APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3307-3314 Vol. 60, No. 90099-2240/94/$04.00+0

Effects of Methane Metabolism on Nitrification and NitrousOxide 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 HamiltonHarbour, Ontario, Canada, a highly polluted ecosystem. Aerobic slurry experiments showed a high potentialfor aerobic N20 production in some sites. It was suppressed by C2H2, correlated to N03- production, andstimulated by NH4 concentration, supporting the hypothesis of a nitrification-dependent source for this N20production. Diluted sediment slurries supplemented with CH4 (1 to 24 ,uM) showed earlier and enhancednitrification and N20 production compared with unsupplemented slurries (.l IFM CH4). This suggests thatnitrification 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 competitionfor 02 between methanotrophs and NH4+-oxidizing bacteria and/or competition for mineral N between thesetwo 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 concludethat the impact of CH4 metabolism on N cycling processes in freshwater ecosystems should be given moreattention.

Nitrification, which is the successive oxidation of NH4' toN02- and NO3- by NH4+- and N02--oxidizing bacteria (3),plays a key role in the cycling of mineral N in aquaticecosystems. It leads to loss of fixed N to the atmosphere whencoupled to denitrification, the dissimilatory reduction of NO3-to N2 (27). Nitrate availability often limits denitrification infreshwater sediment (26, 48), which often makes nitrificationthe rate-limiting step in the recycling of mineral N in eutrophicaquatic systems. Nitrification has wide implications for theenvironmental 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 lakewater, when 02 diffusion is prevented by some barrier such aswinter ice (31) or summer thermal stratification in dimicticlakes. It is also well known that N20 is produced during theoxidation of NH4+ to NO2 by NH4+-oxidizing bacteria (7, 15,20, 28). Therefore, nitrification represents a biological sourceof atmospheric N20, a "greenhouse" gas (13) also involved instratospheric ozone depletion (12). The increase in atmo-spheric N20 concentrations in the recent past and especially inthe last decade (23, 34) has generated great interest inpotential microbiological sources and sinks of this gas, as wellas those of CH4, in order to better understand the globalbudget of these gases on the scale of the biosphere.

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

* Corresponding author. Mailing address: Microbiology Unit, De-partment of Natural Resource Sciences, Macdonald Campus of McGillUniversity, 21111 Lakeshore Rd., Ste-Anne-de-Bellevue, Quebec,Canada H9X 3V9. Phone: (514) 398-7751. Fax: (514) 398-7624.Electronic mail address: EH13@musica.mcgill.ca.

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 beoxidized to CH30H by the ammonia monooxygenase ofNH4+-oxidizing bacteria (2). From kinetic studies on Nitro-somonas europaea, it appears that CH4 binds at the sameammonia 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 ofcompetition between methanotrophs and nitrifiers for 02 andmineral 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 ofnitrification and N20 production and, more generally, in the Ncycle in natural environments.

Until now, very few studies have focused on the interactionof CH4 and NH4+ oxidation in natural systems. Megraw andKnowles (38-40) carried out such a study with a humisol butdid not try to relate the findings from the slurry experimentsand enrichment cultures to field conditions. Studies performedwith aquatic sediments have focused on metabolism of eitherCH4 or NH4+. Only in a few studies are pore water NH4' andCH4 concentrations reported (10, 11, 16, 21, 49). The effect ofCH4 on nitrification and its impact on the overall N cycle wasnot considered. A recent article reported the inhibitory effectof 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 thecritical role of CH4 in nitrification. It is hoped that this workwill contribute to a better understanding of factors controllingnitrification in freshwater sediments and provide importantinformation on the dynamics of nitrogen cycling in Hamilton

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3308 ROY AND KNOWLES

Harbour to help in the design of an appropriate remedialaction plan.

In this work we address four specific objectives. First, wepresent 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 anitrification-dependent source of such N20 production inslurries. Third, we show that CH4 is critical in the regulation ofnitrification: it can suppress or stimulate production of NO3-and N20. Fourth, to assess what is most likely to occur underfield conditions, we discuss the spatial variations of CH4 andNH4' concentrations in sediment pore water of HamiltonHarbour in relation to nitrification and compare it with otheraquatic ecosystems.

MATERIALS AND METHODS

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

Microbial assays. Potential microbial activities were mea-sured by dispensing sediment (5 ml) from a given site in eithera 50-ml Erlenmeyer flask with an equal volume of steriledistilled water to obtain 1:1 slurry dilution or a 125-ml Erlen-meyer flask with 45 ml of sterile distilled water to obtain 1:10slurry dilution. Flasks were capped with serum stoppers (Suba-Seal Works, William Freeman and Co., Barnsley, UnitedKingdom) as already described (44). For potential nitrificationassays, 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 dissolvedgases in the pore water. For potential CH4 oxidation, pure CH4(Matheson, Montreal, Canada) was added by syringe afterwithdrawal of an equivalent volume of the gas phase. Forpotential CH4 production, flasks were backfilled with ultrapurehelium (Linde, Union Carbide, Montreal, Canada) instead ofair with no addition of NH4'. Acetylene (Linde, UnionCarbide) was added by syringe as an inhibitor of nitrification orCH4 oxidation. All flasks were wrapped with aluminum foil toavoid light exposure, which may stimulate CH4 oxidation (25).Flasks were incubated on a gyratory shaker at 250 rpm and atroom temperature (-25°C) except for experiments measuringpotential CH4 production, in which case flasks were incubatedstatically at 20°C (44). Nitrification was measured by NH4'disappearance and N02 and NO3- production in the porewater with time of incubation. Methane oxidation and N20and CH4 production were measured by gas disappearance orproduction in the headspace of experimental flasks with timeof incubation.

Analytical procedures. Gases were analyzed by gas chroma-tography (GC) as described previously (44), except that CH4was also determined with a GC (HP 5750; Hewlett-Packard,Montreal, Canada) equipped with a Durapak column and aflame 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 conductivitydetectors (8). Gas standards were as described previously (44).Gas concentrations were calculated, taking into account thedissolved 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 125-ml Erlenmeyer flasks (1:10 slurries).Pore water was extracted from sediment samples by centrif-

ugation (44). Pore water was extracted from sediment slurry(1:1) by centrifugation at 10,000 x g (20 min at 4°C). Then thesupernatant was membrane filtered (pore size, 0.45 ,um) andstored at 4°C or frozen until further chemical analysis. Porewater was extracted from diluted sediment slurry (1:10) bycentrifuging 2-ml aliquots in microcentrifuge tubes at 12,000 xg for 10 min. Nitrogen ion concentrations were determined byautomated colorimetric methods either on a TRAACS-800instrument (44) or on an autoanalyzer (Chemlab Instruments,Hornchurch, United Kingdom) (38). Dissolved organic carbon(DOC) was determined by infrared detection of CO2 releasedfrom acidic digestion of the predigested water samples (44).Particulate carbon (PC) was determined on the dried andground pellet of the centrifuged sediment by a combustionmethod on a LECO automated carbon-sulfur analyzer (44).The methane level in sediment was determined by dispens-

ing sediment (5 ml) in a 50-ml Erlenmeyer flask with an equalamount of distilled water. Flasks were immediately cappedwith serum stoppers at the time of sampling on the ship. Aftervigorous shaking and an equilibration time of 1 to 2 h, CH4 inthe headspace was determined by GC. Flasks containing 10 mlof distilled water but no sediment were capped at the time ofsampling and analyzed by GC as controls. Concentrations werecalculated by dividing the amount of CH4 in the flask by theamount of sediment on a volumetric basis. Then the concen-trations were corrected by dividing by the average sedimentporosity (0.6) to give CH4 concentrations in the pore water.Sediment porosity was determined by gravimetry after over-night drying at 110°C.

Numerical analyses. A sampling campaign performed on 20September 1991 generated a matrix of 21 objects (sites) by 9variables. The nine variables included three dependent vari-ables (aerobic N20 production and CH4 oxidation and pro-duction) and six independent variables (water column depth,dissolved-02 concentration in the water column [1 m above thesediment-water interface] [DO], PC in the sediment, and CH4,NH4', and DOC in the sediment pore water). Preliminarystatistics (position and dispersion), test for the normality of thedistribution, the Box-Cox method to find the best normalizingtransformation, and the correlation coefficients (Pearson andSpearman) were computed (44).To study the interplay of environmental factors and micro-

bial activities, we performed a canonical redundancy analysis(52) on the transformed data as previously described (44). Thematrix of dependent variables contained the microbial activi-ties, and the matrix of independent variables contained the sixenvironmental variables mentioned above. To establish howmuch of the variance of aerobic N20 production and CH4oxidation and production could be explained by the environ-mental factors only or by the spatial component only, we usedthe method of Borcard et al. (4), which relies on constrainedordination (here the canonical redundancy analysis) (44).Three matrices were used: (i) a matrix of dependent variables(the three microbial activities); (ii) a matrix of the mostsignificant environmental variables (four), which included PC,NH4', depth, and DO, as selected by a forward procedureincluded in the CANOCO program (50); and (iii) a matrix ofgeographical coordinates (four) for each sampling station. Thisthird matrix included the most significant terms (underlined)for a cubic trend surface regression of the form z = blx + b2y+ b3x2 + b4y + b5y2 + b,x3 + b x2y + b8xy2 + bgy3, alsoselected by the same forward procedure (44).

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FIG. 1. N20 production in aerobic sediment slurries (1:1) from twolocations in Hamilton Harbour, a profundal (hypolimnetic) site (site906) and a littoral (epilimnetic) site (site 910). Slurries were incubatedat 25°C and 250 rpm in the dark under initial CH4 concentrations of 0,uM (O), 13 ,uM (V), 25 ,uM (V), 47 ,uM (O), 122 F.M (0), and 47 ,uMplus C2H2 (10 kPa) (-). SEM, standard error of the mean.

RESULTS

Spatial variation of N20 production, CH4 oxidation, andCH4 production. Potential aerobic N2O production was highenough to be detected even with a thermal conductivitydetector in sediment slurries at a profundal site (site 906) butnot at a littoral site (site 910) (Fig. 1) (for locations of the sitesin Hamilton Harbour, see reference 44). There was a muchhigher initial N2O production in flasks with lower initial CH4concentrations. The disappearance of N2O after 1, 2, or 4 daysof incubation is probably related to reduction to N2 bydenitrifying bacteria occurring with the onset of anoxic condi-tions. We found that in such an experimental slurry fromsediment showing high N2O production, only NH4'-N (26 + 1,umol fl-') was detected after a 1:1 dilution. As expected,NO2 and NO3 were not detectable at the beginning of theincubation, since the concentrations of these ions are very lowto undetectable in sediment pore water (<11.3 ,uM) (44).After an aerobic incubation of 48 h, there was a very smallaccumulation of NO2- (1.8 ± 0.6 ,umol fl- or 10% of Noxide products), but most NH4+ was converted to NO3 (122 ,umol * fl-1 or 60%). N20-N (6.4 1.7 ,umol - fl-1 or 30%)accounted for a rather significant portion of oxidized products.Considering the total amount of inorganic N at the end of the

incubation less the initial amount of NH4+-N, we calculate that11 ,umol of NH4+-N fl-' was produced by mineralizationwithin 48 h. This represents 30% of the final inorganic N.

Next we determined N20 production in such aerobic slurriesof sediment collected from 21 sites. We also measured poten-tial CH4 production and oxidation rates and six environmentalvariables. A statistical summary of these data is given in Table1. The high coefficient of variation of N20 production and pore

water NH4' concentrations indicates a great dispersion ofthese variables as a result of data from a few sites which hadmuch higher rates or concentrations than most of the sites. TheCH4 concentrations were higher than the NH4' concentra-tions, as can be seen from the average of both variables (Table1). As expected, CH4 concentrations were higher in the deepcenter of Hamilton Harbour, where sites become depleted in02 during summer stratification. These sites had a lowerpotential CH4 oxidation as well as a higher potential CH4production when compared with shallow sites. Results of a

canonical redundancy analysis performed on these field dataare summarized in Fig. 2. A Monte Carlo permutation testindicated that the overall analysis was highly significant (P0.01). The environmental variables explained 62.3% of thevariance of the dependent variables (aerobic N20 productionand CH4 oxidation and production). The graph describes thenegative correlation between CH4 oxidation and both N20 andCH4 production and their relationship with the environmentalvariables. None of the environmental factors measured showeda significant correlation to aerobic N20 production or poten-tial CH4 oxidation rates. The graph indicates that the first axis(P c 0.06), which explained 38.7% of the total variance,corresponded to the variation of PC, CH4 concentration,depth, and DOC (all positively correlated) and of DO (nega-tively correlated to these variables). The second axis, whichexplained 19.2% of the total variance, corresponded to varia-tion in NH4+ concentration. This analysis suggests that poten-tial CH4 production rates were a function mainly of PC (r =0.836). CH4 concentrations in sediments were closely related todepth (r = 0.920) and potential CH4 production rates (r = 0.605).These were higher in sites exhibiting low DO. The main axis ofvariation (axis I) appears to correspond well to the water columndepth variation between littoral and profundal sampling sites.To measure how much of the variance of each microbial

activity could be accounted for by the variation of environmen-tal factors alone without the spatial component, we partitionedout space as a component of the variance by using the methodof Borcard et al. (4). A summary of these analyses (Table 2)shows the amount of the variance of each microbial activityaccounted for by the environmental factors alone (fraction a),

TABLE 1. Statistical data for the nine variables measured at 21 locations in Hamilton Harbour on 20 September 1991

ValueVariable (unit) Location CV (%)

Minimum Mean Maximum

Aerobic N20 production (nmol - cm-3 * day-') Sediment 0 100 536 149CH4 oxidation (RMmol cm-3 day-l)a Sediment 0.74 2.26 3.35 31CH4 production (,umol - cm-3 * day-') Sediment 0.061 0.278 0.634 54Depth (m) 3 15 24 37DO (mg liter-) Water columnb 0.36 3.53 7.09 81PC (% drywt) Sediment 2.56 7.15 10.75 28CH4 sediment (mM) Pore water 1.02 4.04 6.83 42NH4' sediment (mM) Pore water 0.26 1.72 11.2 133DOC (mM) Pore water 0.83 1.28 2.63 34

Measured by adding CH4 (1 kPa) to 50-ml flask assay mixtures.b Water column 1 m above sediment.

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Sa m pling sites ± 20.2% = 56.6%). In contrast, the variation of potential CH4production between sites was very well accounted for by the

3.0 -1.5 0.0 1.5 3.0 variation of the measured environmental factors as shown byAerobic O prod . . 3.0 the highly significant fraction (47.4%). For this variable, only a2*Aerobic N20 prod. very small and insignificant fraction of the variance was

o 4 oxidation v explained by the spatial structure only (1.1%). Concerning the* OH4 production potential CH4 oxidation rates, most of the explained variance

1.5 cannot be attributed to either spatial or environmental com-DO ponents individually.

v Nitrification-dependent N20 production, nitrification, and

vveDfects of CH4. To determine the nature of the biologicalv Depth t 0 source of N20 in sediment slurries for sites showing high

. [CH4] potential N20 production, we tested the effect of C2H2 on theN20 production in aerobic slurries (243 + 21 and 394 + 102

'0 \ DOC Pc nmol of N20* cm-3 * day-' for sites 926 and 906, respectively).v Added C2H2 completely suppressed the N20 production in.\* 1.5 aerobic slurries of the two sites tested. This suggests stronglyv Littoral < 1 1 m |[NHthat N20 production in such slurries is nitrification dependent.v Profundal >11 m 4 This experiment did not rule out the possible contribution of

v N20 from denitrification of nitrification products. Indeed, in..., -3.0 another experiment, addition of C2H2 after an initial incuba-

I.0 -0.5 0.0 0.5 1 .0 tion of 12 h did not completely suppress the N20 production inicrobial activities/Environ mental factors aerobic slurries (results not shown).

To further support the hypothesis of a nitrification-depen-Axis dent source of N20 and to study the oxidation of NH4+ to

N02-, NO3-, and N20 in the presence of different initialOrdination diagram resulting from a canonical redundancy concentrations of CH4, we used more-diluted slurries (1:10)rformed as previously described (44) with the CANOCO spiked with NH4+-N (final concentration, 4 mM). Productionia matrix of three microbial activities (dependent variables) rates of N20 and NO3- were strongly correlated (r = 0.975, n'ironmental variables (independent) measured at 19 loca- = 24) (Fig. 3). At a comparatively low CH4concentration (1obial activities are shown by large symbols. Environmental Fg 3) At a0comaratvely low C4cneratioe(re shown by vectors: DO (dissolved oxygen 1 m above the

e ,no NOd or atn vr int erm e measured,ater interface), PC (sediment particulate carbon), DOC even after 11 days of incubation. At intermediate CR4 concen-r dissolved organic carbon), [CH4] (pore water methane trations (24 ,uM), NH4+ was completely oxidized to NO3 andons), and [NH4I] (pore water ammonium concentrations). N20 after 7 days of incubation. Very little NO2- accumulated.ications are indicated by the smaller symbols. Sites 926 and 929 At comparatively high dissolved-CH4 concentrations (84 ,uM),led because of extreme values and missing data, respectively. nitrification was completely suppressed and no NO2 or NO3

was detected in the slurries. Low N20 concentrations weredetected in the gas phase.

of interaction between environmental factors and To reduce 02 limitations, a similar experiment with dilutedction b), the spatial structure alone (fraction c), and slurries (1:10) was carried out, but the gas phases of thee unexplained fraction (fraction d). The significance experimental flasks were changed periodically. We found thatis a and c was tested by a Monte Carlo permutation under such conditions the rates of nitrification were similar tovariation of aerobic N20 production was poorly those obtained earlier in this study (Fig. 4). However, compar-by the variation of the environmental factors se- atively high CH4 concentrations (119 ,uM) did not completelyowever, much of this variation was related to the suppress NO3 production, although it was lower than thatucture, as indicated by the significant fraction (36.4% measured at lower CR4 concentrations. Note that NH4+

disappearance was more rapid at relatively high CH4 concen-trations (119 ,uM) than at any other CH4 concentrations. After

2. Partitioning of the variance of the three microbial the disappearance of NH4' (6 days) at 119 jM CR4, NO3ities studied in fractions representing environmental disappeared also. The absence of added CH4 led to a much-factors, spatial structure, environmental-spatial delayed NH4+ consumption and N03 production. Slurries with

interaction, and unexplained fractiona CH4 (109 ,uM) and C2H2 (10 kPa) did not show NO3- produc-tion, but NH4I disappeared after an initial lag of 3 days. Since

Aerobic N20 CH4 CH4 C2H2 was consumed in these slurries (data not shown), NH4+raction production oxidation production may have been immobilized by C2H2-oxidizing bacteria (29).

Sterile controls showed no NH4( consumption and no NO3-imental factorsb 18.2 22.1 47.4c production. In no treatment did N02- accumulate significantly.:nvironment 20.2 43.3 31.5 Effects of NH4+ on CH4 oxidation and field interpretation.stucurd We also investigated the effect of NH4+ on the production ofained 25.2 26.9 2010 N oxides and on CH4 oxidation in 1:1 sediment slurries (Fig.*ined 5). Rates of N20 and NO2 + NO, production increased

are expressed as a percentage of the total variance of each with NH4' concentration. In contrast, CH4 oxidation ratesivity. decreased by almost half at 10 mM NH4' and by two-thirds at+, depth, andDO.20MNR TIrrni-tnificant (P 20 mM NH4+. This negative correlation between N2 produc-dinates. tion and CH4 oxidation rates supports what was suggested byIt (P c 0.05). the field data (Fig. 2). The NH4+ concentration in pore water

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250 rpm in the dark. NH4' (0), NO2- (C), N03 (V), N20 (A), 02 (-), and CH4 (O) data are shown. Each datum point is the average oftriplicate flasks. Error bars represent + standard error of the mean.

sediment at site 926, located at the Burlington sewage treat-ment plant outfall, was about 10 times higher (11.2 mM) thanat other locations, and this may explain the lowest potential ofCH4 oxidation measured at this site. When site 926 is excluded,we find a highly significant (P . 0.01) positive correlation (r =0.585, n = 19) between NH4' and CH4 concentrations insediment pore water. When NH4' and CH4 concentrationsmeasured in sediment of other aquatic systems are considered(Table 3), a highly significant (P c 0.01) linear relationship (r= 0.952, n = 7) between CH4 and NH4' concentrations isobserved. It can be formalized by the equation y = 0.552x +0.130, where x is the CH4 concentration and y is the NH4'concentration, both in sediment pore water.

DISCUSSION

Spatial variation of N20 production is important, as sug-gested by the initial experiment on slurries from littoral andprofundal sites, and by the study of 21 sites. As shown by thecanonical redundancy analysis, none of the seven environmen-tal variables selected explained significantly the observed vari-ation of N20 production. This is quite unlike the variation ofCH4 production, which is well accounted for by the variation ofPC, NH4+, depth, and DO (44). However, it is of interest thatthe variation in N20 production is significantly explained bythe spatial polynomial in Hamilton Harbour. This means thatvariation of N20 production in this system is closely related toa spatially structured variable. A good candidate for such avariable could be toxicity to enzymes involved in the produc-tion or consumption of N2O. Heavy metals (unpublished data)and organic chemicals such as polycyclic aromatic hydrocar-bons (9) are located mostly in the eastern basin of HamiltonHarbour, which is subject to industrial and municipal wastewaterdischarges. This possibility is currently being studied (43).The ratio (30%) of N2O production to total nitrogen oxides

produced in Hamilton Harbour sediment slurries at site 926was more than similar ratios (N2O/NO2J = 0.3 to 15%)

reported for pure cultures of N. europaea (15) or for sterile soilinoculated with N. europaea (20). Biological sources of N2O inaquatic sediments are diverse and may include nitrification,denitrification (48), methanotrophic nitrification (54), andheterotrophic nitrification (1). Although at this stage wecannot rule out any of these processes as a contributing sourceof N20, it appears that it is dependent on a nitrification source,either chemolithotrophic or methanotrophic. Given the lowNO3- concentration in Hamilton Harbour sediment porewater (44), the high N2O production observed could not besupported solely by denitrifying bacteria. It is more likely thatdenitrification contributes to a certain extent by acting onNO3- produced from nitrification. C2H2 is a very stronginhibitor of the ammonia monooxygenase of nitrifying bacteria(19, 22) but not of heterotrophic nitrification (46). Therefore,acetylene inhibition of N20 production in Hamilton Harboursediment slurries suggests that the N20 is produced not fromheterotrophic nitrification but rather from chemolithotrophicor methanotrophic nitrification. However, Megraw andKnowles (40) showed that heterotrophic nitrifiers such asPseudomonas spp. may ultimately depend on methanotrophsfor a carbon source. If this is the case, heterotrophic N20production would be suppressed by C2H2 following the inhibi-tion of methanotrophs. The strong correlations between N20and NO3 production, and the stimulation of N20 productionby increased NH4' concentrations in slurries, further suggest anitrification source of N20.

Interaction between the nitrogen and the methane cyclesmay occur at several levels, of which nitrification is critical (30).We found that in sediment slurries, 6.04 kPa of CH4 (84 ,uM)inhibited nitrification completely, in contrast to 26 to 56%inhibition of nitrification by 10 kPa of CH4 reported for soilslurries (37). On the basis of the 02 and CH4 data, it seemsthat this inhibition of nitrification is the outcome of the 02depletion caused by CH4 oxidation. Such a suppression wasalso found in a humisol by Megraw and Knowles (38). Theypointed out that the higher 02 demand, coupled with the low

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FIG. 5. Effect of NH4' on initial potential rates of nitrification andN20 production (A) and CH4 oxidation (B) measured in sedimentslurries (1:1) under an initial 1 kPa of CH4 in the headspace (13.9 ,uMdissolved), at 25°C and 250 rpm in the dark. N02- + N03- productionrates (0 to 48 h) (0), N20 production rates (0 to 24 h) (V), and CH4oxidation rates (0 to 24 h) ([1) are shown. Datum points are average oftriplicate flasks, with error bars indicating ± standard error of the mean.

20

FIG. 4. (A and B) Effect of CH4 on NH4' consumption (A) andNO3- production (B) under nonlimiting 02 conditions. (C) Cumula-tive oxidized CH4 for each treatment. Aerobic conditions were main-tained by periodic evacuation and backfilling of the flask with air, asindicated by the arrows. Diluted sediment slurries (1:10) from site 906were incubated at 25°C and 250 rpm in the dark. CH4 concentrationsmaintained by periodic injections at the time of evacuation (arrows)were as follows: 0.62 + 0.13 ,uM (Li) (initial endogenous concentra-tion; in this treatment only, CH4 was not added at any time), 1.39 ±

0.09 ,uM (V), 10.8 ± 0.3 p.M (V), 119 ± 2 p.M (0), 109 ± 4 ,uM plusC2H2 (10 kPa) (M), and 104 ± 1 ,uM sterile (A). Each datum point isthe average of triplicate flasks, with error bars indicating + standarderror of the mean.

apparent K,(02) of methanotrophs, could suppress the activityof ammonium oxidizers under certain environmental condi-tions. Our results support this view. Under limiting 02 condi-tions and a dissolved-CH4 concentration of 84 ,uM, a completesuppression of nitrification, probably linked to the drasticdepletion of 02 due to CH4 oxidation, was observed. Undernonlimiting 02 conditions, complete suppression of nitrifica-tion did not occur even at a dissolved-CH4 concentration of119 ,uM, although NO3- production at this concentration waslower and the NO3- eventually disappeared, in contrast to thepattern observed at lower CH4 concentrations. This fact, inaddition to a more rapid disappearance of NH4' in slurrieswith 119 F.M dissolved CH4, supports the idea that under high

02 tension methanotrophs may compete with nitrifiers fornitrogen (38). Disappearance of N03- in highly aerobic sedi-ment slurries under a high-CH4 (119 ,uM) regime may beexplained on the basis of assimilation by active methanotrophicpopulations limited in nitrogen after the disappearance ofNH4+. Megraw and Knowles (38) showed that NO3 con-sumption occurred with CH4 oxidation in soil slurries.

Nitrification and N20 production in Hamilton Harboursediment slurries were clearly stimulated by intermediateconcentrations of CH4 (between 1 and 24 FM) under bothlimiting and nonlimiting 02 conditions. This is similar to theCH4-dependent nitrification reported for Methylosinus tricho-sporium OB3b (32). This suggests that methanotrophic nitrifi-cation may be important in Hamilton Harbour sediment eventhough specific nitrification rates by methanotrophs are gen-erally much lower than those reported for chemolithotrophicnitrifiers (2, 39). The most likely outcome of methanotrophicsuppression or stimulation of nitrification will depend verymuch on the in situ CH4 concentrations. In sediment ofHamilton Harbour, the observed CH4 concentrations (1.02 to6.83 mM) (Table 1) would suppress nitrification rather thanstimulate it. Such a reservoir of CH4 in the sediment clearlyfavors NH4' immobilization to organic N over oxidation toN03 by methanotrophs and subsequent loss to the atmo-sphere by denitrifying bacteria.

Increasing NH4' concentration would in turn reduce CH4

A

B

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METHANE METABOLISM AND NITRIFICATION IN SEDIMENT 3313

TABLE 3. Comparison of CH4 and NH4' concentrations in sediment of some aquatic ecosystems

Ecosystem Location Sampling Concn (,umol/liter) of": Referencesite' CH4 NH4_

FreshwaterEutrophic Blelham Tarn P 50-400 250-1,000 21

L 1-90 50-200Vechten P 60-1,125 75-425 49

L 50-250 41Mendota P 1,144c 892 10

L 687 11Geneva P 0-1,560 6Hamilton Harbour P 3,020-6,830 570-3,060 This study

L 1,020-2,880 260-1,230

Mesotrophic Constance L 200-1,200 51Washington P 11-330 35

P 0-330 33

Oligotrophic Superior P 0-6 42

Marine Cariaco Trenche (14-32) x 103 3,250-9,600 16Long Island Sound 3-16 36Davies Reef (3-390) x 10-3 <1-84 45Checker Reef (5-840) x 10-3 5.2-76 45

"P (profundal) and L (littoral) indicate the location of the sampling site when mentioned in the article.b The data represent variation in the top 5 cm of sediment except for the marine sediments (Cariaco Trench, 40 m below sediment-water interface; Long Island Sound,

top 50 cm; Davies and Checker Reefs, 200 cm within the reef framework).c Calculated from the CH4 concentration in gas bubbles collected at the sediment surface (P = 75% and L = 45% [vol/vol]), and assuming a temperature of 20°C

and the Ostwald coefficient for CH4 of 0.03668 (53).dSite 926 with an unusually high NH4' concentration (11.2 mM) is excluded.'NH4+ values were interpolated assuming [HCO3-- alkalinity.

oxidation and contribute to CH4 accumulation in sediment.Such an inhibition of CH4 oxidation was reported to becomplete at NH4' concentrations above 20 mM in sedimentslurries from Lake Constance (5), whereas we observed partialinhibition (65%) at 20 mM NH4' in sediment slurries fromHamilton Harbour. When compared with other aquatic sedi-ments, Hamilton Harbour sediments contain unusually highCH4 and NH4' concentrations (Table 3). The fact that bothconcentrations are higher than those reported for any otherlakes suggests the reciprocal inhibition of CH4 and NH4'oxidation by, respectively, NH4' and CH4 accumulations inthis sediment. This is supported by the positive correlationbetween CH4 and NH4' concentrations in sediment porewater throughout Hamilton Harbour, at 19 locations when 926and 929 are excluded, and also when compared with otheraquatic ecosystems. Methane concentrations in other eutro-phic and mesotrophic lakes would suppress nitrification. Inoligotrophic lakes, such as Lake Superior, or in marine sediment,such as Long Island Sound, CH4 concentrations may stimulatenitrification. The outcome in this case would depend on theNH4+ concentration. At a low NH4 concentration (<0.5 mM),NH4' may be immobilized by active methanotrophs (38).

In summary, our study shows the critical role played by CH4metabolism in the nitrogen cycle, especially at the nitrificationlevel, which is the rate-limiting step in the net loss of fixednitrogen. This is particularly important in Hamilton Harbour,where very high NH4' concentrations may be toxic to mem-bers of benthic communities. High CH4 concentrations insediments are likely to prevent oxidation of NH4( and may alsocontribute significantly to 02 depletion in the hypolimnion duringsummer stratification through high methanotrophic activity.

ACKNOWLEDGMENTSThis research was supported by a grant from the Great Lakes

University Research Fund of the Natural Sciences and EngineeringResearch Council of Canada (NSERC) to R.K. and by postgraduatefellowships from NSERC and FCAR (Fonds pour la Formation deChercheurs et l'Aide a la Recherche) to R.R.We thank Pierre Legendre and Shirley Richards for critical reading

of the manuscript; Murray N. Charlton for providing laboratory spaceat the National Water Research Institute (NWRI), EnvironmentCanada, and the initial interest in Hamilton Harbour; Robin LeSagefor technical assistance; and the crew members of the TechnicalOperation Division at the NWRI for assistance in the fieldwork.

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