Ethanol production from seaweed extract

Extracts from Laminaria hyperborea could possibly be fermented to ethanol commercially. In particular, seaweed harvested in the autumn contains high levels of easily extractable laminaran and mannitol. Four microorganisms were tested to carry out this fermentation, one bacterium and three yeasts. Only Pichia angophorae was able to utilise both laminaran and mannitol for ethanol production, and its substrate preferences were investigated in batch and continuous cultures. Laminaran and mannitol were consumed simultaneously, but with different relative rates. In batch fermentations, mannitol was the preferred substrate. Its share of the total laminaran and mannitol consumption rate increased with oxygen transfer rate (OTR) and pH. In continuous fermentations, laminaran was the preferred substrate at low OTR, whereas at higher OTR, laminaran and mannitol were consumed at similar rates. Optimisation of ethanol yield required a low OTR, and the best yield of 0.43 g ethanol (g substrate)⁻¹ was achieved in batch culture at pH 4.5 and 5.8 mmol O₂ l⁻¹ h⁻¹. However, industrial production of ethanol from seaweed would require an optimisation of the extraction process to yield a higher ethanol concentration. Journal of Industrial Microbiology & Biotechnology (2000) 25, 249–254. Received 25 February 2000/ Accepted in revised form 05 August 2000     

Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Analysis and optimization of a two-substrate fermentation for xylitol production using Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose using Candida tropicalisATCC 13803. A two-substrate fermentation was designed in order to increase xylitol yield and volumetric productivity. Glucose was used initially for cell growth followed by conversion of xylose to xylitol without cell growth and by-product formation after complete depletion of glucose. High glucose concentrations increased volumetric productivity by reducing conversion time due to high cell mass, but also led to production of ethanol, which, in turn, inhibited cell growth and xylitol production. Computer simulation was undertaken to optimize an initial glucose concentration using kinetic equations describing rates of cell growth and xylose bioconversion as a function of ethanol concentration. Kinetic constants involved in the equations were estimated from the experimental results. Glucose at 32 g L⁻¹ was estimated to be an optimum initial glucose concentration with a final xylose concentration of 86 g L⁻¹ and a volumetric productivity of 5.15 g-xylitol L⁻¹ h⁻¹. The two-substrate fermentation was performed under optimum conditions to verify the computer simulation results. The experimental results were in good agreement with the predicted values of simulation with a xylitol yield of 0.81 g-xylitol g-xylose⁻¹ and a volumetric productivity of 5.06 g-xylitol L⁻¹ h⁻¹. Received 16 June 1998/ Accepted in revised form 28 February 1999     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Production of butanol from starch-based waste packing peanuts and agricultural waste

We examined the fermentation of starch-based packing peanuts and agricultural wastes as a source of fermentable carbohydrates using Clostridium beijerinckii BA101. Using semidefined P2 medium containing packing peanuts and agricultural wastes, instead of glucose as a carbohydrate source, we measured characteristics of the fermentation including solvent production, productivity, and yield. With starch as substrate (control), the culture produced 24.7 g l⁻¹ acetone–butanol–ethanol (ABE), while with packing peanuts it produced 21.7 g l⁻¹ total ABE with a productivity of 0.20 g l⁻¹ h⁻¹ and a solvent (ABE) yield of 0.37. Cell growth in starch, packing peanuts, and agricultural wastes medium was different, possibly due to the different nature of these substrates. Using model agricultural waste, 20.3g l⁻¹ ABE was produced; when using actual waste, 14.8 g l⁻¹ ABE was produced. The use of inexpensive substrates will increase the economic viability of the conversion of biomass to butanol, and can provide new markets for these waste streams. Journal of Industrial Microbiology & Biotechnology (2002) 29, 117–123 doi: 10.1038/sj.jim.7000285 Received 14 November 2001/ Accepted in revised form 07 June 2002     

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l⁻¹ (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l⁻¹ h⁻¹ for SSF compared to 0.313 g l⁻¹ h⁻¹ for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192. Received 02 December 1999/ Accepted in revised form 20 July 2000     

The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus

Summary The ability of a Candida shehatae and a Pachysolen tannophilus strain to ferment D-xylose to ethanol was evaluated in defined and complex media under different levels of aeration. Aeration enhanced the ethanol productivity of both yeasts considerably. C. shehatae maintained a higher fermentation rate and ethanol yield than P. tannophilus over a wide range of aeration levels. Ethanol production by C. shehatae commenced during the early stage of the fermentation, whereas with P. tannophilus there was a considerable lag between the initiation of growth and ethanol production. Both yeasts produced appreciable quantities of xylitol late in the fermentation. P. tannophilus failed to grow under anoxic conditions, producing a maximum of only 0.5 g · l^-1 ethanol. In comparison, C. shehatae exhibited limited growth in anoxic cultures, and produced ethanol much more rapidly. Under the condition of aeration where C. shehatae exhibited the highest ethanol productivity, the fermentation parameters were: maximum specific growth rate, 0.15 h^-1; maximum volumetric and specific rates of ethanol production, 0.7 g (l · h)^-1 and 0.34 g ethanol (g cells · h)^-1 respectively; ethanol yield, 0.36 g (g xylose)^-1. The best values obtained with P. tannophilus were: maximum specific growth rate, 0.14 h^-1; maximum volumetric and specific rates of ethanol production, 0.22 g (l · h)^-1 and 0.07 h^-1 respectively; ethanol yield coefficient, 0.28. Because of its higher ethanol productivity at various levels of aeration, C. shehatae has a greater potential for ethanol production from xylose than P. tannophilus.     

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose

The gene mel1, encoding α-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and α-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanolproducing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular α-galactosidase and β-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing 2 genes could achieve 0.29 OD600 h⁻¹ and a biomass yield up to 7.8 g l⁻¹ dry cell weight on medium containing 10.0 g l⁻¹ cellobiose and 10.0 g l⁻¹ melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l⁻¹ ethanol was produced from 100 g of cellulose supplied with 5 g l⁻¹ melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Aeration strategy: a need for very high ethanol performance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> fed-batch process

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions—oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)—on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l^–1), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l^–1 in 45 h) and average productivity (3.3 vs 2.6 g l^–1 h^–1) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l^–1. We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l^–1 in 45 h) in order to achieve a highly competitive dynamic process.     

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Its metabolic characteristics suggest that Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into Z. palmae, where their expression was driven by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant Z. palmae strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using Z. palmae expressing E. coli xylose catabolic enzymes.     

Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification–fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 °C and 37 °C, while the activity of cellulolytic enzymes is highest at around 50 °C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, which successfully converts a cellulosic β-glucan to ethanol directly at 48 °C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of β-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Aeration alleviates ethanol inhibition and glycerol production during fed-batch ethanol fermentation

In this study, we investigated the effects of aeration on ethanol inhibition and glycerol production during fed-batch ethanol fermentation. When aeration was conducted at 0.13, 0.33, and 0.8 vvm, the ethanol productivity, specific ethanol production rate, and ethanol yield in the presence of greater than 100 g/L of ethanol were higher than when aeration was not conducted. In addition, estimation of the parameters (α and β) in a model equation of ethanol inhibition kinetics indicated that aeration alleviated ethanol inhibition against the specific growth rate and the specific ethanol production rate. Specifically, when aeration was conducted, the glycerol yield and specific glycerol production rate decreased approximately 50 and 70%, respectively. Finally, the results of this study indicated that aeration during fed-batch ethanol fermentation may improve the ethanol concentration in the final culture broth, as well as the ethanol productivity.     

Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 7.5 g l⁻¹ and 0.36 g (g xylose consumed)⁻¹. These parameters improved to 13.4 g l⁻¹ and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l⁻¹ glucose and 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 29.8 g l⁻¹ and 0.42 respectively, in the presence of borate. Temperature modulation from 30 °C to 35 °C during fermentation further enhanced the above parameters to 39 g l⁻¹ and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield. Received: 14 May 1999 / Received revision: 27 September 1999 / Accepted: 2 October 1999     

Overexpression of the OLE1 gene enhances ethanol fermentation by Saccharomyces cerevisiae

 The fermentation characteristics of Saccharomyces cerevisiae strains which overexpress a constitutive OLE1 gene were studied to clarify the relationship between the fatty acid composition of this yeast and its ethanol productivity. The growth yield and ethanol productivity of these strains in the medium containing 15% dextrose at 10 °C were greater than those of the control strains under both aerobic and anaerobic conditions but this difference was not observed under other culture conditions. During repeated-batch fermentation, moreover, the growth yield and ethanol productivity of the wild-type S. cerevisiae increased gradually and then were similar to those of the OLE1-overexpressing transformant in the last batch fermentation. However, the unsaturated fatty acid content (77.6%) of the wild-type cells was lower than that (86.2%) of the OLE1-recombinant cells. These results suggested that other phenomena caused by the overexpression of the OLE1 gene, rather than high unsaturated fatty acid content, are essential to ethanol fermentation by this yeast. Received: 11 June 1999 / Received last revision: 12 November 1999 / Accepted: 28 November 1999     

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.