Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Poplar wood was treated with peracetic acid, KOH, and ball milling to produce 147 model lignocelluloses with a broad spectrum of lignin contents, acetyl contents, and crystallinity indices (CrIs), respectively. An empirical model was identified that describes the roles of these three properties in enzymatic hydrolysis. Lignin content and CrI have the greatest impact on biomass digestibility, whereas acetyl content has a minor impact. The digestibility of several lime-treated biomass samples agreed with the empirical model. Lime treatment removes all acetyl groups and a moderate amount of lignin and increases CrI slightly; lignin removal is the dominant benefit from lime treatment.

Fundamental factors affecting biomass enzymatic reactivity.

The ability and, consequently, the limitations of various microbial enzyme systems to completely hydrolyze the structural polysaccharides of plant cell walls has been the focus of an enormous amount of research over the years. As more and more of these extracellular enzymatic systems are being identified and characterized, clear similarities and differences are being elucidated. Although much has been learned concerning the structures, kinetics, catalytic action, and interactions of enzymes and their substrates, no single mechanism of total lignocellulosic saccharification has been established. The heterogeneous nature of the supramolecular structures of naturally occurring lignocellulosic matrices make it difficult to fully understand the interactions that occur between enzyme complexes and these substrates. However, it is apparent that the efficacy of enzymatic complexes to hydrolyze these substrates is inextricably linked to the innate structural characteristics of the substrate and/or the modifications that occur as saccharification proceeds. This present review is not intended to conclusively answer what factors control polysaccharide biodegradation, but to serve as an overview illustrating some of the potential enzymatic and structural limitations that invariably influence the complete hydrolysis of lignocellulosic polysaccharides.

Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis

Lignocellulosic biomass is recalcitrant to biodegradation due to the rigid and compact structure of plant cell wall The recalcitrance of biomass is mainly constructed by its chemical compositions that build a spatial network as a protective bulwark Generally, the factors affecting the accessibility of biomass cellulose can be divided into direct and indirect factors The direct factors refer to the accessible surface area, and the indirect factors include biomass structure-relevant factors (pore size and volume, particle size, and specific surface area), chemical compositions (lignin, hemicelluloses, and acetyl group), and cellulose structure-relevant factors (cellulose crystallinity and degree of polymerization) Pre-treatment is actually the process to alter indirect factors and improve direct factors thus enhancing the accessibility of cellulose In this review, we summarize the effects of chemical compositions and physical structures on the enzymatic digestibility of lignocellulosic biomass We suggest that future work should be focused on but not limited to the molecular mechanisms of biomass recalcitrance by investigating the microscale and nanoscale features as well as hydrogen bonds network of lignocellulosic biomass © 2012 Society of Chemical Industry and John Wiley & Sons, Ltd

Biomass recalcitrance. Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose

Pretreatment of corn stover by aqueous ammonia.

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H/sub 2/SO/sub 4/, Cadoxen, and DMSO-P-formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hours by Aspergillus niger cellulase containing mainly 1,4-Beta-glucan glucanohydrolase without action of 1,4-beta-glucan cellobiohydrolase. On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

The preparation and regeneration of mycelial protoplasts from Collybia veltipes and Pleurotus ostreatus were examined. Composition of liquid medium for cultivation, culture aging of mycelium, and the combination of enzymes had important effects on the isolation of protoplasts. Three characteristic patterns were observed in the regeneration of hyphae from protoplasts.

Preparation and regeneration of mycelial protoplasts of Collybia veltipes and Pleurotus ostreatus

Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated CH-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.260281215

Properties of cellulase immobilized on agarose gel with spacer.

Fruiting bodies were induced from mycelium regenerated from Pleurotus ostreatus protoplasts. Mycelia originated from protoplasts conformed to parental strain mycelia in morphology. Six strains selected at random from the dikaryotic regenerants were able to form normal fruiting bodies, yielding 18% more than the parent.

Fruiting body formation from regenerated mycelium of Pleurotus ostreatus protoplasts

Aspergillus niger cellulase was imobilized on cyanogen bromide activated dextran of varying molecular weights. The effect of different concentrations of cyanogen bromide used for the activation process was also studied. About 50% conjugation and 70% retention activity was achieved in the immobilized cellulase. The pH activity of immobilized enzyme was unchanged, but exhibited more stable activity at acidic pH than the free enzyme. Higher resistance to heat inactivation was also observed.

Takashi Sasaki

Papers

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Preparation and regeneration of mycelial protoplasts of Collybia veltipes and Pleurotus ostreatus

Properties of cellulase immobilized on agarose gel with spacer.

Fruiting body formation from regenerated mycelium of Pleurotus ostreatus protoplasts

Cellulase immobilized on a soluble polymer