Toward a biophysical theory of organogenesis: Birefringence observations on regenerating leaves in the succulent,Graptopetalum paraguayense E. Walther

Polarity shifts occur during organogenesis. The histological criterion for polarity is the direction of cell division. The biophysical criterion is the orientation of reinforcing cellulose microfibrils which lie normal to the organ axis and which determine the preferred growth direction. Using cell pattern to deduce cell lineage, and polarized light to study cellulose alignment, both aspects of polarity were examined in the epidermis of regenerating G. paraguayense. In this system new leaves and a stem arise from parallel cell files on a mature leaf. Large (90°) shifts in polarity occur in regions of the epidermis to give the new organs radial symmetry in the surface plane (files radiating from a pole). Study of the shifts in the epidermis showed that, during certain stages, shifts in the division direction are accompanied by shifts in the cellulose deposition direction, as expected. The new cellulose orientation is parallel to the new cross wall. During normal organ extension, however, shifts in division direction do not bring on changes in cellulose pattern. Thus the coupling between the two kinds of polarity is facultative. This variable relation is used in a biophysical model which can account for the reorganization of cell file pattern and cellulose reinforcement pattern into the radial symmetry of the new organ.     

The complexities of hydrolytic enzymes from the termite digestive system

Abstract

The main challenge in second generation bioethanol production is the efficient breakdown of cellulose to sugar monomers (hydrolysis). Due to the recalcitrant character of cellulose, feedstock pretreatment and adapted hydrolysis steps are needed to obtain fermentable sugar monomers. The conventional industrial production process of second-generation bioethanol from biomass comprises several steps: thermochemical pretreatment, enzymatic hydrolysis and sugar fermentation. This process is undergoing continuous optimization in order to increase the bioethanol yield and reduce the economic cost. Therefore, the discovery of new enzymes with high lignocellulytic activity or new strategies is extremely important. In nature, wood-feeding termites have developed a sophisticated and efficient cellulose degrading system in terms of the rate and extent of cellulose hydrolysis and exploitation. This system, which represents a model for digestive symbiosis has attracted the attention of biofuel researchers. This review describes the termite digestive system, gut symbionts, termite enzyme resources, in vitro studies of isolated enzymes and lignin degradation in termites.     

Application of extended Kalman filter to identification of enzymatic deactivation

A recursive estimation scheme, the Extended Kalman Filter (EKF) technique, was applied to study enzymatic deactivation in the enzymatic hydrolysis of pretreated cellulose using a model previously developed by the authors. When no deactivation model was assumed, the results showed no variation with time for all the model parameters except for the maximum rate of cellobiose-to-glucose conversion (r'(m)).The r'(m) variation occurred in two zones with a grace period. A new model of enzymatic hydrolysis of pretreated cellulose deactivation was proposed and validated showing better behavior than the old deactivation model. This approach allows one to study enzyme deactivation without additional experiments and within operational conditions.     

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady-state expressions that can be used to describe the pseudo-steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme-substrate affinity at which the pseudo-steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes.     

CMC-modified cellulose biointerface for antibody conjugation

In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulose-based biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir-Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

Cellulose-binding domains: cellulose associated-defensive sensing partners?

The cellulose-binding domains (CBDs) in the Phytophthora cellulose-binding elicitor lectin (CBEL) are potent elicitors of plant defence responses. Induction of defence has also been reported in various cellulose-deficient mutants of Arabidopsis thaliana. Based on these observations, we propose a model linking cellulose alteration to defence induction. This integrates the fast increase in cytosolic calcium recorded in response to CBEL, mechano-stimulated calcium uptake mechanisms, and proteins that interact functionally with the cellulose synthase complex. In this context, CBDs emerge as new tools to decipher the signalling cascades that result from cell wall-cellulose perturbations.     

The physical action of cellulases revealed by a quartz crystal microbalance study using ultrathin cellulose films and pure cellulases

The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.     

Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes

Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme's ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.     

3D Simulation of Internal Tablet Strength During Tableting

This study presents a new approach to model powder compression during tableting. The purpose of this study is to introduce a new discrete element simulation model for particle–particle bond formation during tablet compression. This model served as the basis for calculating tablet strength distribution during a compression cycle. Simulated results were compared with real tablets compressed from microcrystalline cellulose/theophylline pellets with various compression forces. Simulated and experimental compression forces increased similarly. Tablet-breaking forces increased with the calculated strengths obtained from the simulations. The calculated bond strength distribution inside the tablets showed features similar to those of the density and pressure distributions in the literature. However, the bond strength distributions at the center of the tablets varied considerably between individual tablets.     

Characteristics and bending performance of electroactive polymer blend made with cellulose and poly(3-hydroxybutyrate)

This paper describes a novel cellulose/poly(3-hydroxybutyrate) blend based electroactive polymer. The fabrication process, bending actuation test and its characteristics are investigated. To prepare this new EAP, cellulose and PHB were dissolved in trifluoroacetic acid. The solution was cast to form a film followed by depositing thin gold electrode on both sides of the film. The characteristics of the cellulose/PHB film were investigated by Fourier transform infrared spectra, scanning electron microscopy, X-ray diffraction differential scanning calorimetry, tensile test and dynamic mechanical analysis. The bending performance was evaluated in terms of free bending displacement, electrical power consumption output and lifetime test under ambient conditions. Primary results show that this cellulose/PHB blend EAP is less sensitive to humidity and it shows higher bending displacement and longer lifetime than pure cellulose EAP at room humidity condition. These results indicate that this new cellulose/PHB blend EAP has potential for many biomimetic applications.     

Modeling microbial dynamics in heterogeneous environments: growth on soil carbon sources

We have developed a new kinetic model to study how microbial dynamics are affected by the heterogeneity in the physical structure of the environment and by different strategies for hydrolysis of polymeric carbon. The hybrid model represented the dynamics of substrates and enzymes using a continuum representation and the dynamics of the cells were modeled individually. Individual-based biological model allowed us to explicitly simulate microbial diversity, and to model cell physiology as regulated via optimal allocation of cellular resources to enzyme synthesis, control of growth rate by protein synthesis capacity, and shifts to dormancy. This model was developed to study how microbial community functioning is influenced by local environmental conditions in heterogeneous media such as soil and by the functional attributes of individual microbes. Microbial community dynamics were simulated at two spatial scales: micro-pores that resemble 6-20-μm size portions of the soil physical structure and in 111-μm size soil aggregates with a random pore structure. Different strategies for acquisition of carbon from polymeric cellulose were investigated. Bacteria that express membrane-associated hydrolase had different growth and survival dynamics in soil pores than bacteria that release extracellular hydrolases. The kinetic differences suggested different functional niches for these two microbe types in cellulose utilization. Our model predicted an emergent behavior in which co-existence of membrane-associated hydrolase and extracellular hydrolases releasing organisms led to higher cellulose utilization efficiency and reduced stochasticity. Our analysis indicated that their co-existence mutually benefits these organisms, where basal cellulose degradation activity by membrane-associated hydrolase-expressing cells shortened the soluble hydrolase buildup time and, when enzyme buildup allowed for cellulose degradation to be fast enough to sustain exponential growth, all the organisms in the community shared the soluble carbon product and grew together. Although pore geometry affected the kinetics of cellulose degradation, the patterns observed for the bacterial community dynamics in the 6-20 μm-sized micro-pores were relevant to the dynamics in the more complex 111-μm-sized porous soil aggregates, implying that micro-scale studies can be useful approximations to aggregate scale studies when local effects on microbial dynamics are studied. As shown with examples in this study, various functional niches of the bacterial communities can be investigated using complex predictive mathematical models where the role of key environmental aspects such as the heterogeneous three-dimensional structure, functional niches of the community members, and environmental biochemical processes are directly connected to microbial metabolism and maintenance in an integrated model.     

Microbial cultures in the utilization of cellulosic materials

This review elaborates on the most recent microbial development in saccharification of cellulose and cellulase formation. A particular highlight is a new genetic-immunochemical approach investigating the mechanism of adhesion of bacterial cellulase to cellulose during cellulose conversion. New developments and recent reviews in hemicellulose and lignin degradation are also covered.     

Characterization of the crystalline structure of cellulose using static and dynamic FT-IR spectroscopy

The cellulose structure is a factor of major importance for the strength properties of wood pulp fibers. The ability to characterize small differences in the crystalline structures of cellulose from fibers of different origins is thus highly important. In this work, dynamic FT-IR spectroscopy has been further explored as a method sensitive to cellulose structure variations. Using a model system of two different celluloses, the relation between spectral information and the relative cellulose Ialpha content was investigated. This relation was then used to determine the relative cellulose Ialpha content in different pulps. The estimated cellulose I allomorph compositions were found to be reasonable for both unbleached and bleached chemical pulps. In addition, it was found that the dynamic FT-IR spectroscopy technique had the potential to indicate possible correlation field splitting peaks of cellulose Ibeta.     

Kinetic modeling of rapid enzymatic hydrolysis of crystalline cellulose after pretreatment by NMMO

Pretreatment of cellulose with an industrial cellulosic solvent, N-methylmorpholine-N-oxide, showed promising results in increasing the rate of subsequent enzymatic hydrolysis. Cotton linter was used as high crystalline cellulose. After the pretreatment, the cellulose was almost completely hydrolyzed in less than 12 h, using low enzyme loading (15 FPU/g cellulose). The pretreatment significantly decreased the total crystallinity of cellulose from 7.1 to 3.3, and drastically increased the enzyme adsorption capacity of cellulose by approximately 42 times. A semi-mechanistic model was used to describe the relationship between the cellulose concentration and the enzyme loading. In this model, two reactions for heterogeneous reaction of cellulose to glucose and cellobiose, and a homogenous reaction for cellobiose conversion to glucose was incorporated. The Langmuir model was applied to model the adsorption of cellulase onto the treated cellulose. The competitive inhibition was also considered for the effects of sugar inhibition on the rate of enzymatic hydrolysis. The kinetic parameters of the model were estimated by experimental results and evaluated.     

Role of contact in bacterial degradation of cellulose

Abstract Bacterial cells can adhere to cellulose fibres, but it is not known if cell-to-fibre contact is necessary for cellulose degradation. This problem was explored using aerobic cellulolytic bacteria, including known species and new isolates from soil. These were tested on plates containing Avicel, Solka floc, CF11 cellulose, carboxymethyl cellulose, or phosphoric acid-treated cellulose. Cellulose degradation was measured both by formation of clearing zones and by growth when cellulose was the only carbon source. The bacteria tested were either inoculated directly on the cellulose-containing agar, or separated from it by a pure agar layer or by membrane filters (not containing cellulose). Even when separated from the cellulose-containing agar all strains grew well. Clearing zones, best seen in phosphoric acid-treated cellulose, were larger under colonies separated from cellulose by an agar layer than under those in direct contact with cellulose. Such zones could also appear under filters. Our results show that bacterial degradation of cellulose does not depend on cell-to-fibre contact and suggest that when cellulose is at a greater distance from the cell, the removal of end products reduces catabolite repression of cellulose formation.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.