Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria |
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Authors: | Gerda Harms Ralf Rabus F Widdel |
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Institution: | (1) Max-Planck-Institut für Marine Mikrobiologie, Celsiusstrasse 1, D-28359 Bremen, Germany e-mail: fwiddel@mpi-bremen.de, Tel.: +49-421-2028-702, Fax: +49-421-2028-790, DE |
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Abstract: | The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated
with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater
mud. Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks). Enrichment of denitrifiers 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, which has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval
cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively. Strain pCyN1, but
not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., α-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids. Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic
conditions. Adaptation 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 on p-cymene under oxic conditions was observed only with strain pCyN1. Strains pCyN1 and pCyN2 are closely related to members
of the Azoarcus-Thauera cluster within the β-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis. This cluster encompasses
several described denitrifiers that oxidize toluene and other alkylbenzenes.
Received: 15 July 1998 / Revision received: 29 July 1999 / Accepted: 2 August 1999 |
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Keywords: | Anaerobic degradation Denitrifying bacteria Aromatic hydrocarbons Alkylbenzenes p-Cymene Alkylbenzoates Intermediates 16S rRNA sequence analysis |
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