Oil-removal enhancement in media with keratinous or chitinous wastes by hydrocarbon-degrading bacteria isolated from oil-polluted soils.

The aim of this work was to isolate oil-degrading bacteria that use chitin or keratin as carbon sources from oil contaminated soils; and additionally to study if oil removal by these bacteria is enhanced when a chitinous or a keratinous waste is added to the culture media. To isolate the above-mentioned bacteria, 12 soil samples were collected close to an oil-well. Such soils showed unsuitable nutrients content, but their counts of heterotrophic bacteria ranged within 10(5)-10(8) CFU g(-1) soil, of which 0.1-77% corresponded to oil hydrocarbon-degrading ones. By sampling on plates, 109 oil-degrading bacterial isolates were obtained. Their keratinase and chitinase activities were then screened by plate assays and spectrophotometric methods, resulting in 13 isolates that were used to integrate two mixed cultures, one keratinolytic and the other chitinolytic. These mixed cultures were grown in media with oil, or oil supplemented with chicken-feathers or shrimp wastes. The oil-hydrocarbon removal was measured by gas chromatography. Results showed that keratinolytic bacteria were better enzyme producers than the chitinolytic ones, and that oil removal in the presence of chicken-feathers was 3.8 times greater than with shrimp wastes, and almost twice, in comparison with oil-only added cultures. Identification of microorganisms from the mixed cultures by 16S rDNA, indicated the presence of seven different bacterial genera; Stenotrophomonas, Pseudomonas, Brevibacillus, Bacillus, Micrococcus, Lysobacter and Nocardiodes. These findings suggest that the isolated microorganisms and the chicken-feather wastes could be applied to the cleaning of oil-contaminated environments, whether in soil or water.

Chitosanase activity in Bacillus thuringiensis.

The ability to produce extracellular chitosanase (EC 3.2.1.132) was found by plate assays in 18 (23%) out of 77 crystalliferous strains of Bacillus thuringiensis. The best chitosanase producer was selected after the growth chosen in a liquid medium with colloidal chitosan as carbon source. Enzyme production was optimized (a 4-d incubation at 32 degrees C with shaking in a medium of pH 6.5 with 4% colloidal chitosan) and the enzyme was partially characterized. This is the first report on the chitosanase of B. thuringiensis.

Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases.

A simple and sensitive method based on the use of colloidal chitin stained with Remazol Brilliant Blue R (RBB) is proposed to evaluate chitinase activity. If this colloidal-stained substrate is included as a carbon source in a liquid medium, this technique allows the selection or the comparison of chitinolytic microorganisms. The colloidal substrate is proportionally solubilized and the dye released is spectrophotometrically quantified at 595 nm. The procedures used for the staining and fixing of RBB in the colloidal chitin, and a comparison with the commercial substrate chitin-azure, are presented. The influence of several physicochemical and enzymatic parameters on the release of dyes is also shown. Both stained substrates were used for studying the effect of pH, substrate concentration, temperature and time on the chitinase reaction of Bacillus thuringiensis Bt-107.