A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbonlimitation in continuous culture. 1.3 mmoles of citrate were consumed per 450 mg of biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry weight. The inclusion bodies were stained with Sudan Black and Nile Red, suggesting a lipid material which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by gas chromatography and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with Nile Red fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increased overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.
A species of Serratia bacteria produces nano-crystalline hydroxyapatite (HA) crystals by use of a cell-bound phosphatase enzyme, located both periplasmically and within extracellular polymeric materials. The enzyme functions in resting cells by cleaving glycerol-2-phosphate (G-2-P) to liberate free phosphate ions which combine with calcium in solution to produce a cell-bound calcium phosphate material. Bacteria grown as a biofilm on polyurethane reticulated foam cubes were challenged with calcium and G-2-P in a bioreactor to produce a 3-D porous bone-substitute material. The scaffold has 1 mm macropores and 1 microm micropores. XRD showed the crystallites to be 25-28 nm in size, resembling HA before sintering and beta-tricalcium phosphate (beta-TCP, whitlockite) after. When biofilm was grown on titanium discs and challenged with calcium and G-2-P, a calcium phosphate layer formed on the discs. Biomineralisation is therefore a potential route to production of precursor nanophase HA, which has the potential to improve strength. The scaffold material produced by this method could be used as a bone-filler or as an alternative method for coating implants with a layer of HA.
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