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Applied and Environmental Microbiology, November 2004, p. 6551-6558, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6551-6558.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bacterium-Based NO2– Biosensor for Environmental Applications

Michael Nielsen,1* Lars Hauer Larsen,2 Mike S. M. Jetten,3 and Niels Peter Revsbech1

Department of Microbiology, University of Aarhus,1 Unisense A/S, Aarhus, Denmark,2 Department of Microbiology, University of Nijmegen, Nijmegen, The Netherlands3

Received 26 March 2004/ Accepted 7 July 2004

A sensitive NO2– biosensor that is based on bacterial reduction of NO2– to N2O and subsequent detection of the N2O by a built-in electrochemical N2O sensor was developed. Four different denitrifying organisms lacking NO3– reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO2– tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO3– and N2O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N2O reductase. The macroscale biosensors constructed exhibited a linear NO2– response at concentrations up to 1 to 2 mM. The detection limit was around 1 µM NO2–, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO2–, and interference was observed only with NH2OH, NO, N2O, and H2S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO2– biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO2– biosensor were constructed and used to measure NO2– profiles in marine sediment.

* Corresponding author. Mailing address: Department of Microbiology, University of Aarhus, Bd. 540, Ny Munkegade, 8000 Aarhus C, Denmark. Phone: 45 89423329. Fax: 45 86127191. E-mail: mnielsen{at}biology.au.dk.

Applied and Environmental Microbiology, November 2004, p. 6551-6558, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6551-6558.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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