Transformations of fixed nitrogen and N2O in the Cariaco Trench

Deep-SeaResearch,Vol.30, No. 6A, pp. 575 to 590, 1983. Printedin Great Britain.

0198-0149/83/$3.00+ 0.00 ~ 1983PergamonPressLtd.

Transformations of fixed nitrogen and N20 in the Cariaco Trench L. K. HASHIMOTO,* t W . A . KAPLAN,* S. C . WOFSY* a n d M. B. MCELROY*

(Received 22 February 1982; in revisedform 5 November 1982; accepted 15 December 1982) Abstract--The cycle of nitrogen in the Cariaco Trench was studied using stable isotope techniques with vertical profiles of nutrients, oxygen, and N20. The rate of nitrification was found to be zero order with respect to the concentration of NH~ at levels above 0.1 laM. The associated halfsaturation constant is about 0.15 IxM, much lower than in laboratory cultures of nitrifying bacteria. The result could explain efficient oxidation of NH~ in the deep sea, despite low numbers of nitrifying bacteria and low concentrations of NH~. Nitrification ceased in the trench for O 2 concentrations below 10 to 15 ltM, coincident with the appearance ofS 2-, possibly reflecting inhibition by reduced sulfur compounds. Nitrous oxide is a by-product of nitrification and is depleted where O 2 is <65 gtM, probably by denitrification. Denitrification and net loss of fixed N affected the concentrations of oxidized species (NO~, N20) at O 2 levels between about 10 and 65 IxM.

INTRODUCTION

THE Cariaco Trench contains a wide range of environmental conditions in a compact, stable system, providing an excellent setting for the study of nitrogen transformations. This paper reports results from experiments to elucidate factors controlling rates for nitrification, denitrifieation, and the cycle of N20 in waters of the trench. Previous studies using laboratory cultures indicated that nitrifying bacteria require high concentrations of NH~ (> 100 labl) for efficient growth (WATSON, 1965; C~tLUCCI and S~CXLAND, 1968). We sought to determine if organisms in natural systems have similar requirements and to examine the influence of O 2 on rates for nitrification and denitrification. We report observations between 28 March and 1 April 1979 at 10°30'N, 64°38'W on cruise EN-34 of the R.V. Endeavor. The data include measurements of NH~, NO~, NO~, and 02 as functions of depth at close intervals. Concentrations are complemented by studies of microbial oxidation of NH~ carried out using additions of is NH~ to water samples from various depths. EXPERIMENTAL DETAILS

The Cariaco Trench has two deeps, east and west, separated by a saddlepoint at 900 m. Stations selected for discussion (Stas 5, 11, and 13) were in the eastern deep where the total depth is 1400 m (Fig. 1). The trench is isolated from the Caribbean by a sill at its western end with maximum depth 146 m. Mixing of waters below about 150 m with the exterior is inhibited by the sill and by the * Center for Earth and Planetary Physics, Harvard University, Cambridge, MA 02138, U.S.A. t Present address: Montgomery Engineers, Pasadena, CA 91109, U.S.A. 575

576

L.K. HASrIIMOTOet al.

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Fig. 1. Map of the Cariaco Trench, Venezuela (MALONEY, 1966), showing the locations of Stas 5, 11, and 13 in the trench, and Sta. 14 in the Caribbean north of the trench. Depths are in meters.

strong pycnocline between 150 and 300 m. Temperature, density, and salinity are nearly uniform below 300 m and 02 is absent, in contrast to the more extensive thermocline and oxygenated deep waters of the Caribbean (see Fig. 2). [See ~CrlARDS (1975) for a comprehensive review of previous work in the basin.] Samples for nutrient analysis were acquired using 5-1 Niskin bottles equipped with reversing thermometers. Samples for 15N-labelled incubations were taken with 30-1 Niskin bottles washed with 0.1 N HCI immediately prior to use. Concentrations of NH~, NO~, and NO~ were determined promptly using methods of STRICKLANDand PARSONS(1972) scaled for 5-ml samples (MCCARTHY,TAYLORand TAFT, 1977). Salinity was determined using an inductive salinometer. Nitrous oxide was determined after the cruise using a gas chromatograph equipped with an electron capture detector (ELKINS, 1980). Samples for N20 analysis were poisoned by addition of 1 ml of saturated solution of HgCl2 and were stored in 60 ml BOD bottles. Sulfide concentrations were determined by the method of PACHMAYR(1960). Oxygen was measured on board ship using the standard Winkler technique (SrRICKLAND and PARSONS, 1972) except for Sta. 11, where a polarographic oxygen probe was used. Concentrations determined by the two methods agreed to better than + 5 BM in the range 5 to 150BM. Atmospheric contamination contributed <7 BM of O 2 to samples from the oxic-anoxic interface, as inferred from measurements of N 2 0 in the samples. Systematic errors in the Winkler procedure could add about 5 ~tM to the measured O2 concentration (CLINE and ~CHAgVS, 1972). However, sulfide was present in waters with 02 below about 10 ~M, which would tend to produce errors in the opposite sense (INovORS~N and JORGENSEN, 1979). Concentrations of sulfide were <2 BM for the important range of 02 between 10 and 20 ~tM, and associated errors in the Winider determinations should be < 1 BM

577

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Larger errors are possible with the polarographic technique (Sta. 11), depending on diffusion rates for sulfides through the Teflon membrane. Incubations with added ]SNH~ were conducted in 4-1 Pyrex bottles rinsed with 0.1 N HCI and with several increments of sample. Ammonium was added in increments ranging from 0.1 to 5 ~tM to examine the influence of substrate concentration on the rate of nitrification. Light was excluded from incubations by wrapping the bottles with several layers of black plastic f'dm and tape (BocK, 1965). Samples were placed in a water bath flushed with surface seawater at a temperature of 27°C. Incubations lasted between 6 and 8 h and were terminated by filtering the sample through Reeve-Angel 984H glass fiber filters having a mesh size of approximately 0.1 ~tm. Pressure across the fdters was not allowed to exceed 25 cm Hg. Filtrates were stored at approximately - 3 0 ° C . Samples were analyzed for ]5 NO~ by adaptation (HAsrnMOXO, 1981) of methods described by SCHELL (1978) and OLSON (1981). Samples were purged of NH~ by addition of base, followed by distillation of one third of the sample volume (BRE~N~ and KEENEr, 1966). Nitrite was diluted by addition of 3 pM of unlabelled NO~ to provide adequate sample size for the mass spectrometer. The sample was transferred to a 5-1 separatory funnel and NO~ was reacted with reagents to form a diazo compound. The sample was acidified and the dye was extracted into 1,1, l-trichloroethane. The extract was evaporated onto a glass fiber filter, tombusted (BARSDATE and DUGDALE, 1965) to form N 2, and the isotopic ratio (]SN]4N :I4NI4N) was recorded on a mass spectrometer. The rate for NH~ oxidation was determined by dividing ~5NO~2 by the duration of the incubation period. Concentrations of ]5 NO] and other pools of ]5 N were not determined. The value obtained represents, therefore, a lower limit to the rate of nitrification. The correction for ]~NO~ should be small. Less than 4 nM of NO~ was formed during the incubations, implying turnover times for NH~ in excess of 200 h due to nitrification. Dilution of ]5 NH~ by other processes could be important in a few eases (see Table 1). Turnover times were also long for NO~2 in those samples (about 50%) where initial 0 2 •

L K. HASHIMOTO et al.

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Cycle of nitrogen in the Cariaco Trench

579

NO~2 concentrations could be measured, and we have no reason to expect more rapid turnover of NO~2 in the other samples. RESULTS

Distributions of nutrients, oxygen, and nitrous oxide

Prof'des of salinity, temperature, and density (a t) are shown in Fig. 2, while results for 02, N2O, NH~, and NO~3 are shown in Fig. 3. Data for the adjacent Caribbean shelf waters are shown for comparison. The nutrient data agree with results obtained previously at lower spatial resolution (OKt~A, BENITEZand FERNANDEZ,1969; OKt~A, RuIz and GARCIA, 1974; RJCHARDS, 1975; CLINE, 1973). Prof'des for NO~3 and N 20 have maxima near 200 m, about 50 m below sill depth, indicating in situ production of these species in the trench. The maximum N20 appears to occur slightly below that for NO~3 (220 vs 190 m). Nitrate and N20 were undetectable below 290 m (Fig. 3). The disappearance of NO~3 and N 20 was abrupt, with concentrations falling from one third of peak values to zero in < 10 m. Concentrations of NH~ were very low or undetectable above 290 m, increasing steadily below that level to about 18 I~M near the bottom. The profde of inorganic N (NH~ + NO 2 + NO~) has a pronounced minimum near 300 m (Fig. 4). Organic N was nearly constant with depth in the trench (OKuDA et al., 1969, 1974) and consequently the behavior indicated by Fig. 4 should hold also for total N. The decline in N from 200 to 300 m is associated with disappearance of NO~ ; the increase below 300 m is due to increasing concentrations of NH~. Concentrations of NO~2 were below the limit of measurement (0.03 ~tM) in most samples. However, samples from depths between 254 and 277 m contained nitrite at levels up to 0.70 ~M. In one case, samples from Niskin bottles 1 m apart at 275 m yielded concentrations of 0.43 and 0.19 ~tM, indicating vertical layering and steep gradients in the NO 2 distribution. Detectable NO~2 was confined to the vicinity of the minimum for inorganic N in waters with concentrations ofO 2 in the range 13 to 20 I~M. Figure 5 shows data for NO~, NO~, NH~, N20, and S2- plotted as functions of 02 concentration. Oxidized forms of fixed N disappear abruptly and simultaneously at 02 levels near 10 ~M at all stations, coincident with the minimum in mineral N. The same result was obtained with Winkler and polarographic oxygen determinations and agrees with data given by OKUDA et al. (1969, 1974). Oxygen was undetectable 10 to 20 m below the level where NO~ and N 20 disappear. Reduced species such as NH~ and S2- were virtually absent from waters with significant concentrations of NO~ or N 20. Reduced and oxidized species were separated by a relatively sharp boundary at about 290 m (10 to 15 ~M 02). Figures 6a and b show strong correlations between concentrations of N20 and NO~ and between NH~ and S2-. The molar ratio N20-N :NO~ is produced as a by-product of nitrification (GOREAU, KAPLAN, WOFSY, MCELROV, VALOlS and WATSON, 1980; F~raNs, WOFSY, MCELROY, KOLBand KAPLAS, 1978; COHEN and GORDON, 1978, 1979). The ratio NH~ :S2is about 0.45 near the 02 interface, decreasing slightly to 0.35 at depth. The intercept S2- = 0 corresponds to about 0.2 IJM NH~. The quantity of fixed N removed from trench waters by denitrification can be inferred from observations of N compounds and 02. CLINE(1973) estimated net removal of f'LXedN, AN, by calculating the difference between the observed concentrations of inorganic N and the concentration expected from a regression between 02 and inorganic N (R~FIELD, KETCHUMand RICHARDS, 1963). He showed that N deficits calculated in this manner agree with observed

580

L.K. HASHIMOTOet al.

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Fig. 3. Profiles of O z, N20, NH~, and NO3 at Stas 5, 11, and 13 in the Cariaco Trench (panels a, b, and c, respectively). Note the disappearance of NOT, NOT, and N20 where the 02 concentration reaches 13 pM. Panel d shows the same profiles for the Caribbean (Sta. 14). Concentrations of NH~ and NO~ were very low at all depths for this station.

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58 3

Cycle of nitrogen in the Cariaco Trench

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584

L . K . HASHIMOTO et al.

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excesses of dissolved N2, which he derived independently from measurements of the N 2 :Ar ratio. OKUDA et aL (1974) estimated AN using observed concentrations of inorganic PO~and assuming a value of 16:1 for the regeneration ratio N:P. This approach gives results similar to CLINE'S(1973). We calculated AN from the present observations using CLINE'S(1973) regression equation with results shown in Fig. 7. Nitrogen removal increases as the O2 level declines, but removal ceases where.NO~3 and N20 disappear, near [02] = 13 ~tM. Measurements of the rate of nitrification obtained using 15N are given in Table 1, together with a summary of conditions under which samples were incubated. Rates are plotted in Figs 8a and b as functions of the total initial concentration of NH~ (including label). Errors in individual rates were estimated using analyses of standard mixtures of ~5NO2. Rates of nitrification increased with NH~, with the sharpest rise between 0 and 0.1 ~tM NH~. The EJ

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most complete set of data, for samples from 225 and 275 m, suggest Monad kinetics with a half-saturation constant of about 0.15 ~tM. Asymptotic (maximum) rates for nitrification were between 0.8 and 6 x 10-4 I~M 1-1 hq , with no obvious dependence on depth. Samples from 300 and 400 m were contaminated by small amounts of 02 introduced in transferring water to the incubation flasks and during the incubation. It is probable that nitrification rates inferred for these samples are too high as a consequence. Asymptotic rates observed for high levels of NH~ (> 1 BM) are similar to results from coastal waters elsewhere (Table 2). Rates for the Caribbean are lower than values observed in the mid-Pacific. Apparent half-saturation constants observed in the present experiments are lower by factors of 103 to 104 than those inferred for cultures of nitrifying bacteria (WATSON, 1965; CARLUCCl and SrRICKLAND, 1968; PAINT~, 1970), indicating that marine nitrifying organisms can utilize very low concentrations of NH~. The result is consistent with recent data by OLSON(1981). DISCUSSION

It is instructive to consider a simple model for vertical transport in the trench as an aid in interpretation. Assume that water from the Caribbean enters the deep part of the trench during brief episodes when the Caribbean pycnoeline is elevated by meteorological events, usually in February (RaCHARDSand VACCARO, 1956; PaCHARDS, 1975). Trench waters most of the time are quiescent and vertical mixing is slow, allowing steep gradients to persist. The water column acquires N from sediments and from mineralization of organic material in sinking debris. Ammonium and phosphate accumulate in deep water where 02 is absent, and the oxic-anoxic interface rises slowly with time (BAcoN, BREWER, SPENCER, MUNOZ and

586

L.K. HASHIMOTOet al. Table2.

Previous estimates of the potential rate of nitrOqcation

Study and site MIYAZAKI,WADAand HATTORI(1973) Sagami Bay, Japan

MIYAZAKI,WADAand HATTOR1(1975) East China Sea Philippine Sea West Pacific HATTORI,GOERINGand BOISSEAU(1978) Skan Bay, Alaska

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Rate (10-4 I~M h-~)

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Values represent rate of appearance of nitrite from incubations with labeled ammonium, in dark bottles. * OLSON(1981) did several incubations with samples from several stations and with differing concentrations of added ammonium.

GODDARD, 1980). The evolution continues until the system is perturbed by fresh input of oxygenated water from the Caribbean. The present results can be interpreted readily in the framework o f this model. We distinguish three zones o f biological activity with boundaries defined by the concentration o f 02 as illustrated schematically in Fig. 9. Mineralization and nitrification represent the principal transformations of N at 02 levels above about 65 laM (2 mg 1-1) (zone I). Nitrification is quite efficient, and consequently ambient NH~ is very low. Nitrate accumulates and 02 declines in a manner consistent with REDFIELD et al. (1963). Nitrous oxide builds up in proportion to NO~3, as observed for other marine systems (ELKINS et al., 1978), with about 2 x 10 -3 moles N in N 2 0 per mole of NO~. Nitrification and denitrification proceed simultaneously at O 2 concentrations between 10 and 65/aM (zone II). Nitrous oxide and nitrate are removed from the water column as O 2 decreases and fixed N is lost. Nitrification continues but rates decline as the level of O 2 is reduced. Nitrification ceases abruptly near 300 m where O 2 concentrations fall below 10 to 15 ~tM (zone III). A m m o n i u m accumulates in the water and fixed N is conserved. The concentration

Cycleof nitrogenin the CafiacoTrench

587

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of 02 appears to exceed 10 pM at the interface between oxidized and reduced species, although we cannot exclude a contribution to this value from systematic errors or from atmospheric contamination. If we accept the data at face value, it seems unlikely that nitrification should be inhibited solely by low concentrations of 02 , because nitrification and growth of nitrifying bacteria have been observed in the laboratory at O2 levels as low as 5 pM (CARLUCCIand MCNALLY,1969; PAINTER, 1970; GOREAUet al., 1980). The sharp boundary separating NO~3 from S2- and NH~ (Figs 5 and 7) raises the possibility that oxidation of NH~ could be affected by the presence of reduced sulfur compounds. Nitrification is known to be inhibited by S2-, COS, and CS 2 (BREMNERand BUNDY, 1974). Loss of N, due presumably to denitrification, is observed at 02 concentrations of about 65 ~tM (2 mg 1-1). The threshold is higher than might be expected if activity were confined to the water column. WUHRMANN(1964) proposed that denitrification in the water column should begin typically at about 30 ~tM 02, and GOEmNG and CLINE(1970)observed the onset of denitrification in water from the northeast tropical Pacific at 02 levels of about 20 ~M. The discrepancy could reflect the influence of anoxic microsites associated with decay of organic material (JANNASCH, 1960) or the role of denitrification in anoxic sediments at the trench boundaries. An important role for sediments would be consistent with observations from Chesapeake Bay (ELKINS et al., 1978; ELKINS, 1980) and Harrington Sound, Bermuda (BRowN, 1981). Concentrations of N20 and NO~ drop sharply in such systems when O 2 levels decline below about 60 ~tM. Previous studies (FANNING and PILSON, 1972; CUNE, 1973) USed an eddy diffusion formulation to fit distributions of trace species below and above the oxic-anoxic transition, whereas we have assumed that vertical mixing is normally slow, especially in the transition zone itself. Our view is motivated by observations of sharp gradients near the interface, with vertical scales of 10m or less (Figs 3 and 4). BROENKOW(1969, p. 169) observed sharp gradients near the oxic-anoxic transition in Lake Nitanat, and he inferred a vertical eddy diffusion coefficient close to that for molecular diffusion. A similar situation may apply to the Cariaco Trench, although more data are clearly needed to resolve the issue. Efficient nitrification at low NH~ may be the key to resolution of a long-standing puzzle. Nitrate concentrations in deep waters of the ocean average about 30 pM, most of which was

588

L.K. HASHIMOTOel aL

generated by in situ oxidation after last contact with the surface (REDFIELD el al., 1963). If one adopts 500 y for the turnover time of the ocean, the average rate for nitrification would be about 2 x 10-15 mole 1-1 s-1. However, it is difficult to account for a rate of this magnitude using kinetic parameters and numbers of organisms reported in the literature. The rate R for nitrification by active growing bacteria is given by (CARLUCCIand SrRICKLAND,1968) R = anKm~[Nn+4]/(K ' + [NH~]),

where (NH~ ] is the ambient ammonium concentration (about 0.1 ~M in deep waters), n is the average number of cells (103 1-1 in deep waters, WATSON, 1965), a is the amount of NH~ utilized per cell doubling (1.5 x 10-12 moles; PAINTER, 1970; GOREAUet aL, 1980), KmaXis the growth rate at high NH~ (10 to 100 x 10-3 h-1 ; WATSON, 1965; PAINTER, 1970; GOREAU et aL, 1980), and K ' is the half-saturation constant. If K' is adopted from laboratory observations (100 to 500 ~M; CAP~UCCIand STRICKLAND,1968; PAINTER, 1970), the calculated value for R would be <10 -17 mole 1-1 s-1 , a discrepancy exceeding two orders of magnitude (CARLUCCl and STRICKLAND, 1968). However, if we adopt K' from present results (0.1 to 0.2 laM), calculated values for R are 1 to 10 x 10-15 mole 1-1 S-1 , close to the mean global rate. Hence the discrepancy may be an artifact of experiments using laboratory cultures, in which bacteria are adapted to high levels of NH~. CONCLUSION

Measurements from the Cariaco Trench show that nitrification is efficient for concentrations of NH~ below 0.1 pM so long as ambient 02 concentrations exceed about 10 laM. Nitrification appears to terminate abruptly at oxygen concentrations below that level and the transition corresponds to the appearance of detectable S2-. Such behavior may reflect inhibition of nitrification by reduced sulfur compounds. Nitrous oxide is produced as a by-product of nitrification and is consumed at low levels of 02, presumably by denitrifieation. The influence of denitrification is apparent for prof'des of NO~ and N 20 at 02 concentrations below 65 laM. Denitrification may occur either in sediments or in the water column. Acknowledgements--We thank Dr. IAN MORRIS, chief scientist on cruise EN-34, the crew of the R.V. Endeavor, and the Republic of Venezuela for making possible our work in the Cariaco Trench. We are indebted to D. P. KELLY for permission to quote the results on sulfide concentrations and to J. J. MCCARTHY for use of the MS-9 mass spectrometer. The work was supported by NASA Grant NASW-2952 to Harvard University. REFERENCES BACON M. P,, P. G. BREWER, D. W. SPENCER, J. W. MUNOZ and J. GODDARD (1980) Lead-210, polonium-210, manganese and iron in the Cariaco Trench. Deep-Sea Research, 27, 119-135. BARSDATE R. J. and R. C. DUGDALE (1965) Rapid conversion of organic nitrogen to N 2 for mass spectrometry: an automated Dumas procedure. A nalytical Biochemistry, 13, 1-5. BOCK E. (1965) Vergleichende Untersuchung uber die Wirkung sichtbaren Lichtes auf Nitrosomonas europaea und Nitrobacter wingoradskyi. Archiv ffir Mikrobiologie, 51, 18-41. BREMNER J.M. and L.G. BUNDY (1974) Inhibition of nitrification in soils by volatile sulfur compounds. Soil Biology and Biochemistry, 6, 161-165. BREMNER J. M. and D. R. KEENLY (1966) Determination and isotope ratio analysis of different forms of nitrogen in soils--III. Exchangeable ammonium, nitrate and nitrite by extraction--distillation methods. Proceedings of the Soil Science Society of America, 30, 577-582. BROENKOW W. W. (1969) The distributions of non-conservative solutes related to the decomposition of organic material in anoxic marine basins. Ph.D. Thesis, University of Washington, Seattle, 207 pp.

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