Ethanol Production from Hydrolyzed Soybean Molasses - American

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Ethanol Production from Hydrolyzed Soybean Molasses Betânia B. Romaõ ,* Francielle B. da Silva, Miriam M. de Resende, and Vicelma L. Cardoso Faculty of Chemical Engineering, Federal University of Uberlândia, Post Office Box 593, Avenida João Naves de Á vila, 2121, Campus Santa Mônica, Bloco 1K, 38408-100 Uberlândia, Minas Gerais (MG), Brazil ABSTRACT: The aim of this work was to produce ethanol using the acid hydrolysis of soybean molasses followed by alcoholic fermentation via submerged Saccharomyces cerevisiae. The influence of the acid type, pH, and absolute pressure of the hydrolysis on the ethanol yield and the total residual sugar concentration was evaluated using a factorial design (FD). The absolute pressure ranged from 1 to 2 atm; the pH ranged from 3 to 5; and three different acids were studied in the hydrolysis process: sulfuric, hydrochloric, and nitric acids. The experiments were conducted in an Applicon batch reactor with a useful volume of 1.5 L at a stirring speed of 230 rpm and with an inoculum concentration of 30 g/L. The inoculum volume used was 30% of the total volume. The best results, as determined by FD, were obtained at pH 4 and an absolute pressure of 1.5 atm for all of the acids studied. The highest ethanol yield was 46% for sulfuric acid, 48% for hydrochloric acid, and 54% for nitric acid. After the concentration of inoculum and the fermentation kinetics profiles were investigated, a 62% yield relative to the initial sugar content was obtained under optimum conditions after 14 h of fermentation and an inoculum concentration of 35 g/L.

1. INTRODUCTION Worldwide interest in the use of bioethanol as an energy source has stimulated economic studies and research toward improving the industrial processes of ethanol production. Ethanol production from renewable sources is a promising contributor to the reduction of environmental impacts.1 Ethanol is produced from raw materials, such as sugar cane, sugar beet, molasses, corn, and cellulose (bagasse and wood).2−4 In recent years, the use of agricultural residues to obtain renewable fuels, such as bioethanol, has gained significant interest. Although a large volume of this fuel is produced from sugar cane sucrose, ethanol production from alternative sources can be interesting, especially when associated with existing industries.5 The soybean is one of the world’s primary agricultural products because of its high productivity, adaptive capacity, and nutritional qualities. It is one of the most important food sources for men and animals, mainly because of its high nutritional content of lipids and proteins.6,7 It is widely consumed in the form of oil and its derivatives, such as margarine and hydrogenated fats.8 It contains high-quality protein and a high caloric content. Soybeans contain a large fat percentage (15−25%) and are rich in protein (30−45%); they also contain all of the essential amino acids. Moreover, the grain contains carbohydrate levels that range from 20 to 35%, which imparts a high energy value to this food.9 Brazil is responsible for approximately 22% of global soybean exports and is the second largest exporter of soybeans in the world. On average, 68% of the total soybean production in Brazil is exported, mainly to the European Union, Japan, and the U.S.10 According to the Brazilian Association of Vegetable Oil Industries,10 soybean production in 2011 was estimated to exceed 71 million tons and the total volume of soybeans and derivatives that were exported exceeded 45 million tons. Soybean molasses is a co-product obtained during the extraction of soybean proteins. It contains high concentrations © 2012 American Chemical Society

of sugar (57% dry weight), nitrogen, and other macro- and micronutrients. The sugars in molasses that can be converted into ethanol are sucrose, glucose, and fructose, while approximately 47% of the sugars in soybean molasses cannot be fermented by Saccharomyces cerevisiae. With respect to the oligosaccharides raffinose and stachyose, only the fructose unit is consumed. The molecular structures of these sugars are shown in Figure 1. The residual sugar oligomers are connected by an α-1,6 linkage.11 Because S. cerevisiae does not produce the enzyme α-1,6galactosidase, the hydrolysis of these sugars is necessary prior to fermentation. The hydrolysis can be performed as an acidic or enzymatic hydrolysis. Many researchers have been working with acid-hydrolyzed molasses to obtain simpler sugars that can be easily metabolized by microorganisms. Lee et al.12 used hydrolyzed sugar cane molasses as a carbon source in the biological removal of nitrogen. The hydrolysis was performed using sulfuric acid at 90 °C over 2 h. Several pH values were investigated, including 1.9, 2.7, 3.6, 4.3, and 4.6. The highest reducing sugar concentrations were found for pH 3.6, increasing from 170 to 520 g/kg of molasses. Morsy13 hydrolyzed molasses using hydrochloric acid at 120 °C over 20 min with the aim of obtaining fermentable sugars that were used by Escherichia coli HD701 to produce biohydrogen. Therefore, this paper aims to study the feasibility of ethanol production from acid-hydrolyzed soy molasses by submerged fermentation using S. cerevisiae.

2. MATERIALS AND METHODS 2.1. Raw Materials. The substrate used in the fermentation to produce ethanol was obtained as a co-product generated from the extraction of soy protein, which is referred to as soy molasses. The soy Received: October 26, 2011 Revised: March 10, 2012 Published: March 12, 2012 2310

dx.doi.org/10.1021/ef201908j | Energy Fuels 2012, 26, 2310−2316

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Figure 1. Molecular structure of the main soybean molasses sugars: (1) stachyose, (2) raffinose, (3) sucrose, (4) glucose, and (5) fructose. the hydrolysis were the parameters varied, and a FD was conducted for each type of acid studied. The responses evaluated were ethanol production, ethanol yield, and concentration of total residual sugar. Statistical calculations were performed using the software Statistica 7.1 StatSoft. An absolute pressure of 2 atm and a pH of 5 were established as the highest levels for the variables in the hydrolysis and assigned a value of +1. The lowest levels for the chosen hydrolysis variables were an absolute pressure of 1 atm and a pH of 3, which were assigned values of −1. The values used were selected according to a literature review.11,14 Batch fermentation tests were performed without mineral supplements under stirring in a reactor (Applikon) that contained a total volume of 1.5 L. The volumes of the added inoculum were 30% of the total volume of the medium. Experiments were performed at 35 ± 0.5 °C, a pH of 4.5, and a stirring speed of 230 rpm, and the initial microorganism concentration was approximately 30 ± 0.2 g/L. 2.5. Evaluation of the Inoculum Concentration. The microorganism concentrations tested in the inoculums were 25, 30, 35, 40, and 45 g/L; the microorganisms were held in a volume of inoculum equal to 30% of the total volume of the medium. Only the concentration of microorganisms in the inoculum was varied, and the inoculum volume and the soybean molasses concentration were kept constant. The hydrolysis with nitric acid and fermentation were performed under the optimum conditions established during the previous experiments. 2.6. Kinetic Study To Evaluate the Optimal Conditions. A kinetic study was conducted to validate the experimentally determined optimum conditions. Fermentation was performed by varying the absolute hydrolysis pressure and the pH of hydrolysis in the range of the best responses obtained in the FD. The kinetic study was performed in a reactor (Applikon) with a working volume of 1.5 L. Aliquots of 30 mL were taken every 2 h, and the alcohol concentration, total residual sugar, and cell concentration required to stabilize the production of ethanol were analyzed. Graphical results of the kinetic study were constructed with Origin Graph 8.0 software. The maximum volume of the samples taken from the reactor consisted of 10% of the total volume of the reactor. 2.7. Soybean Molasses Consecutive Fermentation Followed by Ethanol Extraction Using a Rotary Evaporator. A test was conducted to evaluate the use of a lower concentration of the substrate (SM) to reduce water consumption and energy expenditure. The test consisted of conducting a fermentation of the SM hydrolyzed with a concentration of 1:2. The fermentation time and the hydrolysis conditions were investigated under the optimal conditions established by the previous experiments. The fermentation volume was 1.5 L, of which 0.45 L consisted of inoculum; the remainder consisted of acidhydrolyzed soy molasses. After fermentation, centrifugation was performed to remove the yeast, and the supernatant was then placed in a rotary evaporator (Fisatom) at 90 °C for 2 h to remove the ethanol produced. After the ethanol was removed, another fermentation was performed using the same fermented broth under conditions identical to those previously used to evaluate the reuse of the fermented broth and the yeast in consecutive fermentations. 2.8. Determination of Biomass, Total Sugar, Ethanol, Furfural, and Hydroxymethylfurfural Concentration. The biomass concentration (cells/mL) was quantified with a Neubauer counting chamber.15 The ethanol concentration and the total sugars were quantified using HPLC (Varian Liquid Chromatography System)

molasses was donated by the Selecta soybean factory, located in Araguari, MG (Brazil). The donated samples were placed into 2 L bins and stored in a freezer (Electrolux, model H300) for subsequent use during the experiments. Sulfuric, hydrochloric, and nitric acids were used, all of which were of analytical grade (AG). The S. cerevisiae strain was Y904, donated by Mauri Brasil. 2.2. Characterization of Soybean Molasses. The protein concentration of the soybean molasses was determined by the Kjeldahl method. The carbohydrate composition of the soybean molasses was determined by high-performance liquid chromatography (HPLC; see section 2.8). Lipid concentrations were determined by a total hydrocarbon analyzer, model 404, after solvent extraction with hexane. Ashes were quantified by gravimetric analysis after burning samples at 550 °C for 5 h. 2.3. Preliminary Tests. Preliminary tests were conducted with the three acids to determine the optimum molasses dilution, hydrolysis time, and fermentation time to promote the highest yield of ethanol in relation to the consumed sugars for each acid studied. The dilution factor and hydrolysis time were varied in the first test. The conditions tested were dilutions of 1:2, 1:3, and 1:4 (grams of molasses/gram of medium) and hydrolysis times of 10, 20, 30, and 40 min. For the hydrolysis tests, a dilute acid solution (1.2 N) was added to the medium until the pH of the medium was 3. The flasks that contained soy molasses and acid solution were autoclaved at an absolute pressure of 1.5 atm (111 °C). The fermentations were performed in a reactor (Applikon) with a working volume of 1.5 L under controlled temperature and agitation. The medium contained the hydrolyzed soybean molasses solution and inoculum without nutrient supplements. Fermentations performed after the acid hydrolysis of the soy molasses were conducted at a temperature of 35 ± 0.5 °C, a pH adjusted to 4.5, a stirring speed of 130 rpm, a fermentation time of 12 h, and a microorganism concentration of 30 ± 0.2 g/L. Inoculum were prepared by weighing dry yeast and completing the flask volume with distilled water to obtain a specific concentration. Before initiating fermentation, the inoculums were incubated in a shaker at 130 rpm for 2 h to ensure hydration. The inoculum volume was 30% of the total volume of the medium. After the optimum dilution factor of the molasses and the optimum time for the acid hydrolysis were established, a kinetics study was performed using each acid to determine the optimum fermentation time. The hydrolysis and fermentation conditions were the same as those used in the dilution test; the hydrolysis time and dilution factor selected were those that showed the best yield of ethanol based on the results obtained in the previous test. Aliquots of 30 mL were withdrawn at 2 h intervals to measure the ethanol and total residual sugar concentrations. The kinetic study was performed in three batches and analyzed at intervals from 0 to 6 h, from 6 to 12 h, and from 12 to 16 h. In all of the analyses, the maximum amount of sample withdrawn did not exceed 10% of the reactor volume. To study the fermentation of the soybean molasses without hydrolysis, fermentation was performed at the optimum dilution factor, as determined by the preliminary tests; the fermentation was then performed under the same conditions as those previously described. 2.4. Design of Experiments. A statistical approach within a factorial design (FD) was used to select the acid used for hydrolysis and to check the tendencies of some variable processes in the production of ethanol. The FD was performed in three levels with two variables (32) for a total of nine experiments. The pressure and pH of 2311


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equipped with a Varian model 240 solvent delivery module. The sample was diluted with ultrapure water, filtered through a hydrophilic polyvinylidene fluoride (PVDF) membrane (0.22 μm pore size and 13 mm diameter, Millipore), and injected into a chromatographic system (Shimadzu model LC-20A Prominence, Ca Supelcogel column), where the components were separated and detected by refractive index. A deionized water carrier solution, pump flow of 0.5 mL/min, oven temperature of 80 °C, and an injection volume of 20 μL were used. Concentrations were calculated by means of standard curves that related the individual concentrations to the peak area. The total sugar (TS) concentration was calculated by the sum of the individual sugars: stachyose, raffinose, sucrose, glucose, and fructose. The furfural and hydroxymethylfurfural concentrations were quantified using HPLC. The sample was injected into a chromatographic system (Shimadzu model LC-20A Prominence, Supelcogel C-610H column), where the components were separated and detected by ultraviolet (UV). A phosphoric acid carrier solution, pump flow of 0.7 mL/min, oven temperature of 40 °C, and an injection volume of 20 μL were used. 2.9. Yield. The ethanol yield in grams per gram of total initial sugar concentration (TSI) and per gram of the total concentration of sugar consumed at the end of fermentation was determined using eqs 1 and 2, respectively, with a theoretical yield of 0.511 gethanol/gRTS as 100%

Y ′P/S0 = YP/S =

EC × 100 TSI × 0.511

Table 2. Yields Obtained after 12 h of Fermentation as a Function of the Dilution and Hydrolysis Time Using H2SO4 in the Hydrolysisa

3. RESULTS AND DISCUSSION 3.1. Characterization of Soybean Molasses. The composition of soybean molasses is shown in Table 1. Table 1. Composition of Soybean Molassesa percentage on a dry basis

glucose fructose sucrose raffinose stachyose proteins lipids ash a

4.59 2.93 25.99 11.74 15.5 6.44 15.6 7.88

TSI (g/L)

EC (g/L)

TSF (g/L)

YP/S (%)

Y′P/S0 (%)

10 10 10 20 20 20 30 30 30 40 40 40

1:4 1:3 1:2 1:4 1:3 1:2 1:4 1:3 1:2 1:4 1:3 1:2

156.1 207.2 306.6 156.1 207.2 306.6 156.1 207.2 306.6 156.1 207.2 306.6

19.40 23.90 27.20 35.80 45.60 55.80 35.70 46.40 57.10 35.50 45.30 56.90

87.60 117.90 193.90 54.80 74.30 145.80 55.90 77.10 142.10 55.40 75.80 143.90

55.4 52.4 47.2 69.2 67.1 67.9 69.7 69.8 67.9 69.0 67.5 68.4

24.3 22.6 17.4 44.9 43.1 35.6 44.8 43.8 36.4 44.5 42.8 36.3

Table 3. Yields Obtained after 12 h of Fermentation as a Function of the Dilution and Hydrolysis Time Using HCl in the Hydrolysisa

(2)

where Y′P/S0 is the ethanol yield produced in relation to the total initial sugar concentration (%), YP/S is the ethanol yield produced in relation to the total consumed sugar concentration (%), EC is the ethanol concentration (g/L), TSF is the total residual sugar concentration (g/ L), and TSI is the total initial sugar concentration (g/L).

component

dilution (g/g)

a EC, ethanol concentration; YP/S, ethanol yield produced in relation to the total consumed sugar concentration; TSI, initial total sugar concentration; TSF, total residual sugar concentration; and Y′P/S0, ethanol yield produced in relation to the total initial sugar concentration.

(1)

EC × 100 (TSI − TSF) × 0.511

time (min)

standard deviation 0.15 0.09 1.24 0.65 0.81 0.21 0.76 0.15

time (min)

dilution (g/g)

TSI (g/L)

EC (g/L)

TSF (g/L)

YP/S (%)

Y′P/S0 (%)

10 10 10 20 20 20 30 30 30 40 40 40

1:4 1:3 1:2 1:4 1:3 1:2 1:4 1:3 1:2 1:4 1:3 1:2

155.7 206.3 310.2 155.7 206.3 310.2 155.7 206.3 310.2 155.7 206.3 310.2

21.9 25.8 30.4 36.6 47.3 58.2 35.9 46.6 59.3 36.5 46.9 57.9

80.8 113.4 191.2 56.3 77.1 144.8 58.3 79.7 143.3 56.1 76.7 145.2

57.2 54.3 50.0 72.1 71.6 68.9 72.1 72.0 69.5 71.7 70.8 68.7

27.5 24.4 19.2 46.0 44.9 36.7 45.1 44.2 37.4 45.9 44.5 36.5


Average of three different samples.

in the ethanol yield was observed; consequently, the hydrolysis times for the sulfuric, hydrochloric, and nitric acids were set to 20 min at a dilution of 1:4 (g/g). According to Letti,16 soybean molasses at a concentration of 20 °Bx yields a larger ethanol production than a concentration of 30 °Bx; i.e., a reduction in ethanol production occurs with an increase in the concentration of soybean molasses. This reduction may be related to an inhibition of the substrate and reduced water activity with more concentrated molasses. Figures 2, 3, and 4 show the kinetics of molasses fermentation with respect to the responses studied after hydrolysis with sulfuric, hydrochloric, and nitric acids, respectively. As evident in Figures 2−4, ethanol was produced from the initial moment of fermentation, and its production stabilized after 12 h of fermentation in nitric acid and after 14 h of

Carbohydrates, representing 60.8% of the dry mass of the molasses, include mainly sucrose (25.99%), which is a fermentable disaccharide for yeasts, and also stachyose (15.5%) and raffinose (11.74%), complex tetra- and trisaccharides, respectively. The molasses also contains a significant amount of proteins (6.44%), lipids (15.6%), and ash (7.88%). 3.2. Preliminary Tests. Results obtained in the experiments performed to determine the optimum dilution and the time of soybean molasses hydrolyzed for sulfuric, hydrochloric, and nitric acids are presented in Tables 2, 3, and 4, respectively. An analysis of the results from Tables 2−4 shows that, for all hydrolysis times, the 1:4 (g/g) dilution exhibited the highest ethanol yield relative to the total sugars consumed and the lowest concentration of total residual sugar at the end of the fermentation. After 20 min of hydrolysis, no significant increase 2312


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Table 4. Yields Obtained after 12 h of Fermentation as a Function of the Dilution and Hydrolysis Time Using HNO3 in the Hydrolysisa time (min)

dilution (g/g)

TSI (g/L)

EC (g/L)

TSF (g/L)

YP/S (%)

Y′P/S0 (%)

10 10 10 20 20 20 30 30 30 40 40 40

1:4 1:3 1:2 1:4 1:3 1:2 1:4 1:3 1:2 1:4 1:3 1:2

154.2 205.3 308.2 154.2 205.3 308.2 154.2 205.3 308.2 154.2 205.3 308.2

23.4 27.9 32.8 40.7 50.9 60.6 40.2 51.7 59.3 39.7 50.9 60.9

77.1 108.9 181.4 45.4 72.5 135.6 48.3 68.3 140.6 48.4 73.5 135.9

59.4 56.6 50.6 73.2 73.0 68.7 74.3 73.8 69.2 73.4 73.1 69.2

29.7 26.6 20.8 51.7 48.5 38.5 51.0 49.3 37.7 50.4 47.9 38.7

Figure 3. Evolution of the concentrations of ethanol (▲), total sugar (■), and cells (●) as a function of the fermentation time in trials where hydrochloric acid was used in the hydrolysis of sugars under fermentation conditions of pH 4.5, temperature of 35 ± 0.5 °C, dilution of 1:4, and yeast concentration of 30 ± 0.2 g/L.


Figure 4. Evolution of the concentrations of ethanol (▲), total sugar (■), and cells (●) as a function of the fermentation time in trials where nitric acid was used in the hydrolysis of sugars under fermentation conditions of pH 4.5, temperature of 35 ± 0.5 °C, dilution of 1:4, and yeast concentration of 30 ± 0.2 g/L.

Figure 2. Evolution of the concentrations of ethanol (▲), total sugar (■), and cells (●) as a function of the fermentation time in trials where sulfuric acid was used in the hydrolysis of sugars under fermentation conditions of pH 4.5, temperature of 35 ± 0.5 °C, dilution of 1:4, and yeast concentration of 30 ± 0.2 g/L.

total concentration of sugars consumed and the total residual sugar concentration at the end of fermentation. The equations of the empirical model adjusted to the ethanol yield (YP/S) and to the total residual sugar concentration (TSF) using sulfuric acid as a function of the significant variables are presented in eqs 3 and 4, respectively. The correlation coefficients (R2) obtained after adjustment were 0.97 for the ethanol yield (meaning that the results were explained by the proposed empirical equation with 97% of the variability of the data) and 0.95 for the concentration of the total residual sugar.

fermentation in both sulfuric and hydrochloric acids. The fermentation process was conducted with a high initial cell concentration to provide higher ethanol production and to ensure that the microorganisms did not exhibit high growth. The ethanol yield in relation to the total sugar consumed in the fermentation of soybean molasses with no hydrolysis using a 1:4 dilution was 64.7%, which resulted in a production of 31.2 g/L ethanol from an initial total sugar concentration of 155.2 g/L. The results of the soybean molasses acidic hydrolysis after 20 min using a 1:4 dilution (Tables 1−3) shows an increase in the ethanol yield relative to the results of the fermentation without hydrolysis. Thus, a FD was used to improve the results by evaluating the effects of pH and absolute pressure on the yield. 3.3. Experimental Design for Selection of the Acids Studied. One FD was used for each type of acid studied. The variables were the pH and absolute hydrolysis pressure. The responses evaluated were the ethanol yield in relation to the

YP/S = 74.29 + 5.16X1 − 10.88X12 − 1.29X 2 − 2.69X 2 2 (3) 2

TSF = 55.21 − 11.533X1 + 18.97X1 + 6.87X 2

2

(4)

The equations of the empirical models adjusted for the ethanol yield and total residual sugar concentration using hydrochloric acid (eqs 5 and 6) and nitric acid (eqs 7 and 8) as a function of the significant variables are presented below. The correlation coefficients (R2) obtained after adjustment were 0.99 for the 2313


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1.5 atm (because 1.5 atm was the lowest pressure obtained within the optimization area). 3.4. Analysis of the Inoculum Concentration Influence on the Ethanol Yield. Table 5 shows the ethanol

ethanol yield and 0.92 for the total residual sugar concentration for hydrochloric acid, and R2 values of 0.95 for both responses were obtained for nitric acid. YP/S = 75.27 + 6.13X1 − 13.34X12 − 3.24X 2 2

(5)

TSF = 53.78 − 13.31X1 + 21.07X12

(6)

YP/S = 76.14 + 5.51X1 − 13.81X12 − 3.36X 2 2

(7)

inoculum concentration (g/L)

TSI (g/L)

TSF (g/L)

EC (g/L)

YP/S

Y′P/S0

(8)

25 30 35 40 45

156.7 156.7 156.7 156.7 156.7

47.5 37.1 42.9 42.2 43.4

38.9 43.7 47.3 47 46.2

69.7 71.5 81.3 80.3 79.8

48.6 54.6 59.1 58.7 57.7

2

TSF = 40.53 − 13.46X1 + 23.42X1 + 8.32X 2

2

Table 5. Experimental Results of Variations in the Inoculum Concentrationa

The central point (1.5 atm and pH 4.0) showed the best results for all three acids. The ethanol yields in relation to the total consumed sugars for sulfuric, hydrochloric, and nitric acids were 74.3, 75.1, and 76.4%, respectively, and 49.1, 50.8, and 45.5 g/L, respectively, for the total residual sugar concentration. The ethanol yield was slightly higher when nitric acid was used. Hence, this acid was chosen for subsequent experiments. The ethanol yield may have been affected in the H2SO4 hydrolysis by the formation of sulfite. According to Amaral,17 sulfite interferes with the yield of ethanol. Nitric acid, in contrast, exhibits a higher antioxidant capacity than the other acids and most likely promotes a lower inhibitory effect on the fermentation process. The boundary curve for nitric acid is shown in Figure 5; this represents the pH and pressure range where the ethanol yield

a EC, ethanol concentration; YP/S, ethanol yield produced in relation to the total concentration of consumed sugar; TSI, initial total sugar concentration; TSF, total residual sugar concentration; and Y′P/S0, ethanol yield produced in relation to the total initial sugar concentration.

concentration, yield (YP/S and Y′P/S0), and total residual sugar concentration in relation to variations in the yeast concentration in the fermentation medium. An analysis of the results shown in Table 5 does not show a significant increase in the ethanol yield for inoculum concentrations greater than 35 g/L; this concentration was therefore selected for further kinetic profile studies. 3.5. Kinetic Study To Define the Best Fermentation Time Using the Optimized Conditions with Nitric Acid. A kinetic study was performed to verify the optimum fermentation time. Afterward, the pH, hydrolysis pressure, and inoculum concentration were optimized with the total residual sugar (TSF) and ethanol yield (YP/S) responses. Samples of 30 mL were withdrawn at 2 h intervals for further analysis; a maximum of 10% of the useful reactor volume was withdrawn. Table 6 shows the percentage of each sugar before Table 6. Percentage of Each Sugar Analyzed before Hydrolysis, at the Beginning of Fermentation, and after Fermentation Was Concludeda component glucose fructose sucrose raffinose stachyose

percentage before hydrolysis 4.59 2.93 25.99 11.74 15.5

percentage at the beginning of fermentation 23.64 13.44 11.84 9.68 3.28

percentage after fermentation was concluded 2.32 0.56 0.92 5.8 0.76

Figure 5. Boundary curves for responses to the ethanol yield (%) (- - -) and total residual sugar concentration (g/L) () as a function of the pH and pressure of hydrolysis when nitric acid was used.

a

response is maximized and the total residual sugar concentration is minimized. On the basis of an analysis of the boundary curves, the absolute pressure must be between 1.5 and 1.7 atm to achieve the best ethanol yield and total residual sugar results. In addition, the pH must be between 3.5 and 4.5 for both responses. On the basis of the response range and considering the technical and economic criteria, the conditions for performing the experiments were established as pH 4.5 (because 4.5 is the pH used in alcoholic fermentation) and an absolute pressure of

hydrolysis, at the beginning of fermentation, and after fermentation was concluded. Figure 6 shows the kinetic profile of the fermentation in relation to the total consumed sugar, ethanol produced, and cell growth. As evident in Figure 6, the ethanol concentration increases significantly at the beginning of fermentation. After 14 h, the ethanol production stabilizes, and the total sugar consumed undergoes a slow decrease. The yield relative to the total sugar

Hydrolysis conditions of pH 4.5, hydrolysis time of 20 min, and hydrolysis pressure of 1.5 atm and fermentations conditions of pH 4.5, temperature of 35 ± 0.5 °C, dilution of 1:4, and yeast concentration of 35 ± 0.2 g/L were employed.

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Table 7. Experimental Results Using the Rotary Evaporatora experiment first fermentation after first evaporation second fermentation after second evaporation third fermentation

TSI (g/L)

TSF (g/L)

ECI (g/L)

ECF (g/L)

YP/S

Y′P/S0

312.8 150.9

150.9 238.4

0 61.8

61.8 22.7

74.8

38.7

166.9

70.2

15.8

53.3

75.9

44.0

70.2

87.7

53.3

23.6

60.5

24.2

16.3

25.8

51.2

30.7

ECI, initial ethanol concentration; ECF, final ethanol concentration; YP/S, ethanol yield produced in relation to the total concentration of consumed sugar; TSI, initial total sugar concentration; TSF, total residual sugar concentration; and Y′P/S0, ethanol yield produced in relation to the total initial sugar concentration. a

Figure 6. Evolution of the concentrations of ethanol (▲), total sugar (■), and cells (●) as a function of the fermentation time when nitric acid was used in the hydrolysis of sugars using the following fermentation conditions: pH 4.5, temperature of 35 ± 0.5 °C, dilution of 1:4, and yeast concentration of 35 ± 0.2 g/L.

An analysis of the results shown in Table 7 reveals that the medium is fermented by yeast after the ethanol has been removed, which enables the consumption of residual sugars in consecutive fermentations. Moreover, the yields obtained after the second fermentation were higher than those obtained after the first fermentation. This result may indicate a low inhibition because of the high sugar concentration during the first fermentation. Additionally, the yield decreases significantly after the third fermentation. This behavior might be related to the inhibition because of the subproducts produced during the previous fermentation processes. According to Bobbio,19 two chemical reactions occur with carbohydrates at high temperatures, which result in the degradation of the carbohydrates and the formation of volatile compounds. These reactions are known as the Maillard reaction and caramelization. The Maillard reaction can produce compounds, such as acetaldehyde, benzaldehyde, formaldehyde, and lactic aldehyde. Caramelization can produce formic acid, acetic acid, and hydroxymethylfurfural, which may inhibit the fermentation process. Furfural and hydroxymethylfurfural concentrations were measured in the experiments. According to Delgenes,20 0.5 g/L furfural was required to obtain 41 and 43% inhibition of yeast growth and ethanol production, respectively. This author also reported that 1 g/L hydroxymethylfurfural inhibited the growth (65%) and ethanol fermentation (71%) of S. cerevisiae. The concentration of these compounds in the beginning of fermentation were found to be 0.17 g/L furfural and 0.07 g/L hydroxymethylfurfural. Hence, ethanol production may have been inhibited by furfural. These results indicate that the use of concentrated SM is viable for ethanol production in consecutive fermentations. These results are noteworthy because extractive alcoholic fermentation can be used with high concentrations of molasses. Furthermore, extractive alcoholic fermentation reduces the amount of water used for dilution, consequently reducing the energy consumed during the distillation process and the amount of residue produced at the end of the fermentation. The results also show that the yeast can be reused during the consecutive fermentation process. The total ethanol concentration obtained at the end of consecutive fermentations was 108.8 g/L, and the total yield in relation to the substrate consumed was 73.8%. There was not a complete removal of ethanol, and the second and third fermentations therefore started with ethanol in the medium. This fact is related to the difficulty in removing the high

consumed (eq 4) was 78%. Cell growth was very low (ranging from 5.2 × 108 to 1.2 × 109 cells/mL), which was expected because the fermentation was begun with a high cell concentration. Therefore, a time of 14 h was chosen for the alcoholic fermentation using nitric acid for the hydrolysis of soybean molasses. Machado14 has obtained 33.9 g/L ethanol after 34 h of fermentation by adding sulfuric acid to soybean molasses and starting with 114.6 g/L total sugar. According to Machado,14 the acidic hydrolysis process provides a high concentration of reducing sugars but the substrate does not achieve a conversion higher than that from soybean molasses enriched with MgSO4. Letti16 has studied the acidic hydrolysis of soybean molasses using sulfuric, hydrochloric, and phosphoric acids followed by Zymomonas mobiliz fermentation. In his work, the ethanol concentration obtained was approximately 28 g/L and the difference between the ethanol yields obtained with the three acids was not significant. The average total sugar concentration was 90 g/L. Siqueira11 has concluded that the acidic hydrolysis of soybeans provides an increase of 17% in the production of ethanol. Furthermore, the use of an enzymatic hydrolysis results in an increase of 20%. When the substrate is comprised of sucrose with trace amounts of glucose and fructose, such as the substrates used in the Brazilian ethanol industry, the sugar is defined as the total reducing sugars (TRS) and the stoichiometric yield of fermentation is 0.511 g of ethanol/g of TRS. When the stoichiometric yield is calculated on the basis of sucrose, the value is 0.538 g of ethanol/g of sucrose. In industry, when alcoholic fermentation is conducted well, the yields can reach 90−92% of the stoichiometric yield because some sugars are consumed to build biomass cells and subproducts. If contamination occurs in the medium, this yield is lower.18 3.6. Consecutive Fermentation Using a Rotary Evaporator To Remove Ethanol. Three consecutive fermentations were performed in which the ethanol was removed by a rotary evaporator, and the fermentation medium was used in the next fermentation. Table 7 shows the results obtained in each fermentation and evaporation step. 2315


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by Saccharomyces cerevisiae cells immobilized in agar agar and Caalginate matrices. Appl. Energy 2010, 87, 96−100. (3) Hatano, K.; Kikuchi, S.; Nakamura, Y.; Sakamoto, H.; Takigami, M.; Kojima, Y. Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses. Bioresour. Technol. 2009, 100, 4697−4703. (4) Yuji, O.; Nakamura, K. Production of ethanol from the mixture of beet molasses and cheese whey by a 2-deoxylucose-resistant mutant of Klyveromyces marxianus. FEMS Yeast Res. 2009, 9, 472−478. (5) Neureiter, M.; Danner, H.; Thomasser, C.; Saidi, B.; Braun, R. Dilute-acid hydrolysis of sugarcane bagasse at varying conditions. Appl. Biochem. Biotechnol. 2002, 98, 49−58. (6) Sediyama, T.; Pereira, M. G.; Sediyama, C. S.; Gomes, J. L. L. Cultura da Soja; Universidade Federal de Viçosa (UFV): Viçosa, Brazil, 1989; p 75. (7) Morais, A. A. C.; Silva, A. L. Soja: Suas Aplicaçoẽ s; MEDSI Editora ́ Médica e Cientifica: Rio de Janeiro, Brazil, 1996; p 259. (8) Khare, S. K.; Krishna, J. Entrapment of wheat phytate in polyacrylamide gel and its application in soymilk phytate hydrolysis. Biotechnol. Appl. Biochem. 1994, 19, 193−198. (9) Moreira, A. M. Programa de melhoramento genético da qualidade ́ da soja desenvolvido na UFV. An.Congr. Bras. de óleo e proteina Soja, Londrina, PR 1999, 99−104. (10) Associação Brasileira das Indústrias de Ó leos Vegetais (ABIOVE). Complexo SojaExportaçoẽ s; http://www.abiove.com.br/ exporta_br.html (accessed April 28, 2011). (11) Siqueira, P. F. Production of bio-ethanol from soybean molasses by Saccharomyces cerevisiae. Master’s Dissertation, Federal University of Paraná/Universities of Provence and of the Mediterranean, Curitiba, Paraná, Brazil, 2007. (12) Lee, S. T.; Quan, Z. X.; Jin, Y. S.; Yin, C. R.; Lee, J. J. Hydrolyzed molasses as an external carbon source in biological nitrogen removal. Bioresour. Technol. 2005, 96, 1690−1695. (13) Morsy, F. M. Hydrogen production from acid hydrolyzed molasses by the hydrogen overproducing Escherichia coli strain HD701 and subsequent use of the waste bacterial biomass for biosorption of Cd(II) and Zn(II). Int. J. Hydrogen Energy 2011, 36, 14381−14390. (14) Machado, R. P. Produçaõ de etanol a partir de melaço de soja. Master’s Dissertation, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 1999. (15) Madigan, M. T.; Martinko, J. M.; Parker, J. Microbiologia de Brook, 10th ed.; Prentice Hall: São Paulo, Brasil, 2004. (16) Letti, L. A. J. Production of bioethanol by soybean molasses fermentation by Zymomonas mobilis. Master’s Dissertation, Federal University of Paraná, Curitiba, Paraná, Brazil, 2007. (17) Amaral, F. S. Influência conjunta do pH, temperatura e concentraçaõ de sulfito na fermentaçaõ alcoólica de mostos de sacarose. Master’s Dissertation, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil, 2009. (18) Lima, U. A.; Basso, L. C.; Amorim, H. V. Biotecnologia Industrial: Processos Fermentativos e Enzimáticos; Edgard Blücher: São Paulo, Brazil, 2001; Vol. 3, pp 1−43. ́ (19) Bobbio, P. A.; Bobbio, F. O. Quimica do Processamento de Alimentos, 3rd ed.; Livraria Varela: São Paulo, Brazil, 2001; pp 47−78. (20) Delgenes, J. P.; Moletta, R.; Navarro, J. M. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzyme Microb. Technol. 1996, 19, 220−225.

concentrations of ethanol under laboratory conditions, where the experiments were performed. However, our results show that a higher yield is possible if a more thorough ethanol extraction is performed. The yields obtained in this study were lower than those achieved using sugar cane molasses for ethanol production. However, this substrate should be studied more thoroughly because ethanol production using sugar cane is already wellknown and has been performed on an industrial scale for more than 30 years. It is noteworthy that the yeast used in this study was developed for ethanol production using sugar cane substrates as the raw material. Furthermore, the hydrolyzed product obtained is comprised of numerous sugars, including stachyose, raffinose, sucrose, glucose, and fructose as the main sugars. A comparison of the results with and without hydrolysis reveals that an increase of 13% was obtained in the YP/S response and that a reduction of 57.5% was obtained in relation to the total residual sugar. The results presented in this paper show the potential of using soybean molasses to produce bioethanol.

4. CONCLUSION On the basis of the results, the fermentation of soybean molasses represents a potential method for the production of ethanol. A 1:4 dilution of molasses with a hydrolysis time of 20 min showed the best performance for all of the acids studied. For sulfuric, hydrochloric, and nitric acids, hydrolysis conditions, including a pH of 4.0 and an absolute pressure of 1.5 atm, resulted in the highest ethanol yield and lowest total residual sugar concentration. Nitric acid produced the highest yield of ethanol. Under the optimal conditions found from the pH (4.5) and absolute pressure (1.5 atm), nitric acid hydrolysis resulted in 50.1 g/L ethanol produced, with a 60% yield of ethanol relative to the initial total sugar concentration and a 78% yield of ethanol relative to the total concentration of sugar consumed. The fermentation time under the optimum conditions was 14 h. Acid hydrolysis increased the ethanol yield by 13.3% compared to nonhydrolyzed soybean molasses. The study of the consecutive molasses fermentation of soybeans after the extraction of ethanol showed that it is possible to work with larger concentrations of MS without a substantial loss in the ethanol yield relative to the amount of substrate consumed.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Federal University of Uberlândia, FAPEMIG, CNPq, and CAPES for financial support. REFERENCES

(1) Nikolic, S.; Mojovic, L.; Pejin, D.; Rakin, M.; Vukasinovi, M. Production of bioethanol from corn meal hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var. ellipsoideus. Biomass Bioenergy 2010, 34, 1449−1456. (2) Behera, S.; Kar, S.; Mohanty, R. C.; Ray, R. C. Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.) flowers 2316


Ethanol Production from Hydrolyzed Soybean Molasses - American

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