Growth and PHA production of wild-type Ralstonia eutropha H16 using glucose and fructose as single carbon substrates

Ralstonia eutropha H16 is a bacterial species of considerable industrial importance for the production of polyhydroxyalkanoates (PHA) as renewable and biodegradable substitutes of plastic materials. The study presented here is part of a larger Systems Biology project on PHA metabolism using a combination of mathematical modeling and bioreactor experiments. In a series of cultivation experiments for deriving the initial data sets, some interesting observations were made about biosynthesis and degradation of PHA and particularly concerning the utilizable carbohydrate substrates. Cultivations were performed with either fructose and glucose as single growth substrates, respectively. While fructose is well known to be an appropriate carbon source for R. eutropha H16, the utilization of glucose by the wild-type strain was quite surprising. A deficiency of utilizing glucose is commonly considered as a drawback for the industrial application of the R. eutropha wild-type strain H16 (DSMZ 428) employed during our study. Hence, in commercial processes, mutated glucose-utilizing strains obtained spontaneously or after mutagenic treatment were applied previously to overcome this limitation. The unexpected growth of the wild-type with glucose was initially observed only after prolonged incubation (> 100 h) at high (2%) glucose concentrations but could be sustained by sub-culturing these cultures also at low (0.2 %) glucose concentrations. While the specific growth rate was much lower than with fructose, the final cell densities and biomass/substrate yields were similar with both substrates. Control experiments were performed to rule out obvious explanations for the unprecedented growth with glucose such as spontaneous mutants and contaminated cultures as well as to confirm the identity of the employed strain. After repetitive sub-culturing in glucosefree complex medium, glucose utilization was again severely attenuated. In conclusion, the observed growth pattern showed the characteristics of a reversible adaptive response to the presence of glucose. The effect proved to be reproducible with three independent strains obtained from different culture collections. In addition to the unusual growth pattern, dynamic responses of PHA biosynthesis as well as degradation to variations of the ammonia/glucose supply are presented