing Agent in the Very High Gravity Ethanol Fermentation with Fresh Cassava

S533.099Using Viscosity Reducing Enzyme as Annexing Agent in the Very High Gravity Ethanol Fermentation with Fresh Cassava

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

BackgroundVery high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15% (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production.ResultsBatch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 ± 3.1, 252 ± 2.9 and 298 ± 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at −100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 ± 3.1 and 252 ± 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 ± 1.3 and 120.0 ± 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 ± 0.4 and 17.0 ± 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 ± 1.8 g/L ethanol was produced at 72 h when ORP was controlled at −150 mV for the VHG fermentation with medium containing 298 ± 3.8 g/L glucose, since yeast cell viability was improved more significantly.ConclusionsNo lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at −150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at −100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.

Optimization of Agitation and Aeration for Very High Gravity Ethanol Fermentation from Sweet Sorghum Juice by Saccharomyces cerevisiae Using an Orthogonal Array Design

Optimization of three parameters: agitation rate (A; 100, 200 and 300 rpm), aeration rate (B; 0.5, 1.5 and 2.5 vvm) and aeration timing (C; 2, 4 and 6 h), for ethanol production from sweet sorghum juice under very high gravity (VHG, 290 g L−1 of total sugar) conditions by Saccharomyces cerevisiae NP 01 was attempted using an L9 (34) orthogonal array design. The fermentation was carried out at 30 °C in a 2-L bioreactor and the initial yeast cell concentration was approximately 2 × 107 cells mL−1. The results showed that the optimum condition for ethanol fermentation should be A2B3C2 corresponding to agitation rate, 200 rpm; aeration rate, 2.5 vvm and aeration timing, 4 h. The verification experiments under the optimum condition clearly indicated that the aeration and agitation strategies improved ethanol production. The ethanol concentration (P), productivity (Qp) and ethanol yield (Yp/s) were 132.82 ± 1.06 g L−1, 2.55 ± 0.00 g L−1h−1 and 0.50 ± 0.00, respectively. Under the same condition without aeration (agitation rate at 200 rpm), P and Qp were only 118.02 ± 1.19 g L−1 and 2.19 ± 0.04 g L−1h−1, respectively while Yp/s was not different from that under the optimum condition.

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.

Very High Gravity Fermentation Using Sweet Potato for Fuel Ethanol Production

Sweet potato (Ipomoea batatas) was a wildely planted root crop in most area of China and considered as a good feedstock for fuel ethanol production. Very high gravity ethanol fermentation technology exhibited promising industrial application for advantages including productivity improvement, polluted water output reduction and energy consumption saving. In this study very high gravity liquefied sweet potato mash containing 260 g/kg glucose (after fully saccharified) was used for fuel ethanol fermentation. 0.8 g/kg (dry matter weight) was proved as the optimum glucoamylase adding dosage in simultaneous saccharification and fermentation. Datas analysis indicated that the osmotic pressure was controlled strictly exhibited by high growth rate of yeast and high rate of ethanol formation comparing with other dosages, and 119.78 g/kg (15.07 %, v/v) ethanol equivalent to 90.16 % of theoretical yield was achieved in 64 hours.

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L(-1) glucose and 6.75 g L(-1) each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8-14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L(-1) h(-1), much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.

Application of full permeate recycling to very high gravity ethanol fermentation from corn

A ceramic membrane with pore size of 0.2 μm was used to percolate grain stillage of very high gravity (VHG) ethanol fermentation from corn, and the micro-filtration permeate was completely recycled for the cooking step in the next fermentation process. The concentrations of solids, sugars, total nitrogen and Na+ in the grain stillage and permeate reached a relative steady state after two or three batches of filtration and recycling process. There are no negative effects of by-products on VHG ethanol fermentation, and the final ethanol yield was above 15% (v/v). The conditions of filtration were examined to determine the optimum conditions for the process and included an initial flux of clean water above 550 L·m−2·h−1 (0.1 MPa), an operating differential pressure of 0.15 MPa, an operating temperature above 70 °C, and a permeation flux greater than 136 L·m−2·h−1. It could be concluded that full permeate recycling during ethanol production was an efficient process that resulted in less pollution and less energy consumption.

Very high gravity ethanol fermentation by repeated batch operation using a flocculating fusant yeast strain

Very high gravity ethanol fermentation by repeated batch operation using a flocculating fusant yeast strain

High gravity and high productivity ethanol fermentation with self-flocculating yeast cells

High gravity and high productivity ethanol fermentation with self-flocculating yeast cells

Selection of Saccharomyces cerevisiae and Investigation of its Performance for Very High Gravity Ethanol Fermentation

Selection of Saccharomyces cerevisiae and Investigation of its Performance for Very High Gravity Ethanol Fermentation

Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain

Ethanol was produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions. The effects of glucoamylase dosage, pre-saccharification and Saccharomyces cerevisiae strain on the growth characteristics such as the ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration from 106 to 126 g/kg although the maximal specific growth rate was decreased. Ethanol production was not only growth related, as more than half of the total saccharides were consumed and more than half of the ethanol was produced during the stationary phase. Furthermore, a high stress tolerance of the applied yeast strain was found to be crucial for the outcome of the fermentation process, both with regard to residual saccharides and final ethanol concentration. The increased formation of cell mass when a well-suited strain was applied increased the final ethanol concentration, since a more complete fermentation was achieved.

Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae

The final ethanol concentration achieved was increased by 17% (to 103 g ethanol/l) when excess assimilable nitrogen was added to the batch very high gravity (VHG) ethanolic fermentations by Saccharomyces cerevisiae. The supplementation of the media with 12 g yeast extract l−1, 0.3 g cell walls l−1, 3 g glycine l−1 and 20 g soya flour l−1 led to halving reduction of the fermentation time to 28 h. The ethanol productivity was enhanced by more than 50% (to achieved value 3.3 g l−1 h−1).