High cell density culture by Halobacterium salinarium mutant D96N and effective purification for bacteriorhodopsin production

Abstract

The cell membranes of a number of extremely halophilic bacteria, such as Halobacterium salinarium, contain highly organized patches of a purple membrane (PM) which functions as a light-driven proton pump. The purple membrane contains a single protein, - bacteriorhodopsin (MW, 26,500), this has recently been suggested to have several applications in the design of molecular electron devices and optical computers since it possesses desirable photophysical/chemical properties. To find out characteristics for cell growth and the productivity of BR, several batch fermentations, including 50L pilot scale, were performed by Halobacterium salinarium wild type strain. The average productivity of BR was 0.110 mg/Lㆍh in batch mode and there was little increase of productivity (0.145 mg/Lㆍh) in 1L batch fermentation when used high purity oxygen as supplied gas. Instead of fed-batch fermentation, repeated batch was selected as alternative for BR production. The result for productivity, in that case, was 10 times higher than batch one, but has not lasted after 72 hours (for 3-steps medium-feeding periods). The final average productivity of BR was 0.207 mg/Lㆍh in repeated batch mode. To remove inhibitory-product and give more stable condition to cell, continuous cell recycle culture with external membrane and air was carried out by Halobacterium salinarium wild type strain. The final amount of BR and productivity was 177mg/L, 0.96mg/Lㆍh, respectively. This amount of BR was 10 times higher than in a batch mode. Also experiment by Halobacterium salinarium mutant D96N with high purity oxygen was done and obtained the result, which was 21.5 times higher than in a batch mode and 2.41 times higher than in continuous mode with air. The purification process of protein from cells contain a lot of phases such as harvesting, disruption of cell, concentration of protein, polishing for high purity final product, removal of salts or impurities. The most important phase of them is a concen...