Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

Production of biobutanol from acid-pretreated corncob using Clostridium beijerinckii TISTR 1461: Process optimization studies

Corncob is a potential feedstock in Thailand that can be used for fermentable sugar production through dilute sulfuric acid pretreatment and enzymatic hydrolysis. To recover high amounts of monomeric sugars from corncob, the sulfuric pretreatment conditions were optimized by using response surface methodology with three independent variables: sulfuric acid concentration, temperature, and time. The highest response of total sugars, 48.84 g/L, was found at 122.78°C, 4.65 min, and 2.82% (v/v) H₂SO₄. With these conditions, total sugars from the confirmation experiment were 46.29 g/L, with 5.51% error from the predicted value. The hydrolysate was used as a substrate for acetone–butanol–ethanol fermentation to evaluate its potential for microbial growth. The simultaneous saccharification and fermentation (SSF) showed that C. beijerinckii TISTR 1461 can generate acetone–butanol–ethanol products at 11.64 g/L (5.29 g/L acetone, 6.26 g/L butanol, and 0.09 g/L ethanol) instantly using sugars from the hydrolysed corncob with Novozymes 50013 cellulase enzyme without an overliming process.     

Butanol production from wheat straw hydrolysate using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation 48.9 g L⁻¹ glucose (initial sugar 62.0 g L⁻¹) was used to produce 20.1 g L⁻¹ ABE with a productivity and yield of 0.28 g L⁻¹ h⁻¹ and 0.41, respectively. In a similar experiment where WSH (60.2 g L⁻¹ total sugars obtained from hydrolysis of 86 g L⁻¹ wheat straw) was used, the culture produced 25.0 g L⁻¹ ABE with a productivity and yield of 0.60 g L⁻¹ h⁻¹ and 0.42, respectively. These results are superior to the control experiment and productivity was improved by 214%. When WSH was supplemented with 35 g L⁻¹ glucose, a reactor productivity was improved to 0.63 g L⁻¹ h⁻¹ with a yield of 0.42. In this case, ABE concentration in the broth was 28.2 g L⁻¹. When WSH was supplemented with 60 g L⁻¹ glucose, the resultant medium containing 128.3 g L⁻¹ sugars was successfully fermented (due to product removal) to produce 47.6 g L⁻¹ ABE, and the culture utilized all the sugars (glucose, xylose, arabinose, galactose, and mannose). These results demonstrate that C. beijerinckii P260 has excellent capacity to convert biomass derived sugars to solvents and can produce over 28 g L⁻¹ (in one case 41.7 g L⁻¹ from glucose) ABE from WSH. Medium containing 250 g L⁻¹ glucose resulted in no growth and no ABE production. Mixtures containing WSH + 140 g L⁻¹ glucose (total sugar approximately 200 g L⁻¹) showed poor growth and poor ABE production. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

Fermentation of cheese whey by a mixed culture ofClostridium beijerinckii andBacillus cereus

Summary Fermentation of cheese whey to produce butanol and butyric acid was carried out using a mixed culture ofClostridium beijerinkii andBacillus cereus. Fermentation selectivities were studied by controlling the pH of the system. Controlled pH values higher than 6.5 as well as those below 5.0 were not conducive to butanol production. Maximum product formation was obtained by controlling the pH at 5.5. When compared with the results obtained using the pure culture ofC. beijerinckii, a higher butanol concentration was obtained in the mixed culture without sacrificing the level of butyric acid formed.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

The sugar,biomass and biofuel potential of temperate by tropical maize hybrids

The demand for biofuels has created a market for feedstocks to meet future energy requirements. Temperate × tropical maize (Zea mays L.) hybrids, which combine high biomass and fermentable stalk sugars, have yet to be considered as a biomass feedstock. Our objective was to evaluate biological potential, genetic variability and impact of nitrogen (N) on biomass, stalk sugar, and biofuel potential of temperate × tropical maize (TTM) hybrids. Twelve TTM hybrids, two grain and silage hybrids were grown in 2008, followed in 2009 by seven earshoot‐bagged TTM hybrids. In both years, they were grown with and without supplemental N (202 kg ha^?1) in Champaign, IL. Plants were sampled for total and partitioned biomass, and analyzed for concentration and content of sugar. The TTM hybrids were 40% taller, exhibited later reproductive maturity, greater flowering asynchrony, and remained green longer. All hybrids responded to supplemental N by producing more biomass and grain, a lower percent of biomass partitioned to stalk and leaf, whereas TTM also had a decreased concentration of sugar. Total average biomass yields were 24 Mg ha^?1 for both the TTM and grain hybrids. However, TTM partitioned 50% more biomass to the stalk and produced 50% more sugar, and had less than half the grain of the commercial hybrids, indicating grain production and sugar accumulation are inversely related. When grain formation was prevented by earshoot bagging, TTM hybrids produced, without supplemental N fertilizer, an average of 4024 kg ha^?1 of sugar, which was three‐ to four‐fold greater than the non earshoot‐bagged TTM and ear removed hybrid. Calculated estimates for ethanol production, considering the potential from sugar, stover and grain, indicate TTM can yield nearly the amount of ethanol per hectare as modern grain hybrids, but with a decreased requirement for supplemental fertilizer N.     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

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Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5–5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-P_thl-augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

The acetone butanol fermentation on glucose and xylose. II. Regulation and kinetics in fed-batch cultures

The kinetics in fed-batch cultures of acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The final conversion yield of sugars into solvents always increases with the sugar feeding rate. At low feeding rates, the sugar concentration in the medium becomes limiting, which results in a slower cellular growth, a slower metabolic transition from an acid to a solvent fermentation and, thus, a higher accumulation of acids. It is only at sufficiently high feeding rates that fed-batch fermentations yield kinetic results comparable to those of batch fermentations. With mixtures of glucose and xylose, because of a maintained low glucose level, both sugars are taken up at the same rate during a first fermentation period. An earlier accumulation of xylose when the fermentation becomes inhibited suggest that xylose utilization is inhibited when the catabolic flux of glucose alone can satisfy the metabolic activity of the cell. Kinetic results with batch and fed-batch fermentations indicate several important features of the regulation of C. acetobutylicum metabolism: an early inhibition by the produced acids; an initiation of solvent formation between 4 and 6 g/L acetic and butyric acid depending on the metabolic activity of the cell; a metabolic transition from acids to solvents production at a rate closely related to the rate of sugar uptake; during solvent production, a reassimilation of acids above a minimal rate of sugar consumption of 0.2 h(-1); a final inhibition of the fermentation at a total butanol and acids concentration of ca. 20 g/L.     

Decanter cake waste as a renewable substrate for biobutanol production by Clostridium beijerinckii

The aim of this study was to determine if decanter cake waste from a palm oil mill could be used as a renewable substrate for biobutanol production. Decanter cake waste was first hydrolyzed to fermentable sugars by nitric acid and detoxified by activated-charcoal. The detoxified hydrolysate supplemented with whey protein and ammonium sulfate as cheap nitrogen sources, was used for butanol production by growing cells of Clostridium beijerinckii. The detoxified hydrolysate was also used as a co-substrate for direct conversion of butyric acid to butanol in a nitrogen-free medium. By these two steps, C. beijerinckii produced 3.42 g/L of butanol with a yield of 0.28 C-mol butanol/C-mol carbon in the first step and produced 6.94 g/L of butanol with a yield of 0.47 C-mol butanol/C-mol carbon in the second step. This study has showed that decanter cake waste could serve as a low-cost substrate for biobutanol production.     

Hydrogen production conditions from food waste by dark fermentation with Clostridium beijerinckii KCTC 1785

The optimum conditions for biological hydrogen production from food waste by Clostridium beijerinckii KCTC 1875 were investigated. The optimum initial pH and fermentation temperature were 7.0 and 40°C, respectively. When the pH of fermentation was controlled to 5.5, a maximum amount of hydrogen could be obtained. Under these conditions, about 2,737 mL of hydrogen was produced from 50 g COD/L of food waste for 24 h, and the hydrogen content in the biogas was 38%. Hydrogen production rate and yield were about 108 mL/L·h and 128 mL/g COD_degraded, respectively. High concentrations of acetic (< 5,000 mg/L) or butyric acid (< 3,000 mg/L) significantly inhibited hydrogen production.     

Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol

The study was focused on developing a continuous method to produce an alcohol mixture suitable to be used as a gasoline supplement. The immobilized column reactor with wood pulp fibers was successfully used for the continuous production of butanol and isopropanol using Clostridium beijerinckii DSM 6423. A sugar mixture (glucose, mannose, galactose, arabinose and xylose) representing lignocellulose hydrolysate was used as a substrate for the production of solvents. The effect of dilution rate on solvent production was studied during continuous operation. The maximum total solvent concentration of 11.99 g/l was obtained at a dilution rate of 0.16 h^?1. The maximum solvent productivity (5.58 g/l h) was obtained at a dilution rate of 1.5 h^?1. The maximum solvent yield of 0.45 g/g from sugar mixture was observed at 0.25 h^?1. The system will be further used for the solvent production using wood hydrolysate as a substrate.     

The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures

The kinetics in batch culture of the acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The fastest initial growth and transition from an acid to a solvent metabolism occurs on glucose, with a final 62 g/L glucose conversion. On xylose, an initial slower growth rate and a longer metabolic transition result in higher cellular and acids concentration, thus in a level of fermented sugar limited to 47 g/L. Batch fermentations on mixtures of glucose and xylose show that both sugars can be fermented, with a higher rate for glucose. However, xylose fermentation is inducible and inhibited at glucose level above 15 g/L. Mixtures of glucose and xylose yield the highest amount of fermented sugars, up to 68 g/L, as a result of both a fast metabolic transition on glucose and a strong acid reconsumption on xylose. In all cases, solvent production is triggered at a total acid concentration between 4 and 5 g/L, whereas the final inhibition of the fermentation takes place at a total butanol and acid concentration between 18 and 20 g/L.     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Evaluation and optimization of ethanol production from carob pod extract by <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> using response surface methodology

In this research, ethanol production from carob pod extract (extract) using Zymomonas mobilis with medium optimized by Plackett–Burman (P–B) and response surface methodologies (RSM) was studied. Z. mobilis was recognized as useful for ethanol production from carob pod extract. The effects of initial concentrations of sugar, peptone, and yeast extract as well as agitation rate (rpm), pH, and culture time in nonhydrolyzed carob pod extract were investigated. Significantly affecting variables (P = 0.05) in the model obtained from RSM studies were: weights of bacterial inoculum, initial sugar, peptone, and yeast extract. Acid hydrolysis was useful to complete conversion of sugars to glucose and fructose. Nonhydrolyzed extract showed higher ethanol yield and residual sugar compared with hydrolyzed extract. Ethanol produced (g g⁻¹ initial sugar, as the response) was not significantly different (P = 0.05) when Z. mobilis performance was compared in hydrolyzed and nonhydrolyzed extract. The maximum ethanol of 0.34 ± 0.02 g g⁻¹ initial sugar was obtained at 30°C, initial pH 5.2, and 80 rpm, using concentrations (g per 50 mL culture media) of: inoculum bacterial dry weight, 0.017; initial sugar, 5.78; peptone, 0.43; yeast extract, 0.43; and culture time of 36 h.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101

Spray-dried soy molasses (SDSM) contains the sugars dextrose, sucrose, fructose, pinitol, raffinose, verbascose, melibiose, and stachyose. Of the 746 g kg⁻¹ total sugars in SDSM, 434 g kg⁻¹ is fermentable using Clostridium beijerinckii BA101. SDSM was used to produce acetone, butanol, and ethanol (ABE) by C. beijerinckii BA101 in batch cultures. Using 80 g l⁻¹ SDSM, 10.7 g l⁻¹ ABE was produced in P2 medium. Higher concentrations of SDSM resulted in poor solvent production due to the presence of excessive salt and inhibitory components. C. beijerinckii BA101 in SDSM at 80 g l⁻¹ concentration produced 22.8 g l⁻¹ ABE when supplemented with 25.3 g l⁻¹ glucose. SDSM contains 57.4 g kg⁻¹ mineral ash and 2% tri-calcium phosphate. Tri-calcium phosphate up to 43.1 g l⁻¹ was not inhibitory and at a tri-calcium phosphate concentration of 28.8 g l⁻¹, the culture produced more solvents (30.1 g l⁻¹) than the control experiment (23.8 g l⁻¹). In contrast, sodium chloride was a strong inhibitor of C. beijerinckii BA101 cell growth. At a concentration of 10 g l⁻¹ sodium chloride, a maximum cell concentration of 0.6 g l⁻¹ was achieved compared to 1.7 g l⁻¹ in the control experiment. The effects of two salts on specific growth rate constant (μ) and specific rate of ABE production (ν) for C. beijerinckii BA101 were examined. Journal of Industrial Microbiology & Biotechnology (2001) 26, 290–295. Received 20 September 2000/ Accepted in revised form 16 February 2001     

Fermentation of molasses by Zymomonas mobilis: effects of temperature and sugar concentration on ethanol production

Fermentations utilizing strains of Zymomonas mobilis, in place of the traditional yeasts, have been proposed due their ethanol yields being close to theoretical. Ethanol production from sugar cane molasses was analyzed under different culture conditions using Z. mobilis in batch fermentation. The total reducing sugars (TRS) concentrations in the molasses, temperature, agitation and culture time effects were studied simultaneously through factorial design. The best conditions for ethanol production were 200 g L(-1) of total reducing sugars in the molasses, temperature of 30 degrees C and static culture and time of fermentation of 48 h, achieving 55.8 g L(-1). The pH of the medium was kept constant during the experiments, showing that molasses presents a buffering effect.