Characterization of Dicarboxylic Acids for Cellulose Hydrolysis

Transcription

1 474 Biotechnol. Prog. 2001, 17, Characterization of Dicarboxylic Acids for Cellulose Hydrolysis Nathan S. Mosier,, Ayda Sarikaya,, Christine M. Ladisch, and Michael R. Ladisch*,,, Department of Agricultural and Biological Engineering, Laboratory of Renewable Resources Engineering, Department of Biomedical Engineering, and Textile Science, Department of Consumer Sciences and Retailing, Purdue University, West Lafayette, Indiana In this paper, we show that dilute maleic acid, a dicarboxylic acid, hydrolyzes cellobiose, the repeat unit of cellulose, and the microcrystalline cellulose Avicel as effectively as dilute sulfuric acid but with minimal glucose degradation. Maleic acid, superior to other carboxylic acids reported in this paper, gives higher yields of glucose that is more easily fermented as a result of lower concentrations of degradation products. These results are especially significant because maleic acid, in the form of maleic anhydride, is widely available and produced in large quantities annually. Introduction Limited fossil fuel supplies and rising oil prices along with increasing concern about the environmental impact of their use has prompted emphasis on research and development for the use of renewable resources for the generation of fuels and other chemicals now produced from petroleum. Biomass materials, consisting largely of cellulose, are a promising renewable resource for the production of fuels and industrial chemicals. A number of processes for hydrolyzing cellulose into glucose have been developed over the years. The two most common processes utilize either cellulolytic enzymes harvested from filamentous fungi such as Trichoderma sp. or sulfuric acid of varying strengths from dilute to concentrated. Historically, enzymes have been too expensive for economical production of fuel ethanol from biomass (1). Sulfuric acid itself is less expensive than cellulolytic enzymes, although disposal costs associated with the use of sulfuric acid significantly increase its cost. However, the single largest drawback to using sulfuric acid is that it also readily degrades glucose at the high temperatures required for cellulose hydrolysis (2-4). Glucose degradation not only lowers the yield of fermentable sugars from biomass but forms the degradation products hydroxymethyl furfural, levulinic acid, and formic acid, which themselves are inhibitory to yeast fermentation (5-8). Although concentrated mineral acids at lower temperatures have been used with some success (9, 10), the cost of acid recovery has impeded their widespread use. This paper addresses the use of carboxylic acids as cellulose-hydrolyzing catalysts as part of a larger research effort to develop organic molecules that mimic the specificity of enzymes (11). It is known that strong mineral acids hydrolyze cellulose more effectively than weak acids (12) and that carboxylic acid alone is a weak acid (high pk a ). However, compounds with multiple * ladisch@ecn.purdue.edu. Department of Agricultural and Biological Engineering. Laboratory of Renewable Resources Engineering. Department of Biomedical Engineering. Textile Science, Department of Consumer Sciences and Retailing. Table 1. pk a for Acid Catalysts at 20 C pk a acid catalyst molecular wt 1 2 sulfuric acid maleic acid succinic acid acetic acid water carboxylic acid moieties are stronger acids than monocarboxylic acids, both in number of protons available for donation to a base and lowered pk a s for the individual carboxylic acid moieties (compare the pk a of acetic acid to succinic and maleic acids, Table 1). The dicarboxylic acids, maleic and succinic acids, were evaluated by hydrolysis of the cellulose disaccharide repeat unit cellobiose. Results were compared against acetic acid, a monocarboxylic acid, and sulfuric acid, a mineral acid, as well as water alone. Maleic and sulfuric acids gave the largest extents of hydrolysis. These were then evaluated for the hydrolysis of the microcrystalline cellulose Avicel. Materials and Methods All chemicals used in these experiments were purchased from Sigma-Aldrich, St. Louis, MO. General lab and HPLC supplies were obtained from Fisher Scientific, Pittsburgh, PA. Stainless steel tubing and Swagelok fittings were purchased from Indianapolis Valve and Fitting Co., Indianapolis, IN. Carbohydrate HPLC Analysis. Sample analysis utilized a Bio-Rad HPX-87H organic acid column (Bio- Rad Laboratories Inc., Hercules, CA) in a HPLC system consisting of a Milton Roy minipump (Milton Roy Co., Ivyland, PA), Waters 717 plus autosampler, Waters R401 differential refractometer (Waters Corp., Milford, MA), Hewlett-Packard 3396 series II integrator (Hewlett- Packard, Palo Alto, CA), and a personal computer for data storage. The mobile phase was 5 mm sulfuric acid in distilled, deionized water filtered to 0.2 µm. The operating conditions for the HPLC column were 60 C with a flow rate of 0.6 ml/minute. Complete sample elution could be accomplished within 35 min per injection /bp010028u CCC: $ American Chemical Society and American Institute of Chemical Engineers Published on Web 05/01/2001

2 Biotechnol. Prog., 2001, Vol. 17, No Figure 1. Heat-up profile for 200-mL autoclave reactor to 160 C in 60 min with 30 min hold time. Standard curves were generated for glucose and cellobiose with pure samples dissolved in mobile phase. Chemical samples were dried at 70 C for 24 h before weighing to eliminate moisture error. Fractional dilutions of the standard solution, 5.00 mg/ml, were made to give a standard curve range of mg/ml. Glucose Analyzer. Glucose concentrations for all samples were confirmed with an enzymatic analysis using a Beckman glucose analyzer 2 (Beckman Coulter, Inc., Fullerton, CA). All samples from a given experiment were equally diluted when necessary to give maximum concentrations below 2.5 mg/ml to keep the measurements within the linear range of the instrument. Instrument calibration was carried out using a 150 mg/dl glucose standard purchased from Beckman. ph Measurement. The ph of each carbohydrate and catalyst solution was measured using a Calomel ph electrode from the Cole-Parmer Instrument Company (Vernon Hills, IL) attached to a Corning ph/ ion meter 150 (Corning Medical and Scientific Instruments, Halstead, Essex, England). Before each use, the ph meter was calibrated at two points using ph 7.00 and 4.00 buffer solutions obtained from the Fisher Chemical Company (Pittsburgh, PA). Each carbohydrate and catalyst solution was equilibrated to room temperature (23 C) before the ph probe was inserted into the solution. The solution was gently stirred until the ph reading stabilized, and the result was recorded to the nearest ph (0.01. Initial Screening of Varying Acid Catalyst Concentrations. Initial screening of different catalysts used different concentrations in order to determine reasonable concentrations for more detailed kinetic analysis. Aqueous solutions of cellobiose at 10 g L -1 were hydrolyzed with varying concentrations of acid. A concentration of 10gL -1 represents the maximum of the linear range for detection by HPLC without sample dilution for both cellobiose and glucose. Experiments utilized samples of 100 ml in a 200-mL continuous-stir autoclave reactor (Autoclave Engineers Inc., Erie, PA). All experiments followed the same temperature profile (Figure 1) in which the target temperature of 160 C was reached in 60 min and then held constant for an additional 30 min. Cool down at the end of each experiment was achieved by running tap water through cooling coils in the reactor. Reactor temperature fell below 100 C in less than 60 s for each experiment. In addition to the carboxylic acid catalyst candidates, a control with no acid (water with 10 g L -1 cellobiose) and varying concentrations of sulfuric acid, the standard acid for cellulose hydrolysis, were used. Each experiment was duplicated to give two data points per concentration. Reactant and product concentrations before and after hydrolysis were determined using HPLC and Beckman glucose analyzer 2. Glucose concentrations in solutions containing maleic acid were determined solely by Beckman glucose analyzer 2 because maleic acid and glucose coelute (Figure 2). Cellulose Hydrolysis. Avicel (FMC Corporation, Philadelphia, PA), sieve cut (53-75 µm), was used as the microcrystalline cellulose for hydrolysis. To 200 ml of 50 mm maleic acid and sulfuric acid aqueous solutions was added 8.0 g of the sieved Avicel, giving an approximated cellulose concentration of 35 g L -1. This concentration of Avicel was selected to give measurable glucose within 30 min of hydrolysis at 175 C. The solution ph was measured before and after the addition of the cellulose. The Avicel and acid solution mixture was vigorously shaken immediately before 4.0 ml of liquid was withdrawn by pipet to fill the reactor tubes. Reactor tubes were constructed from in in. 316 stainless steel tubing and in. Swagelok caps (Swagelok Cos., Solon, OH). Each reactor tube was 6 in. in length giving a total volume of 5.5 ml. However, a sample volume of only 4.0 ml (at room temperature) was used in each tube to allow for liquid expansion from heating. Temperature control was achieved utilizing a Tecam SBL-1 fluidized sand bath. As a result of size constraints and temperature variation on the vertical axis of the sand bath, only two reactor tubes could be immersed at one time. Internal reactor tube temperatures during heatup for varying bath temperatures were determined using an Omega thermowell with thermocouple (Omega Engineering, Inc., Stamford, CT). The thermowell and thermocouple were inserted into the reactor tube to the vertical and axial center and held in place with a Swagelok fitting and ferule to maintain pressure. Heatup times were determined using two reactor tubes each filled with 4 ml of water, one blank and one with thermocouple inserted. A replicate of each temperature experiment was performed (data shown in Figure 3). Heat-up times of 2 min were required to achieve 175 C at the corresponding saturation water vapor pressure

3 476 Biotechnol. Prog., 2001, Vol. 17, No. 3 Figure 2. Chromatograms of solutions containing cellobiose and glucose, cellobiose and 0.05 M maleic acid, and all three compounds. Maleic acid and glucose coelute, and thus all glucose concentrations in maleic acid solutions determined solely by Beckman glucose analyzer 2. Figure 3. Heat-up profiles for reactor tubes filled with water. (930 kpa), so that the contents of the tube were maintained in a liquid state. The reactor tubes were cooled by immersion in ice water. Internal tube temperature dropped below 100 C in less than 10 s for all tested sand bath temperatures (data not shown). Four time points were tested, including a zero time, which represented heat-up of the reactor tube to the desired temperature and immediate cool-down in ice water. The order of reaction times was randomized for each experiment. In addition, all time point experiments were repeated. The two replicates gave four data points per reaction time (two sets of two tubes). After cool-down, the contents of the reactor tubes were filtered through Whatman PVDF centrifuge filters, 0.2 µm pore size, 6.5 mm diameter (Fisher Scientific, Pittsburgh, PA) that were dried at 60 C for 24 h and individually weighed prior to filtration. The supernatant was retained for analysis. The sample bottles were rinsed to recover residual Avicel that was then filtered through the same filters. The recovered Avicel in the centrifuge filters was then dried at 60 C for 72 h and weighed. The initial mass of Avicel was determined by averaging the recovered Avicel masses from the four zero-time samples. Concentrations of dissolved saccharides, oligosaccharides, and degradation products were determined by HPLC and Beckman glucose analyzer 2 as described above. Theory Modeling Cellulose Saccharification by Dilute Sulfuric Acid. Cellulose is a heterogeneous substrate that makes modeling cellulose hydrolysis difficult. Cellulose is composed of chains of glucose connected by β- (1-4) glycosidic bonds. One chain end is termed the reducing end because the hemiacetal is able to open to expose the reducing aldehyde. The other chain end is called the non-reducing end because the 1-carbon in the hemiacetal is involved in the β(1-4) bond, preventing ring opening. Like starch chains, this gives cellulose

4 Biotechnol. Prog., 2001, Vol. 17, No chains directionality. Unlike starch, having glucose as its repeat unit, the repeat unit for cellulose is cellobiose, a glucose dimer. This is because the β(1-4) bond causes the glucose molecules to align with alternating directionality, unlike the R(1-4) bound glucose molecules in starch (13). Beginning with Saeman in 1945 (2) and confirmed by many others (12, 14-18) cellulose hydrolysis by acid catalysts has been modeled as a pseudo-first-order homogeneous sequential reaction of cellulose hydrolysis followed by glucose degradation: k hyd C98 k deg G98 HMF + H 2 O f LA + FA (1) where C ) cellulose, G ) glucose, HMF ) hydroxymethyl fufural, LA ) levulinic acid, FA ) formic acid, k hyd ) kinetic constant of cellulose hydrolysis, and k deg ) kinetic constant of glucose degradation. However, cellulosic materials are not homogeneous solids. Their physical and chemical properties change over the course of hydrolysis, which makes effective generalized modeling of cellulose hydrolysis difficult. The cellulose hydrolysis kinetic constants must be determined empirically for each biomass material, acid catalyst, and set of reaction conditions. Cellobiose Hydrolysis and Glucose Degradation. Cellobiose, the repeat unit of cellulose, was chosen for the screening and initial kinetic experiments of the acid catalysts. The use of cellobiose served to simplify the experiments since there was only one hydrolyzable bond for each molecule of reactant. Furthermore, cellobiose is soluble and can be obtained in greater purity (>99.9%) than plant cellulose (97-99%), simplifying interpretation of the data. There is also less homogeneity for reaction slurries containing cellulose than cellobiose because the crystallinity and accessibility of the cellulose to the catalyst can vary substantially between solid particles of cellulose, as well as within the individual particles themselves. It must also be noted that direct utilization of cellobiose hydrolysis kinetics for predicting cellulose hydrolysis is limited by the varying accessibility and crystallinity of the cellulose, which changes the reactivity of the individual β(1-4) glycosidic bonds. However, cellobiose hydrolysis data show important trends that help focus further study with purified cellulose and biomass materials of possible industrial importance. Hydrolysis of cellobiose occurs by the following formula where G 2 ) cellobiose and G ) glucose: k G 2 + H 2 O 98 2G (2) Glucose degradation has been shown to generate products by k deg G98 HMF + H 2 O f LA + FA (3) where G ) glucose, HMF ) hydroxymethyl furfural, LA ) levulinic acid, FA ) formic acid, and k deg ) kinetic constant of glucose degradation. Disappearance of cellobiose from the reaction liquid is reported in this paper as a percent of the measured original concentration. The measured glucose concentrations can be interpreted in two different ways. The first expression of the glucose concentration data is glucose yield. Glucose yield is the observed final glucose concentration over the stoichiometric maximum glucose from Figure 4. Cellobiose (10 g L -1 ) hydrolysis (a) and glucose yield (b) for varying acid concentrations at 160 C, 30 min hold time. the measured disappearance of cellobiose: Y G ) [G] 100% (4) 2([G 2,0 ] - [G 2,f ]) where Y G ) percent expected glucose yield, [G] ) molar glucose concentration, [G 2,0 ] ) initial molar cellobiose concentration, and [G 2,f ] ) final molar cellobiose concentration The second expression, theoretical glucose yield, is calculated as percent final glucose concentration over total theoretical glucose from initial cellobiose: Y G/G2 ) [G] 100% (5) 2[G 2 ] where, Y G/G2 ) percent glucose yield, [G] ) molar concentration of glucose, and [G 2 ] ) molar concentration of cellobiose. Results and Discussion Cellobiose Hydrolysis. Maleic acid hydrolyzed 95-99% of the cellobiose (Figure 4a) with a maximum glucose yield of 90% achieved at 50 mm acid concentration (Figure 4b). The glucose yield gives a measure of the quantity of hydrolyzed cellobiose that is found as glucose at the end of the reaction. Succinic acid gave conversions of 60-80% with glucose yields of 85-90%. Sulfuric acid gave close to 100% hydrolysis but only 80% glucose yield in the best case (compare Figure 4a and b). The monocarboxylic acid, acetic acid, resulted in the lowest conversion for all examined acids, with glucose yields falling between sulfuric acid and the dicarboxylic acids. When cellobiose is cooked in water alone, 15% is hydrolyzed

5 478 Biotechnol. Prog., 2001, Vol. 17, No. 3 Figure 5. Measured glucose yield from decomposed cellobiose vs acid concentration at 160 C, 30 min hold time. with a glucose yield of 55%. Water alone is capable of both hydrolyzing cellobiose and degrading glucose, consistent with the literature results (19, 20). Chromatographic analysis of all controls and acids tested showed the presence of significant levels of glucose degradation products such as levulinc acid, formic acid, and hydroxymethyl furfural in the samples with low glucose yield. The results show striking differences among the catalytic ability of the four acids tested. The percent hydrolysis of cellobiose (Figure 4a) shows some important trends. Although the acetic acid and succinic acid solutions buffer water to approximately the same ph, , the percent hydrolysis is substantially different. For the other group, both maleic acid and sulfuric acid achieve near complete hydrolysis for the reaction temperature and time. The baseline hydrolysis of cellobiose in water alone, shown for comparison, is by far the lowest. Both succinic acid and maleic acid show very high (82-95%) glucose yield in the final solutions (Figure 4b). Acetic acid had moderate levels of expected gluocose as did lower concentrations of sulfuric acid. However, water alone and higher concentrations of sulfuric acid showed significant loss of the desired product. For both maleic and sulfuric acid, the theoretical glucose yields (Figure 5) have a local maximum at approximately 50 mm concentrations. Theoretical glucose yield (eq 5) describes percent total conversion to glucose of the initial cellobiose in solution. This shows that, for the reaction duration and temperature profile, 50 mm acid concentration is near optimal since near 100% cellobiose hydrolysis was achieved. These results led to the selection of 50 mm maleic and sulfuric acid as concentrations for initial kinetic studies of cellobiose hydrolysis and glucose degradation. For succinic and acetic acids, the upward trend in yield closely mirrors the upward trend in percent hydrolysis for increasing concentrations (Figure 4a). This can be interpreted that the reaction conditions, temperature and time, as well as acid concentrations, are far from optimized. In summary, maleic acid produced the highest glucose yields of all of the tested acids. Although sulfuric acid produced slightly higher cellobiose hydrolysis than maleic acid, less subsequent glucose degradation by the maleic acid produced higher glucose yields than sulfuric acid. Succinic acid gave superior glucose yields compared with sulfuric acid when both acids were at the highest tested concentrations. Although succinic and maleic acid had high glucose yields from hydrolyzed cellobiose (Figure 4b), HPLC results also show small levels of glucose degradation products that account for the lost glucose. These results suggest that the reaction rates for cellobiose hydrolysis are greater than the rates of glucose degradation for succinic acid and maleic acid when compared to acetic acid and sulfuric acid. The results for maleic acid are especially significant since it is a chemical commodity widely used in the form of maleic anhydride. Maleic anhydride will hydrolyze into maleic acid at room temperature in aqueous solution (21). Although maleic anhydride itself has no consumer uses, its derivatives are universally known and used. Maleic anhydride is a required reactant for the production of unsaturated polyester resins used to produce fiberglass, casting resins, and auto repair putty. The US. production of maleic anhydride was in excess of 189,000 tons in 1992 with the major producers being Amoco and Huntsman Specialty Chemicals, formerly of Monsanto (21). It is currently sold as a bulk commodity for $ per pound (22). Hydrolysis of Cellulose (Avicel). Although hydrolysis of cellobiose is useful for screening catalysts for cellulose hydrolysis, further testing of the promising candidates with cellulose is required to confirm the cellobiose hydrolysis results. Avicel was chosen as the cellulose substrate. Avicel is a highly crystalline form of cellulose produced by acid reflux hydrolysis of wood. The high crystallinity of Avicel makes it resistant to hydrolysis (23). Cellulolytic enzymes are able to achieve a maximum conversion of 90% (10% not degradable by the enzymes) after more than 100 hours at 50 C (24). Of the acids screened, maleic acid was chosen as the most promising carboxylic acid because of the high theoretical glucose yields obtained from cellobiose hydrolysis. Maleic acid at 50 mm concentration was chosen as the test case since it gave the highest glucose yield at 160 C (Figure 4b). As a control, Avicel was also hydrolyzed by sulfuric acid at 50 mm, the sulfuric acid concentration that gave the highest glucose yield at 160 C. However, the hydrolysis was carried out at 175 C because microcrystalline cellulose requires pretreatment at temperatures above 160 C to open the crystalline structure to hydrolysis (19, 25). Hydrolysis was carried out for three hold times, 30, 60, and 180 min, at 175 C. To better control the temperature, these reactions were carried out in 5.5- ml, 316 stainless steel reactor tubes placed in a fluidized sand bath that allowed the reaction mixture to reach 175 C in 2 min, as described in Materials and Methods. After the hold time, the reaction tubes were quenched in ice water to reach ambient temperature in less than 10 s. Table 2 shows the solid cellulose and dissolved glucose results from four replicates. The final percent hydrolysis is nearly identical for both acids. However, there is slightly more cellulose hydrolyzed at 60 min in maleic acid (13.8%) than in sulfuric acid (8.23%). The results of greatest importance are % glucan as glucose (i.e., % yield column). This number represents the molar mass of measured glucose divided by the change in molar mass of cellulose. The molar mass is calculated by describing cellulose as glucan, or anhydroglucose, to simplify the varying degrees of polymerization of the individual cellulose chains within each particle: MW glucan ) MW glucose - MW water ; ) (6) The remaining percent change in cellulose molar mass is lost as glucose degradation products. Although the percent hydrolysis is nearly identical for both acids, the

6 Biotechnol. Prog., 2001, Vol. 17, No Figure 6. Comparison of measured Avicel disappearance and glucose appearance from hydrolysis at 175 C. Table 2. Results of Avicel Hydrolysis at 175 C time (min) compound concn (g/l) -glucan (g/l) std dev % hydrolysis % yield 50 mm Maleic Acid 0 cellulose cellulose cellulose cellulose glucose n/a 30 glucose glucose glucose mm Sulfuric Acid 0 cellulose cellulose cellulose cellulose glucose n/a 30 glucose glucose glucose percent glucose yielded from hydrolysis is much higher for maleic acid. The data suggest that the rate of Avicel hydrolysis is nearly identical for both 50 mm maleic and sulfuric acids, whereas the rate of glucose degradation is much lower for maleic acid than sulfuric acid. This is clear evidence of the superiority of maleic acid for the hydrolysis of cellulose compared with sulfuric acid. Figure 6 shows this large difference in glucose from the nearly equivalent hydrolysis of Avicel between the two acids. At 30 min, 65.6% of the decrease in cellulose molar mass is recovered as glucose through maleic acid hydrolysis compared with 25.7% for sulfuric acid hydrolysis. At the 60 and 180 min intervals the percent recovered as glucose decreases for both acids. This is presumably because hydrolysis rate slows as the more easily hydrolyzed cellulose is removed leaving increasingly recalcitrant material. Although the rate of hydrolysis that generates glucose is reduced, the rate of glucose degradation remains unchanged. At the point the rate of glucose generation is less than the rate of glucose degradation, the glucose concentration decreases (note the drop in glucose concentration after reaching a peak in Figure 6). However, the large variability in glucose concentrations at 60 and 180 min within the four replicates prompted further investigation. To determine if sulfuric acid degraded glucose more significantly than maleic acid, the same experimental apparatus was used to test four replicates of each acid on solutions of pure glucose. After 30 min at 175 C, between 84% and 93% of the glucose (5 g L -1 initial concentration) was degraded in the presence of 50 mm sulfuric acid, whereas only between 13% and 17% of the glucose was degraded in the presence of 50 mm maleic acid. Conclusion Dilute maleic acid, a dicarboxylic acid, has been shown to hydrolyze cellobiose and cellulose as effectively as dilute sulfuric acid. Higher glucose yields from cellulose hydrolysis also suggest that maleic acid does not degrade glucose as easily as sulfuric acid. This is significant because less glucose degradation by maleic acid would result in higher glucose yields from a cellulosic biomass, which would be more easily fermented as a result of lower concentrations of fermentation-inhibiting degradation products. Further work into the kinetics of cellobiose and cellulose hydrolysis and the kinetics of glucose degradation for these acids is needed to confirm these hypotheses and to develop a model for predicting glucose yields from biomass hydrolysis. Such work is required for optimizing the cost of biomass conversion using maleic acid. Acknowledgment We would like to thank Craig Keim and Kyle Beery for their helpful comments on this manuscript. This material is based upon work supported by the National Science Foundation under grant BES References and Notes (1) Lynd, L. R.; Elander, R. T.; Wyman, C. E. Likely Features and Costs of Mature Biomass Ethanol Technology. Appl. Biochem. Biotechnol. 1996, 57-8, (2) Saeman, J. F. Kinetics of Wood Saccharification: Hydrolysis of Cellulose and Decomposition of Sugars in Dilute Acid at High Temperatures. Ind. Eng. Chem. 1945, 37(1),

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