Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria

The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xyl ene, ethyl toluene, and isopropyltoluene (cymene) in enrichment cultures in oculated with freshwater mud. Denitrifying enrichment cultures developed mo st readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon o ccurring in plants, and with In-xylene (within 6 weeks). Enrichment of deni trifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months. Anaerobic degradation of p-cymene, wh ich has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were is olated; they grew on p-cymene (diluted in an inert carrier phase) and nitra te with doubling rimes of 12 and 16 h, respectively. Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew w ith some alkenoic monoterpenes structurally related to p-cymene, e.g., alph a-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mo no- and dicarboxylic aliphatic acids. Determination of the degradation bala nce of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions. Adapt ation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene. Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack. Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth o n p-cymene under oxic conditions was observed only with strain pCyN1. Strai ns pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera c luster within the beta-subclass of the Proteobacteria, as revealed by 16S r RNA gene sequence analysis. This cluster encompasses several described deni trifiers that oxidize toluene and other alkylbenzenes.