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Continuous System of Combined Columns of Ion Exchange Resins and Activated Charcoal as a New Approach for the Removal of Toxics from Sugar Cane Bagasse Hemicellulosic Hydrolysate Júlio C. Santos,*,† José M. Marton,‡ and Maria G. A. Felipe† †

Departamento de Biotecnologia, Universidade de São Paulo−Escola de Engenharia de Lorena, Estrada Municipal do Campinho sn 12602-810, Lorena, São Paulo, Brazil ‡ Agência Ambiental de São José dos Campos, CETESB−Companhia Ambiental do Estado de São Paulo, Av. Olivo Gomes no. 100 12211-115, São José dos Campos, São Paulo, Brazil ABSTRACT: A continuous system of combined columns of ion exchange resins and activated charcoal was proposed as a new approach for the removal of toxics from sugar cane bagasse hemicellulosic hydrolysate. A factorial design was carried out to evaluate the influence of temperature and feed flow rate in the performance of the detoxification procedure. By using a temperature of 30 °C and a flow rate of 2.5 VB/h, the total removal of furfural and 5-(hydroxymethyl) furfural was observed, as well as a large reduction in the color and concentration of phenolic compounds. Removal of metals was also observed, with 50, 63, and 23% reduction in the concentration of copper, chromium, and nickel, respectively. The product of the detoxification process was a hydrolysate with a reduced concentration of inhibitors that can be used as a raw material for a number of processes to obtain products of economic and social interest.

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ammonium hydroxide treatment. In another work, Okuda et al.15 investigated the detoxification of hydrolysate of waste house wood by the bacterium Ureibacillus thermosphaericus. In that work, when the hydrolysate was biologically detoxified, the ethanol production rate was comparable to that for a hydrolysate treated by overliming. As observed, even using new techniques of detoxification, traditional methods were utilized to generate comparison data. Additionally, traditional techniques could be enhanced by using new designs or alternative possibilities. Among the possibilities to detoxify hydrolysates, the use of activated charcoal and ion exchange resins is an interesting approach. Activated charcoal is a well-established detoxification method for sugar cane bagasse hydrolysates.16 It is a cheap material with a large interfacial area to adsorb a quantity of toxic compounds. A drawback to the utilization of activated charcoal is the fact that it is difficult to reuse, especially in its powdered form. Thus, the use of columns with the granular form of activated charcoal could be advantageous. Ion exchange resins possess the capacity to remove organic and inorganic compounds, an advantage over other detoxification methods. Moreover, other advantages of ion exchange resins include the possibility of regeneration and reuse.13 Aiming to detoxify the hemicellulosic hydrolysate utilized in fermentation processes, Rao et al.18 used a combination of activated charcoal with ion exchange resins for the detoxification of corn fiber and sugar cane bagasse hydrolysates to produce xylitol in a process using the yeast Candida tropicalis. However, in that study, the activated charcoal adsorption was a batch pathway in the process. Considering that a continuous process

INTRODUCTION The use of nonrenewable resources to meet the global demand for energy and chemicals has a number of problems, mainly related to the political instability in oil-producing countries and environmental issues, such as global warming, which is related to CO2 emissions from fuels produced from those sources.1,2 In this context, the wide range of products which can be produced from lignocellulosics and the interest in its exploitation as an alternative to fossil carbon sources2,3 indicate their use as raw material in a biorefinery is suitable.4,5 Considering the biorefinery concept, hemicellulose is a fraction of lignocellulosics that merits research attention.6 Hemicellulose is a complex macromolecule7 and represents, in general, 15−35% of plant biomass. Some possible applications of hemicellulose include its hydrolysis and subsequent use in biotechnological processes to produce ethanol6 or other interesting products, such as xylitol, 2,3-butanediol, organic acids, ferulic acid, vanillin, and others.8,9 However, fermentation of hemicellulosic hydrolysates is limited by the release or generation of inhibitor compounds of microbial metabolism during the process of hydrolysis,10,11 such as sugar-derived byproducts (furfural and hydroxymethylfurfural), acetic and levulinic acids, aromatic and polyaromatic compounds from lignin degradation and metals, or others.12,13 Thus, detoxification methods that aim to reduce the concentration of these inhibitors to acceptable levels have been evaluated; these methods include physical, chemical, or biological techniques or a combination of techniques, e.g.: membrane extraction,14 microorganisms,15 and activated charcoal or vegetal polymers.16,17 In the work of Grzenia et al.,14 for instance, microporous hollow fiber membranes were used to remove toxic compounds from hydrolysate of corn stover to produce ethanol. Yields obtained using detoxified hydrolysates were approximately 10% higher than those from hydrolysates detoxified utilizing an © 2014 American Chemical Society

Received: Revised: Accepted: Published: 16494

July 7, 2014 September 23, 2014 September 26, 2014 September 26, 2014 dx.doi.org/10.1021/ie502712j | Ind. Eng. Chem. Res. 2014, 53, 16494−16501

Industrial & Engineering Chemistry Research

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by Hipperquı ́mica Comercial Cientı ́fica Ltda. (Santo André/ SP, Brazil). Before each experiment, a new sample of activated charcoal was washed extensively with distilled water and used to fill the column. The manufacturer’s instructions for preparation of the ion exchange resins were followed during the procedures. Before the first use of each resin, they were kept immersed in distilled water for 24 h. After that, the following sequence was always carried out throughout the experiments for each run of the experimental design: 1. Regeneration procedure: a total of 2.5 VB of a descendent flow of regenerating solution was passed through the column beds at a flow rate of 1 VB/h. The regenerating solution was a 10% NaCl solution for the anionic A-860S and A-500PS resins and a 6% HCl solution for the cationic C-150 resin. 2. Washing 1: a total of 2.5 VB of a descendent flow of deionized water was passed through the column beds at a flow rate of 1 VB/h. 3. Detoxification: concentrated hydrolysate was passed through the column beds according to each experimental design condition. 4. Washing 2: a total of 2.5 VB of a descendent flow of deionized water was passed through the column beds at a flow rate of 1 VB/h. Although it would be possible to work with all of the columns connected, in the experiments, each one was percolated in separate, collecting its effluent hydrolysate and using it in the next pathway of the detoxification procedure. The total volume of concentrated hydrolysate used for the first column in the system set up was 1400 mL. For each column, the first 150 mL of solution that passed through the bed was always dismissed due to the dilution with the water used in the washing procedure explained above. The remaining treated hydrolysate was then used in the next column of the set up. The total volume used in each bed of the system was below the maximum capacity of the bed, as determined in previous tests in our lab (data not shown). Analytical Methods. The water content of the sugar cane bagasse was measured in an infrared moisture balance ID 50 manufactured by Marte Balanças e Aparelhos de Precisão Ltd.a (Santa Rita do Sapucaı ́/MG, Brazil). Xylose, glucose, arabinose, and acetic acid concentrations were measured using an HPLC provided with a Bio-Rad HPX87H (300 × 7.8 mm) column and a refractive index (RI) detector at 45 °C. H2SO4 (0.01 N) was used as the mobile phase at a flow rate of 0.6 mL/min, and the injection volume was 20 μL. Concentrations of furfural and 5-(hydroxymethyl) furfural (5-HMF) were determined with a Hewlett−Packard RP18 column at 25 °C with a UV−VIS detector (SPD-10A UV−VIS) and eluted with a solution of acetonitrile:water (1:8) and 1% acetic acid at a flow rate of 0.8 mL/min. Total phenolic compounds concentration was estimated via spectrophotometry using a Beckman DU 640B Spectrophotometer (Beckman Coulter, Inc., Santana de Parnaiba/SP, Brazil) in a procedure that utilized Folin and Ciocalteu reagents and a method that was described by Singleton et al.22 The color of the hydrolysates was evaluated with an absorbance measurement at 420 nm in a Beckman DU 640B Spectrophotometer. Before the absorbance measurements, samples were diluted by a factor of 10 and the pH was

is an interesting approach better suited for industrial applications and that sugar cane bagasse is an important raw material abundant in some countries such as Brazil, the present work reports a study that was conducted for a detoxification procedure of sugar cane bagasse hemicellulosic hydrolysate that combined columns of granular activated charcoal and ion exchange resins. The hydrolysate was characterized in relation to the presence of potential inhibitors before and after detoxification, and experimental design was used as a tool to evaluate the process.

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MATERIALS AND METHODS Hemicellulosic Hydrolysate Preparation. Sugar cane ́ bagasse was obtained from Usina Guarani S. A. (Olimpia, SP, Brazil), and it was previously dried in sunlight until it reached approximately 10−15% of water content before its use in the experiments. A diluted acid hydrolysis of sugar cane with sulfuric acid was conducted in a jacketed 250 L AISI316 stainless steel reactor located in the Chemical Engineering Department of the Engineering School of the LorenaUniversity of São Paulo (Lorena, SP, Brazil). The conditions for the hydrolysis were the same as those established by Pessoa et al.19 and are summarized as follows: temperature of 121 °C, 10 min, 100 mg of H2SO4 per gram of dry mass of bagasse, and a solid:liquid ratio of 1:10. The obtained hydrolysate was vacuum filtered using qualitative filter paper and kept at a temperature of 4 °C until its use. Hydrolysate was vacuum concentrated until a xylose concentration of approximately 65 g/L in a 4 L AISI316 stainless steel evaporator at 43 °C/70 mbar, as described by Rodrigues et al.20 Samples were taken for characterization before and after the concentration procedure. Detoxification Procedure. First, the pH of the concentrated hydrolysate was adjusted to 7.0 using NaOH, and the solution was kept at 4 °C for 24 h and then centrifuged at 2000 g for 30 min to remove the formed precipitate. Next, the liquid was sequentially passed through a series of columns containing activated charcoal and ion exchange resins. Anionic ion exchange resins were of strong base A-860S and A-500PS, both type 1 in their Cl− form, and cationic ion exchange resin was C-150 in its H+ form, all of them manufactured by Purolite do Brasil Ltda. (São Paulo, SP, Brazil). Granular activated charcoal was Alfa LU 10 × 30 obtained from Brasilac (Brazil). Similarly to the study by Rao et al.,18 the activated charcoal column was used before all of the ion exchange resins, and the sequence of the ion exchange resins was based on the study by Viñals21 and proceeded in the following order: (i) anionic A-860S (ii) anionic A-500PS and cationic C-150. A 22 full factorial design with triplicate runs at the center point was used as a tool to evaluate the influence of the variables temperature and feed flow rate in the columns in the detoxification procedure. The statistical analysis of the obtained data was performed using STATISTICA for Windows 5.1 (StatSoft, Inc., Tulsa, OK, USA). Jacketed columns with an internal diameter of 3.8 cm and a height of 60 cm were filled with a bulk volume of 400 mL of ion-exchange resin or activated charcoal (VB). The feed flow rate was descendent and obtained by using a metering pump Gamma/L manufactured by Prominent (Heidelberg, Germany). Temperature in the columns was maintained by recirculating water from a thermostatic bath SP-281 Sppencer manufactured 16495

dx.doi.org/10.1021/ie502712j | Ind. Eng. Chem. Res. 2014, 53, 16494−16501


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adjusted to 10 with pellets of NaOH p.a. and filtered in cellulose ester through a 0.45 μm membrane using a Swinnex Millipore filter holder (Millipore Industria e Comercio Ltd.a, São Paulo, Brazil). Soluble solids concentration (SSC, °Brix) was determined using a refractometer Eloptron Schmidt + Haensch (SCHMIDT + HAENSCH GmbH & Co., Berlin, Germany). pH measurements were determined using a B-474 Micronal pH meter. Metal concentrations in the hydrolysate were analyzed via atomic absorption spectroscopy as described elsewhere.23

sugars increased following this ratio, whereas this behavior was not observed for the other compounds. The ratios between the concentrations after and before this procedure were approximately 2.7, 1.5, 0.2, and 3.5 for the phenolic compounds, acetic acid, furfural, and 5-HMF, respectively. These ratios can be attributed to the volatility of these compounds in the concentration conditions, and in the case of the acetic acid, its removal was favored with the low pH value of the hydrolysate. Actually, in low pH values, acetic acid is in its noncharged form and thus the intermolecular forces of attraction are comparatively weak.20 About the metals, some of them could precipitate during the concentration procedure and others could be solubilized, increasing or decreasing the concentration in different ways. Some metals, however, saw the concentration increase more than expected. This can be explained by the liberation of some of the metals from the solid raw material particles present in the liquid (magnesium and manganese, e.g.)26 or from the stainless steel equipment used in the concentration procedure (as chromium, nickel, and manganese). With relation to the color, the values of absorbance at 420 nm increased in this step (Table 1). The color of the hydrolysate is related to the presence of furfural and 5-HMF, as well as by lignin derivatives, like phenolic compounds. Before passing the hydrolysate through the columns containing the ion exchange resins and activated charcoal, the pH was increased to 7.0 using NaOH. This step was necessary considering that in preliminary experiments in our laboratory, a high quantity of precipitate was generated in the columns if a hydrolysate with a lower pH value was directly introduced into the columns. This precipitate can quickly clog the column bed, impairing the detoxification procedure. Table 2 shows the concentration of the sugars, acetic acid, phenolic compounds, furfural, and 5-HMF measured after the

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RESULTS AND DISCUSSION The sugar cane hemicellulosic hydrolysate was first obtained and vacuum concentrated, as described in the previous section. Table 1 shows the results of the hydrolysate characterization before and after the concentration procedure. Table 1. Sugarcane Bagasse Hemicellulosic Hydrolysate Characterization before and after the Concentration Procedure value characteristic pH D-xylose

sugars (g/L)

toxic compounds (g/L)

L-arabinose D-glucose phenols acetic acid

furfural 5-HMF SSC (°BRIX)

metals (mg/L)

color

sodium (Na) potassium (K) magnesium (Mg) calcium (Ca) chromium (Cr) manganese (Mn) iron (Fe) nickel (Ni) copper (Cu) zinc (Zn) absorbance (420 nm)

original hydrolysate

concentrated hydrolysate

1.10 17.02 1.03 0.84 4.25 1.83

0.70 64.50 4.35 3.31 11.34 2.76

0.120 0.006 3.50 67.00 131.60 27.50 42.80 66.85 6.50 832.00 43.12 0.52 2.50 0.1875

0.021 0.021 14.30 243.81 310.00 132.50 113.60 418.48 29.70 3195.00 241.47 1.50 10.50 0.4256

Table 2. Concentrations of the Sugars, Acetic Acid, Phenolic Compounds, Furfural, and 5-HMF after the Neutralization of the Hydrolysate with NaOH compound D-xylose L-arabinose D-glucose

phenols acetic acid furfural 5-HMF

concentration (g/L)a 57.89 ± 1.77 4.15 ± 0.07 3.19 ± 0.09 0.21 ± 0.04 2.70 ± 0.04 0.012 ± 0.001 0.010 ± 0.001

removal (%)a,b 10.25 4.60 3.63 98.15 2.17 42.86 52.38

± ± ± ± ± ± ±

2.74 1.63 2.85 0.32 1.47 5.09 3.37

a

As observed in Table 1, xylose was the predominant sugar in the obtained hydrolysate, with low concentrations of arabinose and D-glucose. This composition is in accordance with the related by other authors for diluted acid hydrolysis of hemicellulose from sugar cane bagasse,24,25 especially when natural variation due to different sources of raw material from different region, climate, soil, etc., is taken into account. In addition to sugars, compounds normally identified as toxic for microorganisms in the fermentation process were obtained, and in the present work, the phenols had the highest concentration (4.25 g/L), followed by acetic acid (1.83 g/L). Among metals, iron had the highest concentration (832 mg/L) in the original hydrolysate. The concentration procedure was carried out using a ratio of 4 between the initial and final volumes of the hydrolysate used in this step. As shown in Table 1, the concentrations of the

Data correspond to an average of all neutralization procedures carried out in this work in the hydrolysate before its detoxification in the combined activated charcoal/ion exchange columns system (average ± standard deviation). bWith relation to the concentrated hydrolysate.

neutralization of the concentrated hydrolysate with NaOH. This evaluation was carried out before the experimental design with the aim of evaluating the result of neutralization on the medium composition. The data showed that the hydrolysate neutralization could act as an initial detoxification step itself. As shown in Table 2, there was a removal of some toxic compounds after the use of NaOH to increase the pH value. The analysis of the neutralization results shows a reduction of 10% in the concentration of the xylose, with lower reduction for the other sugars (Table 2). However, this step was efficient 16496



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Table 3. Matrix of the 22 Full Factorial Design Used for the Evaluation of the Influence of the Temperature and Feed Flow Rate in the Percent Removal of D-Xylose, Phenolic Compounds, Acetic Acid, and Color from the Hydrolysate Detoxified through the Combined Treatment of Activated Charcoal and Ion Exchange Resinsa levels of the studied variables (coded values in parentheses) runs

temperature (X1), °C

1 2 3 4 5 6 7

25 (−1) 35 (+1) 25 (−1) 35 (+1) 30 (0) 30 (0) 30 (0)

feed flow rate (X2), VB/hc 1.5 1.5 3.5 3.5 2.5 2.5 2.5

(−1) (−1) (+1) (+1) (0) (0) (0)

response variablesb removal of D-xylose, %

removal of phenolic compounds, %

removal of acetic acid, %

removal of color−absorbance 420 nm

37.64 18.26 27.26 24.50 20.03 23.73 22.02

94.17 92.24 95.27 87.01 92.04 94.29 94.35

87.04 52.59 58.52 68.52 47.48 53.33 47.41

92.78 92.55 87.80 81.75 97.26 97.52 98.39

Percent removal is in relation to the concentrated hydrolysate concentration. bData measured in the outlet flow of the cationic C-150 column. VB is the bulk volume of the ion-exchange resin or activated charcoal bed into the column.

a c

negative, indicating that the minimization of the loss of xylose can be reached with higher temperatures. The effect of temperature was also significant and negative, at least at a 90% confidence level, for all response variables evaluated, indicating that the removal of inhibitors or compounds that give color to the hydrolysate is also higher at higher temperatures. The effect of temperature can be related to the interaction phenomena among the different compounds and each component of the combined activated charcoal/ion exchange resins system. Temperature is an indicator for the adsorptive nature of the system, whether it is an exothermic or endothermic process.30,31 If the compound removal increases with increasing temperature, then the adsorption is an endothermic process.30 For the feed flow rate, the use of higher values could be useful to increase the process productivity, but this is limited considering the residence time of the particles in each column in the system. The feed flow rate can also interact with temperature, considering that particle mobility between the liquid and solid phases in the columns is related to the temperature and fluid velocity. The interaction between temperature and feed flow rate was also significant for the removal of xylose, acetic acid and color (p < 0.05), indicating that these variables have to be studied together in the evaluated range. For this last response variable, the main effect of feed flow rate was also significant (p < 0.05) and negative. The interaction between the studied variables could also be considered significant for the removal of phenolic compounds, although with a lower significance level (p < 0.15). To analyze the results considering both the temperature and feed flow rate, a modeling work was carried out to determine the responses to changes in these variables. Table 5 shows the analysis of variance for the linear models with an interaction term between the studied variables. The models for the removal of acetic acid and color were not adequate to fit the data with a high percentage of explained variation, considering that the R2 values were low for both responses (Table 5). On the other hand, the R2 values were ≥0.80 for the removal of xylose and the removal of phenolic compounds. These two response variables can be more important in a fermentative process in comparison with the removal of acetic acid or color. Acetic acid can be consumed by some microorganisms during the fermentation,32 and the inhibitory effect is related to the pH of the medium.33 For xylitol production using Candida guilliermondii, for instance,

in reducing the concentration of some of the inhibitors of microbial metabolism, particularly the phenolic compounds as seen by a decrease in the concentration of more than 98%. For furfural and 5-HMF, the decrease in concentration was 43% and 52%, respectively. The visual observation of the hydrolysate after neutralization showed that it had become darker, with an absorbance value (at 420 nm) of 0.7802 ± 0.0076 (average of all neutralization procedures carried out in this work ± standard deviation). This browning of the hydrolysate can be explained by the oxidation of the phenolic compounds during the hydrolysate neutralization, which results in brown-colored pigments.27 The neutralized hydrolysate was then treated with columns of activated charcoal and ion exchange resins. Experimental runs were carried out according to a 22 full factorial design with a triplicate at the center point, as shown in Table 3. For the response variables, only the removal of D-xylose was considered and, among the inhibitors, only phenolic compounds and acetic acid concentrations were considered, but furfural and 5-HMF were not considered because these compounds were totally removed from the hydrolysate in the first column of the system that was filled with activated charcoal. This is interesting because research into furfural and 5-HMF has revealed that the cell growth inhibition is a function of their concentrations in the medium.28 Thus, for the efficient utilization of hemicellulose hydrolysates, measures have to be taken to adapt the organism to the inhibitors or to remove the compounds to a noninhibitory level.29 In Table 3, all results corresponded with the data obtained after passing the hydrolysate through the complete combined system of activated charcoal/ion exchange resins. The compound removal data for each used column demonstrated a similar behavior in all of the experiments and this will be discussed below for chosen conditions. As shown, a large removal of the phenolic compounds and the color was observed in all experiments−normally a removal of greater than 90% was obtained in almost all of the runs. In the case of acetic acid, the use of lower levels of the evaluated variables resulted in a removal of approximately 90%. On the other hand, the undesirable removal of the sugar of interest, xylose, varied from 18 to 38% in these runs. Table 4 shows the analysis of the significance of the effects of temperature and feed flow rate in the evaluated responses. As can be observed, for the removal of xylose, the main effect of the change in the temperature was significant (p < 0.05) and 16497


0.70 1.85 1.85 1.85

standard error

t

35.4009 −5.9781 −1.1179 4.4876

effect 92.77 −5.10 −2.07 −3.16

p 0.0008c 0.0269c 0.3799 0.0462c 0.50 1.32 1.32 1.32

standard error

t 186.4044 −3.8695 −1.5683 −2.4037

p 0.0000c 0.0608d 0.2573 0.1381

effect 59.2700 −12.2250 −6.2950 22.2250

1.284280 3.397887 3.397887 3.397887

standard error 46.15036 −3.59782 −1.85262 6.54083

t

removal of acetic acid, %

Coded levels of the temperature. bCoded levels of feed flow rate. cSignificant at 95% confidence level. dSignificant at 90% confidence level.

24.78 −11.07 −2.07 8.31

mean X1a X2b X1X2

removal of phenolic compounds, % p 0.0005c 0.0693d 0.2051 0.0226c 92.58 −3.14 −7.89 −2.91

effect 0.22 0.59 0.59 0.59

standard error

413.8860 −5.3058 −13.3321 −4.9172

t

p 0.0000c 0.0337c 0.0056c 0.0390c


16498

a

122.54 4.28 69.06 42.66 6.86 245.40 0.80

1 1 1 1 2 6

degrees of freedom

122.54 4.28 69.06 42.66 3.43

mean square

removal of D-xylose, %

35.7374 1.2496 20.1386 12.4401

F c

0.0269 0.3799 0.0462c 0.0718d

p 25.96 4.26 10.02 3.30 3.47 47.01 0.86

sum of square 1 1 1 1 2 6

degrees of freedom 25.96 4.26 10.02 3.30 1.73

mean square 14.9732 2.4596 5.7779 1.9036

F

removal of phenolic compounds, %

d

0.0608 0.2573 0.1381 0.3017

p

149.45 39.63 493.95 510.75 23.09 1216.87 0.56

sum of square

1 1 1 1 2 6

149.45 39.63 493.95 510.75 11.55

mean square

12.9443 3.4322 42.7825 44.2373

F

removal of acetic acid, % degrees of freedom

Coded levels of the temperature. bCoded levels of feed flow rate. cSignificant at 95% confidence level. dSignificant at 90% confidence level.

X1 X2b X1X2 lack of fit pure error total R2

a

source

sum of square

d

0.0693 0.2051 0.0226c 0.0219c

p

9.86 62.25 8.47 138.96 0.70 220.24 0.37

sum of square

1 1 1 1 2 6

degrees of freedom

9.86 62.25 8.47 138.96 0.35

mean square

28.1515 177.7446 24.1785 396.7641

F


0.0337c 0.0056c 0.0390c 0.0025c

p

Table 5. Analysis of Variance (ANOVA) and Coefficient of Determination (R2) for the Fitted Models for the Removal of D-Xylose, Phenolic Compounds, Acetic Acid, and Color from the Hydrolysate Detoxified by the Combined Treatment with the Activated Charcoal and Ion Exchange Resins as a Function of the Studied Variables

a

effect

factor

removal of D-xylose, %

Table 4. Estimates of the Effects of the Studied Variables, Standard Error e Values of “t” and “p” for the Removal of D-Xylose, Phenolic Compounds, Acetic Acid, and Color from the Hydrolysate Detoxified by the Combined Treatment with Activated Charcoal and Ion Exchange Resins

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Figure 1. Response surface for the removal of xylose (a) and phenolic compounds (b) in the combined system as a function of the temperature and feed flow rate.

inhibition of fermentation is observed only for concentrations higher than 3−5 g/L, depending on the medium used.34,35 Color is indicative of the presence of some compounds, and although this parameter could be very important for some possible applications of the hydrolysate, it is not necessarily related to process inhibition. Moreover, Table 4 shows similar signs of the effects of temperature and feed flow rate for the removal of color and phenolic compounds, indicating that similar analyses could be considered for these two response variables. In relation to the other responses, the importance of reducing the removal of the substrate xylose is obvious, and the necessity of removing phenolic compounds is important considering that phenolic compounds have been suggested to exert a considerable inhibition in the fermentation process due to their ability to affect cell membranes that serve as selective barriers and enzyme matrices.36 The reduction of the negative effect of phenolic compounds in fermentative processes was verified when phenolic acid monomers were significantly removed from the hydrolyzate.12 Thus, the models for the removal of xylose and the phenolic compounds were used to evaluate the process by tracing the response surfaces shown in Figure 1. Although the model for xylose removal has shown a lack of fit that could be considered significant at the 90% confidence level, it was used to trace the surface (Figure 1a) and to facilitate the understanding about the combined effect of the behavior of the temperature and the feed flow rate. As observed, the minimization of the removal of xylose could be reached at higher temperatures and lower feed flow rates, but the opposite was observed for the desirable maximization of the removal of phenolic compounds. Therefore, a process of optimization should be carried out that consider other aspects, with an example being the economic impact for an integrated biorefinery. However, these aspects are not included in the objectives of the present work and thus the potential of the system of the combined activated charcoal/ion exchange columns to detoxify the hydrolysate was evaluated in an intermediary condition using temperature and feed flow rate values of the centerpoint of the experimental design. At the chosen conditions, a deeper analysis was carried out that focused on the concentrations of the sugars and the inhibitors. The results are shown in Table 6. Figure 2 shows the removal of the sugars and the inhibitors, as well as of the color in each column of the combined system.

Table 6. Sugarcane Bagasse Hemicellulosic Hydrolysate Characterization after the Detoxification Procedure in the System of the Combined Activated Charcoal and Ion Exchange Resins characteristic

value 1.20

pH D-xylose

sugars (g/L)

L-arabinose D-glucose

toxic compounds (g/L)

50.31 2.84 2.75

phenols acetic acid furfural 5-HMF

0.02 1.42 nd nd 12.18

sodium (Na) potassium (K) magnesium (Mg) calcium (Ca) chromium (Cr) manganese (Mn) iron (Fe) nickel (Ni) copper (Cu) zinc (Zn) absorbance (420 nm)

456.98 88.25 45.00 167.60 156.70 1.40 84.56 186.38 0.75 0.70 0.0593

SSC (°BRIX)

metals (mg/L)

color

As can be observed in Table 6 and Figure 2, a complete removal of the total furfural and 5-(hydroxymethyl) furfural was observed in the activated charcoal column. Removal of xylose at the chosen conditions was 22% for the entire system, with the largest removal occurring in the activated charcoal column. In this column, the removal of L-arabinose and D-glucose was also high. The adsorption of compounds by the activated charcoal is related to van der Waals forces, and it can act on all molecules of the hydrolysate, including either dissociated or nondissociated forms. For sugars, the adsorption by the activated charcoal was also observed in other studies.13,37 The removal of sugars also occurred in the ion exchange columns (Figure 2), mainly at the ionic A-860S and A-500PS resins. In relation to the phenolic compound concentrations, 16499



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Figure 2. Removal of sugars, acetic acid, phenolic compounds, color, furfural, and 5-HMF in the neutralization step and in each column of the combined system. The removal in a step is in relation to the previous one. Total removal is in relation to the concentrated hydrolysate.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo− FAPESP (2002/13348-0), CNPq, and CAPES.

the column containing the anionic A-500PS resin resulted in the highest removal (Figure 2). The largest removal of acetic acid in relation to the concentrated hydrolysate was also observed in the column containing the anionic A-500PS. In this case, the cationic resin was not very effective. This behavior was similar to the one observed for the anionic resin by Nilvebrant et al.38 These authors, working with anionic and cationic ion exchange resins for the detoxification of Chipped Norway Spruce (Picea abies) hydrolysate, have observed that the anionic resins demonstrated a good performance in the detoxification, especially with the removal of phenolic compounds, furan aldehydes, and aliphatic acids and that the negatively charged groups of the cation exchanger resulted in a resin providing only a poor detoxification effect. Table 6 shows the removal of metals with the system. When comparing the metal concentrations in this table with the metal concentrations in the concentrated hydrolysate (Table 1), the iron concentration possessed the highest decrease in concentration, but all metals showed a reduction from the hydrolysate after the proposed treatment, especially the metals reported as toxic for the microbial metabolism by Nies39 such as copper, chromium, and nickel, which were removed by 50, 63, and 23%, respectively.

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CONCLUSIONS

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AUTHOR INFORMATION

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REFERENCES

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The conditions for a continuous detoxification procedure that is able to be scaled up were determined. For the chosen conditions (30 °C and 2.5 VB/h), the combined system of the activated charcoal and ion exchange resins was adequate to decrease the concentration of known inhibitors of microbial metabolism in the hemicellulosic hydrolysate, such as phenolic compounds and some metals. The color of the hydrolysate could be important for some applications (like downstream steps) and was also reduced by the procedure. The obtained product was a hemicellulosic hydrolysate with reduced concentrations of inhibitors, which could be used as raw material for a number of fermentations useful for biorefineries.

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