Ethanol production from agroindustrial biomass using a crude enzyme complex produced by Aspergillus niger

This study investigates ethanol production from simultaneous fermentation and saccharification (SFS) and separated hydrolysis and fermentation (SHS) using enzyme complexes produced by Aspergillus niger strains (ATCC 16404, ATCC 1057, ATCC 9029). The enzyme complexes were produced by solid-state fermentation (SSF) on inexpensive and readily available agroindustrial products: rice byproduct (composed of AFEX-treated rice rust and rice bran), whey and sugarcane bagasse. The ethanol was produced by Saccharomyces cerevisiae Y904 using whey and rice byproduct as the substrate and the enzyme complex produced by A. niger. The best result for solid-state fermentation (40 U/g of dry substrate, A. niger ATCC 16404) was obtained in a 0.5 L rotating drum bioreactor at 40 °C filled half filled with solid biomass composed of rice byproduct (86% wt/wt), whey (12% wt/wt) and CaCl₂ (2.0% wt/wt). The best result for ethanol fermentation (11.7 g/L of ethanol) was obtained after 12 h of SFS at pH 4.5 and 35 °C. A comparative study of ethanol production by Trichoderma reesei CCT 2768 and A. niger ATCC 16404 complexes under the same optimised SFS and SSF conditions was also performed, revealing that ethanol production by the A. niger enzyme complex was 2.25 times higher than that by T. reesei. These findings suggest that the ethanol production using crude enzymatic complexes produced by A. niger and agroindustrial biomass described in this paper is very promising in terms of disposal of the whey produced by cheese-making and other dairy food processing.     

A novel staggered hybrid SSF approach for efficient conversion of cellulose/hemicellulosic fractions of corncob into ethanol

The following study reports bioconversion of corncob into ethanol using hybrid approach for co-utilization of dilute acid hydrolysate (pentose rich stream) and hexose rich stream obtained by enzymatic saccharification employing commercial cellulase Cellic CTec2 as well as in-house cellulase preparations derived from Malbranchea cinnamomea, Scytalidium thermophilium and a recombinant Aspergillus strain. Acid hydrolysis (1% H₂SO₄) of corncob at 1:15 solid liquid ratio led to removal of 80.5% of hemicellulosic fraction. The solid glucan rich fraction (63.5% glucan, 8.3% pentosans and 27.9% lignin) was hydrolysed at 10% substrate loading rate with different enzymes for 72 h at 50 °C resulting in release of 732 and 535 (mg/g substrate) total sugars by Cellic CTec2 and M. cinnamomea derived enzymes, respectively. The fermentation of enzyme hydrolysate with co-culture of Saccharomyces cerevisiae and Pichia stipitis added in sequential manner resulted in 3.42 and 2.50% (v/v) ethanol in hydrolysate obtained from commercial Cellic CTec2 and M. cinnamomea, respectively. Employing a hybrid approach, where dilute acid hydrolysate stream was added to solid residue along with enzyme Cellic CTec2 during staggered simultaneous saccharification and fermentation at substrate loading rate of 15% resulted in 252 g ethanol/kg corncob.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Simultaneous saccharification and fermentation (SSF) of non-starch polysaccharides and starch from fresh tuber of Canna edulis ker at a high solid content for ethanol production

Canna edulis ker is a potential feedstock for ethanol production because of its low nutrition requirements and the high starch content of its tubers. The processing of C. edulis is limited by the high viscosity of the biomass. In this study, cell wall degrading enzymes (CWDEs) containing acid xylanase and β-glucanase were successful in reducing the viscosity (from 167.30 Pa s to 8.66 Pa s) at 50 °C for 120 min. The effect of CWDEs on simultaneous saccharification and fermentation (SSF) was investigated. Addition of CWDEs before SSF, resulted in an increase in total sugar and fermentable sugar. Meanwhile, the viscosity decreased sharply from approximately 200.00 Pa s to 2.98 Pa s, thereby improving the fermentation parameters and the mass fraction of the theoretical ethanol yield was 94.5%. Only special demand of nutritional ingredients was nitrogen, urea at 750 mg kg⁻¹ was found to be suitable for this purpose. In the verification experiments, the mass fraction of the theoretical ethanol yield in a 5 L fermentor was 98.3%. In conclusion, the pretreatment with CWDEs has significant effect on high level ethanol production using roots and tubers on an industrial scale from the biomass utilization efficiency and economic standpoint.     

Effect of nutrient supplementation on ethanol production in different strategies of saccharification and fermentation from acid pretreated rice straw

The effect of nutrient supplementation on ethanol production by recently selected thermotolerant yeast (Kluyveromyces marxianus NRRL Y-6860) was investigated in different strategies of saccharification and fermentation employing rice straw pretreated by dilute acid. Among the evaluated strategies, similar ethanol yields (Y_P/S ∼ 0.23 g g⁻¹) were obtained with or without nutrient addition. However, considering the whole process time, the strategy based on simultaneous saccharification and fermentation (SSF), without pre-hydrolysis, was assigned as the most suitable configuration due to the highest ethanol volumetric productivity (1.4 g L⁻¹ h⁻¹), about 2-fold higher in relation to the others. The impact of enzymatic preparation employed in this study was also evaluated on glucose fermentation in semi-synthetic medium. The enzymatic preparation affected both glucose consumption and ethanol production by K. marxianus NRRL Y-6860, but just in the absence of nutrients. Therefore, the enzyme type and loading should be carefully defined, not only by the capital costs involved, but also by the possibility of increasing the fermentation inhibitors.     

Sweet sorghum as a whole-crop feedstock for ethanol production

The potential of sweet sorghum as an alternative crop for ethanol production was investigated in this study. Initially, the enzymatic hydrolysis of sorghum grains was optimized, and the hydrolysate produced under optimal conditions was used for ethanol production with an industrial strain of Saccharomyces cerevisiae, resulting in an ethanol concentration of 87 g L⁻¹. From the sugary fraction (sweet sorghum juice), 72 g L⁻¹ ethanol was produced. The sweet sorghum bagasse was submitted to acid pretreatment for hemicellulose removal and hydrolysis, and a flocculant strain of Scheffersomyces stipitis was used to evaluate the fermentability of the hemicellulosic hydrolysate. This process yielded an ethanol concentration of 30 g L⁻¹ at 23 h of fermentation. After acid pretreatment, the remaining solid underwent an alkaline extraction for lignin removal. This partially delignified material, known as partially delignified lignin (PDC), was enriched with nutrients in a solid/liquid ratio of 1 g/3.33 mL and subjected to simultaneous saccharification and fermentation (SSF) process, resulting in an ethanol concentration of 85 g L⁻¹ at 21 h of fermentation. Thus, from the conversion of starchy, sugary and lignocellulosic fractions approximately 160 L ethanol.ton⁻¹ sweet sorghum was obtained. This amount corresponds to 13,600 L ethanol.ha⁻¹.     

Simultaneous enzymatic saccharification and ABE fermentation using pretreated oil palm empty fruit bunch as substrate to produce butanol and hydrogen as biofuel

Simultaneous saccharification and acetone–ethanol–butanol (ABE) fermentation was conducted in order to reduce the number of steps involved in the conversion of lignocellulosic biomass into butanol. Enzymatic saccharification of pretreated oil palm empty fruit bunch (OPEFB) by cellulase produced 31.58 g/l of fermentable sugar. This saccharification was conducted at conditions similar to the conditions required for ABE fermentation. The simultaneous process by Clostridium acetobutylicum ATCC 824 produced 4.45 g/l of ABE with butanol concentration of 2.75 g/l. The butanol yield of 0.11 g/g and ABE yield of 0.18 g/g were obtained from this simultaneous process as compared to the two-step process (0.10 g/g of butanol yield and 0.14 g/g of ABE yield). In addition, the simultaneous process also produced higher cumulative hydrogen (282.42 ml) than to the two-step process (222.02 ml) after 96 h of fermentation time. This study suggested that the simultaneous process has the potential to be implemented for the integrated production of butanol and hydrogen from lignocellulosic biomass.     

Enzymatic saccharification of pretreated rice straw by cellulase produced from Bacillus carboniphilus CAS 3 utilizing lignocellulosic wastes through statistical optimization

A marine bacterium, Bacillus carboniphilus CAS 3 was subjected to optimization for cellulase production utilizing cellulosic waste through response surface methodology. Plackett – Burman and Central composite design was employed and the optimal medium constituents for maximum cellulase production (4040.45 U/mL) were determined as rice bran, yeast extract, MgSO₄·7H₂O and KH₂PO₄ at 6.27, 2.52, 0.57 and 0.39 g/L, respectively. The cellulase produced was purified to the specific activity of 434.94 U/mg and 11.46% of recovery with the molecular weight of 56 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 50 °C, 9 and 30% and more than 70% of its original activity was retained even at 80 °C, 12 and 35% respectively. Further, enzymatic saccharification of pretreated rice straw yielded about 15.56 g/L of reducing sugar at 96 h, suggesting that the purified cellulase could be useful for production of reducing sugars from cellulosic biomass into ethanol.     

Dilute oxalic acid pretreatment for biorefining giant reed (Arundo donax L.)

Biomass pretreatment is essential to overcome recalcitrance of lignocellulose for ethanol production. In the present study we pretreated giant reed (Arundo donax L.), a perennial, rhizomatous lignocellulosic grass with dilute oxalic acid. The effects of temperature (170-190 °C), acid loading (2-10% w/w) and reaction time (15-40 min) were handled as a single parameter, combined severity. We explored the change in hemicellulose, cellulose and lignin composition following pretreatment and glucan conversion after enzymatic hydrolysis of the solid residue. Two different yeast strains, Scheffersomyces (Pichia) stipitis CBS 6054, which is a native xylose and cellobiose fermenter, and Saccharomyces carlsbergensis FPL-450, which does not ferment xylose or cellobiose, were used along with commercial cellulolytic enzymes in simultaneous saccharification and fermentation (SSF). S. carlsbergensis attained a maximum ethanol concentration of 15.9 g/l after 48 h at pH 5.0, while S. stipitis, at the same condition, took 96 h to reach a similar ethanol value; increasing the pH to 6.0 reduced the S. stipitis lag phase and attained 18.0 g/l of ethanol within 72 h.     

Evaluation of fuel ethanol production from aqueous ammonia-treated rice straw via simultaneous saccharification and fermentation

Rice straw (RS) has been considered a promising feedstock for ethanol production in Asia. However, the recalcitrance of biomass, particularly the presence of lignin, hinders the enzymatic saccharification of polysaccharides in RS and consequently decreases the ethanol yield. Here, we used aqueous ammonia pretreatment to remove lignin from RS (aRS). The reaction conditions were a solid:liquid ratio of 1:12, an ammonia concentration of 27% (w w⁻¹), room temperature, and a 2-week incubation. We evaluated enzymatic digestibility and the ethanol production yield. A 42% reduction in lignin content increased the glucan conversion of aRS to glucose from 20 to 71% using a combination of Cellic Ctec2 cellulases and Cellic Htec2 xylanases at enzyme loads of 15 FPU +100 XU g⁻¹ solid. Scanning electron microscopy highlighted the extensive removal of external fibres and increased porosity of aRS, which aided the accessibility of cellulose for enzymes. Using the same enzyme dosage and a solid load of 100 g L⁻¹, simultaneous saccharification and fermentation using a monoculture of Saccharomyces cerevisiae and co-culture with Candida tropicalis yielded ethanol concentrations of 22 and 25 g L⁻¹, corresponding to fermentation efficiencies of 96 and 86% fermentation, respectively. The volumetric ethanol productivities for these systems were 0.45 and 0.52 g L⁻¹ h⁻¹. However, the ethanol yield based on the theoretical glucose and xylose concentrations was lower for the co-culture (0.44 g g⁻¹) than the monoculture (0.49 g g⁻¹) due to the low xylose consumption. Further research should optimise fermentation variables or select/improve microbial strains capable of fermenting xylose to increase the overall ethanol production yield.     

Biological hydrogen production from steam-exploded straw by simultaneous saccharification and fermentation

Hydrogen was produced by simultaneous saccharification and fermentation from steam-exploded corn straw (SECS) using Clostridium butyricum AS1.209. Effect of various process parameters, such as solid to liquid ratio, enzyme loading and initial pH, etc., were examined with respect to maximum hydrogen productivity which was obtained by fitting the cumulative hydrogen production data to a modified Gompertz equation. Maximum specific hydrogen production rate and maximal hydrogen yield were 126 ml/g VSS d and 68 ml/g SECS, respectively. The yield of soluble metabolites was 197.7 mg/g SECS. Acetic acid accounted for 46% of the total was the most abundant product and this shows that hydrogen production from SECS was essentially acetate-type fermentation. Hydrogen production by simultaneous saccharification and fermentation of SECS has the predominance of short lag-stage and high maximum specific hydrogen production rate and it was a promising method for hydrogen production and straw biomass conversion.     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.     

Optimization of concomitant production of cellulase and xylanase from Rhizopus oryzae SN5 through EVOP-factorial design technique and application in Sorghum Stover based bioethanol production

Current study deals with the production of cellulases and xylanases from the Rhizopus oryzae SN5 isolated from composed soil of Himalayan pine forest, in order to meet the challenges of lignocellulosic biomass based biorefineries. Culture parameters for concomitant production of cellulase and xylanase were optimized through EVOP-factorial design technique under solid state fermentation. And maximum yield of cellulase and xylanase were obtained 437.54 U/gds and 273.83 U/gds, respectively at 30 °C and pH 6.0 after 5 days of incubation. On applying these enzymes for the saccharification of the dilute acid pretreated Sorghum Stover (SS), 0.407 g/g sugar was yielded. This hydrolysate on fermentation, yielded 0.411 g/g ehanol with Saccharomyces cerevisiae (NCIM 3288), which could be considered a good conversion. Therefore, Rhizopus oryzae SN5 was found as potent strain for the production of the cocktail of lignocellulosic biomasss hydrolytic enzymes and would be promising tool in the area of lignocellulose based bio-refineries.     

Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes

A new solvent-producing Clostridium has been isolated from soil used in intensive rice cultivation. The 16S rRNA analysis of the isolate indicates that it is closely related to Clostridium acetobutylicum, with a sequence identity of 96%. The new isolate, named C. acetobutylicum YM1, produces biobutanol from multiple carbon sources, including glucose, fructose, xylose, arabinose, glycerol, lactose, cellobiose, mannitol, maltose, galactose, sucrose and mannose. This isolate can also utilize polysaccharides such as starch and carboxylmethyl cellulose (CMC) for the production of biobutanol. The ability of isolate YM1 to produce biobutanol from agro-industrial wastes was also evaluated for rice bran, de-oiled rice bran, palm oil mill effluent and palm kernel cake. The highest concentration of biobutanol (7.27 g/L) was obtained from the fermentation medium containing 2% (w/v) fructose, with a total acetone–butanol–ethanol (ABE) concentration of 10.23 g/L. The ability of isolate YM1 to produce biobutanol from various carbon sources and agro-wastes indicates the promise of the use of this isolate for the production of biobutanol, a renewable energy resource, from readily available renewable feedstocks.     

Ethanol production from food residues

Food residues were converted to ethanol by simultaneous saccharification with an amylolytic enzyme complex (a mixture of amyloglucosidase, ??-amylase, and protease), and fermentation (SSF) with the yeast, Saccharomyces cerevisiae. About 36 g dm⁻³ of ethanol was obtained from 100 g dm⁻³ food residue in 48 h of fermentation. In the SSF with no nitrogen supplements, 25 g dm⁻³ of ethanol was produced from 100 g dm⁻³ food residues. In addition, none of the nutrient components except yeast extract from the SSF medium were found to affect ethanol production from food residues. This result indicates that food residues could be a good economic bioresource for ethanol production.     

Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production

Production of reducing sugar by hydrolysis of corncob material with Streptomyces sp. cellulase and ethanol fermentation of cellulosic hydrolysate was investigated. Cultures of Streptomyces sp. T3-1 improved reducing sugar yields with the production of CMCase, Avicelase and ??-glucosidase activity of 3.8, 3.9 and 3.8 IU/ml, respectively. CMCase, Avicelase, and ??-glucosidase produced by the Streptomyces sp. T3-1 favored the conversion of cellulose to glucose. It was recognized that the synergistic interaction of endoglucanase, exoglucanase and ??-glucosidase resulted in efficient hydrolysis of cellulosic substrate. After 5 d of incubation, the overall reducing sugar yield reached 53.1 g/100 g dried substrate. Further fermentation of cellulosic hydrolysate containing 40.5 g/l glucose was performed using Saccharomyces cerevisiae BCRC 21812, 14.6 g/l biomass and 24.6 g/l ethanol was obtained within 3 d. The results have significant implications and future applications regarding to production of fuel ethanol from agricultural cellulosic waste.     

Valorization of Eucalyptus wood by glycerol-organosolv pretreatment within the biorefinery concept: An integrated and intensified approach

The efficient utilization of lignocellulosic biomass and the reduction of production cost are mandatory to attain a cost-effective lignocellulose-to-ethanol process. The selection of suitable pretreatment that allows an effective fractionation of biomass and the use of pretreated material at high-solid loadings on saccharification and fermentation (SSF) processes are considered promising strategies for that purpose. Eucalyptus globulus wood was fractionated by organosolv process at 200 °C for 69 min using 56% of glycerol-water. A 99% of cellulose remained in pretreated biomass and 65% of lignin was solubilized. Precipitated lignin was characterized for chemical composition and thermal behavior, showing similar features to commercial lignin. In order to produce lignocellulosic ethanol at high-gravity, a full factory design was carried to assess the liquid to solid ratio (3–9 g/g) and enzyme to solid ratio (8–16 FPU/g) on SSF of delignified Eucalyptus. High ethanol concentration (94 g/L) corresponding to 77% of conversion at 16FPU/g and LSR = 3 g/g using an industrial and thermotolerant Saccharomyces cerevisiae strain was successfully produced from pretreated biomass. Process integration of a suitable pretreatment, which allows for whole biomass valorization, with intensified saccharification-fermentation stages was shown to be feasible strategy for the co-production of high ethanol titers, oligosaccharides and lignin paving the way for cost-effective Eucalyptus biorefinery.     

An oil palm-based biorefinery concept for cellulosic ethanol and phytochemicals production: Sustainability evaluation using exergetic life cycle assessment

In this study, thermo-environmental sustainability of an oil palm-based biorefinery concept for the co-production of cellulosic ethanol and phytochemicals from oil palm fronds (OPFs) was evaluated based on exergetic life cycle assessment (ExLCA). For the production of 1 tonne bioethanol, the exergy content of oil palm seeds was upgraded from 236 MJ to 77,999 MJ during the farming process for OPFs production. Again, the high exergy content of the OPFs was degraded by about 62.02% and 98.36% when they were converted into cellulosic ethanol and phenolic compounds respectively. With a total exergy destruction of about 958,606 MJ (internal) and 120,491 MJ (external or exergy of wastes), the biorefinery recorded an overall exergy efficiency and thermodynamic sustainability index (TSI) of about 59.05% and 2.44 per tonne of OPFs' bioethanol respectively. Due to the use of fossil fuels, pesticides, fertilizers and other toxic chemicals during the production, the global warming potential (GWP = 2265.69 kg CO₂ eq.), acidification potential (AP = 355.34 kg SO₂ eq.) and human toxicity potential (HTP = 142.79 kg DCB eq.) were the most significant environmental impact categories for a tonne of bioethanol produced in the biorefinery. The simultaneous saccharification and fermentation (SSF) unit emerged as the most exergetically efficient (89.66%), thermodynamically sustainable (TSI = 9.67) and environmentally friendly (6.59% of total GWP) production system.     

Comparative study on ethanol production from pretreated sugarcane bagasse using immobilized Saccharomyces cerevisiae on various matrices

Microwave alkali pretreated sugarcane bagasse was used as a substrate for production of cellulolytic enzymes, needed for biomass hydrolysis. The pretreated sugarcane bagasse was enzymatic hydrolyzed by crude unprocessed enzymes cellulase (Filter paper activity 9.4 FPU/g), endoglucanase (carboxymethylcellulase, 148 IU/g), β-glucosidase (116 IU/g) and xylanase (201 IU/g) produced by Aspergillus flavus using pretreated sugarcane bagasse as substrate under solid state fermentation. Concentrated enzymatic hydrolyzate was used for ethanol production using Saccharomyces cerevisiae immobilized on various matrices. The yield of ethanol was 0.44 g_p/g_s in case of yeast immobilized sugarcane bagasse, 0.38 g_p/g_s using Ca-alginate and 0.33 g_p/g_s using agar-agar as immobilization matrices. The immobilized yeast studied up to 10 cycles in case of immobilized sugarcane bagasse and up to 4 cycles in case of agar-agar and calcium alginate for ethanol production under repeated batch fermentation study.     

Bioethanol production by a flocculent hybrid,CHFY0321 obtained by protoplast fusion between Saccharomyces cerevisiae and Saccharomyces bayanus

Fusion hybrid yeast, CHFY0321, was obtained by protoplast fusion between non-flocculent-high ethanol fermentative Saccharomyces cerevisiae CHY1011 and flocculent-low ethanol fermentative Saccharomyces bayanus KCCM12633. The hybrid yeast was used together with the parental strains to examine ethanol production in batch fermentation. Under the conditions tested, the fusion hybrid CHFY0321 flocculated to the highest degree and had the capacity to ferment well at pH 4.5 and 32 °C. Simultaneous saccharification and fermentation for ethanol production was carried out using a cassava (Manihot esculenta) powder hydrolysate medium containing 19.5% (w v^?1) total sugar in a 5 l lab scale jar fermenter at 32 °C for 65 h with an agitation speed of 2 Hz. Under these conditions, CHFY0321 showed the highest flocculating ability and the best fermentation efficiency for ethanol production compared with those of the wild-type parent strains. CHFY0321 gave a final ethanol concentration of 89.8 ± 0.13 g l^?1, a volumetric ethanol productivity of 1.38 ± 0.13 g l^?1 h^?1, and a theoretical yield of 94.2 ± 1.58%. These results suggest that CHFY0321 exhibited the fermentation characteristics of S. cerevisiae CHY1011 and the flocculent ability of S. bayanus KCCM12633. Therefore, the strong highly flocculent ethanol fermentative CHFY0321 has potential for improving biotechnological ethanol fermentation processes.