Pyruvic Acid Production Using Thermotolerant Halomonas sp. KM-1

One of most important processes in industrial fermentation is temperature controlling of fermenters, actually huge electric energy is used to cool the fermenters to appropriate temperatures.  While massive consumption of energy can become a factor causing climate change, it must be considered to decrease energy consumption in any fields, including even in the fields known as white-biotechnology, which requires less energy and creates less waste and environmental impact during the fermentative production.  Thereupon we have attempted to develop a fermentation system without or with less temperature controlling using thermotolerant microbes.  Halomonas sp. KM-1, isolated as a contaminant of Spirulina platensis culture, is an alkaliphilic, halophilic, aerobic, thermophilic, and heterotrophic bacterium requesting no organic nutrients including amino acids and vitamins except a carbon source for its cultivation. The KM-1 strain, which characteristics provide useful features, i.e. no requirement of sterilization and less cost for fermentation, accumulates poly-3-hydroxybutyrate (PHB) under aerobic fermentation and secret its monomer, 3-hydroxybutyrate (3-HB) under successive microaerobic one using varied carbon sources.  Besides PHB and 3-HB, 2-oxopropanoic acid (pyruvic acid) was found to be accumulated in media of KM-1 culture using glucose as a carbon source.  Pyruvic acid is known for having numerous benefits as a fine chemical compound to produce medicines and cosmetic products. It is also a relatively new supplement for human to decrease in body weight and fat mass and for athletes as an ergogenic compound to improve endurance during aerobic activity and increase ATP/energy production, but the roles are still controversial.  To produce pyruvic acid at a commercial level using an environmentally benign process, we attempted to optimize the production of pyruvic acid by Halomonassp. KM-1 and selected better mutants derived from the strain mutagenized using UV irradiation.  Under a small-scale culturing of the KM-1 using 10% glucose at 33˚C, pyruvic acid was accumulated up to 2% in the culture medium after 2-day incubation, and vanished within another 2-day culturing.  First we tested a variety of carbon sources, their concentrations, and culturing temperatures.  As a result, we could clarify a condition under which pyruvic acid was accumulated up to 5% in the culture at 45˚C and no diminution of the product was observed even after longer incubation.  We have selected mutants, which could not grow in media including glucose as a sole carbon source.  Based on tests of growth on different carbon sources and productivities of pyruvic acid, it was shown that the mutants carried mutations on sugar transporters and enzymes for the glycolytic pathway. We also isolated several dozens of thermotolerant mutants, which could grow at 47˚C. Those mutants might be useful to produce pyruvic acid or other chemicals in the glycolytic pathway at high temperatures, and capabilities of those mutants will be presented in this meeting.

This work was financially supported by the Advanced Low Carbon Technology Research and Development Program (ALCA), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKEN-HI: 22510222).