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Applied and Environmental Microbiology, November 2008, p. 6672-6681, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00835-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor{triangledown}

Sander A. B. Weelink,1 Nico C. G. Tan,1,{dagger} Harm ten Broeke,1 Corné van den Kieboom,1 Wim van Doesburg,1 Alette A. M. Langenhoff,2 Jan Gerritse,2 Howard Junca,3 and Alfons J. M. Stams1*

Laboratory of Microbiology, Wageningen University, Dreijenplein 10, Building No. 316, 6703 HB Wageningen, The Netherlands,1 TNO Built Environment and Geosciences, Princetonlaan 6, 3584 CB Utrecht, The Netherlands,2 AG Biodegradation, Helmholtz-Zentrum für Infektionsforschung, Inhoffenstrasse 7, D-38124 Braunschweig, Germany3

Received 11 April 2008/ Accepted 30 August 2008

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 µm wide and 1 to 2 µm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.

* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, Building No. 316, 6703 HB Wageningen, The Netherlands. Phone: (31) 317 483101. Fax: (31) 317 483829. E-mail: fons.stams{at}wur.nl

{triangledown} Published ahead of print on 12 September 2008.

{dagger} Present address: NOTOX B.V., Hambakenwetering 7, P.O. Box 3476, 5203 DL 's-Hertogenbosch, The Netherlands.

Applied and Environmental Microbiology, November 2008, p. 6672-6681, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00835-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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