Cooperative Mineralisation of the Fragrance Ingredient 2‐Cyclohexylidene‐2‐Phenylacetonitrile by a Consortium of a <i>Variovorax</i> and an <i>Acidovorax</i> Strain Isolated From Activated Sludge

To be considered ultimately biodegradable, a substance needs to reach ≥ 60% mineralisation in screening tests. Ultimate biodegradability can also be studied with targeted studies, for example, using an inoculum after enrichment or isolated bacteria. Here, degradation of 2-cyclohexylidene-2-phenylacetonitrile (Peonile) was studied. Peonile is composed of biodegradable substructures and does not have typical nonbiodegradable motifs, such as extensive alkyl-branching or quaternary carbon atoms, yet it fails ready biodegradability tests. An adapted sludge was able to mineralise Peonile with a bi-phasic curve. Two Acidovorax strains isolated from this inoculum degrade the phenyl ring of Peonile, generating the metabolite 2-cyano-2-cyclohexylideneacetic acid. A Variovorax strain degrading this metabolite was isolated at the end of a mineralisation experiment with the adapted inoculum. A mixture of both bacteria was shown to mineralise Peonile under OECD 301D and 301F incubation conditions. The need for cooperative action of two bacteria explains the bi-phasic curve in the mineralisation by the adapted sludge and indicates that Peonile is ultimately biodegradable by bacteria originally present in sewage sludge. This is one of the first examples showing cooperative mineralisation of a xenobiotic by specific bacteria in sewage sludge.