Transformational scientific knowledge on bioprocessing is required for developing enabling technologies toward converting renewable biomass to targeted biofuels or biochemicals. A major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall (PCW) so that the carbohydrates can subsequently be hydrolyzed into sugars to be fermented into fuel or chemicals. Existing technologies typically employ thermochemical processes for biomass pretreatment to break down the barriers formed by lignin and lignin-hemicellulose association. Although effective, these processes are energy intensive, environmentally unfavorable and associated with the production of inhibitors that compromise the performance of downstream fermentation. The fact that there is still no successfully demonstrated commercial system in the world for producing biofuels and biochemicals from lignocellulosic biomass underscores the magnitude of the related technical challenges. There is an urgent need for new systems that are more efficient, cost-effective and environmentally-friendly for deconstructing PCW for sugar release.
Nature, through millions of years' evolution, has developed highly efficient biological systems. Wood-feeding termites (Insecta, Isoptera) are among the most effective lignocellulose-recycling invertebrates in terms of the rate and extent of cellulose utilization [1
]. Termites work as a complete PCW deconstruction system and are considered to be highly effective for wood degradation as they requires only biomass, moisture and air to function efficiently. Termites' unique mechanisms could serve as an ideal bioconversion model and a novel source of catalysts for refining biofuels and biochemicals. Recently, their ability to convert recalcitrant PCW into a useable energy source of monomer sugars has attracted much interest in the biofuel area [2
]. Lower termites are believed to utilize lignocellulosic carbohydrates in a step-wise fashion using cellulases and hemicellulases, with help from lignolytic enzymes [3
]. However, the pretreatment mechanism of the lignin matrix during the whole digestive process is as of yet unexplored. In addition, the selective modifications to the lignin-hemicellulose matrix for subsequent cellulose hydrolysis have not yet been explored. Lack of such knowledge is a major barrier to the efficient pretreatment of biomass required to enable substantial biomass utilization.
In exploring the mechanisms of PCW degradation by termites, it has always been speculated that the termite chewing action plays an important role in the initial pretreatment. Fujita et al.
] have already demonstrated one of the characteristics of wood degradation by termites to be the mechanical grinding of food by the mandibles to increase the surface area of the substrates. The termite workers chew wood blocks into small particles at an early stage of the digestion process for better access to carbohydrates. During this mechanical grinding of the wood particles, cellulases append to the wood particles or mix with them to initiate the catalysis process [8
]. Yoshimura demonstrated that the lower termite Coptotermes formosanus
crushes and grinds the wood to exhibit sharp edges on the surface, allowing better accessibility by the functional enzymes [3
]. It has been reported that termite workers in the Rhinotermitidae family excavate the wood for nesting [9
]. When they were moved to a new environment with only wood blocks, they continued their chewing behavior to acquire a large amount of small wood particles for nesting. The nest consisted of ground wood particles pasted together with a special secretion from the salivary glands [9
]. A large proportion of these particles might be re-digested. During the chewing process, the workers released secretions from their salivary and labial glands for the initial digestion and community activity of feeding stimulation [11
Termite saliva, which is used for claying of nesting materials, has been reported in previous studies to contain various digestive enzymes. Fujita et al.
have already cloned two cDNAs that encode premature lysozyme peptides (Rs-Lys1 and Rs-Lys2) from workers of a Japanese damp-wood termite, Reticulitermes speratus
]. Moreover, they demonstrated that the total digestive lysozyme activity is termite in origin and predominates in the salivary glands and, to a minor extent, in the digestive tract. At the same time, Nakashima et al.
found 80.8% of total cellulase activity stemmed from the salivary glands of C. formosanus
]. Cellobiase is also reported to occur in the salivary glands of Mastotermes darwiniensis
and Neotermes koshunensis
]. It has been proposed that cellulose is first degraded to some extent by carboxymethyl cellulase produced in the salivary glands of termites, and then ingested by protozoa, which finally decompose the cellulose to glucose with their own endo-beta-1,4-glucanases, exo-cellobiohydrolase, and β
]. For the digestion of hemicelluloses, Inoue et al.
-1,4-xylanase activity in the salivary glands of the lower termite R. speratus
, however, no β
-xylosidase activity was documented [17
]. For lignin degradation, laccase and phenoloxidase gene expression, as well as benzene-1,2,3-triol oxidation activity, were confirmed in the salivary gland of R. flavipes
In termites, the labial glandular ducts open into the oral cavity, from which the watery labial gland secretion is released onto the wood [11
]. The labial gland secretion, which contributes to the saliva, is reported to have various species-specific functions in nest construction, as a social nutrient and as a source of digestive enzymes [11
]. During communal food exploitation, the labial gland secretion is released onto the food by feeding workers, as demonstrated in the African termite Schedorhinotermes lamanianus
and the French species R. santonensis
], thereby aiding in efficient food exploitation.
It is important to note that current research on lignin modification by termites is mainly focused on the gut process of digestion. Such studies are centered towards the understanding of the symbiont evolution, transcriptomics, meta-transcriptomics and metagenomics of wood-feeding termites, which are necessary for understanding the lignocellulose degradation process. However, there have been no reports nor has attention been drawn towards understanding the combined process of biological lignin modification and physical chewing for cellulose release during the chewing process in termites, which is essential to target the fundamental hurdle in producing cellulosic biofuels-overcoming the barriers of enzymatic hydrolysis of cellulose. In an earlier report, we have already evaluated the possible deconstruction pattern of hardwood lignocellulosics in the clearwing borer system [25
]. Here, we expand our investigation of biological lignocellulosic modification during the chewing stage of wood-feeding termites. The purpose of this study is to determine the associated structural changes of the softwood during the mechanical chewing process of the C. formosanus
termite, and provide new insight into designing new pretreatment processes for biomass conversion. The analysis methods include compositional analysis, enzymatic hydrolysis, pyrolysis gas chromatography mass spectrometry (Py-GC/MS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and thermogravimetry (TG/DTG) analysis.