Intensified biocatalysis for production of fuel and chemicals from lipids

This thesis research aims at developing novel biocatalytic conversion of triglycerides and glycerol for the production of fuels and chemicals. This research explored a unique approach by developing organic-soluble enzymes for a one-pot synthesis-and-use strategy. Specifically, a lipase, lipase AK, was selected and chemically modified to enhance the solubility in organic media in order to catalyze a homogeneous transesterification reaction for maximum reaction velocities. The productivities of the modified lipase in a water-free reaction system were found to be over two orders of magnitude higher than previously reported results in literature. The effects of several key factors including water content, temperature, and solvent were examined for the solubilized enzyme in comparison with several other commercially available lipases.

To simplify purification and immobilization process of biocatalyst and thus reducing overall production cost, a recombinant glycerol dehydrogenase (GDH) tagged with 6xHis was produced. The successful insertion of gldA gene into a pET151/D-TOPO vector with T7 promoter was confirmed through PCR amplification, DNA sequencing, and restriction digestion. Following the transfection, the BL21(DE3) E. coli strain produced GDH dominantly in the induced culture, with yields over 10 mg of enzyme per gram of cell paste. The success in expression of recombinant GDH was evaluated with immunodetection and amino acid sequencing. The specific activity (80 unit/mg) and stability of isolated GDH were also examined. The activity of the recombinant GDH was slightly higher than that of commercially available products.

In this research the development of 3-D carbon material by using carbonized of cellulosic fibers with branched CNT was achieved. The specific surface area of the resulted fibers reached 291 m²/g as determined by using BET method, about 30-fold of improvement over regular carbon fibers. It was also discovered that this novel material showed improved electrochemical activity toward oxidation of NADH. The oxidation potential of NADH decreased from above 0.8 V to 0.6 V as a result of the introduction of CNT. Mediated cofactor regeneration methods were also investigated by using Meldola's blue and poly(methylene green). Both approaches can reduce the oxidation potential of NADH to 0.1 V desired to avoid inactivation and dimerization of the cofactor in addition to energy consumption.

To examine the efficiency of the targeted glycerol oxidation reaction for large scale applications, cofactor regeneration was tested for two reaction processes. In one process, glycerol oxidation reaction with immobilized GDH in a fixed bed bioreactor was operated along with a separated electrochemical cofactor regeneration unit. In a second process, regeneration of cofactor was realized in situ in the enzymatic glycerol transformation reactor.

To demonstrate the vast potentials of using the biorenewable carbon electrodes developed in this work for a variety of bioprocessing, research on using the carbon fibrous electrodes were conducted for biosensing and carbon capture. In one effort, a model microfiber biosensor was constructed to demonstrate the possibility of detecting biochemical compounds and enzymes by taking advantage of the ubiquity of NADH in biocatalysis. In another thrust, the reduction of carbon dioxide to formate was examined by applying the modified carbon material as a working electrode. The results showed that formate concentration was as high as 30 mM with over 50% current efficiency.

One potential advantage of using bioelectrochemical method for cofactor regeneration is the possibility to integrate the biochemical process with biofuel cells for simultaneous chemical production and power generation. That represents an exciting future technology. Toward that, this thesis explored the necessary fundamental issues, including the construction and study of a model glucose/oxygen biofuel cell. The kinetics of the glucose biofuel cell was investigated to determine the limiting factors. (Abstract shortened by UMI.)