Evaluation of pretreatment methods in improving the enzymatic saccharification of cellulosic materials
Introduction
Lignocellulosics, the most abundant biomass available on earth, have attracted considerable attention as an alternative feedstock for the production of various value added products due to their renewable nature and low cost availability (Kuhad & Singh, 2007). Various technological developments have improved the bioconversion of these substrates into bioethanol (Kapoor, Chandel, Kuhar, Gupta, & Kuhad, 2007). Enzymatic saccharification is one of the promising strategy to convert cellulosic biomass into sugars because of low energy requirement and less pollution. However, the primary challenge in enzymatic hydrolysis of cellulose is its low accessibility due to association with lignin. Therefore, efficient pretreatment of lignocellulosic substrates has become a pre-requisite to improve enzymatic saccharification (Zhao, Zhang, & Liu, 2008). The main focus of different pretreatment methods is to remove the lignin content and to decrease the cellulose crystallinity (Mosier et al., 2005). Although various physical (comminution, hydrothermolysis), chemical (acid, alkali, solvents, ozone), and biological pretreatment methods have been investigated over the years (Gupta et al., 2010, Kuhar et al., 2008, Kumar et al., 2009), thermo-chemical pretreatment of biomass has been a pretreatment of choice to enhance substrate accessibility for efficient enzymatic hydrolysis (Himmel et al., 2007).
The thermo-chemical pretreatment strategies such as acid, alkali and oxidation are commonly used for lignocellulosic biomass. The dilute mineral acids have been reported to remove the hemicellulosic fraction from substrates to improve enzymatic saccharification of cellulose (Gupta et al., 2009, Kuhad et al., 2010, Schell et al., 2003). It has dual advantage of solubilizing hemicellulose and subsequently converting it into fermentable sugars. Whereas, the alkali pretreatment remove lignin and various uronic acid substitutions responsible for inhibiting the cellulose accessibility for enzymatic saccharification (Chang & Holtzapple, 2000). Moreover, alkali treatment is also reported to increase the biodegradability of the cell walls due to cleavage of the lignin bonds with hemicellulose and cellulose (Spencer & Akin, 1980). In contrast to acid and alkali treatments, sodium chlorite, a powerful oxidizing agent has been used frequently to delignify wood for cellulose isolation (Sun, Sun, Zhao, & Sin, 2004). The chlorine dioxide produced in this pretreatment method oxidizes lignin to the phenolic compounds and in turn makes cellulose accessible.
Since there is no universal and economically viable pretreatment method available, which could be used to pretreat varied cellulosic biomass, in the present study, it has been attempted to evaluate the suitably used three pretreatment methods (acid, alkali and chlorite treatment) for lignocellulosic feedstocks viz., Prosopis juliflora (PJ, a woody biomass), Lantana camara (LC, a shrub and weed) and Corncob (CC, agricultural residue). The pretreatments of the substrates were attempted at varied chemical dosage and pretreatment time. The pretreated plant materials were enzymatically hydrolysed and the cellulosic saccharification efficiency was determined to evaluate the efficacy of these methods. Irrespective of the substrates used, the chlorite treatment was found to be an efficient method for delignification and producing cellulose rich plant material, which was almost 90% hydrolysable by cellulases into glucose.
Section snippets
Raw materials
The lignocellulosic substrates: Corncob (CC), P. juliflora (PJ) and L. camara (LC) were collected locally, dried in sunlight and then cut into small pieces. The dried material was ground and passed through a 40–60 mesh size screen using a laboratory knife mill (Metrex Scientific Instrumentation, Delhi, India). The processed substrate was thoroughly washed, dried at 60 °C and stored in sealed plastic bags at room temperature.
Acid pretreatment
The dilute sulfuric acid pretreatment of lignocellulosic substrate (100.0
Enzymatic saccharification of pretreated substrates
Cellulase from Trichoderma reesei (ATCC 26921) with an activity of 6.5 FPU/g, supplemented with β-glucosidase from Aspergillus niger (Novozyme 188) having 250 U/g was used for saccharifying the cellulosic material obtained after each pretreatment type.
Enzymatic hydrolysis of each type of pretreated plant materials (10.0 g each) was carried out at 5% (w/v) substrate consistency in 50 mM citrate phosphate buffer (pH 5.0). The substrate with buffer was pre-incubated at 50 °C on a rotatory shaker
Analytical methods
The chemical composition (α-cellulose, klason lignin, pentosans, moisture and ash) of all the three substrates and their residual solid fraction post pretreatment were determined following standard TAPPI (1992), protocols. The reducing sugars released were estimated using the DNS method (Miller, 1959) and the yield of reducing sugars in enzymatic hydrolysate (YRSEH) was calculated as follows:
Statistical analysis
All the experiments were performed in triplicate and the results are presented as mean ± standard deviation.
Compositional analysis of different lignocellulosic substrates
The chemical composition analysis of different lignocellulosic biomass revealed that the holocellulose content was in the range of 61.1–71.6% (w/w), where P. juliflora (PJ) contained maximum cellulose content (47.5 ± 3.27%) followed by L. camara (LC; 44.1 ± 1.72%) and Corncob (CC; 37.4 ± 4.18%). The lignin contents observed in CC, PJ and LC were 19.2 ± 0.83%, 29.1 ± 2.05% and 32.3 ± 1.57% (w/w), respectively. However, the hemicellulose content was maximum in CC (34.2 ± 1.02%) compared to PJ (18.7 ± 1.09%) and
Conclusion
Among different chemical pretreatments studied, the sodium chlorite pretreatment was found to be the most effective in lignin removal and led to the enrichment of the holocellulose content in treated substrates. This method offers the possibility of producing cellulosic material largely free from lignin, which eventually would be a good substrate for bioethanol production. However, there is a need to develop efficient biological delignification methods to make the process environmentally safe.
Acknowledgement
The authors are grateful to Department of Biotechnology, Government of India and Council of Scientific and Industrial Research, India, for the financial support.
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