Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.     

Anaerobic digestion of cellulose by pure and mixed bacterial cultures

Summary By enrichment technique, nine anaerobic mixed bacterial cultures were isolated, five of which showed stable cellulolysis. All cultures fermented cellulose and produced different fermentative products. Mixed culture BOC 25 yielded major levels of acetate and ethanol (39.6 and 12.0 mmol/l, respectively) and minor levels of propionate (2.5 mmol/l) and digested filter paper cellulose to the extent of 32.5% w/v. BOC 25 digested cellulosic and lignocellulosic substrates and produced filter paper cellulase, carboxymethyl cellulase, Avicelase and -glucosidase. Strain DC 25, a cellulolyticClostridium was purified from one of the mixed cultures. The fermentation products of DC 25 from cellulose, cellobiose or glucose were ethanol, acetate, formate, H₂ and CO₂.     

Structure of a cellulose degrading bacterial community during anaerobic digestion

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.     

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.     

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.     

Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion

Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.     

Phenylacetic acid stimulation of cellulose digestion by Ruminococcus albus 8.

The rate of cellulose digestion by Ruminococcus albus 8 grown on a defined medium could be increased by adding a minimum of 6.6% (vol/vol) rumen fluid. Strain 8 was grown on half this concentration, and the culture medium before and after growth was analyzed by gas chromatography-mass spectrometry to determine which components of the rumen fluid were used. Phenylacetic acid was identified as the component needed to make the defined medium nutritionally equivalent to one supplemented with rumen fluid. [14C]phenylacetic acid fed to cultures of strain 8 was primarily incorporated into protein. Hydrolysis of protein samples and separation of the resulting amino acids showed that only phenylalanine was labeled. The results indicate that cellulose digestion by strain 8 was probably limited by phenylalanine biosynthesis in our previously reported medium. The data obtained on the utilization of other rumen fluid components, as well as on the production of metabolites, illustrate the potential usefulness of this method in formulating defined media to simulate those in nature.     

Anaerobic digestion of cellulose fraction of domestic refuse by means of rumen microorganisms

The anaerobic digestion of a cellulose-enriched fraction of domestic refuse by means of rumen microorganisms in an "artificial rumen" digester was studied. Various combinations of solid and liquid retention times and loading rates were applied to establish optimum conditions for the acidogenic phase digestion of the refuse fraction. An optimal substrate conversion of about 72% was obtained at a loading rate of 23.4 g volatile solids (VS)/L d and a solids retention time of 90 h. Variation of dilution rate between 1.04 and 3.14 fermentor volume turnovers per day had no effect on degradation efficiency. At a loading rate of 23.4 g VS/L d a differential removal rate of solids and liquids appeared to be necessary to obtain an effective degradation of the refuse fraction.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Increasing concentrations of phenol progressively affect anaerobic digestion of cellulose and associated microbial communities

Performance stability is a key issue when managing anaerobic digesters. However it can be affected by external disturbances caused by micropollutants. In this study the influence of phenol on the methanization of cellulose was evaluated through batch toxicity assays. Special attention was given to the dynamics of microbial communities by means of automated ribosomal intergenic spacer analysis. We observed that, as phenol concentrations increased, the different steps of anaerobic cellulose digestion were unevenly and progressively affected, methanogenesis being the most sensitive: specific methanogenic activity was half-inhibited at 1.40 g/L of phenol, whereas hydrolysis of cellulose and its fermentation to VFA were observed at up to 2.00 g/L. Depending on the level of phenol, microbial communities resisted either through physiological or structural adaptation. Thus, performances at 0.50 g/L were maintained in spite of the microbial community’s shift. However, the communities’ ability to adapt was limited and performances decreased drastically beyond 2.00 g/L of phenol.     

Performance of a fixed-bed reactor packed with carbon felt during anaerobic digestion of cellulose

The anaerobic digestion of cellulose was assessed in batch and semi-continuous studies using a carbon felt fixed-bed reactor. In the batch operation, the volatile solids reduction (%) and the cumulative methane production during the mesophilic and thermophilic digestion were 52.2% and 15.9%, 96.7 and 49.2 ml/g-total solid fed, respectively. After 99 days of semi-continuous mesophilic digestion, the degradation of cellulose reached its highest level of 67.6% at the hydraulic retention time of 9 days. The methane production and methane concentration of biogas from the bioreactor were maintained at a steady state. The fixed-bed reactor with carbon felt would be suitable for the efficient anaerobic digestion of cellulose. The biomass distribution in the reactor was, in the liquid phase 0.73 g/l-reactor, in the felt 1.59 g/l-reactor, and on the felt surface 9.86 g/l-reactor, which indicated that most of the microbes were immobilized on the carbon felt fixed-bed in the reactor.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Effect of cellulose fine structure on kinetics of its digestion by mixed ruminal microorganisms in vitro

The digestion kinetics of a variety of pure celluloses were examined by using an in vitro assay employing mixed ruminal microflora and a modified detergent extraction procedure to recover residual cellulose. Digestion of all of the celluloses was described by a discontinuous first-order rate equation to yield digestion rate constants and discrete lag times. These kinetic parameters were compared with the relative crystallinity indices and estimated accessible surface areas of the celluloses. For type I celluloses having similar crystallinities and simple nonaggregating particle morphologies, the fermentation rate constants displayed a strong positive correlation (r2 = 0.978) with gross specific surface area; lag time exhibited a weaker, negative correlation (r2 = 0.930) with gross specific surface area. Crystallinity was shown to have a relatively minor effect on the digestion rate and lag time. Swelling of microcrystalline cellulose with 72 to 77% phosphoric acid yielded substrates which were fermented slightly more rapidly than the original material. However, treatment with higher concentrations of phosphoric acid resulted in a more slowly fermented substrate, despite a decrease in crystallinity and an increase in pore volume. This reduced fermentation rate was apparently due to the partial conversion of the cellulose from the type I to the type II allomorph, since mercerized (type II) cellulose was also fermented more slowly, and only after a much longer lag period. The results are consistent with earlier evidence for the cell-associated nature of cellulolytic enzymes of ruminal bacteria and suggest that ruminal microflora do not rapidly adapt to utilization of celluloses with altered unit cell structures.     

Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion

The preparation of biocatalysts based on immobilized trypsin is of great importance for both proteomic research and industrial applications. Here, we have developed a facile method to immobilize trypsin on hydrophobic cellulose-coated silica nanoparticles by surface adsorption. The immobilization conditions for the trypsin enzyme were optimized. The as-prepared biocatalyst was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and elemental analysis. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and denaturants. In addition, the immobilized trypsin showed good durability for multiple recycling. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of two widely used protein substrates: bovine serum albumin (BSA) and cytochrome c. The surface adsorption protocols for trypsin immobilization may provide a promising strategy for enzyme immobilization in general, with great potential for a range of applications in proteomic studies.