Extractive Fed-Batch Ethanol Fermentation with CO2 Stripping in a

Article pubs.acs.org/EF

Extractive Fed-Batch Ethanol Fermentation with CO2 Stripping in a Bubble Column Bioreactor: Experiment and Modeling J. L. S. Sonego, D. A. Lemos, C. E. M. Pinto, A. J. G. Cruz, and A. C. Badino* Graduate Program of Chemical Engineering, Federal University of São Carlos, C.P. 676, 13565-905 São Carlos, São Paulo, Brazil ABSTRACT: The ethanol accumulated in the broth during fermentation is the main component toxic to yeast, causing slower yeast growth and decreased ethanol production. One way of overcoming this inhibition effect is to use extractive fermentation, where the ethanol is removed from the broth during the fermentation process. The present work evaluates ethanol production by extractive fed-batch fermentation with CO2 stripping, under different conditions of substrate concentration in the must feed (CsF), vat filling time (Ft), and start time of ethanol stripping with CO2. First, the process kinetic parameters were estimated by modeling of conventional fed-batch fermentations (without stripping) in a 5 L bubble column bioreactor, with fitting of the model to experimental data. This procedure used a sucrose concentration of 180 g·L−1 in the must feed, temperature of 34.0 °C, and vat filling times of 3 and 5 h. Subsequently, extractive fed-batch ethanol fermentations were performed at 34.0 °C with a sucrose concentration of 180 g·L−1 in the feed, specific CO2 flow rate (ϕ) of 2.5 vol·vol−1·min−1 (vvm), and Ft of 3 or 5 h, starting ethanol stripping with CO2 after 3 or 5 h of fermentation. The hybrid Andrews−Levenspiel model was able to provide accurate descriptions of the behaviors of the conventional and extractive fed-batch ethanol fermentations, considering the removal of ethanol and water from the broth. Use of Ft of 5 h and start of ethanol stripping at 3 h of fermentation substantially reduced the inhibitory effects of the substrate and ethanol on the yeast cells. This condition enabled the extractive fed-batch ethanol fermentation to be performed using substrate concentrations of up to 240 g·L−1 in the feed, with substrate exhaustion occurring after approximately 12 h. The total ethanol concentration reached 110.3 g·L−1 (14 °GL (degrees Gay-Lussac)), around 33% higher than that obtained using conventional fed-batch fermentation without ethanol removal.

■

INTRODUCTION The growing demand for liquid fuels in the transport sector, together with concerns about global warming and the possible depletion of fossil fuels, has led to a move toward alternative, renewable, sustainable, efficient, and cost-effective energy sources that offer lower net emissions of pollutants.1 Bioethanol is viewed as one of the most useful renewable sources of energy.2 Currently, Brazil is the world’s largest ethanol exporter and the second largest producer (after the United States). Brazil is the most competitive producer of ethanol in the world.3 Among all of the forms of ethanol production, the fermentation route using sugar cane as feedstock is the most economically viable process in Brazil. This is due to the geographic location, soil type, variety of feedstock, and possibility of cultivation throughout the country.4 In Brazilian distilleries, ethanol is produced by fermentation of sugar cane musts prepared from the mixing of sugar cane juice and molasses in different proportions in order to obtain a concentration of total fermentable sugars of about 20 °Brix (180 g·L−1).4 Around 85% of distilleries use the fed-batch mode for the fermentation process, while only 15% use the continuous mode.5 The fermentation takes place in largevolume vats, and in the fed-batch process the yeast is recycled, which results in a high cell density in the fermenter (10−15% (w/v)).5 The fermentations are conducted simultaneously in several vats, during a short period of 6−12 h, at temperatures in the range 32−35 °C. The final ethanol concentration in the fermentation broth is up to 11% (v/v), corresponding to an average ethanol yield of 90−92% relative to the theoretical conversion.6,7 © 2016 American Chemical Society

Increasing ethanol concentration in the wine reduces the viability of yeast cells. High cellular viability is essential in fermentation processes with yeast cell recycling, which depend on maintaining yeast cells alive until the end of the fermentation process, because they need to be reused ca. 400−600 times during sugar cane harvest season.6 In industrial fed-batch ethanol fermentation, the inoculum composed of a yeast suspension usually represents around 25− 30% of the total volume of the fermentation vat. In this fermentation mode, the sugar cane must is fed until the vat is full (fed-batch stage), with feeding times that are usually between 4 and 6 h. After filling, the process is completed by batch fermentation up to sugar exhaustion (batch stage).4 Compared to conventional batch mode, use of a fed-batch process results in a higher ethanol concentration in the same time, due to reduction of the inhibitory effect of the sugar on Saccharomyces cerevisiae (S. cerevisiae) growth and ethanol production during the first stages of the fermentation process. The application of fed-batch ethanol fermentation significantly improves results by maintaining low substrate levels while ethanol accumulates in the fermentation broth (up to 10% (v/ v), approximately) and by achieving higher ethanol volumetric productivity.8 According to Thtipamala et al.,9 the inhibitory effects are not observed at substrate concentrations up to 150 g· L−1. In industrial fermentation processes, the use of the fedbatch operational mode results in maximum concentrations of Received: October 5, 2015 Revised: December 15, 2015 Published: January 7, 2016 748

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels substrate in the vat that do not usually exceed 70 g·L−1, hence avoiding the negative effects of substrate inhibition on the yeast.4 The ethanol accumulated in the broth during the course of the fermentation is the main component toxic to yeast, due to its action as a noncompetitive inhibitor of metabolism, resulting in decreases in yeast growth and ethanol production as the ethanol concentration increases in the broth.10 At concentrations above 40 g·L−1, ethanol significantly decreases cell growth, and consequently the ethanol production rate, hence reducing the volumetric productivity of the process.11 According to Lloyd et al.12 and Ly et al.,13 an ethanol concentration in the fermentation broth higher than 8% (v/v) (around 63 g·L−1) can cause damage to the membrane lipids of the cells and other organelles. At concentrations in the region of 95 g·L−1, the cell growth of yeasts is completely inhibited by ethanol.10 In Brazilian distilleries, the low ethanol concentration obtained in the final wine results in the generation of large volumes of vinasse (between 10 and 15 L of vinasse·(L of ethanol)−1)14 and the consumption of around 2.6 kg of steam· (kg of ethanol)−1 produced in the distillation stage.15 One way to overcome the inhibition effect on yeast and obtain a high yield is to use extractive fermentation, where the ethanol is extracted from the broth during the fermentation process.16−20 Gas stripping is a technique that can be used during fermentation and is an attractive method for large-scale production, due to its relative simplicity and the selective removal of volatile compounds in clean forms that are not contaminated with unvolatile material. It does not remove nutrients from the broth and does not harm the cells during the fermentation. Furthermore, it is possible to use a zero-cost stripping gas (carbon dioxide).21,22 Previous studies of continuous ethanol fermentation using glucose produced by the hydrolysis of corn starch have employed inert gases such as carbon dioxide or nitrogen for stripping, achieving high ethanol productivities.23 However, the conversion of glucose was incomplete at higher glucose feeds, and it was reported that yeast growth caused fouling in the packed column utilized for ethanol stripping, which impaired the operation of the system.16,24 More recently, Taylor et al.25 evaluated extractive ethanol fermentation with CO2 stripping, using liquefied maize starch in a 70 L working volume fermenter operated continuously for 60 days. Starch conversion of 95% was achieved, resulting in 88% of the maximum theoretical yield of ethanol. The fermentation and stripping systems were not significantly affected when the CO2 stripping gas was partially replaced by nitrogen or air. Gas stripping in fed-batch fermentations has been used for acetone−butanol−ethanol (ABE) production. Ezeji et al.26 reported an enhancement in ABE production by Clostridium beijerinckii (C. beijerinckii) BA101 in a fed-batch cultivation process with H2 and CO2 stripping, operated for 200 h. In order to reduce substrate inhibition, the process was started with a glucose concentration of 99.9 g·L−1 and culture medium containing glucose and nutrients was subsequently added to the fermentation broth in sufficient amounts that the glucose concentration did not exceed 90 g·L−1. A maximum solvent concentration of 232.8 g·L−1 was achieved, which was 13-fold higher than for conventional batch cultivation (17.6 g·L−1), and a productivity of 1.16 g·L−1.h−1 was 4-fold higher than that obtained for the control (0.29 g·L−1·h−1). Lu et al.22 studied fed-batch fermentation for ABE production by a hyper-butanol-

producing C. acetobutylicum strain, using concentrated cassava bagasse hydrolysate as substrate. A fibrous bed bioreactor was employed, with continuous butanol recovery by gas stripping. Periodic nutrient supplementation resulted in the total production of 108.5 g·L−1 of ABE, and a high butanol yield was obtained over an extended period (263 h), indicating the suitability of the process for industrial production. It is clear that high ethanol productivity can be achieved using continuous extractive ethanol fermentation processes, with CO2 stripping and glucose as substrate. However, this process has limitations related to the complexity of the equipment required, because the stripping operation has typically been carried out in a separate system attached to the vat, and there can be problems of fouling in the packed column used for stripping. Nonetheless, the use of batch/fedbatch fermentation processes integrated with H2 and CO2 stripping for butanol production enables the solvents to be maintained at low levels, hence minimizing the effects of product inhibition on the microorganisms. It is therefore possible to use higher concentrations of substrate in the feed solution. In our recent work,19 sugar cane ethanol production from sucrose (180 g·L−1) using extractive batch fermentation was performed with CO2 stripping in the vat itself. A decrease in the inhibition effect of ethanol on the yeast resulted in an increase in ethanol productivity of approximately 25%, compared to conventional fermentation without ethanol removal. The present work describes an experimental and modeling study of extractive fed-batch ethanol fed-batch fermentation with CO2 stripping, focusing on the influence of the substrate (sucrose) concentration, the must flow rate in the feed vat, and the timing of ethanol extraction from the broth by CO2 stripping. This approach differs from the earlier studies in that it evaluates the extractive ethanol fermentation process using the fed-batch mode widely employed in Brazilian distilleries, with sucrose from sugar cane as feedstock for ethanol production. It is important to mention that the large amounts of carbon dioxide produced during the industrial fermentation process (around 425 L of CO2·(L of ethanol)−1 at the process pressure and temperature) are currently discarded without being used in the industrial plant. An extractive ethanol fed-batch fermentation process, employing the CO2 produced during the ethanol fermentation process itself as stripping gas, has the twin advantages of reducing the substrate inhibition effect that can occur at the beginning of fermentation, which enables the vat to be fed with concentrated must, and minimizing the ethanol inhibition effect by continuous removal of the ethanol accumulated in the fermentation broth.

■

MATERIALS AND METHODS

Equipment. The conventional and extractive fed-batch ethanol fermentations were performed using a jacketed bubble column pneumatic bioreactor with a working volume of 5 L.27 The bioreactor was modified with a mechanical agitation system to ensure adequate mixing of the fermentation broth before the CO2 stripping was started. Influence of Solution Volume and Ethanol Concentration on Ethanol Removal by CO2 Stripping. In order to evaluate the influence of the volume (column height) of the solution in the bioreactor on ethanol removal by carbon dioxide, stripping experiments were performed using different solution volumes (V) and ethanol concentrations (CE), keeping the solution temperature (T) and specific CO2 flow rate (ϕ) fixed at 34 °C and 2.5 vol·vol−1·min−1 (vvm), respectively. The specific flow rate value was defined in our 749

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels

filling times (Ft) of 3 or 5 h, starting the ethanol stripping with CO2 after 3 or 5 h of fermentation. The substrate concentrations in the must feed (CsF) were 180, 200, 220, and 240 g·L−1. The conventional (CF) and extractive (EF) fed-batch ethanol fermentations were performed in duplicate. Analytical Methods. Cell concentrations (on a dry mass basis) were determined after centrifugation of the samples at 10,000 rpm and 4 °C for 10 min. The precipitates were washed twice with distilled water and then dried at 80 °C for 24 h. The concentrations of sucrose, glucose, fructose, and ethanol in the supernatants were determined using an HPLC instrument (Waters, Milford, MA, USA) equipped with a refractive index detector and a Sugar-Pak I column (300 mm × 6.5 mm, 10 μm; Waters) maintained at 80 °C. Ultrapure water was utilized as the eluent, at a flow rate of 0.5 mL·min−1.19 The standards were solutions of sucrose, glucose, fructose, and ethanol at concentrations of between 0.1 and 8.0 g·L−1. At the end of the fed-batch fermentations, samples were taken to assess cell viability. Counting of viable cells was performed using an optical microscope (BX50F-3, Olympus), employing the method of staining with methylene blue.28 Mathematical Modeling of the Conventional Fed-Batch Fermentation. During the stage of must feeding, assuming that the generation of the product (ethanol) was associated with cell growth, and considering the variations in V and F, the mass balances for cells (X), substrate (S, glucose and fructose), and ethanol (E) in the conventional fed-batch fermentation could be described by the following equations:

previous work19 and in preliminary ethanol stripping tests (data not shown). The liquid phase consisted of solutions of ethanol at different concentrations. The gas phase used in the stripping experiments was commercial carbon dioxide (CO2), stored in a cylinder (25 kg) at approximately 60 atm when full. The stripping experiments were carried out as described previously,19 with each experiment lasting 6 h. Samples (20 mL) were withdrawn on an hourly basis in order to determine the changes in CE, and the liquid phase volume (V) was measured at the same time. The experiments were performed using the initial volumes (V0) and ethanol concentrations (CE0) shown in Table 1. The pairs of values of

Table 1. Experimental Conditions for the Ethanol Stripping Experiments Performed Using Hydroalcoholic Solutions at 34 °C and with Specific CO2 Flow Rate (ϕ) of 2.5 vvm condition stripping experiment

V0 (L)

CE0 (g·L−1)

SE1 SE2 SE3 SE4

2.0 3.0 4.0 5.0

45.0 55.0 65.0 75.0

V0 and CE0 were chosen based on typical temporal profiles of broth volume and ethanol concentration for conventional fed-batch ethanol fermentation (without ethanol removal by CO2 stripping). Conventional and Extractive Fed-Batch Ethanol Fermentations. Microorganism and Culture Medium. Commercial lyophilized S. cerevisiae (Fleischmann) was used in this study. The characteristics of the culture medium employed in the fermentations were similar to the sugar cane must used in Brazilian distilleries. The medium was prepared using analytical grade reagents and contained sources of carbon, nitrogen, phosphorus, potassium, and magnesium, simulating the composition of sugar cane molasses used in distilleries and supplying the nutritional requirements of the yeast. Sucrose was dissolved together with the other reagents. The composition of the culture medium was as follows (g·L−1):19 sucrose, 180.0−240.0; KH2PO4, 5.6; MgSO4·7H2O, 1.4; yeast extract, 6.8; urea, 5.32. The initial pH of the fermentation broth was adjusted to 4.6 by adding hydrochloric acid (1 M). In the experiments using 220 and 240 g·L−1 of sucrose, the amounts of KH2PO4, MgSO4·7H2O, yeast extract, and urea were increased by 20%. Experimental Procedure. The initial (inoculum) volume of 1.5 L, which contained 75 g (dry weight) of yeast that had been previously hydrated for 30 min in 500 mL of distilled water, was fed into the vat and agitated at 250 rpm. The composition of the inoculum did not include any substrate or ethanol (CS0 = 0 g·L−1 of total reducing sugars, glucose and fructose; CE0 = 0 g·L−1). A 3.5 L volume of culture medium containing sucrose and nutrients (the must) was then fed for 3 or 5 h. A commercial antifoaming agent (Qualifoam, diluted 1:10) was added the beginning of the process. In the conventional fed-batch fermentation without ethanol stripping, the broth was agitated at 250 rpm from the start of the fermentation until the end of the process. The temperature was maintained at 34.0 °C by circulating water from a water bath through the bioreactor jacket. Samples (30 mL) were withdrawn on an hourly basis. The must was fed at a constant volumetric flow rate (F), so the broth volume (V) increased linearly with time, according to

V = V0 + Ft

dCx 1 = μCx − Cx F V dt dCs 1 1 = (CsF − Cs) F − μCx V YX/S dt

(2) (3)

YE/S dC E 1 = ·μCx − CE · F YX/S V dt

(4)

dV =F dt

(5)

where Cx is the cell concentration (g·L−1), μ is the specific cell growth rate (h−1), Cs is the limiting substrate concentration (g·L−1) obtained from the sum of the glucose and fructose concentrations, CSF is the substrate concentration in the must feed (g·L−1), CE is the ethanol concentration (g·L−1), YX/S is the cell yield coefficient (gX·gS−1), and YE/S is the ethanol yield coefficient (gE·gS−1). The hybrid Andrews−Levenspiel kinetic model29,30 was used to describe cell growth, considering inhibition by both substrate and product: μ = μmax

Cs

(K

S

+ Cs +

Cs2 KIS

)

n ⎛ CE ⎞ ⎟⎟ · ⎜⎜1 − C Emax ⎠ ⎝

(6)

where μmax is the maximum specific cell growth rate (h−1), KS is the saturation constant (g·L−1), KIS is the substrate inhibition constant (g· L−1), CEmax is the maximum concentration of ethanol after which cell growth ceased, and n is a dimensionless constant. In conventional fed-batch fermentation, after feeding of the must into the vat, the fermentation proceeds in batch mode until the substrate is exhausted. At this stage in the fermentation, the mass balance equations for cells, substrate, and ethanol, at constant volume (F = 0), are given by

(1)

where V0 is the initial volume (L) and F is the feed flow rate of the must (L·h−1). The extractive ethanol fed-batch fermentations followed the same procedure described for conventional fermentation. However, the mechanical agitation was turned off after the start of ethanol stripping with CO2. The specific CO2 flow rate (ϕ) was 2.5 vvm, governed by a mass flow controller (GFC 37, Aalborg). Assays were carried out using 750

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels dCx = μCx dt dCs 1 =− μCx YX/S dt YE/S dC E = μCx YX/S dt

differential equations (eqs 12−19) using the Runge−Kutta algorithm, which was implemented in Scilab v. 5.4.1.

(7)

■

RESULTS AND DISCUSSION Effects of Solution Volume and Ethanol Concentration on Ethanol Stripping with CO2. Stripping experiments with CO2 were performed to evaluate the removal of ethanol from a hydroalcoholic solution at 34.0 °C (the temperature of the ethanol fermentation using S. cerevisiae). The ranges of V0 and CE0 utilized in this study were based on typical profiles for fed-batch ethanol fermentations. The CO2 stripping of ethanol and water from a hydroalcoholic solution follows the same behavior found for other organic solvents, and can be represented by a first order model:19

(8) (9)

The global cell and ethanol yield coefficients, YX/S and YE/S, can be determined using

YX/S =

Cx f Vf − Cx 0V0 (Vf − V0)CsF − Cs f Vf

(10)

YE/S =

C Ef Vf (Vf − V0)CsF − Cs f Vf

(11)

where the subscripts ″0″ and “f″ refer to the initial and final times, respectively, and the subscript “F″ refers to the feed. Model Fitting and Numerical Procedure. After determination of YX/S and YE/S, the kinetic parameters (μmax, KS, KIS, CEmax, and n) were estimated using the nonlinear regression method of Nelder and Mead,31 with the Runge−Kutta algorithm used for numerical solving of the differential equations of the model. The criterion used for the best fit and parameter optimization was minimization of the sum of squared residuals (SSR). Mathematical Modeling of the Extractive Fed-Batch Fermentation with CO2 Stripping. The mathematical model of the extractive fed-batch fermentation employed mass balance equations for cells, substrate, and ethanol, considering the removal of ethanol and water (W) by the CO2 stream, as well as changes in V. Based on the literature,19,32−34 the classical first order model valid for any type of extractant was used to describe the removal of ethanol and water from the fermentation broth. The model used for the extractive fed-batch ethanol fermentation can be described by eqs 12−19. During the must feed (fed-batch stage),

dCx 1 = μCx − Cx F V dt dCs 1 1 μCx = (CsF − Cs) F − V YX/S dt YE/S dC E 1 = μCx − CE F − C EkE YX/S V dt (kEC E + k w(ρw − C E))V dV =F− ρw dt

YE/S dC E 1 dV = μCx − CE − C EkE YX/S V dt dt (kEC E + k w(ρw − C E))V dV =− ρw dt

(20)

RW

(21)

where RE is the removal rate of ethanol (g·L−1·h−1), CE is the ethanol concentration in the liquid phase (g·L−1), kE is the ethanol removal rate constant (h−1), RW is the removal rate of water (g·L−1·h−1), kW is the water removal rate constant (h−1), and ρW is the specific mass of water (g·L−1). Figure 1 shows the concentrations of ethanol (CE) and water (CW) during the stripping of hydroalcoholic solutions using

(12) (13) (14)

(15)

where kE is the ethanol removal rate constant (h−1), kw is the water removal rate constant (h−1), and ρw is the specific mass of water (g· L−1). After the must feed (batch stage):

dCx 1 dV = μCx − Cx V dt dt dCs 1 1 dV =− μCx − Cs YX/S V dt dt

dC E = kEC E dt dC W = = k WC W = k W(ρW − C E) dt

RE = −

Figure 1. Concentrations of ethanol and water according to time during the stripping experiments with CO2: ethanol concentration (CE), closed symbols; water concentration (CW), open symbols; lines, simulated data obtained using the first order model.

(16)

different initial volumes (V0) and ethanol concentrations (CE0). The behavior of the water concentration (CW) profile indicated that CO2 preferentially entrained ethanol. The decrease of CE during the course of the stripping consequently caused a very slight increase of CW. However, as the decrease of V over time was more significant than the increase of CW, the water mass in the bubble column (mW = CWV) decreased progressively during the stripping experiments. These features were in agreement with the results reported by Sonego et al.,19 where similar profiles of CW and CE were obtained during the CO2 stripping of ethanol from hydroalcoholic solutions under different conditions of specific flow rate and temperature. The ethanol and water removal rate constants (kE and kW) were determined by the fitting of exponential curves (described

(17) (18)

(19)

Given the range of ethanol concentrations employed in this study (40−80 g·L−1), the specific mass of the hydroalcoholic solution was considered to be very close to that of water at 34 °C (ρW = 994 g· L−1).35 Changes in Cx, Cs, and CE during the course of the fermentation were determined by numerical resolution of the set of 751

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels by eqs 22 and 23) to the experimental CE and CW data according to time (t): C E = C E0e−kEt

(22)

C W = C W0ekWt

(23)

To determine the kinetic parameters of the hybrid inhibition kinetic model, the cell and ethanol yield coefficients (YX/S and YE/S) were first calculated using eqs 10 and 11. Afterward, the kinetic parameters μmax, KS, KIS, CEmax, and n were estimated using the Nelder and Mead31 algorithm to perform the optimization and the Runge−Kutta method for numerical solving of the set of differential equations. Values of the kinetic parameters were obtained based on the best fits between the calculated and experimental values of Cx, Cs, and CE for the conventional fed-batch ethanol fermentations (without ethanol removal) performed in duplicate (CF1 and CF2). Table 3 presents the estimated values of the parameters for the conventional fed-batch fermentations CF1 and CF2, which

where CE0 and CW0 are the initial concentrations of ethanol and water in the solution (g·L−1). It can be observed from Figure 1 that the regression curves closely fitted the experimental data, with correlation coefficient (R2) values greater than 0.99. This showed that eqs 22 and 23 were suitable for description of ethanol stripping with CO2 in extractive fed-batch fermentations. The entrainment factor for ethanol (EFE), defined as the percentage of the initial ethanol amount that became entrained during the stripping, was calculated according to eq 24. This factor and kE were chosen as the criteria for the evaluation of ethanol stripping with CO2 under different conditions of V0 and CE0. C V − C Ef Vf EFE /% = E0 0 × 100 C E0V0

Table 3. Estimated Values of the Kinetic Parameters for the Model of the Conventional Fed-Batch Ethanol Fermentations CF1 and CF2 with Substrate Concentration of 180 g·L−1 in the Must Feed

(24)

where the subscripts “0” and “f” refer to the initial and final times, respectively. The values of kE, kW, and EFE obtained in the stripping experiments are presented in Table 2. Table 2. Values of kE, kW, and EFE Obtained in the Stripping Experiments stripping experiment SE1 SE2 SE3 SE4

kE (h−1) 0.0678 0.0618 0.0656 0.0640

± ± ± ±

0.0018 0.0015 0.0016 0.0008

kW (h−1)

EFE (%)

± ± ± ±

37.6 35.0 39.1 37.3

0.00345 0.00342 0.00443 0.00498

0.00015 0.00005 0.00002 0.00009

a

parameter

value

YX/S (gX·gS−1) YE/S (gE·gS−1) μmaxa (h−1) KSa (g·L−1) KISa (g·L−1) CEmaxa (g·L‑1) na

0.0421 ± 0.0007 0.454 ± 0.015 0.125 ± 0.002 25.1 ± 1.8 131.8 ± 9.3 86.1 ± 1.7 0.22 ± 0.03

95% confidence level.

are within the range of values found in the literature.36 Figure 2 compares the simulated and experimental data for the cell (Cx), substrate (Cs), and ethanol (CE) concentrations during the course of the conventional fed-batch fermentations (CF) with Ft of 3 and 5 h. The simulations provided excellent fits, showing that the hybrid Andrews−Levenspiel model was suitable for describing the kinetic behavior of the conventional fed-batch ethanol fermentation. It can be seen (Figure 2) that there were significant differences between the Cs and CE profiles obtained using filling times of 3 and 5 h. In fermentation with Ft = 3 h (Figure 2a), the cumulative sugar concentration in the vat reached a value of around 120 g·L−1 at the end of filling, while with Ft = 5 h (Figure 2b), the cumulative concentration was around 75 g· L−1. Nevertheless, substrate exhaustion occurred at 9 h in both of these conventional fermentations. Extractive Fed-Batch Ethanol Fermentations Using Different Stripping Start Times and Different Substrate Concentrations in the Must. The values for the stripping parameters kE and kW (Table 2) and the extractive fed-batch fermentation model (eqs 7−9) were used together with the kinetic parameter values (Table 3) to evaluate the effect of ethanol removal on the dynamics of the fed-batch ethanol fermentations with CO2 stripping. The effects of substrate and accumulated ethanol were evaluated in extractive fed-batch fermentations performed with a CsF of 180 g·L−1, temperature of 34.0 °C, ϕ of 2.5 vvm, and Ft of 3 or 5 h, starting the ethanol stripping with CO2 after 3 or 5 h of fermentation. Figure 3 compares the simulated and experimental values of Cs, Cx, and CE during the course of the fermentation. It can be seen that the proposed model was able to predict the behavior of the extractive fermentation with high accuracy. It can also be observed that the behavior of the extractive fermentation was

The results shown in Table 2 demonstrated that the values of kE, kW, and EFE were not significantly affected by changes in either the height of the solution column or the concentration of the hydroalcoholic solution, under the conditions of specific CO2 flow rate (ϕ) and solution temperature (T) employed. The close similarity of the values in Table 2 was possibly due to the fact that, during all the stripping experiments, the specific CO2 flow rate (ϕ) remained fixed at 2.5 vvm. The average residence time of the bubbles in the liquid phase was therefore the same in all cases. The values of kE and kW obtained in the stripping experiments (Table 2) were subsequently adopted for use in the simulations. Conventional Fed-Batch Fermentation with Different Filling Times: Model Fitting and Kinetic Parameter Estimation. During ethanol fermentation, yeast suffers inhibitory effects caused by the substrate as well as by the ethanol formed during the process. The use of fed-batch fermentation makes it possible to regulate the concentration of substrate in the vat in order to minimize the effects of substrate inhibition. Hence, Ft has an important influence on fed-batch fermentation. In order to evaluate the effect of Ft and to perform the mathematical modeling of the conventional fedbatch fermentation process, fermentations were conducted with Ft of 3 or 5 h and a sucrose concentration of 180 g·L−1 in the must feed. 752

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels

Figure 2. Comparative plots of experimental and simulated (−) data for cell concentration in fermentation broth (Cx (green closed boxes)), substrate concentration in fermentation broth (Cs (red closed boxes)), and ethanol concentration in fermentation broth (CE (blue closed circles)) in the conventional fed-batch fermentation with substrate concentration of 180 g·L−1 in the must feed: (a) CF1, filling time (Ft) of 3 h; (b) CF2, Ft of 5 h.

different from that observed in conventional fermentation (Figure 2). The substrate concentration profile in extractive fermentation EF1, with both filling time and stripping start time set at 3 h (Figure 3a), showed the same behavior as the conventional fed-batch fermentation (Figure 2a), with a sugar concentration of approximately 120 g·L−1 at the end of vat filling. However, the substrate uptake rate (rS, g·L−1·h−1) increased after the beginning of ethanol removal by CO2 stripping. The total consumption of the substrate therefore occurred earlier in extractive fermentation EF1 than in the conventional fermentation (CF1), due to the reduction of ethanol inhibition. Total consumption of the substrate occurred approximately 1 h earlier in EF1, compared to CF1, with an ethanol volumetric productivity of 10.0 g·L−1·h−1, representing a gain of about 11.1% relative to the conventional fermentation without ethanol removal (Table 4). In extractive fermentation EF2, with Ft of 5 h, the accumulation of sugar in the fermentation broth was avoided (Figure 3b). However, total consumption of the substrate occurred in about 8 h. This was because the ethanol stripping with CO2 was started after 5 h, when the ethanol concentration in the broth was around 55 g·L−1. Even after having started the

Figure 3. Comparative plots of the experimental and simulated (−) data for cell concentration in fermentation broth (Cx (green closed boxes)), substrate concentration in fermentation broth (Cs (red closed boxes)), and ethanol concentration in fermentation broth (CE (blue closed circles)) in the extractive fed-batch fermentations: (a) EF1, Ft and stripping start time of 3 h; (b) EF2, Ft and stripping start time of 5 h; (c) EF3, Ft of 5 h and stripping start time of 3 h.

gas stripping, the ethanol concentration in the broth continued to increase, reaching a maximum value of 68.6 g·L−1 after 8 h of fermentation, with volumetric productivity of 10.2 g·L−1·h−1. Nonetheless, removal of ethanol during the fermentation significantly decreased the inhibition effect, resulting in an increase in ethanol productivity when compared to the conventional fermentation (Table 4). 753

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels

Table 4. Comparison of Conventional (CF) and Extractive (EF) Fermentations Performed with Substrate Concentration of 180 g·L−1 in the Must Feed and with Filling Times of 3 and 5 h and Start of Ethanol Removal by CO2 Stripping after 3 and 5 h fermentation

a

variable

unit

CF1

CF2

EF1

EF2

EF3

Cs0 or CsF Cs at the final filling time CE at the start of ethanol stripping total CE at the end of fermentation maximum CE in the fermentation broth volumetric ethanol productivity

g·L−1 g·L−1 g·L−1 g·L−1 g·L−1 g·L−1·h−1

182.4 123.8 34.2 81.4 81.4 9.0

183.9 74.6 55.7 83.1 83.1 9.1

178.1 114.3 33.2 80.1a 64.8 10.0b

180.6 63.3 55.7 81.3a 68.6 10.2b

182.7 60.0 46.9 82.2a 65.0 10.3b

Calculated considering Cs0 for each fermentation and YE/S = 0.454 gE·gS−1. bCalculated based on CE at the end of fermentation.

again obtained between the experimental data and the values of Cs, Cs, and CE predicted by the proposed model for extractive fed-batch ethanol fermentation with CO2 stripping. The results for conventional fermentation CF3 (Figure 4a) showed that, with Ft of 5 h, the maximum substrate concentration in the broth was about 90 g·L−1 when the substrate concentration in the feed was 200 g·L−1. However, this substrate concentration resulted in an incomplete fermentation, with 9.2 g·L−1 of residual sugar and an accumulated ethanol concentration of 84.8 g·L−1 after 12 h of fermentation. Figure 4b shows the results for extractive fermentation EF4, in which stripping was started at 3 h (CE = 48.6 g·L−1). With the partial removal of ethanol by CO2 stripping, the maximum concentration of ethanol in the fermentation broth was 69.1 g· L−1. Hence, there was a decrease in ethanol inhibition, and the increased rS resulted in complete substrate consumption after 9 h of fermentation. Figure 4c shows the results for conventional fermentation CF4, performed with a substrate concentration in the feed of 220 g·L−1 and Ft = 5 h. These conditions resulted in a maximum substrate concentration in the broth of around 110 g· L−1. However, after 8 h of fermentation, when the ethanol concentration in the broth reached 75 g·L−1, there was a strong effect of ethanol inhibition and a decrease in the substrate uptake rate, resulting in an incomplete fermentation with 27.8 g·L−1 of residual sugar and a cumulative ethanol concentration of 86.5 g·L−1 at 12 h of fermentation. Figure 4d illustrates the results obtained for extractive fermentation EF5 with a substrate concentration in the feed of 220 g·L−1 and the start of stripping at 3 h of fermentation, when the ethanol concentration was 42.3 g·L−1. The maximum ethanol concentration in the fermentation broth was 75.3 g·L−1 and substrate exhaustion occurred at 10 h of fermentation, demonstrating that the reduction of ethanol inhibition due to CO2 stripping enabled complete consumption of the 220 g·L−1 of substrate fed into the broth during the 10 h of fermentation. The maximum concentration of ethanol obtained in fermentation EF5 was 75.3 g·L−1, which was lower than the maximum concentration of ethanol after which cell growth ceased (CEmax = 86.1 g·L−1), indicating the possibility of performing fermentation with a substrate concentration of 240 g·L−1 in the feed. Figure 4e illustrates the results obtained for extractive fermentation EF6, using a substrate concentration of 240 g·L−1 in the feed, Ft = 5 h, and gas stripping started at 3 h of fermentation, when the ethanol concentration was 37.9 g·L−1. Under these conditions, the maximum substrate concentration in the fermentation broth was 139.3 g·L−1, the maximum

Figure 3c illustrates the results for extractive fermentation EF3, with Ft of 5 h and the start of stripping at 3 h when the ethanol concentration in the broth was 46.9 g·L−1. The substrate uptake rate (rS) increased after the start of ethanol removal by stripping with CO2, due to reduction of the ethanol inhibition. This resulted in total consumption of the substrate occurring around 1 h earlier than in the conventional fermentation (CF2), leading to a productivity of 10.3 g·L−1· h−1 (Table 4). It is clear that the use of ethanol removal by CO2 stripping provided higher productivity values in extractive fermentations EF1, EF2, and EF3 (Table 4). However, when ethanol removal by CO2 stripping was started at 5 h (EF2), the accumulated ethanol concentration was 55.7 g·L−1. Sonego et al.19 reported that starting the stripping at an ethanol concentration around 40 g·L−1 provided greater reduction of inhibition of cell growth by ethanol, resulting in greater fermentation productivity. Therefore, in the subsequent extractive processes (Figure 3a− c) the stripping was started after 3 h of fermentation, when the ethanol concentrations were close to 40 g·L−1. For a substrate concentration of 180 g·L−1, a filling time of 3 h (EF1) provided the highest substrate concentration (114.3 g· L−1) at the end of vat filling (Table 4). When Ft of 5 h (EF2 and EF3) was employed, the substrate concentration at the end of the filling was about 60 g·L−1. Thus, Ft of 5 h and the beginning of CO2 stripping at 3 h (CE ∼ 40 g·L−1) decreased the accumulation of ethanol during the course of the fermentation and consequently reduced the ethanol inhibition, while avoiding possible effects of accumulated substrate in the broth, which could have inhibited cellular growth and ethanol production during the first hours of the fermentation. Based on the results shown in Table 4, a filling time of 5 h was established for use in the subsequent fermentations with higher substrate concentrations of 200, 220, and 240 g·L−1 in the must feed, in order to avoid substrate inhibition during the early fermentation period. The use of this filling time was supported by simulation results showing that feeding of must containing substrate at concentrations of 200, 220, and 240 g· L−1 would result in Cs values in the fermentation broth of up to 180 g·L−1, which would be detrimental to the process. The simulation results also indicated that the highest productivity values were obtained by starting the CO2 stripping at 3 h, with ethanol concentrations of up to 40 g·L−1 in the fermentation broth. Selection was therefore made of a vat filling time of 5 h and the start of ethanol stripping by CO2 at 3 h of fermentation. Figure 4 compares the simulated and experimental values of Cx, Cs, and CE for the conventional and extractive fed-batch ethanol fermentations employing substrate concentrations of 200, 220, and 240 g·L−1 in the feed. Excellent agreement was 754

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels

Figure 4. Comparative plots of experimental and simulated (−) data for cell concentration in fermentation broth (Cx (green closed boxes)), substrate concentration in fermentation broth (Cs (red closed boxes)), and ethanol concentration in fermentation broth (CE (blue closed circles)) in the conventional (CF) and extractive (EF) fed-batch fermentations: (a) conventional fermentation CF3 (CsF = 200 g·L−1); (b) extractive fermentation EF4 (CsF = 200 g·L−1); (c) conventional fermentation CF4 (CsF = 220 g·L−1); (d) extractive fermentation EF5 (CsF = 220 g·L−1), (e) extractive fermentation EF6 (CsF = 240 g·L−1). Extractive fermentations EF4, EF5, and EF6 were conducted with Ft = 5 h and start of stripping at 3 h.

ethanol concentration was 76.9 g·L−1, and substrate exhaustion occurred at 12 h of fermentation. According to Thatipamala et al.,9 substrate inhibition of yeast cells occurs at concentrations exceeding 150 g·L−1. Here, a vat filling time of 5 h was able to maintain the substrate concentration in the fermentation broth below 150 g·L−1, allowing extractive fed-batch ethanol fermentation with CO2 stripping to be performed with substrate concentrations up to 240 g·L−1.

The results shown in Figure 4a,c revealed that there were decreases in the ethanol production rate (rE) and substrate consumption after 8 h of cultivation, due to the strong inhibition effect caused by the high ethanol concentration (>70 g·L−1), resulting in lower ethanol volumetric productivity. A similar effect was reported by Lloyd et al.,12,13,24 who found that ethanol inhibition of the yeast was greatest at ethanol concentrations above 60 g·L−1. In addition, it is worth highlighting that an ethanol concentration above 85.0 g·L−1 755

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels

Table 5. Comparison between Conventional (CF) and Extractive (EF) Fermentations Performed with Ft = 5 h and Start of CO2 Stripping at 3 ha fermentation variable

unit

CF3

CF4

EF4

EF5

EF6

Cs0 or CsF residual Cs at the end of fermentation total CE at the end of fermentation final CE in the fermentation broth volumetric ethanol productivity total ethanol concentration

g·L−1 g·L−1 g·L−1 g·L−1 g·L−1·h−1 °GL

194.4 9.2 87.5 84.8 7.1 11.1

214.1 27.8 96.3 86.5 7.2 12.2

195.1 0.0 87.8b 69.1 9.7b 11.1b

223.6 0.0 100.6b 73.3 10.1b 12.8b

245.0 0.0 110b 76.9 9.2b 14.0b

Conventional fermentation CF3, CsF = 200 g·L−1; conventional fermentation CF4, CsF = 220 g·L−1; extractive fermentation EF4, CsF = 200 g·L−1; extractive fermentation EF5, CsF = 220 g·L−1; extractive fermentation EF6, CsF = 240 g·L−1. bCalculated considering Cs0 for each fermentation and YE/S = 0.454 gE·gS−1. bCalculated based on CE at the end of fermentation.

a

industry uses low substrate concentrations and produces final wines with low ethanol concentrations, resulting in the generation of large volumes of vinasse and requiring high steam consumption to obtain ethanol fuel with a concentration of 96 °GL (vol·vol−1). The use of fed-batch extractive ethanol fermentation with CO2 stripping therefore appears to offer an excellent way of reducing the inhibitory effects of substrate and ethanol on yeast cells, hence delivering better performance compared to the conventional process. Furthermore, the use of a high sucrose concentration in the must feed would result in increased ethanol production in the same industrial installation, together with a decrease in the volume of vinasse generated. This would be reflected in lower costs associated with the distillation step and the transport of vinasse to the sugar cane fields for use in irrigation and as fertilizer. At the end of the fed-batch fermentations, samples were examined using an optical microscope in order to determine the cell viability. In conventional fed-batch fermentations CF1 and CF2 (CS0 = 180 g·L−1), the average viability was 55% (at 9 h of fermentation). In the conventional fermentations with substrate concentrations higher than 200 g·L−1 (CF3 and CF4), the average cell viability was 49% (at 12 h of fermentation). The extractive fed-batch fermentations performed with substrate concentrations of 180 g·L−1 (EF1, EF2, and EF3) showed average cell viability of 64%, and in the extractive fermentations with the highest substrate concentrations (EF4, EF5, and EF6), the average viability was 59%. The high viability values obtained for extractive fermentations EF1, EF2, and EF3 can be explained by the fact that, at the end of these fermentations, the ethanol concentrations in the broth were lower than the values obtained in conventional fermentations CF1 and CF2. These findings confirmed that high ethanol concentrations (>80 g·L−1) had a negative effect on the yeast cells, hindering cell division and reducing viability, as described previously.38 It was also evident that the CO2 used in the extractive technique did not harm the yeast cells during the fermentation, in agreement with Sonego et al.,19 who found that CO2 had no adverse effects on yeast cells and did not decrease the pH of the fermentation broth during extractive batch fermentation with ethanol removal by CO2 bubbling. The results obtained in this study indicate that the extraction of ethanol by CO2, performed in the vat during the fermentation process, can effectively reduce the inhibition of yeast by ethanol, making the process more productive. In conclusion, the findings of this study should contribute to improving the processes used in distilleries. The production of ethanol in Brazil’s distilleries mainly employs fermentation in

virtually halted ethanol production, in agreement with Maiorella et al.,10 who showed that a concentration of around 95 g·L−1 completely inhibited the cell growth of yeasts. According to Basso et al.,4 the substrate concentration in the must usually employed in Brazilian distilleries is around 20 °BRIX (∼180 g·L−1 of total reducing sugars, glucose and fructose), while the final ethanol concentration in the fermentation broth is around 80 g·L−1 (∼10 °GL). However, in this study using the extractive fed-batch ethanol fermentation, it was possible to feed the vat with a must that was 11.1% more concentrated in terms of sugar (EF4; CsF = 200 g·L−1), resulting in an ethanol productivity of 9.7 g·L−1·h−1 (Table 5). When a must that was 22.2% more concentrated in sugar was employed (EF5; CsF = 220 g·L−1), the ethanol productivity was 10.1 g·L−1·h−1 (Table 5). It was still possible to conduct an extractive fermentation with a must that was 33.3% more concentrated (EF6; CsF = 240 g·L−1), with a final ethanol concentration of 76.9 g·L−1 reached in the fermentation broth, which was still below the maximum concentration tolerated by the yeast cells (Table 5). Previous studies have reported increases in substrate utilization and the amount of product (n-butanol) formed during fermentation using fed-batch processes with gas stripping.26,22,37 The present results provide clear evidence that fed-batch extractive fermentation can effectively minimize the substrate inhibition that occurs in the first hours of the process, while reducing ethanol inhibition by removing ethanol from the fermentation broth by CO2 stripping, enabling the fermentation to proceed until complete exhaustion of the substrate. The ethanol concentrations in the broth at the end of the extractive ethanol fermentations EF4, EF5, and EF6 were 69.1, 73.3, and 76.9 g·L−1 (8.8, 9.3, and 9.7 °GL), respectively, very similar to the values obtained in Brazilian distilleries using conventional fermentation (8−10 °GL). This means that the wine resulting from extractive fermentation carried out with concentrated substrate in the must could be sent to the distillation step without compromising the efficiency of the process. An important point is that, in fermentation EF6, with a substrate concentration of 240 g·L−1 in the must feed, a total ethanol concentration of 110.3 g·L−1 (14 °GL) was obtained (Table 5), considering the ethanol in the wine plus the ethanol removed by the CO2 stripping. This value is much higher than typical ethanol concentrations in the final (fermented) wine found in Brazilian distilleries. In conventional ethanol fermentation under industrial conditions, the yeast cells are exposed to substrate and product (ethanol) inhibition. Consequently, the Brazilian bioethanol 756

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757

Article

Energy & Fuels

(10) Maiorella, B.; Blanch, H. W.; Wilke, C. R. Biotechnol. Bioeng. 1983, 25, 103−121. (11) Aiba, S.; Shoda, M.; Nagatani, M. Biotechnol. Bioeng. 1968, 10, 845−864. (12) Lloyd, D.; Morrell, S.; Carlsen, H. N.; Degn, H.; James, P. E.; Rowlands, C. C. Yeast 1993, 9, 825−833. (13) Ly, H. V.; Block, D. E.; Longo, M. L. Langmuir 2002, 18, 8988− 8995. (14) Mohana, S.; Acharya, B. K.; Madamwar, D. J. Hazard. Mater. 2009, 163, 12−25. (15) Morandin, M.; Toffolo, A.; Lazzaretto, A.; Maréchal, F.; Ensinas, A. V.; Nebra, S. A. Energy 2011, 36, 3675−3690. (16) Taylor, F.; Kurantz, M. J.; Goldberg, N.; Craig, J. C. Biotechnol. Prog. 1995, 11, 693−698. (17) Schügerl, K. Biotechnol. Adv. 2000, 18, 581−599. (18) Cardona, C. A.; Sánchez, O. J. Bioresour. Technol. 2007, 98, 2415−2457. (19) Sonego, J. L. S.; Lemos, D. A.; Rodriguez, G. Y.; Cruz, A. J. G.; Badino, A. C. Energy Fuels 2014, 28, 7552−7559. (20) Silva, C. R.; Esperança, M. N.; Cruz, A. J. G.; Moura, L. F.; Badino, A. C. Chem. Eng. Res. Des. 2015, 102, 150−160. (21) Park, C.-H.; Geng, Q. Sep. Purif. Rev. 1992, 21, 127−174. (22) Lu, C.; Zhao, J.; Yang, S.-T.; Wei, D. Bioresour. Technol. 2012, 104, 380−387. (23) Taylor, F.; Kurantz, M. J.; Goldberg, N.; Craig, J. C., Jr. Appl. Microbiol. Biotechnol. 1997, 48, 311−316. (24) Taylor, F.; Kurantz, M. J.; Goldberg, N.; Craig, J. C., Jr. Biotechnol. Bioeng. 1996, 51, 33−39. (25) Taylor, F.; Marquez, M. A.; Johnston, D. B.; Goldberg, N. M.; Hicks, K. B. Bioresour. Technol. 2010, 101, 4403−4408. (26) Ezeji, T. C.; Qureshi, N.; Blaschek, H. P. Appl. Microbiol. Biotechnol. 2004, 63, 653−658. (27) Badino, A. C.; Cerri, M. O.; Hokka, C. O. Sistema reacional pneumático e uso do mesmo. Brazil Patent PI0701608-5, 2007. (28) Lee, S. S.; Robinson, F. M.; Wang, H. Y. Biotechnol. Bioeng. Symp. 1981, 11, 641−649. (29) Andrews, J. F. Biotechnol. Bioeng. 1968, 10, 707−723. (30) Levenspiel, O. Biotechnol. Bioeng. 1980, 22, 1671−1687. (31) Nelder, J. A.; Mead, R. Comput. J. 1965, 7, 308−313. (32) Truong, K. N.; Blackburn, J. W. Environ. Prog. 1984, 3, 143− 152. (33) Ezeji, T. C.; Karcher, P. M.; Qureshi, N.; Blaschek, H. P. Bioprocess Biosyst. Eng. 2005, 27, 207−214. (34) de Vrije, T.; Budde, M.; van der Wal, H.; Claassen, P. A. M.; López-Contreras, A. M. Bioresour. Technol. 2013, 137, 153−159. (35) Perry, R. H.; Green, D. W.; Maloney, J. O. Perry’s chemical engineers’ handbook; McGraw Hill: New York, 1997. (36) Guidini, C. Z.; Marquez, L. D. S.; De Almeida Silva, H.; De Resende, M. M.; Cardoso, V. L.; Ribeiro, E. J. Appl. Biochem. Biotechnol. 2014, 172, 1623−1638. (37) Xue, C.; Zhao, J.; Lu, C.; Yang, S.-T.; Bai, F.; Tang, I.-C. Biotechnol. Bioeng. 2012, 109, 2746−2756. (38) D’Amore, T.; Stewart, G. G. Enzyme Microb. Technol. 1987, 9, 322−330.

fed-batch mode. It must be emphasized that in most installations the fermentation vats are closed and are equipped with systems for collection of the gas stream (containing CO2 and ethanol), which is transferred to a washing tower. In processes employing extractive fed-batch fermentation, it would therefore be possible to use the existing gas collection system, with recovery of the ethanol in the tower or in an absorption system, while the CO2 is recycled to the process in sufficient amounts for the stripping operation.

■

CONCLUSIONS The hybrid Andrews−Levenspiel kinetic model provided an excellent fit to the experimental conventional fed-batch fermentation data. The model proposed for extractive fedbatch fermentation with CO2 stripping was able to accurately predict the behavior of fermentations conducted using different conditions of substrate concentration in the must feed (CsF), vat filling time (Ft), and CO2 stripping start time. The use of a vat filling time of 5 h and the start of stripping at 3 h of fermentation resulted in substantial reductions in the inhibitory effects of substrate and ethanol on the yeast cells. Under these conditions, the extractive fed-batch ethanol fermentation could be performed using substrate concentrations of up to 240 g·L−1 in the feed, with substrate exhaustion after 12 h. A volumetric ethanol productivity of 9.2 g·L−1·h−1 was achieved, and the total ethanol concentration in the process was 110.3 g·L−1 (14 °GL), which was around 33% higher than that obtained in conventional fed-batch ethanol fermentation without ethanol removal by CO2 stripping.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 55 16 3351-8001. Fax: 55 16 3351-8266. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful for the financial support provided by CAPES (National Council for the Improvement of Higher Education, Brazil) and FAPESP (São Paulo State Research Foundation, Brazil, Grant number 2012/50046-4).

■

REFERENCES

(1) Nigam, P. S.; Singh, A. Prog. Energy Combust. Sci. 2011, 37, 52− 68. (2) Bai, F. W.; Anderson, W. A.; Moo-Young, M. Biotechnol. Adv. 2008, 26, 89−105. (3) Ibeto, C. N.; Ofoefule, A. U.; Agbo, K. E. Trends in Applied Sciences Research 2011, 6, 410−425. (4) Basso, L. C.; Basso, T. O.; Rocha, S. N. Biofuel Production−Recent Developments and Prospects; InTech: Rijeka, Croatia, 2011; Vol. 1530, pp 85−100, DOI: 10.5772/959 (5) Godoy, A.; Amorim, H. V.; Lopes, M. L.; Oliveira, A. J. Int. Sugar J. 2008, 110, 175−181. (6) Amorim, H. V.; Lopes, M. L.; de Castro Oliveira, J. V.; Buckeridge, M. S.; Goldman, G. H. Appl. Microbiol. Biotechnol. 2011, 91, 1267−1275. (7) Wheals, A. E.; Basso, L. C.; Alves, D. M. G.; Amorim, H. V. Trends Biotechnol. 1999, 17, 482−487. (8) Sánchez, Ó . J.; Cardona, C. A. Bioresour. Technol. 2008, 99, 5270−5295. (9) Thatipamala, R.; Rohani, S.; Hill, G. A. Biotechnol. Bioeng. 1992, 40, 289−297. 757

DOI: 10.1021/acs.energyfuels.5b02320 Energy Fuels 2016, 30, 748−757