Production of poly(3-hydroxybutyrate) and ectoines using a halophilic bacterium

Abstract: Halomonas boliviensis is a moderate halophilic bacterium, isolated from a soil sample around a hypersaline lake Laguna Colorada in the Andean region of Bolivia. The organism is capable of growing over a wide range of NaCl concentration (0-25 %, w/v), however with an optimum of about 4.5 % (w/v) NaCl. It is able to accumulate two useful products: poly(3-hydroxybutyrate) (PHB) and ectoines (ectoine and hydroxyectoine). H. boliviensis can accumulate PHB from several different carbon sources, e.g., glucose, xylose, sucrose, maltooligosacharides, sodium acetate and butyric acid, as well as from cheap substrate sources such as wheat bran hydrolysate and digested potato waste. A PHB content of 50-90 % of cell dry weight (wt%) can be reached depending on the carbon source and cultivation conditions used. Under nitrogen limitation, a maximum PHB content of 81 wt%, cell dry weight (CDW) of 44 g/L, and PHB volumetric productivity of 1.1 g/L/h were obtained in fed-batch culture with glucose as the carbon source. The PHB content reached by H. boliviensis was comparable to that of some of the known high-PHA producers. The organism accumulates ectoines in response to high salt concentrations in the surrounding medium. Ectoine has been detected at and above sodium chloride concentration of 5 % (w/v) while the presence of hydroxyectoine was observed from 10 % (w/v) NaCl. The content of the ectoines increased on raising the salt concentration. In order to improve the ectoine productivity, a mathematical method was applied with the aim of determining optimal conditions for ectoine production. The optimized conditions were then used for a two-step fed-batch culture, and it was found that the ectoine concentration and productivity were significantly increased, reaching maximum values of 9.2 g/L and 6.3 g/L/d, respectively. A two-step fed-batch culture was also applied for the co-production of ectoine and hydroxyectoine. By increasing the NaCl concentration to 18.5 % (w/v) in the second step, the content of ectoines was augmented to a maximum level of about 27.8 wt% with the relative proportion of hydroxyectoine at 57 %. The productivity of ectoines was also raised to 10 g/L/d, which is among the highest reported so far. Co-production of ectoine and PHB could be achieved by H. boliviensis in a two-step fed-batch culture. H. boliviensis was first grown under optimal conditions in order to achieve a high cell mass. The bacterial cells were then grown in a second fed-batch system at higher salt concentration and under nitrogen limitation for inducing ectoine and PHB accumulations. This process resulted in an ectoine content of 6.1 wt% and a productivity of 2.8 g/L/d, as well as a maximum PHB content of 68.5 wt% and a productivity of 1.06 g/L/h after an overall production time of 40 h. The PHB productivity was similar to that found during the production of PHB alone by H. boliviensis. The present study has demonstrated the protective effect of ectoine and hydroxyectoine on enzymes with respect to pH stress. A xylanase from Bacillus halodurans retained a significantly higher activity in the presence of ectoine and hydroxyectoine, during incubation at low pH (4.5) as well as at high pH (11, 12). It is suggested that the presence of ectoines results in reduced conformational flexibility of the biomolecules.