Local degradation of structural, mechanical, electrical and chemical properties of membrane electrode assembly in polymer electrolyte fuel cell

Polymer electrolyte fuel cell (PEMFC) is promising source of clean power in many applications ranging from portable electronics to automotive and land-based power generation. However, widespread commercialization of PEMFC is primarily challenged by degradation. The mechanisms of fuel cell degradation are not well understood. Even though the numbers of installed units around the world continue to increase and dominate the pre-markets, the present lifetime requirements for fuel cells cannot be guarantee, creating the need for a more comprehensive knowledge of material’s ageing mechanism. The objective of this project is to conduct experiments on membrane electrode assembly (MEA) components of PEMFC to study structural, mechanical, electrical and chemical changes during ageing and understanding failure/degradation mechanism. The first part of this project was devoted to surface roughness analysis on catalyst layer (CL) and gas diffusion layer (GDL) using surface mapping microscopy. This study was motivated by the need to have a quantitative understanding of the GDL and CL surface morphology at the submicron level to predict interfacial contact resistance. Nanoindentation studies using atomic force microscope (AFM) were introduced to investigate the effect of degradation on mechanical properties of CL. The elastic modulus was decreased by 45 % in end of life (EOL) CL as compare to beginning of life (BOL) CL. In another set of experiment, conductive AFM (cAFM) was used to probe the local electric current in CL. The conductivity drops by 62 % in EOL CL. The future task will include characterization of MEA degradation using Raman and Fourier transform infrared (FTIR) spectroscopy. Raman spectroscopy will help to detect degree of structural disorder in CL during degradation. FTIR will help to study the effect of CO in CL. XRD will be used to determine Pt particle size and its crystallinity. In-situ conductive AFM studies using electrochemical cell on CL to correlate its structure with oxygen reduction reaction (ORR) reactivity

Enhancement of heterologous expression of alkaline phytase in Pichia pastors

Phytic acid is the major storage form of phosphorus and inositol in seeds and legumes. It forms insoluble phytate salts by chelating with positively charged mineral ions. Non-ruminant animals are not able to digest phytate due to the lack of phytases in their GI tracks, thus the undigested phytate is excreted leading to environmental contamination. Supplementation with phytases in animal feed has proven to be an effective strategy to alleviate nutritional and environmental issues. The unique catalytic and thermal stability properties of alkaline phytase from lily pollen (LlALP) suggest that it has the potential to be useful as a feed supplement. Our goal is to develop a method for the production of substantial amounts of rLlALP for animal feed and structural studies. rLlALP2 has been successfully expressed in the yeast, Pichia pastoris. However, expression yield was modest (8-10 mg/L). Gene copy number has been identified as an important parameter in enhancing protein yields. Multicopy clones were selected using Zeocin-resistance-based vectors and challenging transformants to high Zeocin levels under different conditions. Data indicate that increasing selection pressure led to the generation of clones with amplification of both rLlAlp2 and Zeor genes and the two genes were not equally amplified. Additionally, clones generated by step-wise methods led to clones with greater amplification. The effects of transgene copy number and gene sequence optimization on expression levels of rLlALP2 were examined. The data indicate that increasing the copy number of rLlAlp2 in transformed clones was detrimental to expression level. The use of a sequence-optimized rLlAlp2 (op-rLlAlp2) increased expression yield of the active enzyme by 25-50%, suggesting that transcription and translation efficiency are not major bottlenecks in the production of rLlALP2. Lowering induction temperature to 20 oC led to an increase in enzyme activity of 1.2 to 20-fold, suggesting that protein folding or post-translational processes may be limiting factors for rLlALP2 production. Cumulatively, optimization of copy number, gene sequence optimization and reduced temperature led to increase of rLlALP2 enzyme activity by three-fold (25-30 mg/L). In an effort to simplify the purification process of rLlALP2, extracellular expression of phytase was investigated. Extracellular expression is dependent on the presence of an appropriate secretion signal upstream of the transgene native signal peptide(s) present in the transgene may also influence secretion efficiency. The data suggest that deletion of both N- and C-terminal signal peptides of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion of LlALP2 by four-fold. The secretion signal peptide of chicken egg white lysozyme was ineffective in secretion rLlALP2 in P. pastoris. To enhance rLlALP2 secretion, effectiveness of the strong inducible promoter (PAOX1) was compared with the constitutive promoter (PGAP). The intracellular yield of rLlALP2 was about four-fold greater under the control of PGAP compared to PAOX1 and extracellular expression level of rLlALP2 was around eight-fold (75-100 mg/L) greater. The successful production of active rLlALP2 in P. pastoris will allow us to conduct the animal feed supplementation studies and structural studies.