Biofilm Formation by Monocultures and Mixed Cultures of Alcaligenes faecalis 2 and Rhodococcus ruber gt 1

Abstract

Biofilm formation by monocultures and mixed cultures of nitrile-hydrolyzing bacteria Alcaligenes faecalis 2 and Rhodococcus ruber gt 1 was studied. Biofilm formation was assessed by staining with Crystal violet, by the energy state of the cells, and by the output of the polymer matrix. A quantitative method for assessment of the output of the polymer matrix by the fluorescence of konA-tetramethylrhodamine was developed. The medium used for obtaining mixed biofilms of Al. faecalis 2 and R. ruber gt 1 contained acetamide and glucose as carbon sources. In mixed biofilms Al. faecalis 2 was shown to act as a primary colonizer, while R. ruber gt 1 was a satellite microorganism. Compared to monoculture biofilms, mixed biofilms exhibited higher output of the polymer matrix and increased biofilm formation by Al. faecalis 2 and R. ruber gt 1 on addition of the culture liquid of the second strain. Daily addition of the toxic substrate acetonitrile to planktonic and biofilm cultures resulted in bacterial adaptation, as was indicated by higher numbers of surviving cells compared to the variants with acetonitrile addition every 10 min, mainly due to nitrile hydratase activity of R. ruber gt 1, which transformed acetonitrile into nontoxic acetamide. Our results show that binary biofilms of amidase-containing Al. faecalis 2 and R. ruber gt 1, a strain with high nitrile hydratase activity, are promising as biocatalysts for acrylic acid production and as the basis for a biofilter for nitrile removal from wastewater.