The effects of the following changes throughout the association of germ-free mice with increasing numbers of anaerobic bacteria were studied: (i) elution patterns obtained by gel-filtration chromatography of caecal diffusates; (ii) concentration of beta-aspartylglycine in caecal and faecal contents; (iii) polypeptide patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of caecal supernatants; (iv) free amino acid content of caecal supernatants; (v) faecal bile acids, analysed by gas-liquid chromatography; (vi) colonization-resistance. The results indicate that monitoring the normalization (association) process can be accomplished in several ways, but the level of colonization-resistance is most easily measured by high-voltage paper electrophoresis of faecal supernatants to determine the concentration of beta-aspartylglycine. During association, the concentration of beta-aspartylglycine decreased and became undetectable after association with 40 to 50 different strains of bacteria. There was a good negative correlation between the level of colonization-resistance and the concentration of beta-aspartylglycine.
Removal of bacteria from wastewater treated with activated sludge was studied by the use of a streptomycin-resistant Escherichia coli strain. The removal appeared to be a biphasic process. A rapid sorption of bacteria to the sludge flocs took place in the first hour after seeding mixed liquor with E. coli. Thereafter, slower elimination of E. coli was observed. The latter process was due to predation on E. coli by ciliated protozoa. This was shown by: (i) appearance of fluorescent food vacuoles of ciliates when fluorescent E. coli cells were added to mixed liquor; (ii) inhibition of predation either in the presence of cycloheximide or under anaerobic conditions; and (iii) absence of predation in bulking and washed sludge.
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