## 5 Biomass Porosity and Cellulase Efficacy
(2012) study, the authors further showed that the _Trichoderma reesei_ cellulases effectively penetrated into the microfibril internal surface and engaged degradation of these microfibrils with digestion holes or pits in the pretreated delignified biomass materials, whereas purified cellulosomes from _Clostridium thermocellum_ were shown to only peel off individual microfibrils from the cell wall surface in these pretreated biomass materials [7]. These results by [PERSON] et al. (2012) [7] would suggest that the dimension size of purified _Clostridium thermocellum_ cellulosomes was likely much larger than the upper pore diameter of 11 nm in the pretreated biomass materials reported by [PERSON] (1985) [95] and [PERSON] et al. (2009) [97], reflecting the fact that cellulosomes are rather large multi-enzyme complexes with molecular weights ranging from several hundreds to over 1 million kDa. [PERSON] et al. (2013) [101] used domain engineering to exploit the advantage of intramolecular and intermolecular proximity synergies by designing mini-cellulosomes to improve cellulase specific activity as well as to enhance their biomass-penetrating feature. Other strategies such as glycosylation by overexpression designer cellulosomes in other microbial platforms could further improve designer cellulosomes' thermostability and activity [102]. Meanwhile, [PERSON] et al. (2013) demonstrated that the multimodar free cellulase CelA cloned from the thermophilic bacterium _Calcidellulosiruptor bescii_ could effectively hydrolyze crystalline cellulose substrates via mechanisms of surface ablation and cavity excavating driven by a combined endo-acting and exo-acting processivity [77]. However, the _Calcidellulosiruptor bescii_ Cel A had a large molecular weight of about 230 kDa [103], and an estimated effective size of 10-35 nm, thus limiting its diffusion into microfibril internal surface for biomass hydrolysis [77]. Furthermore, the large molecular _Calcidellulolosinuptor bescii_ Cel A was associated with multiple carbohydrate-binding modules and its activity would be affected, to a greater extent, by nonproductive adsorption than much lower molecular weight cellulases [77]. Therefore, small molecular weights may be a favorable and advantageous biochemical feature in developing highly active processive free cellulases that may be effective to penetrate into microfibrillar internal surfaces for biomass hydrolysis. Cell wall biomass recalcitrance is collectively referring to the natural resistance of cell wall materials to microbial and enzymatic deconstruction [5; 9]. The biomass recalcitrance is essentially composed of two layers of resistance, including the water-excluding nature of the resistance of the crystalline cellulose bundle core within each of the elementary fibrils as well as the resistance representing the crosslinks around the cellulose bundle core formed by hemicellulose & pectin embedding each elementary fibril within each of microfibrils and the aromatic net further patched by lignin polymers on the exterior of the microfibrils within each of the macrofibril structure primarily in plant cell wall materials [19; 104; 105; 106]. The first layer of biomass resistance as a measure of water-excluding nature associated with the crystalline cellulose bundle core within the elementary fibril is also reflected by the crystallinity index (CrI, ranging between 0 and 1) that is experimentally measured for a number of biofuel materials [19; 107] but in very few plant feed materials for animals. The crystalline cellulose bundle core within each elementary fibril represents about 100 cellulose glucans that are tightly aggregated via extensive interchain and intrachain hydrogen bonds and van der Waals forces in forming a straight and stable supramolecular insoluble quasi-crystalline structure with a typical crystal size of 4-5 nm, being variable dependent upon biomass sources [19; 105; 106; 108].