The influence of pH, temperature and hydrolyzate concentration on the removal of volatile and nonvolatile compounds from sugarcane bagasse hemicellulosic hydrolyzate treated with activated charcoal before or after vacuum evaporation

THE INFLUENCE OF pH, TEMPERATURE AND HYDROLYZATE CONCENTRATION ON THE REMOVAL OF VOLATILE AND NONVOLATILE COMPOUNDS FROM SUGARCANE BAGASSE HEMICELLULOSIC HYDROLYZATE TREATED WITH ACTIVATED CHARCOAL BEFORE OR AFTER VACUUM EVAPORATION

R.C.L.B.Rodrigues¹, M.G.A.Felipe1^*, J.B.Almeida e Silva¹, M.Vitolo² and P.V.Gómez³

¹Faculdade de Engenharia Química de Lorena (FAENQUIL) Departamento de Biotecnologia,

Cx. P. 116,12600-000, Phone [phone], Fax: [phone],Lorena, SP - Brazil.

E-mail: [e-mail]

²Universidade de São Paulo (USP) São Paulo - SP, Brazil

³Instituto Cubano de Investigaciones de los Derivados de la Caña de Azúcar, Habana, Cuba.

(Received: July 10, 2000 ; Accepted: July 11, 2001)

Abstract - This paper analyzes the influence of pH, temperature and degree of hydrolyzate concentration on the removal of volatile and nonvolatile compounds from sugarcane bagasse hemicellulosic hydrolyzate treated with activated charcoal before or after the vacuum evaporation process. Furfural and 5-Hydroxymethylfurfural were almost totally removed in all the experiments, irrespective of pH and temperature and whether the charcoal was added before or after the vacuum evaporation process. Adding activated charcoal before the vacuum evaporation process favored the removal of phenolic compounds for all values of pH. Acetic acid, on the contrary, was most effectively removed when the activated charcoal was added after the vacuum evaporation process at an acid pH (0.92) and at the highest degree of hydrolyzate concentration (f=4). However, addition of activated charcoal before or after vacuum evaporation at an acid pH (0.92) and at the highest degree of hydrolyzate concentration (f=4) favored the removal of both acetic acid and phenolic compounds.

Key words: Sugarcane bagasse, hemicellulosic hydrolyzate, vacuum evaporation, activated charcoal, acetic acid, phenolic compounds

INTRODUCTION

Sugarcane bagasse is a lignocellulosic material and an abundant agricultural residue. This renewable source of cellulose and hemicellulose can potentially serve as a substrate for biotechnological processes. Hydrolysis is required for the bioconversion of this polymeric material, which according to D’arce et al. (1985), is 44 to 48.9 % cellulose, 24 to 28% hemicellulose and 10 to 14% lignin. Cellulose is a homogeneous glucose polymer and once the cellulose is hydrolyzed, the glucose residues can be readily fermented (Vogel-Lohmeier et al., 1998). Hemicellulose, on the other hand, is a heterogeneous pentose (D-xylose and L-arabinose), hexose (D-glucose, D-mannose and D-galactose) and acid (acetic and glucuronic) polymer. The predominant sugar in hemicellulose is aldopentose xylose, which may comprise 20-40% of the total carbohydrates found in the agricultural residues (Bicho et al., 1988). Lignin is a polyphenolic macromolecule whose chemical structure is not yet completely known (Tsao, 1986).

Sugarcane bagasse can be hydrolyzed with dilute sulfuric acid, which is a simple and fast method (Pessoa Jr. 1997). The principal drawback in any chemical process employing lignocellulosic materials is that it is not possible, either technically or on a laboratory scale, to separate these three components (cellulose, hemicellulose and lignin) without changing their chemical structures. It is not only the chemical linkages between lignin and carbohydrates that hinder their separation, but also their macromolecular structure, since because of its three-dimensionally cross-linked structure, native lignin is insoluble in any solvent. Thus, when lignin is extracted from wood or annual plants, it is partially degraded in to lower molecular weight fragments that are soluble in the solvent (Nimz and Casten, 1986) after cleavage of covalent bonds between lignin and hemicellulose. So, together with soluble fermentable sugars, inhibitors that adversely affect microbial growth and fermentation are also found in the hemicellulosic hydrolyzate (Felipe et al., 1996). According to Tran and Chambers (1986), the inhibitors formed during hydrolysis include furfural and hydroxymethylfurfural (degradation products of hemicellulose), phenolic and other aromatic compounds (degradation products of lignin), acetic acid (released from some hemicelluloses) and heavy metals such as chromium, copper, iron and nickel (from the hydrolysis equipment). These toxic compounds need to be removed or to have their concentrations reduced so that the hydrolyzates can be effectively used in the bioconversion process.

The focus of this work is on the partial removal of acetic acid and furfural from sugarcane bagasse hemicellulosic hydrolyzate by vacuum evaporation and with the addition of activated vegetable charcoal. The concentrations of nonvolatile inhibitors also increase after vacuum evaporation, and charcoal is mentioned in the literature as being able to remove phenolic and other compounds (Parajó, et al., 1996; Alves, et al., 1998).

MATERIALS AND METHODS

Sugarcane Bagasse Acid Hydrolysis

The dry weight of sugarcane bagasse was determined in an oven at 105° C. Acid hydrolysis was performed in a 250 L steel reactor at 121° C, using 100 mg sulfuric acid/ g sugarcane bagasse (dry weight) and a reaction time of 10 minutes (Pessoa Jr., 1997). The sulfuric acid was dissolved in water and the solid-to-liquid ratio was 1:10.

Preparation of the Hydrolyzate for Vacuum Evaporation

The hydrolyzate (pH= 0.92) obtained from sugarcane bagasse acid hydrolysis was separated into three portions. The original pH of the first was left unaltered, whereas the pH of the other two was adjusted to 4.76 and 9.5, respectively, by the addition of NaOH pellets. Each of the three portions (pH= 0.92, 4.76 and 9.5) was divided into two equal volumes, both treated with 2.4 % w/v activated vegetable charcoal (refined powder), one before and one after vacuum evaporation. The 2.4% w/v activated charcoal (AC) was mixed with the hydrolyzate by shaking (200 rpm) at 30° C for 1 hour (Alves et al., 1998). The activated charcoal was added to the hydrolyzate at different values of pH and concentrations to verify the influence of these variables on the removal of the volatile and nonvolatile compounds. After each pH alteration, both the precipitate formed and the activated charcoal were removed by vacuum filtration.

Vacuum Evaporation Process

The equipment used in the vacuum evaporation process was a rotaevaporator (BÜCHI R – 134) with an internal pressure control ranging from 1 to 1400 mbar. During the vacuum evaporation process, the effects of temperature (T), pH and degree of hydrolyzate concentration (f) on the removal of volatile (acetic acid and furfural) and nonvolatile compounds (5-HMF and some phenolic compounds) were evaluated. The pH values tested were 0.92, 4.76 and 9.5. The degrees of hydrolyzate concentration (f=2, f=3 and f=4) were calculated by dividing the initial volume (before the concentration process) by the final volumes (after the concentration process), and were 7.5° Brix, 11.25° Brix and 15° Brix, respectively. The vapor pressure was stabilized in the system to keep each temperature studied (43, 61.5 and 80 ° C) constant during the entire vacuum concentration process. The internal temperature of the system was monitored by a thermowell immersed in the hydrolyzate being concentrated. The acetic acid and furfural removal rates were calculated from the mass balance between the evaporated liquid and the condensed liquid.

Statistical Analysis

The amount of acetic acid in the hydrolyzate is larger than the amount of any other volatile compound. In order to minimize the number of experiments and model the vacuum evaporation of the hydrolyzate to reduce the acetic acid content as much as possible, a 2⁴ full factorial design with a centered face and four replicates at the center point (Box et al., 1978) was used. This design also allowed us to evaluate the effects of the variables studied (pH, temperature, degree of hydrolyzate concentration and addition of activated charcoal) on acetic acid removal before using the response surface regression procedure. Table 3 shows the variables studied and their respective maximum (+1), minimum (-1) and intermediary (0) coded levels. Table 2 shows the concentrations of phenolic compounds (r-hydroxybenzoic, vanillic, syringic, r-coumarilic and ferulic acids, vanillin and syringaldehyde) determined in trials 01 to 16 and 33 to 36 (Table 3), according to the 2⁴ full factorial design with four replicates at the center point.

The experiments were conducted in an aleatory order to minimize systematic errors. The statistical analysis of the data was performed using the STATGRAPHICS statistical software, version 6.0 and the STATISTICA program, version 5.0 (Alves et al., 1998). The results were expressed in estimated effects, standard errors, Student’s t distribution tables and tables of analysis of variance (degree of freedom, sum of squares, medium squares, F value (Fisher variance ratio) and significance level (p-value)).

Analytical Methods

The concentrations of D-xylose, D-glucose, L-arabinose, xylitol and acetic acid were determined with a Shimadzu (Kyoto, Japan) high-performance liquid chromatograph (HPLC), using a refractive index (RI) detector and Bio Rad (Hercules, CA) Aminex HPX-87H column (300x7.8 mm) at 45° C and 0.01N H₂SO₄ as the eluent at a flow rate of 0.6 ml min^-1 and an injection volume of 20 ml.

The concentrations of furfural and 5-HMF were determined with a Shimadzu high-performance liquid chromatograph (HPLC), using a dual l absorbance detector (SPD-10^Auv-vis) at 276 nm wavelength and a Hewlett-Packard RP 18 (200 mm) column at 25° C, a 1:8 acetonitrile-to-water ratio and 1% acetic acid as the eluent at a flow rate of 0.8 ml min^-1 and a sample volume of 20 ml.

The phenolic compounds derived from the soluble lignin (r-hydroxybenzoic, vanillic, syringic, r-coumarilic and ferulic acids, vanillin and syringaldehyde) were also analyzed by HPLC (Knauer), using a dual l absorbance detector (UV) at 28 nm wavelength, a Hypersil 50 DS column and 0.01N H₂SO₄ with 16.67% methanol and 2.78% propanol as the eluent at a flow rate of 1 ml min^-1 and an injection volume of 20 ml.

The pH of the hydrolyzate was adjusted to 7.0 with NaOH or H₂SO₄, and the total phenolic concentration was determined by spectrophotometry as described by Kim and Yoo (1996), using phenol as the standard compound.

An inductively coupled plasma atomic emission spectrometer (model ARL 3410 ICP) was used to perform an elementary chemical analysis after digestion of a 5 mL hydrolyzate sample and its dilution to 100 ml. For digestion, the 5 mL hydrolyzate sample was mixed with HNO₃, HCl and H₂O₂ at 70° C during 1 hour.

RESULTS AND DISCUSSION

Original Hydrolyzate

The sugarcane bagasse used as the raw material to prepare the hemicellulosic hydrolyzate had a humidity of 12.44%. The hydrolyzate (pH= 0.92 and 3.75 ° Brix) was obtained by acid hydrolysis and its partial composition is shown in Table 1.

The predominant sugar in the hydrolyzate was xylose (18.24 g L^-1) and the concentrations of arabinose (1.71 g L^-1) and glucose (1.20 g L^-1) were quite low (Table 1). According to Felipe et al. (1997) and Kanagachandran et al. (1997), low glucose concentrations in the hydrolyzate are desirable for xylose bioconversion.

Table 1 shows that, in addition to sugars, the hydrolyzate contained toxic compounds that inhibit cellular growth. These compounds, namely furfural, hydroxymethylfurfural (Sanches and Bautista, 1988; Larsson et al., 1999), acetic acid (Felipe et al., 1995; Kusumegi, et al., 1998) and phenolic compounds (Vogel-Lohmeier et al., 1998), result from hydrolysis of the raw lignocellulosic material. Minimum concentrations of furfural and 5-hydroxymethylfurfural (generated from the degradation of pentoses and hexoses, respectively) as well as of syringic, vanillic and r-hydroxybenzoic acids and other compounds were also found in the hydrolyzate. The highest concentration was detected for syringic acid (0.005 g L^-1), followed by vanillic acid (0.0042 g L^-1) and r-hydroxybenzoic acid (0.002 g L^-1); the concentrations of the other compounds were lower than 0.0006 g L^-1 (Table 1). According to Parajó et al. (1998), even low concentrations of the products of lignin degradation (phenolic compounds, aromatic acids and aldehyde) in the hydrolyzate can inhibit the fermentation of the lignocellulosic raw material. Other potential inhibitors are minerals or metals, probably derived from raw lignocellulosic materials or from corrosion of the hydrolysis equipment (Table 1). The concentrations of some ions found in the sugarcane bagasse hemicellulosic hydrolyzate were not in the range considered by Watson et al. (1984) as inhibitory to microbial activity. However, the concentration of chrome ions was higher than the one tested by the same authors. The most concentrated ion was sulfur (3.43 g L^-1), followed by iron (0.43 g L^-1), chrome (0.38g L^-1) and potassium (0.24 g L^-1); the concentrations of the other ions were lower than 0.2 g L^-1 (Table1).

In our work, the content of acetic acid in the hydrolyzate was 3.38 g L^-1. In general, the concentration of acetic acid varies, depending on the kind of raw lignocellulosic material and on the hydrolytic conditions employed.

Vacuum Evaporation Together with Activated Charcoal

Figure 1 shows vapor pressure versus temperature for water (Weast, 1984) and of hydrolyzate with and without charcoal treatment. Low vapor pressure is practically insensitive to a given range of temperatures, above which the more the temperature rises, the more rapidly the vapor pressure increases (Mahan, 1981). This is shown in Figure 1 together with similar water and hydrolyzate profiles. The vapor pressure in hydrolyzate treated with activated charcoal was on average 4.73% lower than that in untreated hydrolyzate. The influence of activated charcoal on vapor pressure was significant at a confidence level of 95%.

During the experiments, 98% of the furfural content (0.084 g L^-1) was removed from the original hydrolyzate by vacuum evaporation before or after treatment with activated charcoal (Table 1). Hydroxymethylfurfural was not detected in the treated and concentrated hydrolyzate. The addition of activated charcoal possibly facilitated its removal, since this compound is not volatile. Alves et al. (1998) observed that 85% of the furfural was removed from sugarcane bagasse hydrolyzate treated with activated charcoal.

Table 2 shows that the concentrations of vanillin, syringaldehyde and acid compounds (r-hydroxybenzoic, vanillic, syringic, r-coumarilic and ferulic acids) in general decreased after the hydrolyzate was treated with activated charcoal. However, addition of activated charcoal before vacuum evaporation favored the removal of these compounds (experiments 9 to 16 and 33 to 34) more than addition of activated charcoal after vacuum evaporation (1 to 8 and 35 to 36). In the former case, this happened irrespective of pH, whereas in the latter case removal was more effective with an acid pH (0.92). The influence of pH on the removal of these compounds can be associated with the influence of the degree of hydrolyzate concentration.

After the vacuum evaporation process, the concentration of sugars (D-glucose, D-xylose and L-arabinose) was proportional to the degree of hydrolyzate concentration, irrespective of pH and temperature. This suggests that the temperatures tested in the experiments (43-80° C) did not contribute to the degradation of the sugar during vacuum evaporation. The percentage of sugar removed after the addition of 2.4% activated charcoal was less than 10%. On testing different wood hydrolyzate treatments, Frazer and McCaskey (1989) observed that activated charcoal, alone or in combination with calcium hydroxide, was able to reduce by 13% the amount of sugar in the hydrolyzate. As shown in Table 3, the acetic acid was partially removed in its undissociated (volatile) form (pH= 0.92), but not in its dissociated (nonvolatile) form (pH= 9.5). The concentration of total acetic acid (dissociated and undissociated forms) in the experiments varied between 3.41 (experiment 1) and 15.45 gL^-1 (experiment 16). The initial statistical matrix (2⁴ full factorial design) was amplified with a centered face and four replicates at the center point to measure removal of the acetic acid more carefully and to determine a statistical model from RSM (Response Surface Methodology Surface).

Statistical Analysis for Removal of Acetic Acid

Table 3 shows the experimental matrix with coded variable levels (-1, +1 and 0) of the 2⁴ full factorial design with a centered face and four replicates at the center point, using quantitative variables, namely pH, degree of hydrolyzate concentration (f); temperature (T) and a qualitative variable, activated charcoal (AC). The AC was added to the hydrolyzate before (+1) and after (-1) the vacuum evaporation process.

The estimated effects, standard errors and Student’s t determination for the removal of acetic acid based on the experimental matrix (Table 3) are shown in Table 4. The results indicate that the first-order effects of pH, f and AC were significant at a confidence level of 95% and that only the interaction between pH and f was significant at a confidence level of 90%. The first-order effect of temperature and its interactions with other variables (pH, f and AC) were not statistically significant. In addition, a change in color was detected when the temperature of the hydrolyzate was increased from 43 to 80 ° C, indicating the formation of a colorful pigment due to some chemical reaction in the hydrolyzate. Hernández et al. (1987) mentioned the possibility of one or more molecules of phenolic compounds occasionally associated with carbohydrates forming colorful polymers due to condensation and oxidation reactions, but, as mentioned earlier, degradation of the sugar was not detected. Maybe the association of high temperature with a degree of hydrolyzate concentration higher than that tested in this work will make it possible to detect degradation of the sugar as well as other chemical reactions.

In order to increase the degrees of freedom so that the effects can be estimated, an analysis of variance (ANOVA) was done using only the terms that were significant in the Student’s t determination (Table 5). The percentage of variance shown in the analysis (R² = 93.7) indicates that the model selected is likely to be adequate for describing the removal of acetic acid as a function of the factors within the range studied. The significant levels (p-values) found for pH and f as well as for the interaction between pH and f confirmed the significant effects of these factors on the removal of acetic acid (Table 5).

Table 6 shows the analysis of variance for full regression of the model determined, where the total error was broken down into lack of fit and pure error, both in the F distribution, indicating that the lack of fit was not significant for this model. The Fisher variance ratio for regression models can also validate the model obtained for removal of acetic acid (Table 6); the higher the value of F is above one, the more certain it is that the factors adequately explain the variation in the data about its mean and that the estimated factor effects are real.

The application of the response-surface methodology offers, on the basis of the parameter estimate, an empirical relationship between the values of acetic acid for removal (Y) and the coded variables tested (Xi), by means of regression equation 1, by replacing the qualitative variable (AC) by its lowest coded level (-1).

where Y represents the acetic acid removed (%), X₁ the initial coded pH value and X₂ the coded degree of hydrolyzate concentration. Solving this mathematical model by means of the coded levels for pH (-1), degree of hydrolyzate concentration (+1) and treatment with activated charcoal after the vacuum evaporation process (-1) makes it possible to predict a 65.4% removal of acetic acid. The model’s optimal region can be observed in Figure 2, which depicts the response surface and contour lines described by the Y model. Frazer and McCaskey (1989) reported that a number of treatments were employed to remove acetate from the hydrolyzate. However, the most effective treatments tested removed only about 27% of the initial acetate.

According to Figure 2, the first-order effects of pH (0.92) and the addition of charcoal after the evaporation process favored the removal of acetic acid. The interaction between pH and f caused a 13.69% increase in the amount of acetic acid removed at the lowest pH level (0.92) when the hydrolyzate concentration was 2 to 4 times the initial concentration. When the pH level +1 (9.5) decreased to –1 (0.92) and the concentration factors were 2 and 4, the acetic acid removed increased by 47.65% and 58.22%, respectively.

Condition for Removing Both Acetic Acid and Phenolic Compounds

Figure 3 shows that at an acid pH (0.92), removal of the acetic acid was not a function of whether treatment with activated charcoal occurred before or after vacuum evaporation. However, the maximum removal value (63.91%) was achieved when activated charcoal was added to medium after vacuum evaporation and the hydrolyzate concentration reached its highest level (f=4). In this case, the average amount of acetic acid left in the hydrolyzate was 5 gL^–1 . At a basic pH (9.5) around 5% of the acid was removed in most cases, due to the addition of activated charcoal and the change in pH during vacuum evaporation. The removal of acetic acid (pK_a =4.76) is related to the balance between its dissociated and undissociated forms regulated by equilibrium, which is affected by pH.

Figure 3

CONCLUDING REMARKS

The vacuum evaporation process is necessary to increase the amount of sugars in the hydrolyzate used for biotechnological purposes. The association of this procedure with the treatment of the hydrolyzate using activated charcoal decreased the concentration of acetic acid, furfural, 5-hydroxymethylfurfural and phenolic compounds. Furfural and 5-hydroxymethylfurfural were almost totally removed in all the experiments, irrespective of pH, temperature and addition of charcoal during the vacuum evaporation process. Adding activated charcoal before the vacuum evaporation process favored the removal of phenolic compounds irrespective of pH. Acetic acid, on the contrary, was most effectively removed when activated charcoal was added after the vacuum evaporation process at an acid pH (0.92) and at the highest degree of hydrolyzate concentration (f=4). However, addition of activated charcoal before or after the vacuum evaporation at an acid pH (0.92) and at the highest degree of hydrolyzate concentration (f=4) favored the removal of both acetic acid and phenolic compounds. The first-order effect of temperature and the effects of its interactions with pH, degree of hydrolyzate concentration and activated charcoal on the removal of acetic acid were not statistically significant. However, to prevent chemical reactions during the evaporation process, it is advisable to use low temperatures when the degree of hydrolyzate concentration is increased.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support of FAPESP and CNP_q. They are also grateful to Maria E.M.Coelho for her revision of this paper.

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To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  10 Oct 2001
• Date of issue
  Sept 2001

History

• Accepted
  11 July 2001
• Received
  10 July 2000