Biodiesel fuel is favored as a type of carbon neutral energy. To popularize its usage, by-product waste glycerol utilization is a critical problem. We tried to isolate waste glycerol utilizing bacteria, and obtained the alkalo-and halophile bacteria Halomonas sp. KM-1. This strain produced bioplastic poly(3-hydroxybutyrate) (PHB) in a simple medium and diluted waste glycerol as a sole carbon source.
Production of (R)-3-hydroxybutyric acid [(R)-3-HB] by strain Halomonas sp. KM-1 under successive aeration conditions was investigated. The first aerobic condition allowed both cell growth and intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB). The second microaerobic condition, achieved by reducing the culture agitation rate, lead to the degradation of PHB to (R)-3-HB. The amount of PHB stored in KM-1 cells after 48-h cultivation under aerobic conditions was 16.4 g/l. In contrast, after a shift from aerobic to microaerobic conditions and a further 18-h cultivation, PHB content in KM-1 cells decreased to 0.9 g/l. Numerous intracellular PHB-containing granules were observed in cells under aerobic conditions by electron microscopy. After the shift to microaerobic conditions, the number and size of granules were significantly reduced, in agreement with the degradation of prestored PHB. On the other hand, under microaerobic conditions, the concentration of (R)-3-HB in the medium reached a maximum of 15.2 g/l, indicating the production and extracellular secretion of (R)-3-HB as a result of PHB digestion. Notably, cell lysis was not observed during the successive aeration conditions as assessed by elution of genomic DNA to the culture supernatant, cell morphology observed by electron microscopy and counts of colony formation. In this simple system utilizing a change of aeration during cultivation of strain Halomonas sp. KM-1, we obtained one of the highest levels of microbiological production of (R)-3-HB reported to date.
The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize both hexose and pentose sugars for the intracellular storage of bioplastic poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. In this study, we investigated the effects of the sodium nitrate concentration on PHB accumulation in the KM-1 strain. Unexpectedly, we observed the secretion of pyruvate, a central intermediate in carbon- and energy-metabolism processes in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for the production of crop-protection agents, polymers, cosmetics, and food additives. We then further analyzed pyruvate productivity following changes in culture temperature and the buffer concentration. In 48-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 63.3 g/L pyruvate at a rate of 1.32 g/(L·h), comparable to the results of former studies using mutant and recombinant microorganisms. Thus, these data provided important insights into the production of pyruvate using this novel strain.
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