Characterisation of a Novel Nitrogen-Fixing, Polyhydroxyalkanoate-Producing Bacterium, Novosphingobium Nitrogenifigens Y88T

The novel sphingomonad Novosphingobium nitrogenifigens Y88T (Y88T) is an obligate aerobe able to grow in nutrient-imbalanced environments where nitrogen is naturally limiting, but carbon is found in abundance. Due to its ability to fix atmospheric nitrogen and produce the bioplastic polyhydroxyalkanoate (PHA), Y88T is well-suited for growth in a nitrogenlimited but carbon-enriched environment. Because of these metabolic abilities, Y88T is of interest as a model organism for PHA production unconstrained by nitrogen-limiting conditions. Growth profiles and PHA production profiles were determined for Y88T under conditions of carbon enrichment, nitrogen sufficiency and depletion to investigate carbon and nitrogen utilisation as well as PHA production in this organism. Also, since the nitrogenase enzyme required for nitrogen fixation is oxygen labile, the effect of DO concentration and the relationship between aerobic metabolism and the nitrogen-fixing and PHA-producing abilities of Y88T was investigated. This study demonstrated: that glucose is the preferred growth substrate for Y88T; that no direct relationship exists between nitrogen fixation and PHB accumulation in Y88T; that Y88T can reliably produce in excess of 80 % of its dry weight as polyhydroxybutyrate (PHB), a type of PHA, from glucose under nitrogenlimiting conditions. Proteomic signatures were determined for the various physiological responses of Y88T to growth, nitrogen utilisation, PHB production and exposure to different levels of DO. More than 250 unique proteins, including the core nitrogen-fixation, PHB-synthetic and glycolytic proteins were identified. Y88T apparently converts glucose to PHB via three interrelated glucose catabolic pathways and proteins likely involved in these pathways were identified. This study revealed that, regardless of growth conditions and despite decreased abundance of the Y88T nitrogenase enzyme, growth and PHB synthesis were not inhibited at DOhigh concentrations. Proteomic characterisation of the Y88T phasin, a PHA granule-associated protein, iii identified an amino-terminal, low complexity alanine and proline rich segment found only in other sphingomonads. The expression level of the Y88T phasin correlated well with PHB yields, suggesting the use of this protein as a biomarker to optimise PHB yield in a production environment. Y88T has the potential to be a useful production strain in pure culture, utilising its natural and robust propensity to metabolise glucose to preferentially produce PHB. Targets for biotechnological improvement and the potential for application of Y88T to biofuel production are discussed.