Hemicellulosic Ethanol Production in Fluidized Bed Reactor from

Research Article pubs.acs.org/journal/ascecg

Hemicellulosic Ethanol Production in Fluidized Bed Reactor from Sugar Cane Bagasse Hydrolysate: Interplay among Carrier Concentration and Aeration Rate Felipe A. F. Antunes,* Anuj K. Chandel, Julio Cesar dos Santos, Thais S. S. Milessi, Guilherme F. D. Peres, and Silvio Silvério da Silva* Department of Biotechnology, Engineering School of Lorena, University of São Paulo, Estrada Municipal do Campinho, s/no Campinho, Lorena, São Paulo 12602-810, Brazil ABSTRACT: Development of an efficient consolidated process is pivotal in order to design industrially viable processes for conversion of lignocellulosic biomass into second generation (2G) ethanol. Aiming to develop process consolidation, here we explored fluidized bed reactor (FBR) for 2G ethanol production from sugar cane bagasse hemicellulosic hydrolysate (SBHH) employing calcium alginate immobilized cells of Schef fersomyces shehatae UFMG-HM 52.2. A 22-full factorial design of experiments was carried out in order to evaluate the effect of aeration rate (0.027, 0.069, and 0.111 min−1) and carrier concentration (55.55, 83.33, and 111.11 g. L−1) on the ethanol yield (YP/S) and productivity (QP). Both process variables, when used at the highest level (aeration, 0.11 min−1; immobilized carrier concentration, 111.11 g. L−1), showed maximum ethanol production (YP/S, 0.26 g/g and QP 0.17 g·L−1·h−1). Results showed the potential to use this immobilized yeast in a fluidized bed reactor for ethanol production from C5 sugar solution. Repeated batch fermentations in FBR showed stable ethanol yield during 6 batches (288 h) followed by a gradual decrease. The use of immobilized cells in FBR could be conducive to the development of viable 2G ethanol production processes. To the best of our knowledge, this is the first report on 2G ethanol production from immobilized S. shehatae cells employing FBR using SBHH. KEYWORDS: Sugar cane bagasse, Hemicellulosic hydrolysate, Fluidized bed reactor, 2G Ethanol, Immobilized cells, Schef fersomyces shehatae

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INTRODUCTION Ethanol produced from sugar cane bagasse, the so-called second generation (2G) ethanol, seems to be a competent choice as a sustainable liquid transportation fuel particularly in countries like Brazil, India, China, Australia, and others.1 Use of 2G ethanol is important not only because of the depletion of oil reserves but also because of the severe environmental concerns such as greenhouse gas emissions and climate change.1,2 Although lignocellulosic materials (e.g., sugar cane bagasse and straw, corn stover, wheat straw, wood and weedy materials, and others) can be converted to biofuels, their large-scale use in commercial ethanol production processes is rare.2−4 Globally Brazil, India, China, Thailand, Pakistan, Mexico, Columbia, Indonesia, Phillipines, and USA produced around 1600000 thousand metric tons of sugar cane annually.5 In Brazil, approximately 684 million tons of sugar cane was produced in the 2016/2017 crop season.6 In general, 140 kg of bagasse is generated from each ton of processed sugar cane7 yielding around 95 million tons of bagasse readily available for 2G ethanol production in sucro-alcohol industries. According to Cerqueira-Leite et al.,8 up to 90% of the bagasse is © 2017 American Chemical Society

combusted to generate electricity, which is used on site or distributed through the grid. Thus, the surplus of sugar cane bagasse (approximately 9.5 million tons) could be used as a carbon source to produce (2G) ethanol and other value-added chemicals. For 2G ethanol production from sugar cane bagasse, pretreatment is necessary to disrupt the highly recalcitrant structure of the material to unlock complex carbohydrate polymers into monomeric sugars.3,9,10 Dilute acid hydrolysis of sugar cane bagasse is an efficient and fast method to depolymerize the hemicellulosic fraction from the cell wall into sugar monomers principally xylose and others sugars along with some inhibitory products which are produced due to sugars and lignin breakdown during the reaction.3,11 While the use of hemicellulosic sugar solution for ethanol production is an important necessity of the overall success of biorefineries, as it represents up to 30% of sugar cane bagasse Received: June 13, 2017 Revised: July 18, 2017 Published: July 25, 2017 8250

DOI: 10.1021/acssuschemeng.7b01916 ACS Sustainable Chem. Eng. 2017, 5, 8250−8259

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ACS Sustainable Chemistry & Engineering

Table 1. Full 22 Factorial Design with Triplicate at Center Point with Values for the Independent Variable Aeration Rate (AR) and Carrier Concentration (CC) for Ethanol Production by Immobilized S. shehatae UFMG-HM 52.2 in a Fluidized Bed Reactor, with Ethanol Yield (YP/S) and Productivity (QP) as Response Variablesa independent variables

a

−1

exp.

(A) aeration rate (min )

1 2 3 4 5 6 7

0.027 (−1) 0.111(1) 0.027 (−1) 0.111(1) 0.069 (0) 0.069 (0) 0.069 (0)

response variables

(B) carrier concentration (g·L 55.55 (−1) 55.55 (−1) 111.11 (1) 111.11 (1) 83.33 (0) 83.33 (0) 83.33 (0)

−1

)

−1

YP/S (g·g )

QP (g·L−1·h−1)

0.276 0.219 0.288 0.265 0.281 0.277 0.276

0.125 0.130 0.132 0.177 0.140 0.138 0.137

Coded values of the studied variables are in parentheses.

composition,7 the microorganism for C5 sugar fermentation are scarce.12,13 Within this context, the search for a viable microorganism that assimilates C5 sugars remains a challenge.13 Among the known C5 fermenting microorganism, Schef fersomyces shehatae UFMG 52.2, isolated from Brazilian forests has been recently reported as a promising ethanol producer.14,15 In addition to the correct selection of raw materials and microorganisms, the determination of processing strategies and process configurations is important for the development of consolidated industrially feasible processes.16,17 Hence, the use of immobilized cells is advantageous due to easier recovery of the biocatalyst, simplified product purification, and the possibility of increased biocatalyst concentrations, which could lead to improved process economics.18 Different bioreactor configurations have been studied using immobilized cells, and among them, column bioreactors operated in FBR mode can be an interesting alternative. Authors have previously reported the use of S. shehatae UFMG 52.2 in free form19 with the goal of determining the correct fermentation medium, as well as in immobilized form,15 with the aim of investigating the effect of cell concentration in a sodium alginate immobilization system. Milessi et al.20 determined the effect of stirring rate and immobilized cell concentration of S. stipitis (Syn Pichia stipitis). It is important to note that all these studies were performed in shake flasks. However, the present study was done at bioreactor level (2 L capacity) using a fluidized bed reactor (FBR), a unique approach toward process consolidation for ethanol 2G production, employing immobilized S. shehatae UFMG-HM 52.2 to evaluate bioreactor parameters such as carrier concentration and aeration. Fluidization provides high homogeneity to the medium inside the reactor throughout its length, with high mass and heat transfer rates along the bed, without mechanical impellers.21 This is desirable because mechanical stirrers require higher energy for operation, and the high shear stress imposed on the reaction medium can disrupt the cell wall of fermenting microorganisms. The proper adjustment of bioreactor parameters, such as aeration rate and the ratio between the biocatalyst mass and the reactor volume, has also presented great importance for optimizing the process. Taking this into account, this work was aimed to investigate the use of FBR with a bed composed of Ca-alginate immobilized cells of S. shehatae UFMG-HM 52.2 for producing ethanol from sugars present in sugar cane bagasse hemicellulosic hydrolysate (SBHH). This is a new approach for 2G ethanol biorefineries, and the design of experiment (DOE) technique was used aiming to evaluate and understand the

influence of process conditions in the performance of this proposed system.

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MATERIALS AND METHODS

Preparation of the Hemicellulosic Hydrolysate of Sugar Cane Bagasse. Sugar cane bagasse was kindly provided by the Usina São Francisco mill, Sertãozinho, Sao Paulo State, Brazil. Hemicellulosic hydrolysate was obtained in a 200 L reactor, by using sulfuric acid solution (100 mg acid/g dry matter). Acid hydrolysis was conducted at 121 °C for 20 min using a solid/liquid ratio of 1:10 for dry sugar cane bagasse/acid solution.20 The hydrolysate was separated from solid material by filtration. The recovered hydrolysate was then concentrated by a vacuum evaporator under reduced pressure at 70 °C in a 32 L capacity concentrator. The concentrated hydrolysate was detoxified (over liming and activated charcoal combination) according to the method of Alves et al.22 The average composition of the detoxified SBHH was 31 g·L−1 xylose, 0.3 g·L−1 glucose, and 1.6 g·L−1 of acetic acid.20 The fermentation medium was prepared by supplementing the sugar cane bagasse hydrolysate with 5.0 g·L−1 (NH4)2SO4, 3 g·L−1 of yeast extract, and 3 g·L−1 of malt extract.19 Inoculum Preparation and Cell Immobilization. S. shehatae UFMG-HM 52.2, yeast isolated from the Brazilian Atlantic Rain Forest, was kindly provided by the Microorganism Culture Collection of the Federal University of Minas Gerais (UFMG), Brazil. Inoculum preparation was performed according to the methodology of Chandel et al.14 The culture was maintained at 5 °C on malt extract agar slants. A loop full of a slant culture was transferred to liquid medium in 125 mL Erlenmeyer flasks, containing 50 mL of growth medium (30 g·L−1 xylose, 20 g·L−1 of peptone, and 10 g·L−1 of yeast extract) at 30 °C, 200 rpm for 24 h in an incubator shaker (Innova 4000 Incubator Shaker, New Brunswick Scientific, Enfield, CT, USA). After growing, cells were centrifugated, washed, and resuspended in sterile distilled water, for use in the immobilization step. Cell Immobilization. S. shehatae UFMG-HM 52.2 were immobilized in calcium alginate beads, according to the methodology of Antunes et al.15 An adequate volume of the cell suspension was added to a solution of sodium alginate (sodium salt of alginic acid from brown algae, Fluka analytical, Switzerland) sterilized at 121 °C for 15 min, in order to obtain a final solution containing 1% of sodium alginate and 3g·L−1 of cells (dry weight). Beads of immobilized cells were produced by dripping this suspension in a 0.2 mol·L−1 solution of calcium chloride. The beads were maintained in the CaCl2 solution at 4 °C for 12 h. After this conditioning time, spheres having cells were washed with sterilized distilled water and used in a fluidized bed reactor for fermentation. Fluidized Bed Reactor Operation. Column reactor of 2 L capacity (Bioengineering AG, PID Fermenter AWS, Wald, Switzerland) was used in all fermentation assays (540 mm × 55 mm column, with a central vertical tube of 9 mm inner diameter). The reactor was filled with 1800 mL of supplemented sugar cane bagasse hemicellulosic hydrolysate added to 1 mL of antifoam (silicon emulsion) and recirculated in flow at 50.8 mL·min−1 (value determined by 8251

DOI: 10.1021/acssuschemeng.7b01916 ACS Sustainable Chem. Eng. 2017, 5, 8250−8259

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ACS Sustainable Chemistry & Engineering experimental observation of proper fluidization of beads, without the need of aeration), by using an external pump. Different aeration rates were provided by an external air pump and filtered before inserting in the reactor, according to conditions shown in Table 1. Different masses of immobilized beads were added to the reactor in order to obtain carrier concentration (CC) of 55.55, 111.11, and 83.33 g. L−1, related to conditions of design of experiments described in Table 1. Figure 1 presents a schematic diagram of the proposed system.

Determination of Volumetric Oxygen Transfer Coefficient (KLa). The volumetric oxygen transfer coefficient (KLa) was determined by the methodology of “gassing out”. Using the different conditions for carrier concentration (Table 1), the oxygen in the culture medium in the reactor was removed by purging of nitrogen gas. The fluidization system was started with recirculation of supplemented SBHH at the determined flow rate (50.8 mL·min−1), and the increase in dissolved oxygen concentration was monitored as a function of time using an oxygen electrode (Mettler Toledo, 341003037/0473401, Columbus, EUA). By integrating the oxygen balance equation in the liquid medium, according to eq 3, value of KLa in each condition was determined. dC /dt = KLa(C* − C)

where C/C*corresponds to the electrode fraction of dissolved O2 concentration in relation to saturation concentration. Repeated Batch Experiments. The repeated batch experiments were carried out in consecutive batches with reuse of the immobilized cells. The reactor was initially filled with stainless steel beads (100 g) to reduce size and increase the surface area of air bubbles, then 200 g of immobilized biocatalyst was added into the reactor, and the aeration system was set up to a flow rate of 200 mL/min. The system was fed with detoxified SBHH (1.8 L) supplemented with medium composition (section 2.1) and recirculated with a flow rate of 50.8 mL/s. In the fermentation medium, 1.5 mL of antifoam (silicone emulsion) and 1.193 g of the antibiotic solution (ceftriaxone sodium hemieptahydrate) were also added. Each repeated batch was carried out for 48 h at 30 °C. At the end of each batch, the reactor was discharged, and a fresh fermentation medium was added to the reactor by a peristaltic pump. The immobilized cells in the reactor bed were used as inoculum for the next batch. Samples were collected periodically to determine the concentrations of sugars and production of ethanol and biomass. Analytical Methods. Sugars (xylose and glucose) and ethanol concentration were analyzed by HPLC (Schimadzu LC-10 AD (Kyoto, Japan) with the column equipped with BIO-RAD Aminex HPX- 87H (300 × 7.8 mm) coupled to a detector of refractive index RID-6A, with 0.005 M sulfuric acid eluent at a flow rate of 0.6 mL· min−1, a column temperature of 45 °C, and an injected volume of 20 mL. Before passing the samples in HPLC, they were filtered through a Sep Pak C18 filter. The concentration of free cells was determined by turbidimetry using a spectrophotometer (Beckman DU 640 B Fullerton, CA, USA) and correlated with the dry weight of cells (g·L−1) through a calibration curve. For the determination of immobilized cell concentrations, the methodology described by Carvalho et al.25 was followed. A known mass of Ca-alginate beads was taken in the initial run and after completing each fermentation assay. The beads were dried on an absorbent paper and dissolved in a stirred 2.0% potassium citrate solution. The suspension was centrifuged (2000g, 15 min); and the released cells from diluted calcium alginate were resuspended in water to determine the turbidity, similar to that of the free cells. Ethanol yield (YP/S) (g·g−1) was defined as the ratio between ethanol production (g·L−1) and available sugar consumption (Xylose + glucose concentration) (g·L−1), while volumetric productivity (QP) (g· L−1·h−1) was the ratio between ethanol production (g·L−1) and fermentation time (h).

Figure 1. Schematic diagram of the fluidized bed reactor for 2G ethanol production from sugar cane bagasse hydrolysate using immobilized cells. To investigate the influence of aeration rate (0.027−0.111 min−1) and carrier concentration (55.55−111.11 g. L−1) for the ethanol production from sugar cane bagasse in a fluidized bed reactor, the main effects and their interactions of these variables were studied following full factorial design of experiments (22) with three replicates at center points. The response variables were ethanol conversion yield (YP/S) and productivity (QP). The analysis of statistical tests was carried out using the software STATISTICA for Windows (StatSoft, Inc. V.5 Tulsa, OK, USA). The process was carried out at 30 °C for 72 h, and samples were periodically collected for determination of concentrations of sugars and ethanol, and biomass. Determination of Minimum Fluidizing Velocity (Umf). Minimum fluidizing velocity (Umf) was determined by experimental data of fluid velocity and bed height, using the values of bed porosity as a function of superficial liquid velocity, according to the relationships proposed by Richardson and Zaki.23

(U /Utc) = ε n

(1)

where U = superficial velocity of the fluid; Utc = corrected terminal velocity of the particle; n = coefficient of expansion; and ε = bed porosity.

ε = (Vt − Vs)/Vt = 1 − Ms /(rs · A · H )

(3)

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RESULTS AND DISCUSSION The detoxified sugar cane bagasse hemicellulosic hydrolysate (composition described in Materials and Methods) was used as a carbon source for the yeast S. shehatae for ethanol production using FBR. FBR has high stability during the process, providing high mass transfer rates, besides being easy to operate and to control the parameters.18,21 For this bioreactor operation in a fluidized bed configuration, a balance between the drag forces and the apparent weight of particles is required.

(2)

where Vt = total volume of the reactor; Vs = bed volume of solid particles; Ms = Mass of solid particles;... rs = density of particle; A = reactor area traversed by the fluid; H = bed height Fermentation assays were performed using supplemented SBHH, in all conditions of carrier concentration, according to Table 1. The experiments were conducted in the reactor section of 23.75 cm2, without aeration. For calculation of the bed porosity (ε), the density of 1 g·mL−1 calcium alginate beads containing the immobilized cells was used.24 8252

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ACS Sustainable Chemistry & Engineering

Figure 2. Pareto chart of for ethanol yield (YP/S) and ethanol volumetric productivity (QP), according the statistical analysis of the 22 full factorial design carried out to evaluate the influence of the aeration rate (Ar) and carrier concentration (CC) in the process of ethanol 2G production from sugar cane bagasse hemicellulosic hydrolysate by using immobilized S. shehatae UFMG-HM 52.2 in fluidized bed reactor.

considering ethanol yield (YP/S) and productivity (QP) as response variables, as shown in Table 1. A range of YP/S (range of maximum and minimum yield) of 0.07 g/g was obtained; the lowest value of 0.22 g/g was calculated in assay 2 (AR of 0.11 min−1 and CC of 55.55 g·L−1), while the highest value, 0.29, was determined in assay 3 (AR of 0.027 min−1 and CC of 111.11 g·L−1). The range of volumetric productivity QP (range of maximum and minimum volumetric productivity) of 0.05 g L h was calculated, where the minimum value of 0.13 g L h was determined in assay 1 (AR of 0.027 min−1 and 55.55 g·L−1 of CC), while the maximum value of 0.18 g L h was obtained with operating parameters as described in assay 4 (AR of 0.11 min−1 and 111.11 g·L−1 g of CC). Carrier concentration and aeration rate are critical process variables in the operation of FBRs18,29 since small variations in supply and distribution of oxygen can influence directly certain parameters in bioreactors, for example, the volumetric oxygen transfer coefficient (KLa). The KLa values of 2.20, 5.44, 2.20, and 4.22 min−1 for experiments 1, 2, 3, and 4, respectively, were verified. For experiments correspondent to the center points, the average value was 4.08 min−1. Experiments at higher aeration (Exps 2 and 4) showed a higher value of KLa compared to that in other assays since the larger aeration rate provided larger contact area between the air and fluid, resulting in increased oxygen transfer in the fermentation medium.30 Molwitz et al.31 also reported the relationship between aeration rate and KLa, observing a reduction of approximately 10 times in KLa values (from 43 h−1 to 4 h−1 when aeration was 10 times reduced), in xylitol production from SBHH by using Candida guilliermondii. In our work, regarding the concentration of carrier in the reactor, this variable did not influence the oxygen transfer in the system, considering its slight influence in KLa values. Rodmui et al.29 reported that aeration is one of the main parameters to be studied in the process involving C5 sugar assimilation by yeasts since their levels may influence and drive the use of substrate in cellular metabolism. In our investigation, sugar consumption was higher than 90% in all experiments. Experiments 2 and 4 showed maximum ethanol concentration after 36 h of fermentation. On the other hand, experiments 1, 3, 5, 6, and 7 presented maximum ethanol concentration after 48 h of fermentation. Time reported to maximum ethanol concentration (Exp 1, 4.7 g·L−1; Exp 2, 6.3 g·L−1; Exp 3, 7.3 g·L−1; Exp 4, - g·L−1 g·L−1; and center points, 6.97 ± 0.58 g· L−1) was related to conditions of AR and CC. In addition to ethanol, xylitol production was detected for all experimental conditions. Polyol concentrations of 7.3, 5.2, 1.2, and 0.7 g·L−1 were achieved in assays 1, 2, 3, and 4, respectively, while the

Operation parameters are influenced by several factors such as the porosity of the particles, ratio between the diameter of the support and the column, and ratio between the density of the fluid and the solids as well as flow characteristics.17,26 Tubular (or cylindrical) reactors can be operated with beds in packed or fluidized configuration. When operating as a packed bed reactor, if the fluid velocity is increased, the drag force will also increase until it surpasses the sum of resistance forces and, then, the particles lose contact with each other and begin to move, taking a fluidized configuration. The velocity level correspondent to the starting of fluidization is known as the minimum fluidization velocity (Umf) and is a key parameter in this type of bioreactor. Umf can be defined from experimental data of bed porosity (e) as a function of superficial liquid velocity.23 Aiming to gain a preliminary understanding of the characteristics of the bed of the reactor and to ensure proper fluidization by using immobilized cells, initial data were obtained by performing experiments of bed expansion as a function of fluid velocity provided by the flow recirculation, without aeration. In this case, we have observed proper correlation of the experimental data using the Richardson and Zaki equation.23 Thus, this equation has been used taking into account its simplicity and the method indicated by Smith27 for particulate fluidization. The values for particle terminal velocity and porosity (ε), as well as Umf for the different carrier concentration were determined according to eqs 1 and 2. Thus, the minimum ascendant flow that allowed fluidization in the system was obtained as 0.146, 0.167, and 0.166 cm·s−1 for carrier (calcium alginate + yeast cells) concentration (CC) of 55.55, 83.33, and 111.11 g. L−1, respectively. Actually, CC did not influence significantly the Umf, presenting proportionality between increase of mass and bed expansion as a function of fluid velocity. Aiming for total homogenization of the FBR to obtain a stable fluidization and proper homogenization in all studied conditions, the fluidization velocity (Uf) of 2.14 cm·s−1 was used in the following experiments, with a value at least 13 times higher than Umf, selected by considering visual observation regarding complete and homogeneous fluidization in all reactor volumes. This kind of observation was also reported by Ram,28 who determined that bubble size increased linearly with distance above the distributor and excess gas velocity, eventually enabling the gross circulation of solids. Within this context, important process parameters, such as aeration rate and carrier concentration, were investigated. The effects of these variables in the proposed system were determined by using statistical design of experiments. Full 22 factorial design with triplicate at center point was carried out 8253

DOI: 10.1021/acssuschemeng.7b01916 ACS Sustainable Chem. Eng. 2017, 5, 8250−8259

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ACS Sustainable Chemistry & Engineering

Table 2. Analysis of Variance (ANOVA) for Fitted Models for the Response Variable Ethanol Yield (YP/S) and Volumetric Productivity (QP) as a Function of Studied Variables Aeration Rate (Ar) and Carrier Concentration (CC) in the Process of Ethanol 2G Production from Sugarcane Bagasse Hemicellulosic Hydrolysate by Using Immobilized S. shehatae UFMG-HM 52.2 in a Fluidized Bed Reactor YP/S variable (Ar) (ICS) (Ar) × (ICS) error total SS R-sqr = 0.85

SS 1.60 8.41 2.89 4.53 3.18

× × × × ×

10−03 10−04 10−04 10−04 10−03

df

MS

1 1 1 3 6

1.60 8.41 2.89 1.51

× × × ×

10−03 10−04 10−04 10−04

F

p

1.05 × 10+01 5.57 × 10+00 1.91 × 10+00

4.72 × 10−02a 9.93 × 10−02 2.60 × 10−01

QP variable (Ar) (ICS) (Ar) × (ICS) error total SS R-sqr = 0.99 a

SS 6.25 7.29 4.00 1.70 1.71

× × × × ×

10−04 10−04 10−04 10−05 10−03

df

MS 6.25 7.29 4.00 6.00

1

× × × ×

10−04 10−04 10−04 10−06

F

p

1.11 × 10+02 1.29 × 10+02 7.11 × 10+01

1.82 × 10−03a 1.45 × 10−03a 3.49 × 10−03a

Significant factors at 95% of confidence level.

Figure 3. Response surface (A) and the corresponding contour lines (B) for the response ethanol volumetric productivity, considering aeration rate (Ar) and carrier concentration (CC) as independent variables. Data generated while studying the ethanol 2G production from sugar cane bagasse hemicellulosic hydrolysate by using immobilized S. shehatae UFMG-HM 52.2 in a fluidized bed reactor.

assays correspondent to the center points showed 6.9 ± 0.58 g· L−1 of this polyalcohol production. Highest xylitol concentrations were obtained when using lower concentrations of carrier. Xylitol production was affected by oxygen availability.15 Thus, the importance of the fermenter operating parameter settings was highlighted. The results of the design of experiments were statistically analyzed, and Figure 2 shows the Pareto diagram, while the analysis of variance (ANOVA) is shown in Table 2, allowing the statistical evaluation of the effects of studied dependent variables, for the response answers variables YP/S and QP. At 95% of confidence level, only the variable aeration rate was significant (p < 0.05) for the response variable YP/S (Figure 2). On the other hand, for the variable response QP, both independent variables and their interaction were statistically significant (p < 0.05). For this response, a first order model (R2 0.99), including independent variables and interactions for this response, was adjusted and is shown in eq 4.

Q P = 0.129803 − 0.000232Ar − 0.000066CC + 0.000003(Ar )· (CC)

(4)

The values of the relationship ln[1 − (C/C*)] and time were plotted, and KLa values were defined as the angular coefficient of the generated fitted linear curve.32 Regarding these profiles and significant interaction between carrier concentration and aeration rate for variable QP, results were also analyzed by response and contour surfaces, presented in Figure 3. According to Figure 3, by analyzing the contour and response surface graphs, high values of QP were achieved when CC and AR were increased. Results shown in Figure 2 indicate that AR set as 0.111 min−1 and CC as 111.11 g L−1 stimulated ethanol productivity; these parameter settings enabled a decreased xylitol production most likely due to cell metabolism directed to maximum ethanol production. Unfortunately, it is difficult to maintain high fluidization conditions, as such conditions often lead to chaotic 8254

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ACS Sustainable Chemistry & Engineering fluidization as well as shifting metabolism toward cell growth instead of ethanol production. Investigations on the influence of aeration rate and concentration of carrier by using fluidized bed reactors in bioprocess have already been reported by other authors, but for xylitol production. For example, Sarrouh et al.33 evaluated xylitol production in a fluidized bed reactor from sugar cane bagasse hydrolysate by using immobilized cells of C. guilliermondii in calcium alginate matrix. In that work, maximum xylitol concentration of 28.9 g·L−1, with YP/S of 0.58 g·g−1 and QP of 0.4 g·L−1h−1, was obtained at a high aeration rate of 0.4 min−1 and 200 g·L−1 of an immobilized carrier. Authors concluded that use of high aeration rate in this system favored better oxygen transfer into the immobilized cells, maximizing xylitol production. However, adequate values of aeration rate depends on the specificity, type of immobilized carrier, microorganism, and conditions of process. Thus, for each bioprocess, studies of the effect of oxygen availability as well as mass of carrier concentration are recommended. For example, Santos et al.18 studied xylitol production from sugar cane bagasse hydrolysate using Candida guilliermondii cells immobilized on porous glass in a fluidized bed reactor (FBR), evaluating the influence of aeration rate (0.031−0.093 min−1) and carrier concentration (62.5−125 g·L−1) in the process. In that study, carrier concentration presented a negative influence on xylitol yield (YP/S) and volumetric productivity (QP), while aeration rate showed positive influence on QP and negative influence on YP/S. In conditions of aeration rate of 0.093 min−1 and carrier concentration of 62.5 g·L−1, 10.6 g·L−1 of xylitol production was found, with YP/S of 0.25 g·g−1 and QP presenting the highest value of 0.44 g·L−1h−1. Thus, for that work, by using a naturally xylitol producing yeast, authors observed that increase of the aeration rate decreased xylitol yield, verifying metabolism deviation, at high oxygen levels, for biomass production. On the other hand, QP increased with aeration rate, suggesting metabolic activity acceleration, followed by xylitol formation, regarding the use of higher carrier concentration notably affected this response. Immobilized cells of C. guilliermondii have already been used for the production of xylitol in FBR employing batch process. However, the current study offers process advantages for ethanol production in repeated batch configurations besides batch process, in turn saving time and reducing process complexities. In all assays of the present study, up to 12 h of fermentation, cells were released from calcium alginate beads and reproduced in free form in external medium. This fact was interesting for the fermentation process, once new cells were generated in the place that cells were released, maintaining the recycling of immobilized carrier inside alginate beads.15 Also, free cells in medium also convert sugars into bioproducts. The profile of free cell concentration during fermentation time is shown in Figure 4. According to Figure 4, the experiment conducted with low aeration resulted in lower values, 4.9 g·L−1 and 2.8 g·L−1 in experiments 1 and 3, respectively, of free cell concentration at 72 h of fermentation. Concomitantly, greater free cell concentration of 7.01 ± 0.7 g. L−1 was reached in the experiments of center points, while assays performed at high aeration level presented 7.4 g·L−1 and 5.8 g·L−1 in experiments 2 and 4, respectively. Thus, high oxygen supply provided high free cell reproduction.

Figure 4. Free cell concentration profile in the assays of ethanol production from sugar cane bagasse hemicellusic hydrolysate in a fluidized bed reactor by using immobilized cells of S. shehatae UFMGHM 52.2, in different conditions of process according to the 22 full factorial design with aeration rate (Ar) and carrier concentration (CC) as independent variables (Exp 1, Ar, 0.027 min−1; CC, 55.55 g·L−1; Exp 2, Ar, 0.111 min−1; CC, 55.55 g·L−1; Exp 3, Ar, 0.027 min−1; CC, 111.11 g·L−1; Exp 4, Ar, 0.111 min−1; CC, 111.11 g·L−1; Exp. 5, 6, and 7, 0.069 min−1; CC, 83.33 g·L−1).

Besides cells releasing into an external medium, cells also grew inside the beads. The immobilized cell concentrations in a bioreactor (ratio between cell mass inside the beads and total fermentation volume) in all assays were measured at 0 and 96 h of fermentation and are presented in Figure 5, while Figure 6 shows an optical photomicrograph (400×) of an immobilized bead with cells of S. shehatae UFMG-HM 52.2.

Figure 5. Immobilized cell concentration (ratio between cell mass inside the beads and total fermentation volume) at 0h and 96h, in the assays of ethanol production from sugar cane bagasse hemicellusic hydrolysate in fluidized bed reactor, by using immobilized cells of S. shehatae UFMG-HM 52.2, in different conditions of process according to the 22 full factorial design with aeration rate (Ar) and carrier concentration (CC) as independent variables (Exp 1, Ar, 0.027 min−1; CC, 55.55 g·L−1; Exp 2, Ar, 0.111 min−1; CC, 55.55 g·L−1; Exp 3, Ar, 0.027 min−1; CC, 111.11 g·L−1; Exp 4, Ar: 0.111 min−1; CC, 111.11 g· L−1; Exp. 5, 6, and 7, 0.069 min−1; CC, 83.33 g·L−1).

According to Figure 5, the yeast S. shehatae UFMG-HM 52.2 was able to multiply inside the beads in all assays. Figure 6 shows cell concentration agglomeration close to the bead border, with the possibility to release in external medium in free form. In initial fermentation time, immobilized cell concentrations in a bioreactor were approximately 0.21 and 0.43 g·L−1 in experiments 1,2 and 3,4, respectively, with assays in center points showing approximately 0.36 ± 0.021 g·L−1. After 96 h of fermentation, higher immobilized biomass concentrations (1.83 and 1.84 g·L−1) were verified in experiments 3 and 4, 8255

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Figure 7. Fermentation parameters, ethanol yield (YP/S), and volumetric productivity (QP) of second generation ethanol production from sugar cane bagasse hydrolysate using repeated batch fluidized bed reactor fermentations by immobilized S. shehatae UFMG-HM 52.2.

Figure 6. Beads of immobilized cells of S. shehatae UFMG-HM 52.2 in calcium alginate matrix.

respectively. Low concentrations of 0.84 and 0.91 g·L−1 were obtained in experiments 1 and 2 (assays conducted with 55.55 g·L−1 of CC), with center points presenting 1.36 ± 0.02 g·L−1 in the final time of fermentation. Different from free cells, those grew at high aeration, and the growth of cells inside the carrier was directed influenced by the initial carrier concentration in the bioreactor, once cells grown in experiments 3 and 4 (assays conducted with high concentration of CC) reproduced more inside beads. This behavior suggests that a high concentration of carrier could favor better nutrients and oxygen transference for cells inside beads because more solids moving inside the reactor may lead to improved medium homogenization. Actually, a higher mass of immobilized carrier probably promoted greater mixing of medium due to the movement of spheres in the fluidized bed, making better accessibility of nutrients by yeast inside beads and favoring high growth of cells inside calcium alginate system. This fact is favorable for bioprocesses, once beads can be reused with high internal concentration of cells in repeated batches fermentations. Moreover, after completing the fermentation, beads were stable, not showing disruption, size reduction, or defragmentation in all assays, indicating the viability and stability of the immobilization system. Statistical analysis was also in agreement with the experiments conducted with high carrier concentration showing higher values of ethanol yield and productivity. Thus, regarding the stability of cells, along with the possibility of recycling of cells, we have shown the process feasibility using immobilized S. shehatae UFMG-HM 52.2 in a fluidized bed reactor. After the determination of FBR parameters (aeration rate of 0.111 min−1, carrier concentration of 111.11 g, and KLa of 4.22 h−1), the performance of the system in repeated batches was evaluated for ethanol production. After 48 h of fermentation, the reactor was discharged and loaded with a fresh detoxified SBHH, and the immobilized cells in the reactor bed were used as inoculum for the next batch, in a process of repeated batch.34 The YP/S and QP values of these batches are shown in Figure 7. The first batch results showed YP/S and QP values of 0.274 g· g−1 and 0.239 g·L−1·h−1, respectively. From the second to the sixth batch, higher values of YP/S were observed (batch 2, 0.374 g·g−1; batch 3, 0.346 g·g−1; batch 4, 0.398 g·g−1; batch 5, 0.361 g·g−1; batch 6, 0.317 g·g−1), demonstrating that the system favored the ethanol yield factor. The highest YP/S value was

obtained in the fourth batch (144 h−192 h). This behavior was possibly due to the greater adaptation of the yeast to the medium as well as the increased amount of the cells in the repeated batches, as the reactor was not washed between the batch exchange, and free cells remained in the broth. The cells productivity showed a nonlinear behavior in the repeated batches (batch 2, 0.209 g·L−1·h−1, batch 3, 0.363 g·L−1·h−1, batch 4, 0.217 g·L−1·h−1, batch 5, 0.246 g·L−1·h−1; and batch 6, 0.34 g·L−1·h−1); however, in all experiments, productivity values were significant for the execution of the repeated batch mode, once QP values were higher than expected (Figure 3) according to the statistical analysis study, demonstrating that the repeated batch system also favored volumetric productivity. The values of ethanol yield and productivity obtained in repeated FBR batch are close to or even higher than the values obtained by the experiments using free cells of S. shehatae UFMG-HM 52.2 and in experiments using the immobilized yeast in simple batch. A similar behavior was also observed by Santos et al.35 in the evaluation of xylitol production from SBHH using C. guilliermondii cells immobilized on porous glass in a fluidized bed reactor. The authors had observed an increase in the process efficiency in repeated batch mode with an increase of yield and productivity values of 26% and 33%, respectively. Sarrouh and Silva36 reported the stability of the repeated batch system in a fluidized bed reactor for the production of xylitol from SBHH using C. guilliermondii cells immobilized in a calcium alginate matrix. Ding37 studied the xylitol production from corn stalk hydrolysate by immobilized Candida sp. ZU04 in repeated batch mode and reported the results of 6 consecutive batches, during 501 h, presenting YP/S of 0.73 g·g−1 and QP of 0.84 g·L−1·h−1. The repeated batch ethanol production using free and immobilized yeast cells growing on glucose-rich media is widely presented in the literature. Riansa-Ngawong et al.38 observed that in synthetic YM medium, immobilized cells of C. shehatae were able to perform 4 replicates without loss of ethanol production performance. Chen et al.39 used immobilized cells of S. cerevisiae cells in a fibrous matrix and achieved stable ethanol yield in a glucose-rich and inhibitor-free synthetic medium in 22 consecutive batches without decrease in yield. However, studies using xylose-rich media for ethanol production in repeated batch fermentation are few. 8256

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Figure 8. Sugar, ethanol, and free biomass profile during the repeated batches for second generation ethanol production from sugar cane bagasse hydrolysate by immobilized S. shehatae UFMG-HM 52.2 in a fluidized bed reactor.

time. Between the first and seventh batch, more than 90% of available sugars were consumed showing stability in ethanol production (each batch run until 48 h). This stability behavior was also confirmed earlier by Perez-Bibbins et al.40 The authors

In this work, the immobilized FBR system presented high stability during ethanol production, with process efficiency reduction only in the seventh repeated batch (288 h−336 h), probably due to cellular stress caused by extended fermentation 8257

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easy scale-up of ethanol production in bio- refineries under a process consolidation approach.

report the viability and potential of reuse of immobilized cells (entrapped on calcium alginate matrix), in fluidized bed reactors, for bioprocesses conducted for extended periods through cell reuse. The fermentation profile of all batches are presented in Figure 8. Although the seventh batch had already presented lower YP/S and QP values, one more repeated batch experiment (batch 8: 384 to 432 h) was performed to verify the values. In this experiment, it was verified that the yeast did not consume more than 90% of sugars in 48 h, besides showing a significant reduction in ethanol production. Xylitol production was observed in all batches. This behavior was expected in all other experiments using yeast S. shehatae UFMG-HM 52.2 immobilized in calcium alginate. Between the first and sixth batch, about 1 g·L−1 xylitol was observed in the fermentation medium, while in the seventh and eighth batches, this value was 2.6 g·L−1. This behavior of the yeast with higher accumulation of xylitol in experiments of high reactor fermentation times is due to oxygen limitations and cellular stress. In all batches, after 48 h of fermentation time, growth in free cells after the initial two batches showed a continuous slow down (fermentation batch 1, 5.2 g·L−1; batch 2, 4.8 g·L−1; batch 3, 4.8 g·L−1; batch 4, 3.9 g·L−1; batch 5, 3.2 g·L−1; batch 6, 3.3 g·L−1; batch 7, 2.1 g·L−1; and batch 8, 3.0 g·L−1). It is noteworthy that unlike the first batch, between the second and the eighth recycle, the initial cell charge consisted of the mass of immobilized cells and free cells present on the walls of the reactor since the experiment was performed only with substrate exchange, without washing. In the beginning of the second to the seventh batch, the initial concentrations of free cells were 0.05 g·L−1; 0.5 g·L−1; 1.12 g·L−1; 0.58 g·L−1; 0.8 g·L−1; and 1.45 g·L−1, respectively. In this way, the inoculum in repeated batches was higher than that in the first batch, in view of the internal cell growth inside the pellets, adding the remaining free cells in the previous batch. The concentration of immobilized cells in the reactor also increased during the subsequent fermentation batches; in the beginning of the process, it was 0.41 g·L−1, and after 384 h of fermentation, it was observed to be 1.51 g·L−1 of cells. Increased number of cells after every consecutive batch enabled higher values of YP/S and QP. During the fermentation time, there was no deformity observed in spheres containing yeast cells. The stability of the experiments, combined with the easy use of the technique and the reactor configuration, as well as the promising yeast, demonstrated the potential of the system for 2G ethanol production in consecutive batches.

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AUTHOR INFORMATION

Corresponding Authors

*(F.A.F.A.) Phone: +55 12 31595146. E-mail: [email protected]. *(S.S.S.) Phone: +55 12 31595308. E-mail: silviosilverio@ gmail.com. ORCID

Silvio Silvério da Silva: 0000-0003-0669-2784 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Research Council for the State of São Paulo (FAPESP) (Award Number 2014/27055-2), Brazilian National Council for Scientific and Technological Development (CNPq), and Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES) for financial support. Authors are thankful to Prof. Dr. Carlos A. Rosa from University of Minas Gerais - MG - Brazil for the donation of the strain Scheffersomyces shehatae UFMG-HM 52.2.

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REFERENCES

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CONCLUSION Second generation ethanol production from SBHH in FBR by using S. shehatae UFMG-HM 52.2 immobilized in calcium alginate matrix was studied. Through chemical and statistical analyses, important conditions for this process were determined, verifying that the use of aeration rate and carrier concentration of 0.111 min−1 and 111.11 g·L−1, respectively, enhanced ethanol productivity. Using these parameters, an ethanol yield of 0.265 g. g−1 and volumetric ethanol productivity of 0.177 g. L−1·h−1 were obtained. Repeated batch fermentations in FBR also showed the stable ethanol yield until 6 consecutive batches. The bioconversion of sugar cane bagasse hemicellulosic hydrolysate into ethanol employing FBR as shown in the present work is a promising system for 2G ethanol production eventually demonstrating the possibilities to 8258

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