The activated sludge treatment of wastewater containing ɛ-caprolactam from a Nylon 6 manufacturing plant was investigated by basic studies and operation of a full-scale treatment plant. Activated sludges were obtained from several municipal and industrial wastewater sources, or were prepared by mixing ɛ-caprolactam-utilizing bacteria (Bacillus sp. and Achromobacter sp.) with a municipal activated sludge. However, bulking phenomena were soon observed in the acclimatization of ordinary activated sludges, even when started from a very low concentration of ɛ-caprolactam. On the other hand, the activated sludge synthesized from ɛ-caprolactam-utilizing bacteria showed better results as regards sludge volume index (SVI), BOD removal, and transparency of treated water. Wastewater from the Nylon 6 manufacturing plant, which contained ɛ-caprolactam, was treated by this synthesized activated sludge in a bench-scale apparatus consisting of a 4 m3 aeration basin and a 1.4 m3 sedimentation basin. The optimum BOD loading was estimated to be 0.35-0.40 kg BOD/kg MLSS/day for this wastewater. Production of excess activated sludge was 10% of the BOD loaded. In 1974, based on this preliminary experiment, a wastewater treatment facility consisting of a 2,500 m3 aeration basin and a 1,250 m3 sedimentation basin was constructed near the Uji Nylon 6 manufacturing plant (Unitika Ltd), to treat 5,000 m3 of wastewater (BOD 300-400 mg/l) per day. The treatment plant operates successfully, producing treated water with a BOD below 10 mg/l.

A basic compound with empirical formula C12H19N2O5 was isolated from Bacillus cereus 102804 fermentations of a soybean meal -glucose medium. The inhibitory activity of compound 102804 on growth of Gram-positive and Gram-negative bacteria growing in a chemically defined medium was reversed by vitamin B12, by L-methionine, and by n-methionine.It has no inhibitory activity for Escherichia coli (Davis 113-3) when grown in media containing L-methionine.The biosynthesis of 102804 coincides with the sporulation of the B. cereus, and the compound is not produced in the absence of sporulation.A search for microorganisms producing substances inhibiting vitamin B12-stimulated growth of Escherichia coli (Davis 113-3) resulted in selection of a strain of Bacillus cereus which had this capability.The vitamin B12 antimetabolite is produced by this culture (designated as 102804 in our collection) when grown under several conditions, and its production seems to coincide with sporulation of the bacillus. We wish to summarize in this communication our studies on the effect of fermentation conditions on production of compound 102804, and some of the chemical and biological attributes of this vitamin B12-antimetabolite. Materials and MethodsIsolation of culture 102804 A soil sample collected in Madison was diluted with sterile water and plated on nutrient agar. Discrete colonies were transferred to tubes of soybean meal -glucose -CaCO3 medium and placed on a shaker at 30°C. After several days' growth aliquots of the fermented medium were centrifuged and the supernatant liquid tested for ability to inhibit growth of E. coli (Davis 113-3) growing in an agar diffusion bioassay in the DAVIS -MINGi0LI1) medium (with added vitamin B12), and to inhibit the growth of Escherichia coli B(ATCC 11303) (growing in nutrient agar) and Staphylococcus aureus FDA 209P (ATCC 6538) (growing in nutrient agar). The supernatant solution from culture 102804 was found to inhibit the E. coli (Davis 113-3) and not the other two organisms under the test conditions, and the inhibition was competitively reversed by vitamin B12 in an agar diffusion bioassay.2) Culture 102804 was maintained for further study by periodic transfers on agar slants and by storage of cell suspensions in liquid nitrogen.Identification of culture 102804 Culture 102804 was grown in a variety of media mentioned in BERGEY'S manual3) and compared with cultures of Bacillus cereus (ATCC 14579), Bacillus mycoides (ATCC 6462), and Bacillus subtilis (ATCC 6633) grown under the same conditions. Culture 102804 is closely related to B. cereus on the basis of: Colonial morphology on glucose-containing agar; Gram staining characteristics; shape of sporangia; vegetative cell and spore size; acid production when grown on glucose and glycerol containing agars, and no acid production when grown on mannitol and xylose containing agars; a positive VOGES-PROSKAUER test and a positive test for reduction of nitrate to nitrite.

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