Very High Gravity Research Articles

Naringenin and caffeic acid increase ethanol production in yeast cells by reducing very high gravity fermentation-related oxidative stress.

Very high gravity (VHG) fermentation is an industrial-scale process utilizing a sugar concentration above 250g/L to attain a significant ethanol concentration, with the advantages of decreased labor, production costs, water usage, bacterial contamination, and energy consumption. Saccharomyces cerevisiae is one of the most extensively employed organisms in ethanol fermentation through VHG technology. Conversely, high glucose exposure leads to numerous stress factors that negatively impact the ethanol production efficiency of this organism. Here, the impact of various phytochemicals added to the VHG medium on viability, glucose consumption, ethanol production efficiency, total antioxidant-oxidant status (TAS and TOS), and the response of the enzymatic antioxidant system of yeast were investigated. 2.0 mM naringenin and caffeic acid increased ethanol production by 2.453 ± 0.198 and 1.261 ± 0.138-fold, respectively. The glucose consumption rate exhibited a direct relationship with ethanol production in the naringenin-supplemented group. The highest TAS was determined as 0.734 ± 0.044 mmol Trolox Eq./L in the same group. Furthermore, both phytochemical compounds exhibited robust positive correlations with TAS (rnaringenin = 0.9986; rcaffeic acid = 0.9553) and TOS levels (rnaringenin = -0.9824; rcaffeic acid = -0.9791). While naringenin caused statistically significant increases in glutathione reductase (GR) and thioredoxin reductase (TrxR) activities, caffeic acid significantly increased TrxR and superoxide dismutase (SOD). Both phytochemicals seem to impact the ethanol production ability by regulating the redox status of the cells. We believe that the incorporation of particularly cost-effective antioxidants into the fermentation medium may serve as an alternative way to enhance the efficiency of bioethanol production using VHG technology.

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

This study optimized ethanol production from sweet sorghum stem juice (SSJ) by Saccharomyces cerevisiae NP01 under very high gravity (VHG) fermentation in 500-mL air–locked flasks at 30 °C. Response surface methodology based on a Box-Behnken design was employed to optimize initial sugar (267 g/L), urea (3.24 g/L), and cell concentration (1.32 × 108cells/mL) for maximization of ethanol concentration (PE), productivity (QP), and sugar consumption (%SC). The experimental values (PE, 119.29 g/L; QP, 2.49 g/L.h and %SC,91.83 %) under optimal conditions were close to the predicted values, verifying the optimization process. Aeration (2.5 vvm for 4 h) increased viable cell counts and decreased glycerol production (a by-product), but not fermentation efficiency. An osmoprotectant (40 mM potassium chloride combined with 10 mM potassium hydroxide, KCl/KOH) at 30 °C had no positive effect on ethanol fermentation efficiency. However, at 25 °C, the osmoprotectant increased PE from 106 to 116 g/L and ethanol yield from 0.46 to 0.49 g/g. At 35–37 °C, it prolonged cell viability, increasing PE by 5–12 g/L and %SC by 3–8 % without affecting ethanol yield. However, at 39 °C, no positive impact occurred on ethanol fermentation efficiency. The findings from this study, particularly the optimized fermentation conditions and stress tolerance strategies, could guide the scale-up to an industrial level of bioethanol production from sweet sorghum stem juice or other feedstocks using VHG fermentation, contributing to the development of more efficient and sustainable biofuel production processes.

Silica Wort Supplementation as an Alternative for Yeast Stress Relief on Corn Ethanol Production with Cell Recycling

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

Ethanol production from the wood and fruit juice of paper mulberry via simultaneous saccharification and juice co-fermentation under normal and very high gravity conditions

A novel batch simultaneous saccharification and juice co-fermentation (SSJcF) process was investigated using mixtures of PHP (phosphoric acid plus hydrogen peroxide) pretreated wood and fruit juice of paper mulberry tree. All the different substrate mixture configurations evaluated under normal gravity SSJcF (total sugar titer of 180 g/L) significantly improved both ethanol concentration and yield compared to sole simultaneous saccharification and fermentation (SSF) of paper mulberry wood. Among the different mixing ratios, the feedstock mixture with equal sugar content from each substrate resulted in maximum ethanol concentration, yield and productivity of 77.1 g/L, 83.9 % and 1.1 g/L/h, respectively. The external additions of mineral nutrients also had no impact on fermentation performance of the mixture. Furthermore, SSJcF was evaluated under different levels of high gravity (HG) and very high gravity (VHG) conditions. The total sugar titer of 350 g/L at equal contributions from each substrate reached very high ethanol concentration of 147.0 g/L, with yield and productivity of 82.3 % and 1.5 g/L/h, respectively. By implication, the PM wood and fruit juice mixture could serve as suitable low-cost and non-food feedstock alternative to the typical use of sugary and/or starchy feedstocks for VHG fermentation.

Evaluation of different nitrogen sources on growth and fermentation performance for enhancing ethanol production by wine yeasts

The utilization of grape juice from low oenological value grape varieties for bioethanol production represent an alternative for diversification and value addition in viticulture. Optimizing Very High Gravity (VHG) fermentation can significantly increase ethanol productivity while reducing water and energy consumption. In this study, the impact of different nitrogen sources on growth and fermentative performance of locally selected yeast strains was investigated. Five yeast strains of species Saccharomyces cerevisiae and Zygosaccharomyces rouxii were cultured in both synthetic culture media and natural grape juice supplemented with ammonium sulfate (NH), yeast extract (YE), Fermaid K (FERM), and urea (U) at varying concentrations. Due to the very low fermentation rate, the Z. rouxii strain was excluded from the selection. The results obtained in synthetic medium showed that nitrogen sources that promoted growth (NH and YE) had minimal effects on fermentative performance and were highly dependent on the specific yeast strain. However, the combination of urea and ammonium favored the rate of sugar consumption. When validated in natural grape juice, urea combined with ammonium (U + NH 300 + 75 mg/L) improved both growth parameters and ethanol yield. Doubling the concentration (U + NH 600 + 150 mg/L) further enhanced sugar consumption and ethanol production while reducing unwanted by-products. The combined use of urea and ammonium exhibited a synergistic effect, making it a cost-effective nitrogen supplement for VHG fermentations.

Breeding of high-tolerance yeast by adaptive evolution and high gravity brewing of mutant.

Ethanol and osmotic stresses are the major limiting factors for brewing strong-beer with high-gravity wort. Breeding of yeast strains with high osmotic and ethanol tolerance and studying very high gravity (VHG) brewing technology is of great significance for brewing strong-beer. This study used an optimized microbial microdroplet culture (MMC) system for adaptive laboratory evolution (ALE) of Saccharomyces cerevisiae YN81 to improve its tolerance to osmotic and ethanol stress. Meanwhile, we investigated the VHG and VHG with added ethanol (VHGAE) brewing processes for the evolved mutants in brewing strong-beer. The results showed that three evolved mutants were obtained; among them, the growth performance of YN81mc-8.3 under 300, 340, 380, 420, and 460 g·L-1 sucrose stresses was greater than that of the other strains. The ethanol tolerance of YN81mc-8.3 was 12%, which was 20% higher than that of YN81. During strong-beer brewing in a 100 L cylindrical cone-bottom tank, the sugar utilization and ethanol yield of YN81mc-8.3 outperformed those of YN81 in both the VHG and VHGAE brewing processes. Measurement of the diacetyl concentration showed that YN81mc-8.3 had a stronger diacetyl reduction ability; in particular, the real degree of fermentation of beers brewed by YN81mc-8.3 in VHG and VHGAE brewing processes was 75.35% and 66.71%, respectively, higher than those of the 2 samples brewed by YN81. Meanwhile, the visual, olfactive, and gustative properties of the strong-beer produced by YN81mc-8.3 were better than those of the other beers. In this study, the mutant YN81mc-8.3 and the VHGAE brewing process were optimal and represented a better alternative strong-beer brewing process. This article is protected by copyright. All rights reserved.

OPTIMIZATION OF NO COOK TECHNOLOGY AT VERY HIGH GRAVITY FOR RICE-BASED ETHANOL PRODUCTION

The application of raw-starch hydrolysis enzymes to ethanol production by no-cook technology at very high gravity (VHG) (311.5 g/L) from rice would reduce the energy consumption compared to traditional process. In this study, the Taguchi design and the desirability function were used to identify the optimized production process for economic and technological targets. Three factors: dosage of alpha- and gluco-amylase enzyme (Stargen 002 – A), dosage of protease enzyme (Fermgen – B) and dosage of yeast (C) were investigated. With the expected Desirability = 0.74, the no-cook process from rice would be optimized with following dosage: Stargen 002, Fermgen and yeast at 700 µL/L (1140 GAU/kg raw material), 151 µL/L (431 SAPU/kg raw material) and 0.25 g/L (6.25 x 106 cells/mL), respectively. After 120h of fermentation, ethanol concentration reached 17.45 ± 0.07 %v/v, corresponding to ethanol yield of 86.08 ± 0.2 %, and the cost of raw material was at 3225.5 VND/L of rice slurry.

Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae

The ethanol fermentation efficiency of sweet sorghum stem juice (SSJ) under a very high gravity (VHG) condition (250 g/L of sugar) was improved by immobilized Saccharomyces cerevisiae SSJKKU01, using a stirred tank bioreactor (STR) coupled with a column bioreactor (CR). Dried rattan pieces (as carriers for cell immobilization) at 50% of the working volume of the CR were suitable for use in a batch ethanol fermentation. The average ethanol concentration (PE) and ethanol productivity (QP) of repeated-batch fermentation in the CR for eight successive cycles were 109.85 g/L and 1.88 g/L⋅h, respectively. Then an STR coupled with a CR was applied for repeated-batch ethanol fermentation in two systems. System I was an STR (1.8 L working volume), and System II was an STR (1 L) coupled with a CR, referred to as a CR-F (0.8 L). Both systems were connected to a new CR, called CR-I, containing sterile dried rattan pieces at 50% of its working volume. Active yeast cells were inoculated only into the STR, and the medium circulation rate between bioreactors was 5.2 mL/min. The results showed that at least eight successive cycles could be operated with an average PE of 108.51 g/L for System I and 109.44 g/L for System II. The average QP and SC values of both systems were also similar, with values of 1.87 to 1.88 g/L⋅h and 93 to 94%, respectively. The morphology of the carriers with and without immobilized cells before and after the fermentation was investigated. The obtained results demonstrated that a repeated-batch fermentation by immobilized cells on rattan pieces, using an STR coupled with a CR, was successfully used to produce high levels of ethanol from SSJ under a VHG condition.

Effect of medium supplementation on very high gravity bioethanol fermentation using sago hampas hydrolysate as a feedstock

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

Protease increases ethanol yield and decreases fermentation time in no-cook process during very-high-gravity ethanol production from rice

No-cook process or Simultaneous Liquefaction, Saccharification, and Fermentation process (SLSF) under very-high-gravity (VHG) condition possesses many advantages for potable ethanol production from rice. The important nutrient that affects the effectiveness of fermentation is nitrogen. Two sources of nitrogen are considered including added urea and indigenous rice protein which can be hydrolysed by an external protease. This study aims to assess the impact of urea and protease on rice conversion, yeast performance including the formation of ethanol and co-products in the SLSF-VHG. The results show that in comparison to the control process (without adding nitrogen), a protease (Fermgen™ 2.5X) supplementation of 600 SAPU/kg rice flour resulted in efficient breakdown of the rice structure along with 2.4% v/v higher ethanol yield and shorter fermentation time of 48 h. Meanwhile, the adding urea at concentration of 16 mM had a slight effect on the structural modification of the rice material without a significant increase (p < 0.05) of final ethanol yield. There was no significant difference (p < 0.05) in ethanol yield between urea and protease addition compared with protease alone. Our results suggested that the addition of protease provided enough nitrogen for yeast by hydrolysing the endogenous rice protein during SLSF-VHG process.

Mathematical Modeling of Fed-Batch Ethanol Fermentation Under Very High Gravity and High Cell Density at Different Temperatures.

The use of more appropriate kinetic models can assist in improving ethanol fermentation under conditions of very high gravity (VHG) and high cell density (HCD), in order to obtain higher amounts of ethanol in the broth combined with high productivity. The aim of this study was to model fed-batch ethanol fermentation under VHG/HCD conditions, at different temperatures, considering three types of inhibition (substrate, ethanol, and cells). Fermentations were carried out using different temperatures (28 ≤ [Formula: see text] (°C) ≤ 34), inoculum sizes (50 ≤ [Formula: see text] (g L-1) ≤ 125), and substrate concentrations in the must (258 ≤ [Formula: see text] (g L-1) ≤ 436). In the proposed model, the cell inhibition power parameter varied with the temperature and inoculum size, while the cell yield coefficient varied with inoculum size and substrate concentration in the must. Hence, it was possible to propose correlations for the cell inhibition power parameter ([Formula: see text]) and for the cell yield coefficient ([Formula: see text]), as functions of the fermentation conditions. Simulations of fed-batch ethanol fermentations at different temperatures, under VHG/HCD conditions, were performed using the proposed correlations. Experimental validation showed that the model was able to accurately predict the dynamic behavior of the fermentations in terms of the concentrations of viable cells, total cells, ethanol, and substrate.

Genetics and metabolic engineering of yeast strains for efficient ethanol production

AbstractBioethanol production from monomeric sugar is performed by several yeasts. But there are several limitations associated with yeast strains such as their low tolerance to ethanol, toxic inhibitors, and high sugar concentration. Genetic and metabolic engineering of potential yeast strains can overcome the above limitations. The present article summarized current genetic and metabolic engineering approaches for the development of yeast strain for efficient ethanol production. The review systematically examined bioethanol generations based on substrate utilization, criteria for strain selections, strategies for strain improvements including randomized mutagenesis, genetic engineering, metabolic engineering, genome editing, whole genome (re)sequencing, promoter engineering, quantitative trait locus analysis, protein engineering, and evolutionary engineering. Different fermentation technologies employed in hydrolysate fermentation including low gravity (LG), high gravity (HG), and very high gravity (VHG) as well as challenges of yeast strains development and its future prospect have been critically evaluated in this article. Significant engineering efforts are imminent for yeast‐based second‐generation biofuel to leave a demonstration phase through strain improvement and become economically competitive with fossil fuel.Practical ApplicationsThis is a comprehensive review of yeast strain development for bioethanol production. The readers should be able to acquire some basic knowledge on: The accompanied substrates for bioethanol generations as well as the technologies and challenges behind them. The criteria to consider in selecting yeast strain for bioengineering development. Different strategies and their reported applications employed in yeast strain development including randomized mutagenesis, genetic engineering, metabolic engineering, genome editing, whole genome (re)sequencing, promoter engineering, quantitative trait locus (QTL) analysis, protein engineering, and evolutionary engineering. Challenges and merits of different fermentation technologies employed in hydrolysate fermentation including LG, HG, and VHG. Possible challenges to encounter in developing yeast strain for bioethanol production. Desirable traits to consider in the selection and development of yeast strains for bioethanol production.

Omics analysis reveals mechanism underlying metabolic oscillation during continuous very-high-gravity ethanol fermentation by Saccharomyces cerevisiae.

During continuous very-high-gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae, the process exhibits sustained oscillation in residual glucose, ethanol, and biomass, raising a question: how do yeast cells respond to this phenomenon? In this study, the oscillatory behavior of yeast cells was characterized through transcriptome and metabolome analysis for one complete oscillatory period. By analyzing the accumulation of 26 intracellular metabolites and the expression of 90 genes related to central carbonmetabolism and stress response, we confirmed that the process oscillation was attributed to intracellular metabolic oscillation with phase difference, and the expression of HXK1, HXT1,2,4, and PFK1 was significantly different from other genes in the Embden-Meyerhof-Parnas pathway, indicating that glucose transport and phosphorylation could be key nodes for regulating the intracellular metabolism under oscillatory conditions. Moreover, the expression of stress response genes was triggered and affected predominately by ethanol inhibition in yeast cells. This progress not only contributes to the understanding of mechanisms underlying the process oscillation observed for continuous VHG ethanol fermentation, but also provides insights for understanding unsteady state that might develop in other continuous fermentation processes operated under VHG conditions to increase product titers for robust production.

Metabonomic analysis reveals enhanced growth and ethanol production of brewer's yeast by wheat gluten hydrolysates and potassium supplementation

Wheat gluten hydrolysates (WGH) and KCl were used to examine their effects on fermentation performances and physiological characteristics of brewer's yeast during very high gravity (VHG) fermentation. Results showed that all supplementations exhibited a notable promotion in yeast growth, glucose utilization, ethanol production, and physiological characteristics. Combination supplementation of 0.3% WGH and 0.5% KCl showed the best promotion effect with 6.38%, 9.41% and 8.68% of increases in biomass, glucose utilization and ethanol production, respectively. Moreover, combination addition of WGH and KCl also enhanced significantly membrane integrity and mitochondrial functionality of brewer's yeast. Amino acid compositions of WGH and intracellular metabolites analysis indicated that active nitrogen metabolism and high levels of stress resistant substances might be responsible for the improvement of growth and fermentation performances in brewer's yeast by nutrient supplementation.

Very High Gravity Bioethanol Revisited: Main Challenges and Advances

Over the last decades, the constant growth of the world-wide industry has been leading to more and more concerns with its direct impact on greenhouse gas (GHG) emissions. Resulting from that, rising efforts have been dedicated to a global transition from an oil-based industry to cleaner biotechnological processes. A specific example refers to the production of bioethanol to substitute the traditional transportation fuels. Bioethanol has been produced for decades now, mainly from energy crops, but more recently, also from lignocellulosic materials. Aiming to improve process economics, the fermentation of very high gravity (VHG) mediums has for long received considerable attention. Nowadays, with the growth of multi-waste valorization frameworks, VHG fermentation could be crucial for bioeconomy development. However, numerous obstacles remain. This work initially presents the main aspects of a VHG process, giving then special emphasis to some of the most important factors that traditionally affect the fermentation organism, such as nutrients depletion, osmotic stress, and ethanol toxicity. Afterwards, some factors that could possibly enable critical improvements in the future on VHG technologies are discussed. Special attention was given to the potential of the development of new fermentation organisms, nutritionally complete culture media, but also on alternative process conditions and configurations.

Wheat gluten hydrolysates promotes fermentation performance of brewer\u2019s yeast in very high gravity worts

The effects of wheat gluten hydrolysates (WGH) and their ethanol elution fractions obtained on XAD-16 resin on physiological activity and fermentation performance of brewer’s yeast during very-high-gravity (VHG) worts fermentation were investigated. The results showed that the addition of WGH and their elution fractions in VHG worts significantly enhanced yeast biomass and viability, and further increased the fermentability, ethanol yield and productivity of yeast. Supplementation with 40% ethanol fraction exhibited the highest biomass (6.9 g/L dry cell), cell viability, fermentability (82.05%), ethanol titer (12.19%, v/v) and ethanol productivity during VHG worts fermentation. In addition, 40% ethanol fraction supplementation also caused the most consumption of amino acid and the highest accumulation of intracellular glycerol and trehalose, 15.39% of increase in cell-membrane integrity, 39.61% of enhancement in mitochondrial membrane potential (MMP), and 18.94% of reduction in intracellular reactive oxygen species (ROS) level in yeast under VHG conditions. Therefore, WGH supplementation was an efficient method to improve fermentation performance of brewer’s yeast during VHG worts.

High Gravity and Very High Gravity Fermentation of Sugarcane Molasses by Flocculating Saccharomyces cerevisiae: Experimental Investigation and Kinetic Modeling.

Substantial progress has been made in ethanol fermentation technology under high gravity (HG) and very high gravity (VHG), which offer environmental and economic benefits. HG and VHG processes increase the productivity of ethanol, reduce distillation costs, and enable higher yields. The aim of the present study was to evaluate the use of sugarcane molasses as the medium component along with flocculating yeasts for fermentation in a fed-batch process employing this promising technology. We evaluated fed-batch fermentation, HG, and VHG involving a molasses-based medium with high concentrations of reducing sugars (209, 222, and 250g/L). Fermentation of 222g/L of total reducing sugars achieved 89.45% efficiency, with a final ethanol concentration of 104.4g/L, whereas the highest productivity (2.98g/(L.h)) was achieved with the fermentation of 209g/L of total reducing sugars. The ethanol concentration achieved with the fermentation of 222g/L of total reducing sugars was close to the value obtained for P'max (105.35g/L). The kinetic model provided a good fit to the experimental data regarding the fermentation of 222g/L. The results revealed that sugarcane molasses and flocculating yeasts can be efficiently used in HG fermentation to reduce the costs of the process and achieve high ethanol titers.

Improvement of ethanol production in fed-batch fermentation using a mixture of sugarcane juice and molasse under very high-gravity conditions.

Ethanol fermentation in very high gravity (VHG) saves energy consumption for ethanol distillation. As the technology offers high ethanol yield and low waste generation and it can be operated at low cost, it could be more efficient at an industrial scale than other ethanol production methods. This work studied ethanol production using a fed-batch bioreactor with a working volume of 1.5L. The main objective of this research was evaluate the effects of temperature, sugar concentration, and cellular concentration using a Central Composite Design (CCD). Experimental conditions were selected using the surface response technique obtained from the CCD, and the results were validated to test the reproducibility. The following operating conditions were selected: temperature of 27.0°C, sugar concentration 300.0g/L, and cell concentration 15.0% (v/v). Under these conditions, after 30h of fermentation the ethanol concentration, productivity and yield were 135.0g/L, 4.42g/(L·h) and 90.0%, respectively. All sugar was completely consumed.

Alcoholic fermentation with high sugar and cell concentration at moderate temperatures using flocculant yeasts

This paper studied bioethanol production at very high gravity (VHG) conditions using flocculent Saccharomyces cerevisiae, evaluating the response yield, ethanol concentration, productivity, and residual sugar through a central composite design (CCD). This CCD was evaluated at 12 and 24 h fermentation times. In the CCD evaluated for 12 h of fermentation, the best condition for alcoholic fermentation was 27 °C, 260 g/L substrate concentration and a 30% v/v cell concentration; a maximum overall desirability of 0.937 was achieved. For CCD at 24 h of fermentation, the best condition was 27 °C, 300 g/L substrate concentration, and a 26% v/v cell concentration. The desirability achieved was 0.811. These conditions allowed us to verify, experimentally, that the CCD models described the fermentation behavior well. VHG alcoholic fermentation in fed-batch with the reuse of cells without chemical treatment was performed using the optimum conditions obtained from the desirability function (27 °C, 300 g/L, 26% v/v). This resulted in favorable alcohol content 132.90 g/L in comparison to the conventional fermentation process.

Very high Gravity (VHG) Bioethanol Production Using Modified Simultaneous Saccharification and Fermentation of Raw Cassava Chips with Molasses by Kluyveromyces marxianus DMKU-KS07

Purpose: The aim of this study was to investigate the enhancement of very high gravity (VHG) bioethanol production using co-substrates of cassava chip hydrolysates and molasses by Kluyveromyces marxianus DMKU-KS07. Methods: The factors effecting cassava chip hydrolysis by raw starch degrading enzyme (RSDE) from the thermophilic bacterium Laceyella sacchari LP175 and commercial glucoamylase were investigated. The obtained raw cassava chip hydrolysate was co-fermented with molasses by the thermotolerant K. marxianus DMKU-KS07 at 42°C under nonsterile system. Results: Factors affecting sugar syrup production from raw cassava chips by synergistic hydrolysis and enzyme saccharification were optimized at temperature, substrate concentration and agitation rate of 50 °C, 250 g/L and 200 rpm, respectively. A yield of 98.6 g/L was obtained at 6 h of incubation, equivalent to 45.9% saccharification and 51.4% hydrolysis of starch, respectively. High bioethanol concentration at 118 g/L, with highest productivity of 2.19 g/L/h and ethanol yield (YP/S) at 0.44 g EtOH/g total sugar, equivalent to 86.3% theoretical yield, was obtained by modified simultaneous saccharification and fermentation (Modified SSF) with co-fermentation of substrates from the enzymatic hydrolysates of raw cassava chips at 42 °C for 12 h. Subsequent addition of molasses increased the final concentration to 100 g total sugar/L at 36 h. Conclusions: Co-fermentation of raw cassava chip hydrolysates with molasses enhanced production of bioethanol at VHG condition, and showed potential for application in ethanol production by enhancing the fermentation process and reducing energy consumption.