The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still tnaintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly p-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forins under limiting cultural conditions. Azospirillum spp. have been shown to be a common inhabitant of tropical and temperate soils and have also been shown to reduce acetylene in culture media and in association with the roots of forage grasses, cereals, and other plants (11, 19, 26). A. brasilense and A. lipoferum are known to grow well in culture media on a variety of carbon sources, including various sugars and organic acids (15, 24, 30, 32). These organisms, under aerobic conditions, also grow well on a variety of combined nitrogen sources, including ammonia, nitrate, or amino acids (20, 21, 22, 24). Recently, special attention has been given to the use and metabolism of fructose by strains of A. brasilense and A. lipoferum grown with N2 or NH4Cl as the nitrogen source (4, 9, 15, 32). During recent investigations in our laboratory on the various carbon sources for nitrate-dependent growth by azospirilla, we observed intense flocculation in media containing fructose and nitrate. A medium containing KNO3 and glucose has been reported to enhance exopolysaccharide synthesis in certain soil bacteria (14). Also, flocculation in liquid culture medium has been reported for several gramnegative bacteria, but azospirilla were not included in those studies (5, 8, 16). Thus, this research was undertaken to
