Transformations to reduce the effect of particle size in mid-infrared spectra of biomass.

Fourier Transform InfraRed (FTIR) spectroscopy is a very powerful technique for the characterization of the chemical composition of biomass and its modifications occurring during thermochemical and chemical pretreatments. However, method development is necessary to generate reproducible signals that can be used in combination with multivariate techniques (such as principal component analysis, PCA) to extract meaningful information on biomass composition and bond cleavage. Particle size is a great source of spectra variability in FTIR of biomass.

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function.

Revisiting size-exclusion chromatography for measuring structural changes in raw and pretreated mixed hardwoods and switchgrass.

The study of the biomass porous structure and its role in defining the accessibility of cell-wall-degrading enzymes (CWDEs) to the substrate is very important for understanding the cellulase-cellulose reaction system. Specific pore volume and specific surface area are two important measures of accessibility and a variety of methods have been used to make these measurements. For this study a size exclusion chromatography system was developed to measure specific pore volume and specific surface areas for raw and pretreated mixed-hardwood and switchgrass.

Modeling enzymatic hydrolysis of lignocellulosic substrates using confocal fluorescence microscopy I: filter paper cellulose.

Enzymatic hydrolysis is one of the critical steps in depolymerizing lignocellulosic biomass into fermentable sugars for further upgrading into fuels and/or chemicals. However, many studies still rely on empirical trends to optimize enzymatic reactions. An improved understanding of enzymatic hydrolysis could allow research efforts to follow a rational design guided by an appropriate theoretical framework.

Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C).

Hierarchical hybrid peroxidase catalysts for remediation of phenol wastewater.

We report a new family of hierarchical hybrid catalysts comprised of horseradish peroxidase (HRP)-magnetic nanoparticles for advanced oxidation processes and demonstrate their utility in the removal of phenol from water. The immobilized HRP catalyzes the oxidation of phenols in the presence of H2 O2 , producing free radicals. The phenoxy radicals react with each other in a non-enzymatic process to form polymers, which can be removed by precipitation with salts or condensation.

Revising the Representation of Fatty Acid, Glycerolipid, and Glycerophospholipid Metabolism in the Consensus Model of Yeast Metabolism.

Saccharomyces cerevisiae , Supplementary Data S1.

Version 6 of the consensus yeast metabolic network refines biochemical coverage and improves model performance.

Updates to maintain a state-of-the art reconstruction of the yeast metabolic network are essential to reflect our understanding of yeast metabolism and functional organization, to eliminate any inaccuracies identified in earlier iterations, to improve predictive accuracy and to continue to expand into novel subsystems to extend the comprehensiveness of the model. Here, we present version 6 of the consensus yeast metabolic network (Yeast 6) as an update to the community effort to computationally reconstruct the genome-scale metabolic network of Saccharomyces cerevisiae S288c. Yeast 6 comprises 1458 metabolites participating in 1888 reactions, which are annotated with 900 yeast genes encoding the catalyzing enzymes.

Investigation of the porous structure of cellulosic substrates through confocal laser scanning microscopy.

At the most fundamental level, saccharification occurs when cell wall degrading enzymes (CWDEs) diffuse, bind to and react on readily accessible cellulose fibrils. Thus, the study of the diffusive behavior of solutes into and out of cellulosic substrates is important for understanding how biomass pore size distribution affects enzyme transport, binding, and catalysis. In this study, fluorescently labeled dextrans with molecular weights of 20, 70, and 150 kDa were used as probes to assess their diffusion into the porous structure of filter paper.

A pore-hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass.

Until now, most efforts to improve monosaccharide production from biomass through pretreatment and enzymatic hydrolysis have used empirical optimization rather than employing a rational design process guided by a theory-based modeling framework. For such an approach to be successful a modeling framework that captures the key mechanisms governing the relationship between pretreatment and enzymatic hydrolysis must be developed. In this study, we propose a pore-hindered diffusion and kinetic model for enzymatic hydrolysis of biomass.

Thermobifida fusca cellulases exhibit limited surface diffusion on bacterial micro-crystalline cellulose.

Elucidation of cellulase-cellulose interactions is key to modeling biomass deconstruction and in understanding the processes that lead to cellulase inactivation. Here, fluorescence recovery after photobleaching and single molecule tracking (SMT) experiments are used to assess the surface diffusion of Thermobifida fusca cellulases on bacterial micro-crystalline cellulose. Our results show that cellulases exhibit limited surface diffusion when bound to crystalline cellulose and that a large fraction of the cellulases remain immobile even at temperatures optimal for catalysis.

Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy.

Understanding the depolymerization mechanisms of cellulosic substrates by cellulase cocktails is a critical step towards optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 β-glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), which was immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A, which served as a reporter to track cellulase binding onto the physical structure of the cellulosic substrate.

Yeast 5 - an expanded reconstruction of the Saccharomyces cerevisiae metabolic network.

Background: Efforts to improve the computational reconstruction of the Saccharomyces cerevisiae biochemical reaction network and to refine the stoichiometrically constrained metabolic models that can be derived from such a reconstruction have continued since the first stoichiometrically constrained yeast genome scale metabolic model was published in 2003. Continuing this ongoing process, we have constructed an update to the Yeast Consensus Reconstruction, Yeast 5. The Yeast Consensus Reconstruction is a product of efforts to forge a community-based reconstruction emphasizing standards compliance and biochemical accuracy via evidence-based selection of reactions.

Two-temperature stage biphasic CO2-H2O pretreatment of lignocellulosic biomass at high solid loadings.

Most biomass pretreatment processes for monosaccharide production are run at low-solid concentration (<10 wt%) and use significant amounts of chemical catalysts. Biphasic CO(2) -H(2) O mixtures could provide a more sustainable pretreatment medium while using high-solid contents. Using a stirred reactor for high solids (40 wt%, biomass water mixture) biphasic CO(2)-H(2) O pretreatment of lignocellulosic biomass allowed us to explore the effects of particle size and mixing on mixed hardwood and switchgrass pretreatment.

Proteomic and physiological experiments to test Thermotoga neapolitana constraint-based model hypotheses of carbon source utilization.

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways.

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization.

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate.

Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy.

Cellulases are enzymes capable of depolymerizing cellulose. Understanding their interactions with cellulose can improve biomass saccharification and enzyme recycling in biofuel production. This paper presents a study on binding and binding reversibility of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A bound onto Bacterial Microcrystalline Cellulose.

Enzymatic transformations of cellulose assessed by quantitative high-throughput fourier transform infrared spectroscopy (QHT-FTIR).

Enzymatic hydrolysis of bacterial microcrystalline cellulose was performed with the thermophile enzyme system of Thermobifida fusca Cel5A (a classical endocellulase), Cel6B (a classical exocellulase), Cel9A (a processive endoglucanase), and a synergistic mixture of endo- and exocellulases. Different concentrations of enzymes were used to vary the extent of hydrolysis. Following standardization, the concentration of cellulose was directly correlated to the absorbance of the cellulose signals.

Integrating mixed microbial population dynamics into modeling energy transport during the initial stages of the aerobic composting of a switchgrass mixture.

A mathematical model which integrates empirically derived microbial growth kinetics with heat and mass transfer phenomena and substrate degradation kinetics has been developed to capture the dynamics of the aerobic composting of a switchgrass and dog food mixture over a period of 64 h. The model incorporated three microbial populations of yeasts, bacteria and fungi that metabolized composting material consisting of sugars and starches, cellulose and hemicelluloses to produce heat and utilize oxygen in a static, cylindrical reactor employing forced aeration. Model predictions captured well the dynamics obtained experimentally between physical and microbial variables and the model has the potential to become a predictive tool for substrate degradation during aerobic composting processes.

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi.

Background: The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T.

High-solids biphasic CO2-H2O pretreatment of lignocellulosic biomass.

A high pressure (200 bar) CO(2)-H(2)O process was developed for pretreating lignocellulosic biomass at high-solid contents, while minimizing chemical inputs. Hardwood was pretreated at 20 and 40 (wt.%) solids.

Labeling and purification of cellulose-binding proteins for high resolution fluorescence applications.

The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores.

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature, oxygen, and pH on hydrogen production.

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H(2)) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H(2) production at various growth temperatures, (2) investigate the effect of oxygen (O(2)) on H(2) production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85 degrees C to determine product yield coefficients and volumetric productivity rates.

An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates.

Immobilization of cellulose fibrils on solid substrates for cellulase-binding studies through quantitative fluorescence microscopy.

Cellulases, enzymes capable of depolymerizing cellulose polymers into fermentable sugars, are essential components in the production of bioethanol from lignocellulosic materials. Given the importance of these enzymes to the evolving biofuel industry considerable research effort is focused on understanding the interaction between cellulases and cellulose fibrils. This manuscript presents a method that addresses challenges that must be overcome in order to study such interactions through high-resolution fluorescence microscopy.