Bacterium-Based NO ₂ ⁻ Biosensor for Environmental Applications

Abstract

A sensitive NO 2؊ biosensor that is based on bacterial reduction of NO 2 ؊ to N 2 O and subsequent detection of the N 2 O by a built-in electrochemical N 2 O sensor was developed. Four different denitrifying organisms lacking NO 3 ؊ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO 2 ؊ 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 NO 3 ؊ and N 2 O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N 2 O reductase. The macroscale biosensors constructed exhibited a linear NO 2 ؊ response at concentrations up to 1 to 2 mM. The detection limit was around 1 M NO 2 ؊ , and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO 2 ؊ , and interference was observed only with NH 2 OH, NO, N 2 O, and H 2 S. 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 NO 2 ؊ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO 2 ؊ biosensor were constructed and used to measure NO 2 ؊ profiles in marine sediment. Nitrite (NO 2Ϫ ) has a central position in the global nitrogen (N) cycle and is involved in many important biological N transformations. With an intermediate oxidation state, NO 2 Ϫ acts as an electron donor in nitrification and serves as an electron acceptor in denitrification, dissimilative nitrite reduction to ammonium, and anammox.In natural ecosystems NO 2 Ϫ is of interest because of its toxicity for microorganisms and higher organisms (20,37). NO 2 Ϫ concentrations in natural bulk water bodies are usually very low, and in freshwater systems the average worldwide NO 2 Ϫ concentration has been estimated to be about 1 g of N liter Ϫ1 (ϳ0.07 M NO 2 Ϫ ) (28). Recently, however, there have been several reports of NO 2 Ϫ accumulation to concentrations of 5 to 140 M in European eutrophic rivers and estuaries (3,8,15,19,44). Studies have indicated that NO 2 Ϫ accumulations can be related to imbalances in NO 2 Ϫ production and consumption rates for either aerobic sediment processes (nitrification) or anaerobic sediment processes (denitrification and dissimilatory nitrate reduction to ammonia). In marine systems NO 2 Ϫ concentrations are normally negligible, but concentrations of 0.3 to 2.5 M have been reported near the oxic-anoxic boundary of stratified marine water bodies (5, 22). The distribution of NO 2 Ϫ in sediments is largely uncharacterized, except for a few studies that have documented NO 2 Ϫ a...

Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake

“…Oxygen gradients were measured with a Clark-type microelectrode (Ox 50 or Ox100; Unisense). Nitrate and nitrite gradients were measured with NO x and nitrite microbiosensors, respectively (35,36), that were constructed at the Max Planck Institute for Marine Microbiology. The NO x and nitrite sensors were calibrated before and after profiling in tap water spiked with aliquots of NaNO 3 and NaNO 2 stock solutions (10 mmol/L each) to nominal concentrations of 0-100 μmol/L NO 3 and 0-20 μmol/L NO 2 .…”

Nitrate, nitrite, and nitrous oxide transformations in sediments along a salinity gradient in the Weser Estuary

“…Microsensors for NO 2 -, NO x -, N 2 O, and O 2 were prepared and calibrated as described previously (Revsbech 1989, Larsen et al 1997, Andersen et al 2001, Nielsen et al 2004). The NO 2 -and NO x -micro-biosensors had tip diameters of about 50 to 80 µm and had 90% response times of between 45 and 75 s. Electrophoretic sensitivity control (ESC) was used to control sensitivity of the biosensors (Kjaer et al 1999), which made it possible to measure NO 2 -and NO x -concentrations down to about 0.2 µM.…”

“…Similarly, sediment incubated with 200 µM NO 3 -and marine sediment incubated with 10 µM NO 3 -were exposed to 6.5 ‰ water with 200 µM NO 3 -. During each perturbation event, single profiles of N 2 O and O 2 were measured at 0, 1, 2, 4, 6, and 9 h. In freshwater sediment in which N 2 O was found beyond 9 h, the duration of the perturbation experiment was extended to 31 h. NO 2 -and NO x -profiles were only measured at 0 and 9 h during salinity perturbations (data not shown) as the biosensor signals are salinity-dependent (Nielsen et al 2004) and thus do not allow accurate analysis in the presence of a steep salinity gradient.…”

Microbiosensors for Measurement of Microbially Available Dissolved Organic Carbon: Sensor Characteristics and Preliminary Environmental Application

“…Bacterium-or yeast-based microbiosensors have been developed for microscale measurement of microbially available dissolved organic carbon (ADOC) (32) in oxic environments, microbially available volatile fatty acids (e.g., acetate, propionate, and lactate) in anoxic environments (29), and nitrate and nitrite (24,25,28,33) and methane (7,8) in the pore water of sediments. All these microbial biosensors were characterized by measuring tips as small as 25 to 100 m. Due to their small size and the efficient diffusional transport of substrates at a micrometer scale, microbiosensors respond much faster to changes in analyte concentrations than macrobiosensors respond.…”