Cyst production and brown pigment formation in aging cultures of Azospirillum brasilense ATCC 29145

Encystation in Azospirillum brasilense ATCC 29145 was observed by using routine laboratory staining and phase-contrast and electron microscopy. Encystment occurred in liquid and in solid or semisolid media containing fructose (8 mM) and KNO3 (0.5 mM). The encysted forms consisted of a central body filled with poly-beta-hydroxybutyric acid granules, an electron-transparent intinelike region, and a thick outer layer. Enlarged giant encysted forms with multiple central bodies were also observed during the germination of a desiccated brown colony. Morphogenetically different forms in an aging culture could be resolved by sucrose density gradient centrifugation. The dense encysted forms along with numerous granules in a fibrillar network pelleted at 70% sucrose, while empty saclike envelopes along with vegetative cells and coccoid bodies pelleted at 55% sucrose. Different media induced various degrees of pigmentation in A. brasilense ATCC 29145 after aging. The pigment possessed several of the properties reported for microbial melanins, including insolubility in water and organic solvents, solubility in cold and hot alkali, and bleaching in hydrogen peroxide. The UV absorption maxima of the alkali extract were at 280 and 310 nm. Electron micrographs of the brown pigment showed that it occurred as aggregated granules surrounding the encysting cells as well as being excreted into the medium in an aging culture. It is concluded that A. brasilense ATCC 29145 produces compounds that form a brown pigment similar to melanin and are expressed under the influence of certain cultural conditions conducive for encystment.