Accumulation of Nitrogen Oxides in Copper-Limited Cultures of Denitrifying Bacteria

Abstract

Three strains of heterotrophic denitrifying bacteria were cultured in artificial seawater medium under trace metal clean conditions to investigate their physiological response to changes in copper concentration. Decreasing the copper concentration in cultures of Pseudornonas stutzeri and Paracoccus denitrificans resulted in accumulation of nitrous oxide (N,O) gas compared to copper-replete cultures and cessation of growthbefore complete denitrificationto dinitrogen. Correspondingly, the activity of the copper enzyme nitrous oxide reductase. measured for P. denitriticans cultures, was high in copper-replete cultures and was below detection in copper-deficient cultures. Addition of copper to copper-deficient cultures following the accun~ulation of N,O resulted in resumption of growth and complete consumption of N,O in solution. Growth of the third strain, WLB20, in copper-deficient medium caused a marked accumulation of nitrite, suggesting that WLB20 has the copper form of nitrite reductase. These observations suggest a role for trace metals in regulating redox cycling of nitrogen and trace gas production in the ocean. The respiratory decomposition of organic matter from highly productive waters leads to near exhaustion of the available oxygen, triggering the use of alternative oxidants as terminal electron acceptors. Under oxygen-limiting conditions, denitrifying bacteria reduce nitrate (NO;) sequentially to nitrite (NO;), nitric oxide gas (NO), nitrous oxide gas (N,O), and dinitrogen gas (N,) (reviewed by Zumft 1997). The global importance of denitrification lies in its distinction as the most important sink for oceanic fixed nitrogen (Codispoti and Christensen 1985). Moreover, denitrification is believed to control oceanic N,O concentrations, serving either as a net sink of N,O gas (e.g., Cohen and Gordon 1978; Codispoti and Christensen 1985) or conversely as a source of N,O (e.g., Yoshida et al. 1989; Law and Owens 1990). Globally, N,O gas contributes to greenhouse warming with a radiative potential substantially greater than CO, on a per molecule basis. Furthermore, N,O is also involved in catalytic destruction of stratospheric ozone (Jackman et al. 1980). In spite of the global importance of oceanic denitrification, the processes that regulate it remain poorly understood. The reduction of nitrogen oxides under oxygen limitation is catalyzed by a series of metalloenzymes located in the cell membrane and periplasm of denitrifying bacteria. The respiratory nitrate reductase, which catalyzes the reduction of NO; to NO;, contains iron and molybdenum at its reaction center. The reduction of NO; is in turn facilitated by the respiratory nitrite reductase, of which there are two types present in different organisms, an iron and a copper enzyme. Nitric oxide (NO) gas is respired to N,O via the nitric oxide reductase, an iron enzyme. And finally, N,O gas is reduced to dinitrogen by the copper enzyme nitrous oxide reductase. The extensive involvement of copper and iron in the denitrification pathway likely imparts to denitrifiers distinctive nutritional requirements for these metals. To investigate these requirements, we studied the physiological response of denitrifying bacteria to reduced copper and iron concentrations. In this paper, we present our observations on the physiology of copper-limited cultures of denitrifiers. Effects of iron limitation on denitrification will be presented elsewhere (Granger and Ward pers. comm.). The data presented here provide insight into a potential regulatory role for copper in oceanic denitrification and nitrous oxide production. Cultivation of denitrihing bacteria under trace metal clean conditioizs-Three stains of facultative aerobic heterotrophic denitrifying bacteria were examined: Pseudolnonas stuzzeri (ATCC 14405), generally associated with eutrophic coastal marine environments; Paracoccus denitrificans (ATCC 19367), ubiquitous in terrestrial and aquatic systems; and WLB20, a psychrophilic isolate from a hypersaline Antarctic lake (Ward and Priscu 1997). Cells were grown in batch culture using trace metal clean techniques in the artificial seawater medium Aquil (Price et al. 198.811989) modified for heterotrophic bacterial cultures (Granger and Price 1999). Synthetic ocean water containing 10 pmol L-' phosphate and 230 to 250 pmol LA'nitrate was purified of trace metals using Chelex 100 ion exchange resin (Bio-Rad Laboratories) following the procedure of Price et al. (19881 1989). Media were first sterilized by microwaving in acidwashed polycarbonate bottles (Keller et al. 1988) and then enriched with trace metals and vitamins (B,,, thiamin, and biotin) that were filtered through metal-free 0.2-pm filters (Acrodisc). The organic enrichments (0.2 g bactopeptone [Difco] and 0.2 g casein hydrolysate) were purified of trace metals with Chelex 100, then autoclaved before being added to sterile media. Trace metal additions were buffered with 100 pmol L-' ethylenediamine tetraacetic acid (EDTA) so that Fe3+, Mn2+, Zn2, and CoZT free ion concentrations approximated 10-l9 mol L-', mol L-', 10-Io9 mol L-I, and 10-lo9 mol L-I, respectively. These activities were computed with the chemical equilibrium program MINEQL (Westall et al. 1976). Total Mo and Se concentrations in the media were lo-' mol L-I and mol L-l, respectively. All the above metal concentrations are generally optimal for growth of phytoplankton (Price et al. 198811989) and marine bacteria (Granger and Price 1999). Premixed Cu-EDTA (1: 1) was added separately at a range of concentrations: 1.2 pmol L-l, 120 nnlol L-', 11 nmol L-l, and no Cu added. Background concentrations of copper in the no-Cu medium were estimated to be 3 nmol L-' (relative SD = 20%, detection limit = 0.2 nmol L-'; measurement by ICPMS; Moens et al. 1995). Cultures were initiated from frozen stocks, acclimated to experimental medium for 10 generations, then inoculated in opaque, trilaminate, polyethylene-lined, gas-tight bags without a headspace. P. stutzeri and WLB20 were grown at 12°C

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@inproceedings{Granger2007AccumulationON, title={Accumulation of Nitrogen Oxides in Copper-Limited Cultures of Denitrifying Bacteria}, author={Julie Granger and Bess Ward}, year={2007} }