ENVIRONMENTAL MICROBIOLOGY Degradation of 2-nitrodiphenylamine, a component of Otto Fuel II, by Clostridium spp. S. Powell 1,2 , P. D. Franzmann 2 * , R. Cord-Ruwisch 1 and S. Toze 2 1 School of Biological and Environmental Sciences, Murdoch University, Perth, WA 6150, Australia 2 Centre for Groundwater Studies, CSIRO Land and Water, Underwood Ave, Floreat Park, WA 6014, Australia (Received 3 July 1997, accepted in revised form 23 December 1997) Key Words: Clostridium, Otto Fuel, 2-nitrodiphenylamine, biodegradation, trinitrotoluene, reductive degradation Otto Fuel II, a propellant in torpedoes, is composed of 76% 1,2 propanediol dinitrate (PGDN), 22.5% di-n-butyl sebacate, and 1.5% 2-nitrodiphenyla- mine (NDPA), and is largely recalcitrant to aerobic microbial degradation. Anaerobic microbial degradation of Otto Fuel II was tested by inoculating anaerobic enrichment media, containing either 2% (vol:vol) complete Otto Fuel II or 2% of a 0.02% solution of Otto Fuel II in methanol, with soil and water from sites contaminated with munitions or with landfill leachate. Anaerobic bacterial growth was completely inhibited by 2% Otto Fuel II. Two mixed bacterial enrichments developed in anaerobic media containing 2% (v/v) of a 0.02% solution of Otto Fuel II in methanol. After incubation, PGDN could not be detected in either enrichment, but was also not detectable in sterile controls, suggesting abiotic degradation of low concentrations of PGDN in reduced anaerobic medium. NDPA did not degrade in either enrichment. Similarly, complete Otto Fuel was recalcitrant to degradation by highly reducing methanogenic biomass collected from an upflow anaerobic sludge blanket bioreactor (UASB). A comparison of the degradative ability of autoclaved and viable biomass showed that low concentrations of PGDN autodegraded, however unlike the autoclaved anaerobic biomass, the viable anaerobic biomass degraded the NDPA component of Otto Fuel II. Two strains of anaerobic clostridia, strains SP3 and SPF, that caused the disappearance of NDPA at its limit of solubility in culture media, were isolated from the UASB bioreactor biomass. SP3 and SPF were shown, by comparison of 16S rDNA sequences, to be most closely related to Clostridium butyricum and Clostridium cochlearium respectively. Although NDPA was lost from cultures of both strains, metabolic endproducts were not identified. Neither strain could degrade NDPA unless supplied with an alternative energy source. In the culture system used, NDPA stimulated the growth of SP3 but it had no appreciable effect on the growth of SPF. Both SP3 and SPF degraded low concentrations of trinitrotoluene (TNT), without the produc- tion of detectable concentrations of aromatic amines. A possible method for the remediation of small spills of Otto Fuel II is suggested. © 1998 Academic Press *Corresponding author. E-mail: Peter.Franzmann@per.clw.csiro.au Anaerobe (1998) 4, 95–102 1075-9964/98/020095 + 08 $25.00/0/an970141 © 1998 Academic Press