Abstract
Formosan subterranean termites (Coptotermes formosanus Shiraki) and other wood-feeding insects have the ability to digest cellulose and structurally modify or degrade lignin. We examined the physical and chemical changes to lignocellulosic components of Chinese red pine (Pinus massoniana) after passing through the termite (C. formosanus) digestive system. The purpose of this research was to evaluate biochemical digestive processes in the C. formosanus gut as potential models for biofuels processing. Results suggest that demethylation, demethoxylation, and propyl side-chain modification are responsible for higher lignin removal and cellulose crystallinity reduction after structural alteration. SEM images also further indicated that unlike the fungus- growing termites Odontotermes formosanus, the lower termites C. formosanus disrupted the lignocellulose structure, and thus resulted in an increase of surface area to cellulase. Comparative enzymatic hydrolysis tests between raw wood and C. formosanus faeces revealed an enhanced level of enzymatic digestibility in digested material. Based on the results, C. formosanus can efficiently modify lignin at ambient temperatures and pressures in contrast to current methods used in biofuels production.
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Physiochemical lignocellulose modification BY the Formosan subterranean termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) and ITS potential uses in the production of biofuels
Hongjie Li,a Jirong Lu,a and Jianchu Moa,*
Formosan subterranean termites (Coptotermes formosanus Shiraki) and other wood-feeding insects have the ability to digest cellulose and structurally modify or degrade lignin. We examined the physical and chemical changes to lignocellulosic components of Chinese red pine (Pinus massoniana) after passing through the termite (C. formosanus) digestive system. The purpose of this research was to evaluate biochemical digestive processes in the C. formosanus gut as potential models for biofuels processing. Results suggest that demethylation, demethoxylation, and propyl side-chain modification are responsible for higher lignin removal and cellulose crystallinity reduction after structural alteration. SEM images also further indicated that unlike the fungus- growing termites Odontotermes formosanus, the lower termites C. formosanus disrupted the lignocellulose structure, and thus resulted in an increase of surface area to cellulase. Comparative enzymatic hydrolysis tests between raw wood and C. formosanus faeces revealed an enhanced level of enzymatic digestibility in digested material. Based on the results, C. formosanus can efficiently modify lignin at ambient temperatures and pressures in contrast to current methods used in biofuels production.
Keywords: Biofuel; Coptotermes formosanus; Cellulose crystallinity; Enzymatic digestibility; Lignin removal; Odontotermes formosanus; Pre-treatment
Contact information: Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Kaixuan Road 268, Hangzhou, Zhejiang 310029, P. R. China Tel/Fax: +86-571-86971695
*Corresponding author: mojianchu@zju.edu.cn
INTRODUCTION
Pretreatment is a critical step in biomass-to-biofuel conversion and refers to processes in which plant lignin is broken down and cellulose crystalline structures are disrupted so that acids or enzymes can hydrolyse the cellulose (Chandra et al. 2007; Kumar et al. 2009). This is also the most expensive and energy-consuming step in biomass-to-biofuel conversion, requiring sufficient mechanical processing, as well as typically requiring high temperatures and pressure (Mosier et al. 2005). Biological pre-treatments offer an alternative and typically use microorganisms such as brown-, white-, and soft-rot fungi to enhance the suitability of lignocellulose for enzymatic hydrolysis (Galbe and Zacchi 2007; Tian et al. 2011). Pretreatment techniques that utilize natural processes have much lower energy requirements and are environmentally friendly (Okano et al. 2005). The downside, however, is that most biological pre-treatment processes are slow because of low hydrolysis rates (Shary et al. 2007; Singh et al. 2008).
Insects that utilize wood as a food source include beetles, cockroaches, and termites. Termites are especially well known for their ability to overcome the lignin barrier and digest polymer carbohydrates (Brune 1998; Hyodo et al. 1999; Watanabe and Tokuda 2010). Several recent studies looking at lignocellulose digestion processes in these insects suggest that novel physiochemical processes may be involved, especially in Coptotermes formosanus Shiraki (Prins and Kreulen 1991; Breznak and Brune 1994; Geib et al. 2008; Ke et al. 2010).
A number of genes that encode candidate pretreatment enzymes have been identified in the termite guts by transcriptome analyses, including lignases and phenolic acid esterases (Tartar et al. 2009; Scharf and Boucias 2010). Furthermore, functional analyses of these enzymes have shown that they play roles in lignocellulosic pretreat- ment, such as lignin modification and hemicellulose solubilisation (Coy et al. 2010; Wheeler et al. 2010). The physiological and morphological information of digestive systems, especially those of microenvironment and masticating organs, which may result in the efficient use of biomass, were also measured (Sharma et al. 1984; Kim and Holtzapple 2006). These characteristics suggest that the termite digestive gut system may have potential as a model for more efficient pre-treatment of plant matter in biofuel production.
Little is known about the complex pretreatment mechanisms of lignocellulose in the termite gut system beyond current knowledge of single enzyme systems. Lignin degradation in the termite gut is well documented (Geib et al. 2008; Ke et al. 2010), but the high level of cellulose utilization despite apparently low lignin modification continues to be a mystery. Changes in the crystalline structure of cellulose and the morphology of cell walls during digestion in the termite gut have not been documented but may be of importance when trying to understand the exact nature of lignocellulose digestion in C. formosanus. In this paper, the extent of physiochemical changes to lignocellulose during digestion in the termite gut and the extent to which lignocellulose is modified after movement through the termite digestive system are presented and discussed.
EXPERIMENTAL
Biomass Preparation
Samples of Chinese red pine Pinus massoniana (Lamb.) obtained from the market in Hangzhou, Zhejiang Province, P. R. China, were cut into blocks (5 cm × 5 cm × 20 cm) of the sapwood (Hyodo et al. 1999). The blocks were divided into two portions: one was used directly as food of termites and the other was milled and ground to pass a 1 mm screen, oven-dried at 60°C for 72 h, and stored in plastic bag at 4°C for all experiments.
Sample Collection and Preparation
A colony of C. formosanus was obtained from the National Center for Termite Control in Hangzhou, Zhejiang Province, P. R. China, in June 2009. The colony was fed sapwood blocks and maintained in a plastic box (40 cm × 40 cm × 50 cm) without soil in complete darkness at a constant temperature of 26oC and 69% RH. Fecal material was collected daily from surfaces of the sapwood food source and the plastic box. Collected material was stored in a sealed plastic bag at 4°C until processing for all experiments. In addition, fungus-growing termitesOdontotermes formosanus (Shiraki), collected from Hangzhou Botanic Garden in Hangzhou, Zhejiang Province, P. R. China, were fed and maintained under the same conditions as described above.
For both species, C. formosanus and O. formosanus, 100 workers were selected at random and dissected to collect wood particles in various stages of digestion from the foregut, midgut, and hindgut of each insect (Fig.1). The contents of the three different gut segments of each termite were squeezed gently, diluted with 10 μL of deionised water, and collected by capillary tubes, using a dissecting microscope. The suspensions were stored in the capillary tubes until observation with scanning electron microscopy (SEM), described in following sections.