BIOFILM FORMATION AND PHENOTYPIC ALTERATIONS ASSOCIATED TO BACILLUS ATROPHAEUS SPORULATION BY SOLID STATE FERMENTATION

Sandra Regina B.r. Sella (CPPI/SESA-PR); Carla Masetti (UFPR); Belquis P. Guizelini (UFPR); Luciana P. S. Vandenberghe (UFPR); João Carlos Minozzo (CPPI/SESA-PR); Carlos Ricardo Soccol (UFPR)

Bacillus sp. spores are usually obtained from laboratory standard strains cultivated in artificial media - fortified agar or liquid media. However, in natural habitats, spores are predominantly formed from bacteria present in highly surface associated communities of cells in extracellular polymers matrix called biofilms. Solid state fermentation (SSF) mimetizes the natural environment of many microorganisms, that grow attached to the surface of solid particle, better than submerse fermentation. This study aimed to confirm if the sporulation through SSF of Bacillus atrophaeus ATCC 9372 standard strain promotes phenotypic changes and biofilm formation, by observing the growth characteristics of the spores formed. Spores were prepared using agar sporulation medium and sugarcane bagasse plus soybean molasses as SSF support and substrate respectively. Four systems for studying biofilm formation were applied: growth in standing cultures; colonies growth on the surface of agar plates; swarming motility and the microtiter plate assay system. The maximum spores' concentration of 1.6 x 10¹⁰ CFU g^-1 dry matter was obtained in SSF and 1.2 x 10⁸ CFU g^-1  in agar medium, suggesting that SSF promotes an increase in productivity of about 100 times. Agar - spores growth in standing TSB cultures revealed the formation of thin, fragile, smooth and structureless pellicles on air-liquid interface and robust pellicles with intricate web-like structures when inoculated with SSF-spores. When spotted on TSA agar plates, the spores produced colonies with morphological features characteristic of their corresponding pellicles: smooth, small and well defined, mucoid, large, undelimited and structurally complicated, respectively. Swarming motility in 0.7% agar culture was identified only in spores produced by solid state fermentation growth. Adherence to the microtiter plates demonstrated that spores from SSF had great ability to form biofilm (++ up to +++) and spores from agar were classified as "no producers". It was confirmed that sporulation through SSF induced phenotypic changes and biofilm development. There are no published reports on B. atrophaeus biofilm formation associated to SSF sporulation, so elucidation of the genes, proteins and molecular mechanisms involved and its effect is necessary due to its potential application in productions improvement or new products development.