Reactive high pressure carbonated water pretreatment prior to enzymatic saccharification of biomass substrates

https://doi.org/10.1016/j.supflu.2012.02.010Get rights and content

Abstract

Pretreatment of biomass substrates is critical in their use for generating monomeric sugars for conversion to transport fuels, such as bioethanol. In this study, high pressure reactive pretreatment and hydrolysis of cellulose- and hemicellulose-containing substrates, principally corn stover and switchgrass, were conducted over the temperature range of 150–190 °C utilizing carbon dioxide pressures ranging from 150 to 450 bar. Experimental protocol was guided by the development of an orthogonal design criteria consisting of temperature, CO2 pressure, time of pretreatment-hydrolysis, and substrate particle size. High pressure carbonation of the subcritical water-biomass mixtures were conducted in a small batch reactor and the resultant hydrolyzate mixtures were analyzed for sugar content using SEC and HPLC-RI detection. Also, the resultant hydrolyzates after carbonated water pretreatment were further hydrolyzed using commercial enzymes to saccharify the remaining oligomeric sugars to xylose and glucose. The resultant % sugar content of the carbonated water – versus dilute mineral acid – pretreated biomass mixtures were compared as well as the corresponding final enzymatically saccharified hydrolyzates for switchgrass and corn stover. High pressure carbonated water pretreatment yielded 9–13% less sugars than the sulfuric acid-derived hydrolyzate and relatively 6–10% less sugars were found in the final carbonated water treated hydrolyzates after further enzymatic treatment. It was also found that carbonated water pretreated switchgrass hydrolyzate required 33% lesser amount of enzyme for saccharification when compared to that obtained using dilute sulfuric acid pretreatment. The supercritical carbon dioxide dissolved in water provides an environmentally benign and “green” pretreatment method for depolymerization of the sugars present in biomass substrates and to facilitate further saccharification without the necessity of base neutralization, or pH adjustment prior to application of enzyme-initiated hydrolysis.

Highlights

► High pressure carbonated water was used to react and hydrolyze the sugars in biomass substrates such as switchgrass and corn stover and the experimental conditions were optimized to maximize glucose and xylose yield. ► The carbonated water reactive pretreatment, though, slightly less effective in biomass hydrolysis when compared to dilute sulfuric acid pretreatment, did allow for 33% savings in the amount of mixed enzymatic cocktail used especially to saccharify switchgrass. ► Carbonated water pretreatment also allowed for an in-situ pH adjustment based on the solubility of supercritical carbon dioxide in water and alleviated the need for neutralization before enzymatic hydrolysis. ► The carbonated water pretreatment was effective in hydrolyzing more than 90% of the total amount of sugars that are present in the biomass.

Introduction

Pretreatment of biomass substrates is considered a critical step in the process of development of sustainable and renewable biofuels, such as bioethanol, which is produced from the fermentation of monomeric sugars derived from polysaccharide constituents, cellulose and hemicellulose, found in the biomass substrates [1]. A number of pretreatment methods have been developed which utilize acid or base-chemically based agents [2], [3], hydrolytic methods [4], gas-based comminution techniques [5], and the well-known AFEX process [6]. In practice, most all of the aforementioned techniques are followed by the application of enzymes to facilitate depolymerization of carbohydrate polymers contained in the treated biomass substrates [7]. These are usually carbohydrase mixtures whose compositions are a proprietary secret [8], and their attendant cost is the major factor for the high cost of bioethanol [9] relative to the petroleum-based fuels which constituent gasoline.

Gas-based pretreatment/comminution methods have employed carbon dioxide [5], sulfur dioxide [10], steam [11] and ammonia [12]. The use of pressurized carbon dioxide has been of interest due to its relative benign properties and low cost, and it has been employed in its sub- and supercritical fluid state [13]. Carbon dioxide can be used under pressure followed by subsequent abrupt pressure reduction facilitating “explosive comminution” of the particulate matter and fiber often times constituting biomass. As with other biomass pretreatment methods, the objective is to prepare the substrate for subsequent application of mixtures of cellulases and hemicellulases to produce the monomer sugars, glucose and xylose, for fermentation to bioethanol. The actual mechanism of many pretreatment processes is poorly understood and inadequately studied; suffice to say the microscopic studies [14] have been used to document the results of various pretreatment methods, and verify that enhanced access to biomass polymeric constituents has been achieved, allowing more effective application of the enzymatic cocktails mentioned previously.

The application of supercritical carbon dioxide (SC-CO2) above its critical pressure and temperature has several benefits aside from its decompression which is the basis of “explosive comminution”. Since most biomass substrates have some nascent moisture content, the introduction of SC-CO2 results in the formation of carbonic acid; the pH of the “final solution” being dependent on pressure and temperature and the quantity of imbibed CO2, i.e., its partial pressure. Thus, highly compressed CO2 in the presence of water has the capability of performing acid-based hydrolysis on the constituent carbohydrate polymers in biomass substrates. This mode of pretreatment is somewhat different from the reported and patented [15], [16] methods for pretreating biomass in the presence of SC-CO2.

Several reports have appeared in the literature in which SC-CO2 has been used to pretreat various biomass substrates. The interesting studies initiated by the van Walsum group [17], [18], [19] on corn stover and aspenwood showed differing results for compressed CO2 with respect to biomass pretreatment. Additional application of CO2 has also been reported by Mosier et al. [13] at Purdue University for the pretreatment of corn stover. Rogalinski and colleagues in the Brunner group [20], [21] have also explored the hydrolysis of lignocellulosic biomass (rye and rice straw) in water under elevated temperatures and pressures, and applied high pressure CO2, to and above its saturation limit in water, to accelerate the depolymerization of oligomeric sugars. They found that up to CO2 pressures of 100 bar that no effect was recorded by the addition of CO2 as judged by the empirical severity factor index [22]. These results are in contrast to the other above cited studies in which the high pressure carbonation of hot subcritical water depends on the biomass substrate being treated and its relative recalcitrance.

Several investigators have measured the dissolution of high pressure CO2 in hot water over a range of temperatures and pressures [23], [24], [25], [26], which has also been previously cited by us [13]. These studies indicates that up to 100 °C, CO2 dissolution in water decreases with increasing temperature and increases with CO2 pressure. However the measurement of CO2 solubility in water above its boiling point by Sabirzyanov et al. [25] indicates that with increasing CO2 pressure and water temperature (to 150 °C), the solubility of CO2 increases. The relationship between the aqueous solution pH and CO2 pressure for various isobars has been calculated and presented by van Walsum [27], Hunter and Savage [28], and Chuang and Johannsen [29]; however, Chuang and Johanssen actually measured the aqueous solution pHs as a function of CO2 pressure over a limited range of higher temperatures. Complimentary studies by the Balaban group at Florida [30], [31] in conjunction with using SC-CO2 as a pasteurization agent in food processing also confirm the presence and effect of carbonic acid in aqueous solution at high CO2 pressures. Fig. 1 indicates the magnitude of the effect of solution carbonation (pH versus CO2 pressure), as calculated and plotted by Dr. Monika Johanssen of TUHH in Harburg, Germany at our request. The agreement is good when comparing the results from Johanssen's calculations and those of van Walsum [27].

Based on the above considerations, we initiated a multi-year study designed to exploit the effect of high SC-CO2 dissolution into subcritical water held at temperatures consistent for hydrolyzing cellulose- and hemicellulose-containing biomass. The specific objectives of this reported study were multi-fold: (1) to further explore the effect of high pressure carbonation in hot water above its boiling point under sufficient pressure to keep it in its subcritical state, on various biomass substrates, (2) to assess the relative degree of biomass hydrolysis to monomeric and lower oligomeric sugar mixtures due to subcritical water hydrolysis process, and (3) the possible benefit of high temperature and pressure aqueous carbonated water catalysis on reducing the amount of post pretreatment enzyme cocktail required for complete conversion of the oligomeric sugar mixtures to glucose and xylose for eventual conversion to bioethanol.

Section snippets

Materials

The corn stover and cob (Zea Mays. Ssp. Mays L.) samples were obtained from Anderson Corporation (Maumee, Ohio). Alamo switchgrass (Panicum virgatum L.) was grown at the University of Arkansas Agricultural Research and Extension Station in Fayetteville, AR. The biomass samples were comminuted using a UDY sampling cyclone mill (Fort Collins, CO) and separated using a standard testing sieve (Arthur H. Thomas Company, PA) with particle sizes ranging between 45 and 180 μm.

Depol™ 692L (Biocatalysis

Results and discussion

As previously discussed in Section 1, studies have indicated that depending on the temperature and pressure conditions, the solubility of carbon dioxide in water results in formation of a carbonic acid, which is a weak acid and can be used to effectively catalyze biomass hydrolysis [28]. This slightly exothermic reaction can be indicated as follows:CO2+H2OH2CO3H++HCO32H++CO32

It can be seen from Fig. 1, that this formation of weak acid can change the pH of the solvent (water) to that

Concluding remarks

The reported studies utilizing supercritical carbon dioxide dissolved in water for the pretreatment and depolymerization of sugars present in biomass substrates such as corn stover and switchgrass offers some additional benefits aside from lowering the required amount of the enzyme needed to saccharify cellulose and hemicellulose such as (a) alleviating the need for neutralization of the acidic medium after pretreatment has been completed; and (b) in situ optimal pH adjustment via carbonic acid

Acknowledgments

This work was supported by the US Department of Energy South Eastern Regional grant: US/DOE/DE-FG36-08G088036. We would like to acknowledge Anderson Inc. for providing samples of corn cob and stover. The authors would also like to thank Dr. Chuck West of the Dale Bumpers College of Agricultural, Food, and Life Sciences, for supplying samples of Alamo switch grass.

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