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.

Background Lignin, nature’s dominant aromatic polymer, is found in most terrestrial plants in the approximate range of 15 to 40% dry weight and provides structural integrity. Traditionally, most large-scale industrial processes that use plant polysaccharides have burned lignin to generate the power needed to productively transform biomass. The advent of biorefineries that convert cellulosic biomass into liquid transportation fuels will generate substantially more lignin than necessary to power the operation, and therefore efforts are underway to transform it to value-added products. Production of biofuels from cellulosic biomass requires separation of large quantities of the aromatic polymer lignin. In planta genetic engineering, enhanced extraction methods, and a deeper understanding of the structure of lignin are yielding promising opportunities for efficient conversion of this renewable resource to carbon fibers, polymers, commodity chemicals, and fuels. [Credit: Oak Ridge National Laboratory, U.S. Department of Energy] Advances Bioengineering to modify lignin structure and/or incorporate atypical components has shown promise toward facilitating recovery and chemical transformation of lignin under biorefinery conditions. The flexibility in lignin monomer composition has proven useful for enhancing extraction efficiency. Both the mining of genetic variants in native populations of bioenergy crops and direct genetic manipulation of biosynthesis pathways have produced lignin feedstocks with unique properties for coproduct development. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery and enables catalytic modifications for desired chemical and physical properties. Outlook Potential high-value products from isolated lignin include low-cost carbon fiber, engineering plastics and thermoplastic elastomers, polymeric foams and membranes, and a variety of fuels and chemicals all currently sourced from petroleum. These lignin coproducts must be low cost and perform as well as petroleum-derived counterparts. Each product stream has its own distinct challenges. Development of renewable lignin-based polymers requires improved processing technologies coupled to tailored bioenergy crops incorporating lignin with the desired chemical and physical properties. For fuels and chemicals, multiple strategies have emerged for lignin depolymerization and upgrading, including thermochemical treatments and homogeneous and heterogeneous catalysis. The multifunctional nature of lignin has historically yielded multiple product streams, which require extensive separation and purification procedures, but engineering plant feedstocks for greater structural homogeneity and tailored functionality reduces this challenge.

https://lifelong.unt.edu/sites/default/files/EmeritusCollege/Spring2015/Handouts/Lignin%20co-products.pdf

Lignin valorization: improving lignin processing in the biorefinery.

Lignin is the generic term for a large group of aromatic polymers resulting from the oxidative combinatorial coupling of 4-hydroxyphenylpropanoids ([Boerjan et al., 2003][1]; [Ralph et al., 2004][2]). These polymers are deposited predominantly in the walls of secondarily thickened cells, making them

Lignin Biosynthesis and Structure

Lignin is an abundant biopolymer with a high carbon content and high aromaticity. Despite its potential as a raw material for the fuel and chemical industries, lignin remains the most poorly utilised of the lignocellulosic biopolymers. Effective valorisation of lignin requires careful fine-tuning of multiple "upstream" (i.e., lignin bioengineering, lignin isolation and "early-stage catalytic conversion of lignin") and "downstream" (i.e., lignin depolymerisation and upgrading) process stages, demanding input and understanding from a broad array of scientific disciplines. This review provides a "beginning-to-end" analysis of the recent advances reported in lignin valorisation. Particular emphasis is placed on the improved understanding of lignin's biosynthesis and structure, differences in structure and chemical bonding between native and technical lignins, emerging catalytic valorisation strategies, and the relationships between lignin structure and catalyst performance.

Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis.

Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H2O2, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The ``randomness'' of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. As such, lignification is seen as a marvelously evolved process allowing plants considerable flexibility in dealing with various environmental stresses, and conferring on them a striking ability to remain viable even when humans or nature alter ``required'' lignin-biosynthetic-pathway genes/enzymes. The malleability offers significant opportunities to engineer the structures of lignins beyond the limits explored to date.

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Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids

Lignification and lignin topochemistry - an ultrastructural view.

Microfibril angle (MFA) is perhaps the easiest ultrastructural variable to measure for wood cell walls, and certainly the only such variable that has been measured on a large scale. Because cellulose is crystalline, the MFA of the S2 layer can be measured by X-ray diffraction. Automated X-ray scanning devices such as SilviScan have produced large datasets for a range of timber species using increment core samples. In conifers, microfibril angles are large in the juvenile wood and small in the mature wood. MFA is larger at the base of the tree for a given ring number from the pith, and decreases with height, increasing slightly at the top tree. In hardwoods, similar patterns occur, but with much less variation and much smaller microfibril angles in juvenile wood. MFA has significant heritability, but is also influenced by environmental factors as shown by its increased values in compression wood, decreased values in tension wood and, often, increased values following nutrient or water supplementation. Adjacent individual tracheids can show moderate differences in MFA that may be related to tracheid length, but not to lumen diameter or cell wall thickness. While there has been strong interest in the MFA of the S2 layer, which dominates the axial stiffness properties of tracheids and fibres, there has been little attention given to the microfibril angles of S1 and S3 layers, which may influence collapse resistance and other lateral properties. Such investigations have been limited by the much greater difficulty of measuring angles for these wall layers. MFA, in combination with basic density, shows a strong relationship to longitudinal modulus of elasticity, and to longitudinal shrinkage, which are the main reasons for interest in this cell wall property in conifers. In hardwoods, MFA is of more interest in relation to growth stress and shrinkage behaviour.

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Microfibril Angle: Measurement, Variation and Relationships – A Review

How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2010.04319.x

Restricted access of proteins to mannan polysaccharides in intact plant cell walls

The organization of wood cell wall components involves aggregates of cellulose microfibrils and matrix known as macrofibrils. A combination of field emission electron microscopy and environmental scanning electron microscopy was used to visualise the organization of macrofibrils in different cell wall types comparing normal and reaction wood of radiata pine and poplar as examples of a typical softwood and hardwood. The size of macrofibrils is shown to vary among cell wall types with the smallest structures occurring in the gelatinous layer of tension wood (14 nm) and the largest structures in the S2L layer of compression wood (23 nm). A positive correlation between macrofibril size and degree of lignification is observed, with macrofibrils apparently increasing in size in more highly lignified cell wall types. The fibrillar structure of the secondary wall varies from microfibril-sized structures of 3–4 nm up to large aggregates of 60 nm diameter. The size of macrofibrils also varies slightly among adjacent cells of the same cell wall type. Macrofibrils occur predominantly in a random arrangement, although radial and tangential lamellae may sometimes be seen in individual cells.

Cellulose microfibril aggregates and their size variation with cell wall type

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a “bonsai tree-like” appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated β-aryl ether (β-O-4 unit) and spirodienone (β-1) levels, accompanied by lower phenylcoumaran (β-5), resinol (β-β), and dibenzodioxocin (5-5/β-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.

https://www.jstor.org/stable/info/25594940

Lloyd Donaldson

Papers

Lignification and lignin topochemistry - an ultrastructural view.

Microfibril Angle: Measurement, Variation and Relationships – A Review

Restricted access of proteins to mannan polysaccharides in intact plant cell walls

Cellulose microfibril aggregates and their size variation with cell wall type

Suppression of 4-Coumarate-CoA Ligase in the Coniferous Gymnosperm Pinus radiata