Engineering thermostable fungal cellobiohydrolases

Meeting the world's growing energy demands while protecting our fragile environment is a challenging issue. Second generation biofuels are liquid fuels like long-chain alcohols produced from lignocellulosic biomass. To reduce the cost of biofuel production, we engineered fungal family 6 cellobiohydrolases (Cel6A) for enhanced thermostability using random mutagenesis and recombination of beneficial mutations. During long-time hydrolysis, engineered thermostable cellulases hydrolyze more sugars than wild-type Cel6A as single enzymes and binary mixtures at their respective optimum temperatures. Engineered thermostable cellulases exhibit synergy in binary mixtures similar to wild-type cellulases, demonstrating the utility of engineering individual cellulases to produce novel thermostable mixtures. Crystal structures of the engineered thermostable cellulases indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by proline substitutions. At high temperature, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A and wild-type Cel6A. The mechanism of thermal inactivation in this cellulase family is consistent with disulfide bond degradation and thiol-disulfide exchange. Enhancing the thermostability of Cel6A also increases tolerance to pretreatment chemicals, demonstrated by the strong correlation between thermostability and tolerance to 1-ethyl-3-methylimidazolium acetate. Several semi-rational protein engineering approaches – on the basis of consensus sequence analysis, proline stabilization, FoldX energy calculation, and high B-factors – were evaluated to further enhance the thermostability of Cel6A.

Earth-abundant materials for solar hydrogen generation

A critical challenge for the 21st century is shifting from the predominant use of fossil fuels to renewables for energy. Among many options, sunlight is the only single renewable resource with sufficient abundance to replace most or all of our current fossil energy use. However, existing photovoltaic and solar thermal technologies cannot be scaled infinitely due to the temporal and geographic intermittency of sunlight. Therefore efficient and inexpensive methods for storage of solar energy in a dense medium are needed in order to greatly increase utilization of the sun as a primary resource. For this purpose we have proposed an artificial photosynthetic system consisting of semiconductors, electrocatalysts, and polymer membranes to carry out photoelectrochemical water splitting as a method for solar fuel generation.

This dissertation describes efforts over the last five years to develop critical semiconductor and catalyst components for efficient and scalable photoelectrochemical hydrogen evolution, one of the half reactions for water splitting. We identified and developed Ni–Mo alloy and Ni₂P nanoparticles as promising earth-abundant electrocatalysts for hydrogen evolution. We thoroughly characterized Ni–Mo alloys alongside Ni and Pt catalysts deposited onto planar and structured Si light absorbers for solar hydrogen generation. We sought to address several key challenges that emerged in the use of non-noble catalysts for solar fuels generation, resulting in the synthesis and characterization of Ni–Mo nanopowder for use in a new photocathode device architecture. To address the mismatch in stability between non-noble metal alloys and Si absorbers, we also synthesized and characterized p-type WSe₂ as a candidate light absorber alternative to Si that is stable under acidic and alkaline conditions.

Sustainable energy solutions for irrigation and harvesting in developing countries

One of the critical problems currently being faced by agriculture industry in developing nations is the alarming rate of groundwater depletion. Irrigation accounts for over 70% of the total groundwater withdrawn everyday. Compounding this issue is the use of polluting diesel generators to pump groundwater for irrigation. This has made irrigation not only the biggest consumer of groundwater but also one of the major contributors to green house gases. The aim of this thesis is to present a solution to the energy-water nexus. To make agriculture less dependent on fossil fuels, the use of a solar-powered Stirling engine as the power generator for on-farm energy needs is discussed. The Stirling cycle is revisited and practical and ideal Stirling cycles are compared. Based on agricultural needs and financial constraints faced by farmers in developing countries, the use of a Fresnel lens as a solar-concentrator and a Beta-type Stirling engine unit is suggested for sustainable power generation on the farms. To reduce the groundwater consumption and to make irrigation more sustainable, the conceptual idea of using a Stirling engine in drip irrigation is presented. To tackle the shortage of over 37 million tonnes of cold-storage in India, the idea of cost-effective solar-powered on-farm cold storage unit is discussed.

Carbon-carbon bond forming reactions from bis(carbene)-platinum(II) complexes and olefin polymerization and oligomerization using group 4 post-metallocene complexes

A long-standing challenge in transition metal catalysis is selective C–C bond coupling of simple feedstocks, such as carbon monoxide, ethylene or propylene, to yield value-added products. This work describes efforts toward selective C–C bond formation using early- and late-transition metals, which may have important implications for the production of fuels and plastics, as well as many other commodity chemicals.

The industrial Fischer-Tropsch (F-T) process converts synthesis gas (syngas, a mixture of CO + H₂) into a complex mixture of hydrocarbons and oxygenates. Well-defined homogeneous catalysts for F-T may provide greater product selectivity for fuel-range liquid hydrocarbons compared to traditional heterogeneous catalysts. The first part of this work involved the preparation of late-transition metal complexes for use in syngas conversion. We investigated C–C bond forming reactions via carbene coupling using bis(carbene)platinum(II) compounds, which are models for putative metal–carbene intermediates in F-T chemistry. It was found that C–C bond formation could be induced by either (1) chemical reduction of or (2) exogenous phosphine coordination to the platinum(II) starting complexes. These two mild methods afforded different products, constitutional isomers, suggesting that at least two different mechanisms are possible for C–C bond formation from carbene intermediates. These results are encouraging for the development of a multicomponent homogeneous catalysis system for the generation of higher hydrocarbons.

A second avenue of research focused on the design and synthesis of post-metallocene catalysts for olefin polymerization. The polymerization chemistry of a new class of group 4 complexes supported by asymmetric anilide(pyridine)phenolate (NNO) pincer ligands was explored. Unlike typical early transition metal polymerization catalysts, NNO-ligated catalysts produce nearly regiorandom polypropylene, with as many as 30-40 mol % of insertions being 2,1-inserted (versus 1,2-inserted), compared to <1 mol % in most metallocene systems. A survey of model Ti polymerization catalysts suggests that catalyst modification pathways that could affect regioselectivity, such as C–H activation of the anilide ring, cleavage of the amine R-group, or monomer insertion into metal–ligand bonds are unlikely. A parallel investigation of a Ti–amido(pyridine)phenolate polymerization catalyst, which features a five- rather than a six-membered Ti–N chelate ring, but maintained a dianionic NNO motif, revealed that simply maintaining this motif was not enough to produce regioirregular polypropylene; in fact, these experiments seem to indicate that only an intact anilide(pyridine)phenolate ligated-complex will lead to regioirregular polypropylene. As yet, the underlying causes for the unique regioselectivity of anilide(pyridine)phenolate polymerization catalysts remains unknown. Further exploration of NNO-ligated polymerization catalysts could lead to the controlled synthesis of new types of polymer architectures.

Finally, we investigated the reactivity of a known Ti–phenoxy(imine) (Ti-FI) catalyst that has been shown to be very active for ethylene homotrimerization in an effort to upgrade simple feedstocks to liquid hydrocarbon fuels through co-oligomerization of heavy and light olefins. We demonstrated that the Ti-FI catalyst can homo-oligomerize 1-hexene to C₁₂ and C₁₈ alkenes through olefin dimerization and trimerization, respectively. Future work will include kinetic studies to determine monomer selectivity by investigating the relative rates of insertion of light olefins (e.g., ethylene) vs. higher α-olefins, as well as a more detailed mechanistic study of olefin trimerization. Our ultimate goal is to exploit this catalyst in a multi-catalyst system for conversion of simple alkenes into hydrocarbon fuels.