Enhanced Conversion of Lignocellulose to Biofuels: Bioprocess Optimization from Cellulose Hydrolysis to Product Fermentation

Lead Project PIs: Douglas S. Clark, Harvey W. Blanch, Frank T. Robb
Lead Campus: UC Berkeley

The goal of the present project is to develop new experimental systems to study cellulosome degradation of cellulosic biomass. At present, it has not been possible to perform structure-function studies on intact cellulosomes due to their large size and heterogeneous composition in available model organisms. It has also been difficult to probe the nature of the synergy between cellulosomal proteins that results in a more efficient breakdown of cellulose when compared to uncomplexed cellulases 1 . Furthermore, since the substrate for cellulosomes is a solid, the cellulosome is not easily studied by standard enzymological approaches. We propose to develop model systems of the cellulosome that will enable us to study its enzymatic properties at a fundamental level. Understanding the molecular mechanisms used by cellulosomes may provide the key insights needed to reconstitute “designer cellulosomes” 2 optimized for the depolymerization of the complex plant biomass envisioned as the feedstock for biofuel production. This work will further an important goal of the EBI, to improve conversion of lignocellulosic biomass into useful liquid biofuels.

We have developed a series of inter-related experiments to study the degradation of cellulose by Clostridium species cellulosomes. The specific aims of the proposal are:

1. Clostridium microbiology and genomics. We will use the genomic sequence of Clostridium species, as well as biochemical and analytical means, to unravel the regulation of cellulosome composition, expression, export, and assembly as a function of growth condition.

2. Enzymology. We will develop model substrates to quantitatively probe the activities of the various cellulosomal components and assemblies. We will also use tandem mass spectrometry to simultaneously determine cellulosome composition and carbohydrate interactions. Finally, we will develop a real-time spectrophotometric assay for cellulase activity.

3. Single molecule and AFM studies. We will track the movement of individual cellulosomes on both cellulose and plant biomass. In parallel, we will image substrate surfaces by atomic force microscopy (AFM) and monitor changes in topography as a function of enzymatic degradation. The combination of cellulosome tracking and AFM analysis of substrate surfaces will be a powerful approach to identify the dynamics of lignocellulose depolymerization.

Our program will address several of the major bottlenecks impeding the practical production of biofuels, such as ethanol and butanol, from cellulosic feedstocks. The program has several inter-related components, which will interface closely with complementary research performed throughout the EBI. The scope of the program spans the discovery and application of new thermophilic organisms as enzyme sources and/or for biofuel production, protein engineering and kinetic modeling of improved cellulases, cellular engineering for improved solvent tolerance, and bioprocess engineering to optimize fermentation. The specific components of the program are summarized below.

Bioprospecting for High-Temperature Conversion of Lignocellulose to Ethanol

Lignocellulose degradation systems from extremely thermophilic microorganisms are ideal candidates for the development of more active, cost-effective enzymes for cellulose processing. Elevated operating temperatures would also be beneficial in fermentations to produce biofuels. In addition to lower risk of microbial contamination, a higher temperature would reduce cooling costs and facilitate ethanol (or, for example, butanol) removal and recovery. To enable translation of these advantages to practice, we propose to isolate and characterize multisubunit extracellular and periplasmic glycolytic enzymes in several extremely thermophilic bacterial strains specifically adapted for cellulose and hemicellulose degradation. We will also isolate,novel extreme thermophiles that produce ethanol and/or butanol from enrichments of hot spring samples previously collected in Eastern Russia and the continental US. Prospecting for cellulose/hemicellulose degradation systems will be assisted by whole genome sequencing of novel isolates. Another component of the proposed effort is the development of a simultaneous saccharification and fermentation process for operation near the boiling point of ethanol. Ethanol production during saccharification of cellulose/hemicellulose at 75ºC will increase process efficiency, minimize contamination, and facilitate evaporative removal of the fuel product. Individual thermophilic bacterial strains with high rates of specific lignocellulose digestion will be sequenced and the cellulose and xylanase genes will be expressed in productive recombinant host strains.

High-Throughput Solid-Substrate Cellulolytic Screens and Directed Evolution of Improved Cellulases

Protein engineering has proven to be a powerful tool in creating enzymes with new and improved properties; however, designing and employing methods to screen or select cellulase mutants using solid cellulosic substrates remains a largely unmet challenge. This proposal seeks to overcome this challenge, as well as that of developing more cost-effective cellulases, by developing high-throughput solid substrate assays and applying them in the directed evolution of thermophilic cellulases (e.g., T opt = 80ºC). The methodology developed will be applicable to the generation and study of improved cellulases that can be used in various process configurations for the production of biofuels from cellulosic biomass.

Mechanistic Kinetic Modeling for Optimal Cellulase Design and Cellulose Hydrolysis

Figure 1. The celluose chain is randomly cleaved by endo-cellulase to provide both reducing and non-reducing free ends. Exocellulases bind to these and release cellobiose which is subsequently hydrolyzed to glucose monomers.

Accurate kinetic models of cellulose hydrolysis by cellulases (Figure 1) are of critical importance for evaluating cellulase-component compositions and for designing and optimizing processes for cellulose conversion to biofuels. Such models will also aid in the development and characterization of improved cellulolytic systems generated by protein engineering and synthetic biology. We propose to develop a comprehensive model of cellulose hydrolysis that can be used to predict cellulase performance, guide cellulase design, and optimize the hydrolysis of various cellulosic substrates, including those obtained from EBI investigators. Reaction rate data will be collected in batch reactors for mesophilic and thermophilic cellulases and analyzed using the model to determine the kinetics of hydrolysis and the corresponding rate law parameters. 

Alleviating Product Toxicity in Biofuel Production
 

Figure 2. Schematic design of experimental apparatus for continuous in situ extraction of biofuels during fed-batch fermentation.