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Effects MethaneMetabolism on Nitrification Nitrous APPLIEDANDENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3307-3314 Vol. 60, No. 9 0099-2240/94/$04.00+0 Effects ofMethaneMetabolism onNitrification

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  • APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3307-3314 Vol. 60, No. 9 0099-2240/94/$04.00+0

    Effects of Methane Metabolism on Nitrification and Nitrous Oxide Production in Polluted Freshwater Sediment

    REAL ROY AND ROGER KNOWLES* Microbiology Unit, Department of Natural Resource Sciences, Macdonald Campus

    of McGill University, Ste-Anne-de-Bellevue, Que'bec, Canada H9X 3V9

    Received 5 April 1994/Accepted 17 June 1994

    We report the effect of CH4 and of CH4 oxidation on nitrification in freshwater sediment from Hamilton Harbour, Ontario, Canada, a highly polluted ecosystem. Aerobic slurry experiments showed a high potential for aerobic N20 production in some sites. It was suppressed by C2H2, correlated to N03- production, and stimulated by NH4 concentration, supporting the hypothesis of a nitrification-dependent source for this N20 production. Diluted sediment slurries supplemented with CH4 (1 to 24 ,uM) showed earlier and enhanced nitrification and N20 production compared with unsupplemented slurries (.l IFM CH4). This suggests that nitrification by methanotrophs may be significant in freshwater sediment under certain conditions. Suppres- sion of nitrification was observed at CH4 concentrations of 84 ,eM and greater, possibly through competition for 02 between methanotrophs and NH4+-oxidizing bacteria and/or competition for mineral N between these two groups of organisms. In Hamilton Harbour sediment, the very high CH4 concentrations (1.02 to 6.83 mM) which exist would probably suppress nitrification and favor NH4' accumulation in the pore water. Indeed, NH4+ concentrations in Hamilton Harbour sediment are higher than those found in other lakes. We conclude that the impact of CH4 metabolism on N cycling processes in freshwater ecosystems should be given more attention.

    Nitrification, which is the successive oxidation of NH4' to N02- and NO3- by NH4+- and N02--oxidizing bacteria (3), plays a key role in the cycling of mineral N in aquatic ecosystems. It leads to loss of fixed N to the atmosphere when coupled to denitrification, the dissimilatory reduction of NO3- to N2 (27). Nitrate availability often limits denitrification in freshwater sediment (26, 48), which often makes nitrification the rate-limiting step in the recycling of mineral N in eutrophic aquatic systems. Nitrification has wide implications for the environmental quality of aquatic systems. It may be an impor- tant process in relation to NH4' toxicity in sediments (17, 47). It may also contribute to the problem of 02 depletion in lake water, when 02 diffusion is prevented by some barrier such as winter ice (31) or summer thermal stratification in dimictic lakes. It is also well known that N20 is produced during the oxidation of NH4+ to NO2 by NH4+-oxidizing bacteria (7, 15, 20, 28). Therefore, nitrification represents a biological source of atmospheric N20, a "greenhouse" gas (13) also involved in stratospheric ozone depletion (12). The increase in atmo- spheric N20 concentrations in the recent past and especially in the last decade (23, 34) has generated great interest in potential microbiological sources and sinks of this gas, as well as those of CH4, in order to better understand the global budget of these gases on the scale of the biosphere.

    Besides NH4+-oxidizing bacteria, other microorganisms have been implicated in the oxidation of NH4+. Since the first report by Hutton and ZoBell (18), evidence has accumulated that methanotrophic bacteria can also oxidize NH4' to N02- (reviewed by Bedard and Knowles [2]). Evolution of N20 also occurs during such methanotrophic nitrification (32, 54). Var-

    * Corresponding author. Mailing address: Microbiology Unit, De- partment of Natural Resource Sciences, Macdonald Campus of McGill University, 21111 Lakeshore Rd., Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9. Phone: (514) 398-7751. Fax: (514) 398-7624. Electronic mail address: [email protected]

    ious heterotrophic bacteria can also oxidize NH4' to N02- with N20 as a by-product (1, 14, 24, 40). Ammonium can be oxidized to NH20H by the methane

    monooxygenase of methanotrophs, and CH4 may also be oxidized to CH30H by the ammonia monooxygenase of NH4+-oxidizing bacteria (2). From kinetic studies on Nitro- somonas europaea, it appears that CH4 binds at the same ammonia monooxygenase active site as does NH3 (22). Meth- ane acts as a competitive inhibitor of NH4+ oxidation. Meth- ane suppression of nitrification may also be the outcome of competition between methanotrophs and nitrifiers for 02 and mineral N (38). Methanotrophic nitrification, although inhib- ited by increasing CH4 concentrations, is dependent on CH4 (32). Methane dependence of nitrification was also demon- strated in a CH4-enriched soil consortium (39). Therefore, CH4 is likely to be an important factor in the regulation of nitrification and N20 production and, more generally, in the N cycle in natural environments.

    Until now, very few studies have focused on the interaction of CH4 and NH4+ oxidation in natural systems. Megraw and Knowles (38-40) carried out such a study with a humisol but did not try to relate the findings from the slurry experiments and enrichment cultures to field conditions. Studies performed with aquatic sediments have focused on metabolism of either CH4 or NH4+. Only in a few studies are pore water NH4' and CH4 concentrations reported (10, 11, 16, 21, 49). The effect of CH4 on nitrification and its impact on the overall N cycle was not considered. A recent article reported the inhibitory effect of NH4+ on CH4 oxidation but not the effect of CH4 on NH4' oxidation (5). Therefore, in this study we intend to demon- strate by field measurements and laboratory experiments the critical role of CH4 in nitrification. It is hoped that this work will contribute to a better understanding of factors controlling nitrification in freshwater sediments and provide important information on the dynamics of nitrogen cycling in Hamilton


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    Harbour to help in the design of an appropriate remedial action plan.

    In this work we address four specific objectives. First, we present a numerical analysis of the spatial variation of poten- tial aerobic N20 production, CH4 oxidation, and CH4 produc- tion in sediment slurries. Second, we present data supporting a nitrification-dependent source of such N20 production in slurries. Third, we show that CH4 is critical in the regulation of nitrification: it can suppress or stimulate production of NO3- and N20. Fourth, to assess what is most likely to occur under field conditions, we discuss the spatial variations of CH4 and NH4' concentrations in sediment pore water of Hamilton Harbour in relation to nitrification and compare it with other aquatic ecosystems.


    Study site and sediment sampling. The top 5 cm of sediment was collected with an Ekman dredge at various locations in Hamilton Harbour, a natural embayment of Lake Ontario, Ontario, Canada. A detailed description of Hamilton Harbour, the sampling procedures, and the field measurements are published elsewhere (44).

    Microbial assays. Potential microbial activities were mea- sured by dispensing sediment (5 ml) from a given site in either a 50-ml Erlenmeyer flask with an equal volume of sterile distilled water to obtain 1:1 slurry dilution or a 125-ml Erlen- meyer flask with 45 ml of sterile distilled water to obtain 1:10 slurry dilution. Flasks were capped with serum stoppers (Suba- Seal Works, William Freeman and Co., Barnsley, United Kingdom) as already described (44). For potential nitrification assays, 0.1 M (NH4)2SO4 was added to a final slurry concen- tration of 4 mM. All flasks were shaken, evacuated three times (15 min each), and backfilled with air to eliminate dissolved gases in the pore water. For potential CH4 oxidation, pure CH4 (Matheson, Montreal, Canada) was added by syringe after withdrawal of an equivalent volume of the gas phase. For potential CH4 production, flasks were backfilled with ultrapure helium (Linde, Union Carbide, Montreal, Canada) instead of air with no addition of NH4'. Acetylene (Linde, Union Carbide) was added by syringe as an inhibitor of nitrification or CH4 oxidation. All flasks were wrapped with aluminum foil to avoid light exposure, which may stimulate CH4 oxidation (25). Flasks were incubated on a gyratory shaker at 250 rpm and at room temperature (-25°C) except for experiments measuring potential CH4 production, in which case flasks were incubated statically at 20°C (44). Nitrification was measured by NH4' disappearance and N02 and NO3- production in the pore water with time of incubation. Methane oxidation and N20 and CH4 production were measured by gas disappearance or production in the headspace of experimental flasks with time of incubation.

    Analytical procedures. Gases were analyzed by gas chroma- tography (GC) as described previously (44), except that CH4 was also determined with a GC (HP 5750; Hewlett-Packard, Montreal, Canada) equipped with a Durapak column and a flame ionization detector. Oxygen in the gas phase was mea- sured on a GC (Fisher-Hamilton 29; Fisher, Montreal, Can- ada) equipped with Molecular Sieve and DEHS (di-2-ethyl- hexylsebacate) columns in series and two thermal conductivity detectors (8). Gas standards were as described previously (44). Gas concentrations were calculated, taking into account the dissolved gas by using the Ostwald solubility coefficient (53). Rates of production of gases were calculated as already described (44). At 25°C, 1 kPa of CH4 is equivalent to 13.9 ,uM or 23.5

    ,umol - fl-' for the 50-ml Erlenmeyer flasks (1:1 slurries) or 60.8 ,umol * fl-' for the

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