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Therefore, anammox in these waters could account for 10–15% of the marine N2 production. The anammox process may be more important than this, with particularly favourable conditions occurring in upwelling areas where anoxic nitrate-rich water reaches the sediment. A good example would be upwelling areas off the Peruvian and Chilean coasts, where sedimentary sulphide is oxidized by sulphide-oxidizing bacteria with nitrate, producing ammonium23. This ammonium is released to the water column, probably enhancing the signi?cance of anammox, and N2 production, in these areas. Thus, the anammox process, with its distinct regulatory characteristics, should be included in studies of nitrogen cycling in the marine environment. A
15. Luther, G. W. I., Sundby, B., Lewis, P. J. & Silverburg, N. Interactions of manganese with the nitrogen cycle: alternative pathways to dinitrogen. Geochim. Cosmochim. Acta. 61, 4043–4052 (1997). 16. Hulth, S., Aller, R. C. & Gilbert, F. Coupled anoxic nitri?cation/manganese reduction in marine sediments. Geochim. Cosmochim. Acta. 63, 49–66 (1999). 17. Murray, J. W., Lee, B., Bullister, J. & Luther, G. W. in Environmental Degradation of the Black Sea: ¨ ¨ ¨ Challenges and Remedies (eds Besiktepe, S. T., Unluata, U. & Bologa, A. S.) 75–91 (Kluwer Academic, Dordrecht, 1999). 18. Nielsen, L. P. Denitri?cation in sediment determined from nitrogen isotope pairing. FEMS Microbiol. Ecol. 86, 357–362 (1992). 19. Van Mooy, B. A. S., Keil, R. G. & Devol, A. H. Impact of suboxia on sinking particulate organic carbon: Enhanced carbon ?ux and preferential degradation of amino acids via denitri?cation. Geochim. Cosmochim. Acta. 66, 457–465 (2002). 20. Codispoti, L. A. & Christensen, J. P. Nitri?cation, denitri?cation and nitrous oxide cycling in the Eastern tropical South Paci?c Ocean. Mar. Chem. 16, 277–300 (1985). 21. Cline, J. D. & Richards, F. A. Oxygen de?cient conditions and nitrate reduction in the Eastern tropical northern Paci?c Ocean. Limnol. Oceanogr. 17, 885–900 (1972). 22. Naqvi, S. W. A. Some aspects of the oxygen-de?cient conditions and denitri?cation in the Arabian Sea. J. Mar. Res. 45, 1049–1072 (1987). 23. Otte, S. et al. Nitrogen, carbon, and sulfur metabolism in natural Thioploca samples. Appl. Environ. Microbiol. 65, 3148–3157 (1999). 24. Braman, R. S. & Hendrix, S. A. Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium (III) reduction with chemiluminescence detection. Anal. Chem. 61, 2715–2718 (1989). 25. Grasshoff, K., Ehrhardt, M. & Kremling, K. Methods of Seawater Analysis (Verlag Chemie, Weinheim, 1983). 26. Hall, P. O. J. & Aller, R. C. Rapid, small-volume, ?ow injection analysis for CO2 and NH? in marine 4 and freshwaters. Limnol. Oceanogr. 37, 1113–1119 (1992).

Methods
Sampling was performed at Station A (88 34.15 00 N, 838 14.69 00 W) and Station B (88 37.99 00 N, 838 20.62 00 W) of Golfo Dulce in November 2001. Pro?les of salinity, temperature and oxygen were measured at 10-m intervals with a DataSonde 4 (Hydrolab). All water samples were retrieved with a 5-l Niskin bottle (KC Denmark). For nutrient pro?les, water was sampled from the Niskin bottle with a plastic syringe and ?ltered through a cellulose acetate ?lter (pore size 0.22 mm) into polypropylene vials that were stored on ice until return to the laboratory where they were frozen for later analysis. For the 15N-labelling experiments, water was sampled at 120, 140, 160 and 180 m depth at Station A, and at 100, 120, 160 and 180 m depth at Station B. Water was transferred from the Niskin bottle via Tygon tubing into the bottom of a 250-ml glass bottle and allowed to ?ow over for half a volume change. The bottle was closed with a Viton stopper taking care to exclude bubbles, and stored on ice until return to the laboratory. Experiments were started no later than 6 h after sampling. For each of the four depths from each station experiments were started by the addition of 15N labelled and unlabelled nitrate and ammonium to the 250-ml bottles to the following ?nal concentrations from concentrated stock solutions: 10 mM 15NO2, 10 mM 3 15 NO2 ? 10 mM 14NH? and 10 mM 15NH?. After addition the amended water was 3 4 4 bubbled with helium for 15 min to facilitate the detection of N2 production, and then transferred to 12-ml glass vials with a 5-mm butyl rubber septum (Exetainers, Labco) through a glass tube leading from the bottle into the bottom of the Exetainer and allowed to over?ow. The Exetainers were incubated at in situ temperature in a water bath kept anoxic with an alkaline sodium ascorbate solution. The oxygen concentration in the Exetainers was measured with a microelectrode designed for measuring low oxygen concentrations (Unisense) and was below the detection limit of 0.2 mM throughout the experiment. At each time point Exetainers were sampled in triplicate by removing 5 ml of water while replacing it with helium and adding 0.1 ml of 50% (w/v) ZnCl to stop biological activity in the Exetainer. The removed water was ?ltered for nutrient analysis and stored as described above. The concentration of NO2 ? NO2 was determined using the vanadium chloride 3 2 reduction method24 (NOx analyser model 42c, Thermo Environmental Instruments Inc.). Nitrite was analysed spectrophotometrically25 and NH? was determined using the ?ow 4 injection method with conductivity detection26. The isotopic composition of the nitrate and ammonium pool was estimated from their concentrations before and after amendment. Concentrations of 14N15N and 15N15N were determined by isotope ratio mass spectrometry and calculated as excess above their natural abundance2.
Received 28 October 2002; accepted 3 March 2003; doi:10.1038/nature01526.
1. Falkowski, P. G., Barber, R. T. & Smetacek, V. Biogeochemical controls and feedbacks on ocean primary production. Science 281, 200–206 (1998). 2. Thamdrup, B. & Dalsgaard, T. Production of N2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl. Environ. Microbiol. 68, 1312–1318 (2002). 3. Gruber, N. & Sarmiento, J. L. Global patterns of marine nitrogen ?xation and denitri?cation. Glob. Biogeochem. Cycles 11, 235–266 (1997). 4. Codispoti, L. A. et al. The oceanic ?xed nitrogen and nitrous oxide budgets: Moving targets as we enter the anthropocene? Sci. Mar. 65 (suppl. 2), 85–105 (2001). 5. Thamdrup, B., Can?eld, D. E., Ferdelman, T. G., Glud, R. N. & Gundersen, J. K. A biogeochemical survey of the anoxic basin Golfo Dulce, Costa Rica. Rev. Biol. Trop. 44, 19–33 (1996). 6. Vargas, J. A. Paci?c coastal ecosystems of Costa Rica with emphasis on the Golfo Dulce and adjacent areas: A synoptic view based on the R.V. Victor Hensen expedition 1993/94 and previous studies. Preface. Rev. Biol. Trop. 44, U1–U4 (1996). 7. Lipschultz, F. et al. Bacterial transformations of inorganic nitrogen in the oxygen-de?cient waters of the Eastern Tropical South-Paci?c ocean. Deep-Sea Res. 37, 1513–1541 (1990). 8. Bange, H. W. et al. A revised nitrogen budget for the Arabian Sea. Glob. Biogeochem. Cycles 14, 1283–1297 (2000). 9. Deutsch, C., Gruber, N., Key, R. M., Sarmiento, J. L. & Ganachaud, A. Denitri?cation and N2 ?xation in the Paci?c Ocean. Glob. Biogeochem. Cycles 15, 483–506 (2001). 10. Richards, F. A. in Advances in Water Pollution Research (ed. Pearson, E. A.) 215–232 (Pergamon, London, 1965). 11. Richards, F. A. in Chemical Oceanography (eds Riley, J. P. & Skirrow, G.) 611–645 (Academic, London, 1965). 12. van de Graaf, A. et al. Anaerobic oxidation of ammonium is a biologically mediated process. Appl. Environ. Microbiol. 61, 1246–1251 (1995). 13. Dalsgaard, T. & Thamdrup, B. Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments. Appl. Environ. Microbiol. 68, 3802–3808 (2002). 14. Knowles, R. Denitri?cation. Microbiol. Rev. 46, 43–70 (1982).

Acknowledgements We thank J. A. Vargas for assistance in arranging ?eld work, and E. Ruiz and D. Morera for assistance during sampling. We also thank L. Salling, P. S?holt, K. G. Lauridsen, E. Frandsen, T. Quottrup, M. V. Skj?rb?k and A. Haxen for analytical work. D.E.C., J.P. and B.T. were supported by the Danish National Research Foundation and the Danish National Science Research Council. Competing interests statement The authors declare that they have no competing ?nancial interests. Correspondence and requests for materials should be addressed to T.D. (e-mail: tda@dmu.dk).

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Anaerobic ammonium oxidation by anammox bacteria in the Black Sea
Marcel M. M. Kuypers*, A. Olav Sliekers?, Gaute Lavik*, Markus Schmid?, ? Bo Barker J?rgensen*, J. Gijs Kuenen?, Jaap S. Sinninghe Damste?, Marc Strous§ & Mike S. M. Jetten§
* Max Planck Institute for Marine Microbiology (MPI), Department of Biogeochemistry, Celsiusstrasse 1, 28359 Bremen, Germany ? Department of Microbiology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands ? Royal Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59, 1790 AB Den Burg, The Netherlands § Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6526 ED Nijmegen, The Netherlands
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The availability of ?xed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions1. The conversion of nitrate to N2 by heterotrophic bacteria (denitri?cation) is believed to be the only important sink for ?xed inorganic nitrogen in the ocean2. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the world’s largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors3. Nutrient pro?les, ?uorescently labelled RNA probes, 15 N tracer experiments and the distribution of speci?c ‘ladderane’ membrane lipids4 indicate that ammonium diffusing
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upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the ?rst time that anammox bacteria have been identi?ed and directly linked to the removal of ?xed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings5–7 indicates that anammox might be important in the oceanic nitrogen cycle. The Black Sea is the world’s largest anoxic basin and is a model for both modern and ancient anoxic environments. It is characterized by a high ammonium concentration in the deep waters (up to 100 mM), whereas only trace amounts of ?xed inorganic nitrogen are present in the ‘suboxic’ zone6,8 where the reduction of nitrate, manganese oxide or iron oxide occurs9. This apparent ammonium sink in the suboxic zone strongly suggests7,10,11 that ammonium is oxidized anaerobically to N2. Indeed, bacteria able to oxidize ammonia anaerobically have recently been discovered in laboratory bioreactors and wastewater treatment systems3,12. These so-called ‘anammox’ bacteria belonging to the order Planctomycetales directly oxidize ammonia to N2 with nitrite as the electron acceptor (Fig 1a, b): NH? ? NO2 ! N2 ? 2H2 O 4 2 ?1? During an R/V Meteor cruise in December 2001 we investigated the role of anammox in the Black Sea water column by using microbiological and biogeochemical techniques. In accord with with earlier studies6,8,11 we observed a nitrate maximum at the bottom of the oxic zone in the western basin (site 7605; 428 30.71 0 N, 308 14.69 0 E; Fig. 2a). This maximum is caused by the mineralization of phytoplankton-derived organic nitrogen coupled to aerobic nitri?cation (Fig 1a). Ammonium concentrations are high in deep waters but decrease to background values above 97 m water depth (Fig. 2a). Aerobic nitri?cation cannot account for the consumption of ammonium because O2 is absent below 80 m (Fig. 2b). However, nitrate penetrates 15 m deeper in the water column, indicating that nitrate could be the oxidizer of ammonium11. Alternatively, anammox bacteria could be using nitrite instead of nitrate to oxidize ammonium. Nitrite is an intermediate of denitri?cation and a nitrite peak is present at the base of the nitrate peak (Fig. 2a). Anammox in the suboxic zone could be coupled to nitrate reduction to nitrite (Fig 1a) by denitri?ers13, similarly to the process in anammox bioreactors14. To check for anammox activity in the suboxic zone we anaerobically incubated water samples from various depths after the addition of [ 14 N]nitrite and [ 15 N]ammonium. Because the anammox process combines 1 mol of [15N]ammonium and 1 mol of [14N]nitrite to form 1 mol of single-labelled dinitrogen gas (14N15N) (equation (1)), the depth distribution of d14N15N (Fig. 2c) expresses the potential anammox activity. The d14N15N record shows a clear peak in the zone of nitrite and ammonium disappearance, whereas no signi?cant anammox activity is observed outside the suboxic zone. Speci?c biomarkers, so-called ladderane lipids, were used to trace anammox bacteria in particulate organic matter collected from various depths across the suboxic zone. Ladderane lipids4 are the main building blocks of a unique bacterial membrane that surrounds the anammoxosome, a special compartment of the anammox cell, in which the anaerobic oxidation of ammonium to N2 takes place (Fig. 1b). Three different ladderane lipids were detected in the saponi?ed total lipid extracts with a depth distribution (Fig. 2d and e) similar to that of the potential anammox activity (Fig. 2c), indicating that anammox bacteria could indeed be responsible for the anaerobic oxidation of ammonium. A clone library was generated from DNA extracted from Black Sea water at the depth of maximum ladderane abundance (90 m), after ampli?cation of the 16S ribosomal RNA gene with primers speci?c for Planctomycetes15. Phylogenetic analysis of the 16S rRNA sequences con?rms that the Planctomycetes, tentatively named Candidatus ‘Scalindua sorokinii’, from the suboxic zone of the Black Sea are related to bacteria known to be capable of the anammox process (87.9% sequence similarity to Kuenenia, 87.6% to Brocadia; Fig. 3). In fact, the sequence obtained from the Black Sea is nearly identical (98.1%) to a sequence recently obtained from a bioreactor shown to have anammox activity (M. Schmid, K. Walsh, R. Webb, W. I. Rijpstra, K. T. van de Pas Schoonen, T. C. J. Hill, B. F. Moffett, J. A. Fuerst, J.S.S.D., J. A. Harris, P. J. Shaw, M.S.M.J. and M. Strauss, unpublished observations). On the basis of the sequence obtained from the Black Sea, we designed an oligonucleotide probe, labelled with Cy3 ?uorochrome, for ?uorescence in situ hybridization (FISH). This probe gave a bright and speci?c signal with cells that have the unusual doughnut shape characteristic for anammox bacteria in bioreactors. Ladderane biomarkers and cells hybridizing with the new FISH probe (Fig. 1c) were also found in the suboxic zone at the shelf break (Station 7617, 438 38.04 0 N, 308 02.54 0 E), indicating that anammox bacteria are not restricted to the strongly strati?ed central basin but are also present in the more dynamic peripheral current8. The combined results clearly indicate that anammox bacteria are abundant and active in the Black Sea. Could these anammox bacteria be responsible for the observed ammonium sink in the suboxic zone of the Black Sea? If we assume that the concentration pro?le of ammonium represents a steady state, an anaerobic ammonium oxidation rate of ,0.007 mM day21 was calculated for the suboxic zone of the central basin by using a reaction diffusion model. This rate is comparable to aerobic ammonium oxidation rates (0.005– 0.05 mM day21) determined for the nitrate maximum of the western central basin of the Black Sea16. An anammox rate of 2–20 fmol ammonium per cell per day was found in laboratory bioreactors3. Assuming a similar range of cell-speci?c activity for the Black Sea, 300–3,000 anammox cells ml21 would be needed to account for the observed ammonium oxidation rates in the suboxic zone. Counts of
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Figure 1 Morphology and physiology of anammox bacteria and their role in the marine nitrogen cycle. a, Simpli?ed marine nitrogen cycle including the anammox ‘sink’. Org.N, organic nitrogen. b, Morphology of the anammox cell and proposed model for the anammox process. HH, hydrazine (N2H4) hydrolase; HZO, hydrazine oxidizing enzyme; NR, nitrite reducing enzyme. c, Fluorescence in situ hybridization of ?lter material from station 7617 (142 m water depth). Green cells are total Eubacteria stained with EUB338 probe; red cells (encircled) are anammox bacteria stained with a new speci?c probe (AmxBS820).
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Figure 2 Chemical zoning and distribution of anammox indicators across the Black Sea chemocline. a, Fixed inorganic nitrogen species; b, water density and oxygen concentrations; c, interface peak of potential anammox activity expressed as anaerobic 15 NH? oxidation by 14NO2 to 14N15N; d, peak of three ladderane membrane lipids 4 2 speci?c for anammox bacteria; e, molecular structures of the three ladderane membrane lipids speci?c for anammox bacteria presented in d. The suboxic zone is indicated by grey

shading. Density (jT, the density of seawater in kg m23 2 1,000), nitrate (NO2), nitrite 3 (NO2), ammonium (NH?) and oxygen pro?les from Station 7605 (428 30.71 0 N, 2 4 0 0 308 14.69 E). Ladderane lipid data from Stations 7605 and 7620 (428 55.56 N, 308 03.65 0 E) were used to create a composite plot for the ladderane glycerol monoether and for the fatty acid methyl esters (FAMEs) 1 and 2.

cells stained with the newly designed FISH probe (Amxbs820) gave an anammox cell density of ,1,900 ^ 800 cells ml21 (0.75% of all cells counted by 4,6-diamidino-2-phenylindole (DAPI)) at the nitrite peak. Although we acknowledge the uncertainty involved in the extrapolation of laboratory-derived anammox activities to the natural environment, the rates of net ammonium and nitrate consumption calculated as a function of depth indicate that nitrate reduction by denitri?ers coupled to anammox accounts for a substantial loss of

?xed inorganic nitrogen. In fact, the downward ?ux of nitrate (,7 mmol m22 h21) is suf?cient to oxidize all the ammonium (,5 mmol m22 h21) diffusing up into the suboxic zone. If we assume that the area (3 ? 105 km2 ) below the shelf break (,200 m)8 represents the total surface area of the suboxic zone, 0.3 Tg of ?xed inorganic nitrogen per year might be lost through nitrate reduction coupled to anammox. For comparison, the annual primary production of phytoplankton in the whole basin is ,80 Tg carbon (ref. 8), which is equivalent to 14 Tg of ?xed organic N if we assume an atomic C/N ratio of 6.6 for phytoplankton17. Because more than 95% of this phytoplanktonic organic nitrogen is recycled in the upper 80 m (ref. 18), anammox might consume more than 40% of the ?xed nitrogen that sinks down into the anoxic Black Sea water. Moreover, these results demonstrate that anammox bacteria are abundant and are important in the nitrogen cycle of the Black Sea. In fact, the widespread occurrence of ammonium consumption in suboxic marine waters as well as in sediments7 suggests that anammox bacteria could have an important but as yet neglected role in the oceanic loss of ?xed nitrogen. A

Methods
Nutrient analyses
Water samples for nutrient analyses were obtained by a pumpcast conductivity– temperature–depth (CTD) system equipped with an oxygen sensor. Before analyses, ZnCl2 was added to the samples from the anoxic part of the water column to precipitate sulphide. Nitrate, nitrite and ammonium concentrations (detection limits 0.1, 0.01 and 0.5 mM, respectively) were determined on board with an autoanalyser, immediately after sampling.
15

Figure 3 Phylogenetic tree of 16S rRNA gene sequences showing the order Planctomycetales and the position of the anammox-af?liated organisms from the Black Sea (indicated by a rectangle). The black triangles indicate phylogenetic groups. The bar represents 10% estimated sequence divergence. Deep-sea sediment clone is from ref. 26; English BC clone EN5 and Candidatus ‘Scalindua brodae’ are from M. Schmid, K. Walsh, R. Webb, W. I. Rijpstra, K. T. van de Pas Schoonen, T. C. J. Hill, B. F. Moffett, J. A. Fuerst, J.S.S.D., J. A. Harris, P. J. Shaw, M.S.M.J. and M. Strauss (unpublished observations).
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N incubations and analysis

Black Sea water collected from speci?c water depths was ?ushed for 1 h with argon and, after the addition of 500 mM 15NH4Cl and 100 mM Na14NO2, incubated for 4 days at in situ 2 temperatures (,8 8C). Subsequently, the samples were stored at 4 8C until analysis. 14 15 14 14 N N: N N ratios were determined by gas chromatography–isotope ratio mass spectrometry and expressed as d14N15N values (d14N15N ? [(14N15N:14N14N)sample]: [(14N15N:14N14N)standard] 2 1; air was used as the standard).
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Lipid analysis
Particulate organic matter for lipid analyses was collected from speci?c water depths by in situ ?ltration of large volumes (,1,000 l) of water through 292-mm diameter precombusted (at 450 8C) glass ?bre ?lters (GFF; nominal pore size 0.7 mm) with in situ pumps. Because ?ltration through 0.7-mm pore-size ?lters could lead to an undersampling of anammox cells, the calculated ladderane lipid concentrations are minimum values. The GFF were extracted for 24 h in a Soxhlet apparatus to obtain the total lipid extracts. Aliquots of the total extracts were saponi?ed after addition of an internal standard and separated into fatty acid and neutral lipid fractions. The fatty acid fractions were methylated and the neutral fractions were silylated and analysed by gas chromatography– mass spectrometry for the identi?cation and quanti?cation of ladderane lipids. Repeated concentration measurements were within ^10%.
sediment pore water. Limnol. Oceanogr. 43, 1500–1510 (1998). 24. Oguz, T., Murray, J. W. & Callahan, A. E. Modeling redox cycling across the suboxic-anoxic interface zone in the Black Sea. Deep-Sea Res. I 48, 761–787 (2001). 25. Lewis, B. & Landing, W. M. The biogeochemistry of manganese and iron in the Black Sea. Deep-Sea Res. 38 (suppl. 2), S773–S803 (1991). 26. Li, L., Kato, C. & Horikoshi, K. Bacterial diversity in deep-sea sediments from different depths. Biodivers. Conserv. 8, 659–677 (1999).

Molecular cloning and phylogeny
DNA extraction, isolation and cloning were performed as described previously19. Phylogenetic analysis was performed with the ARB software package15. The phylogenetic tree is based on a maximum-likelihood analysis of different data sets.

Acknowledgements We thank L. Neretin for discussions; the Romanian and Turkish authorities ¨ for access to their national waters; the crew of the R/V Meteor for collaboration; S. Kruger, ¨ F. Pollehne and T. Leipe (IOW, Warnemunde) for operating the pumpcast and providing the CTD data; J. Eygensteyn, A. Pol, H. op den Camp, K. van de Pas Schoonen and G. Klockgether for analytical assistance. C. Han?and and the AWI (Bremerhaven) provided the in situ pumps. The investigations were supported by the MPG, the University of Nijmegen, the TU Delft and the DFG. M.M.M.K. was ?nancially supported by the EC Human Potential Programme Research Training Networks Activity (CT-net); A.O.S. was supported by a grant of the ALW; M. Schmid was supported by an EU grant. Competing interests statement The authors declare that they have no competing ?nancial interests. Correspondence and requests for materials should be addressed to M.M.M.K. (e-mail: mkuypers@mpi-bremen.de).

FISH and microscopy
Filter material was stained with an oligonucleotide probe speci?c for Planctomycetes (Pla46, S-P-Planc-0046-a-A-18)20, a newly designed Anammox probe (AmxBS820, S-*BS-820-a-A-22 (5 0 -TAATTCCCTCTACTTAGTGCCC-3 0 )), a eubacterial probe (EUB338, S-D-Bact-0338-a-A-18)21 and DAPI to determine the abundance of anammox and total bacteria. FISH and DAPI staining were performed as described22 and the average number of anammox bacteria was determined by analysing 20 different slides.

Flux calculations
Nitrate and ammonium ?uxes and ammonium oxidation rates were calculated from the concentration pro?les and a vertical diffusion coef?cient (K z) with the program Pro?le23. Published estimates of the vertical diffusion coef?cient for the suboxic zone vary over an order of magnitude (0.02–0.7 cm2 s21)8,24,25. However, most calculations of chemical ?uxes24,25 have used a K z value close to the lower end of the range. Accordingly, a K z of 0.04 cm2 s21 was used here. The model predicted zones of net ammonium and nitrate consumption at 106–93 and 88–94 m, respectively.
Received 8 November 2002; accepted 3 February 2003; doi:10.1038/nature01472.
1. Falkowski, P. G. Evolution of the nitrogen cycle and its in?uence on the biological sequestration of CO2 in the ocean. Nature 387, 272–275 (1997). 2. Codispoti, L. A. & Christensen, J. P. Nitri?cation, denitri?cation and nitrous oxide cycling in the eastern tropical South Paci?c Ocean. Mar. Chem 16, 277–300 (1985). 3. Strous, M. et al. Missing litotroph identi?ed as new planctomycete. Nature 400, 446–449 (1999). ? 4. Sinninghe Damste, J. S. et al. Linearly concatenated cyclobutane (ladderane) lipids from a dense bacterial membrane. Nature 419, 708–712 (2002). 5. Bender, M. et al. Organic carbon oxidation and benthic nitrogen and silica dynamics in San Clemente Basin, a continental borderland site. Geochim. Cosmochim. Acta 53, 685–697 (1989). 6. Codispoti, L. A., Friederich, G. E., Murray, J. W. & Sakamoto, C. M. Chemical variability in the Black Sea: implications of continuous vertical pro?les that penetrated the oxic/anoxic interface. Deep-Sea Res. 38, S691–S710 (1991). 7. Thamdrup, B. & Dalsgaard, T. Production of N2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl. Environ. Microbiol. 68, 1312–1318 (2002). 8. Sorokin, Y. I. The Black Sea—Ecology and Oceanography (ed. Martens, K.) 1–875 (Backhuys, Leiden, 2002). 9. Froelich, P. N. et al. Early oxidation of organic matter in pelagic sediments of the eastern equatorial Atlantic: Suboxic diagenesis. Geochim. Cosmochim. Acta 43, 1075–1090 (1979). 10. Richards, F. A. in Chemical Oceanography (eds Ripley, J. P. & Skirrow, G.) 611–645 (Academic, London, 1965). 11. Murray, J. W., Codispoti, L. A. & Frederich, G. E. in Aquatic Chemistry (eds Huang, C. P., O’Melia, C. R. & Morgan, J. J.) 157–176 (American Chemical Society, Washington DC, 1995). 12. Kuenen, J. G. & Jetten, M. S. M. Extraordinary anaerobic ammonium-oxidizing bacteria. ASM News 67, 456–463 (2001). 13. Dalsgaard, T. & Thamdrup, B. Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments. Appl. Environ. Microbiol. 68, 3802–3808 (2002). 14. Mulder, A., van de Graaf, A. A., Robertson, L. A. & Kuenen, J. G. Anaerobic ammonium oxidation discovered in a denitrifying ?uidized bed reactor. FEMS Microbiol. Ecol. 16, 177–184 (1995). 15. Schmid, M. et al. Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst. Appl. Microbiol. 23, 93–106 (2000). 16. Ward, B. B. & Kilpatrick, K. A. in Black Sea Oceanography (eds Izdar, E. & Murray, J. W.) 111–124 (Kluwer Academic, Dordrecht, 1991). 17. Red?eld, A. C., Ketchum, B. H. & Richards, F. A. in The Sea (ed. Hill, M. N.) 26–77 (Interscience, New York, 1963). 18. Karl, D. M. & Knauer, G. A. Microbial production and particle ?ux in the upper 350 m of the Black Sea. Deep-Sea Res. 38, S921–S942 (1991). 19. Schmid, M., Schmitz-Esser, S., Jetten, M. & Wagner, M. 16S–23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection. Environ. Microbiol. 3, 450–459 (2001). 20. Neef, A., Amann, R., Schlesner, H. & Schleifer, K. H. Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology 144, 3257–3266 (1998). 21. Amann, R. I. et al. Combination of 16S rRNA-targeted oligonucleotide probes with ?ow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 6, 1919–1925 (1990). ¨ ¨ 22. Pernthaler, J., Glockner, F. O., Schonhuber, W. & Amann, R. in Methods in Microbiology (ed. Paul, J. H.) 207–226 (Academic, London, 2001). 23. Berg, P., Risgaard-Petersen, N. & Rysgaard, S. Interpretation of measured concentration pro?les in

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Catastrophic ape decline in western equatorial Africa
Peter D. Walsh*, Kate A. Abernethy??, Magdalena Bermejo§, Rene Beyersk, Pauwel De Wachter{, Marc Ella Akou{, Bas Huijbregts{, Daniel Idiata Mambounga#, Andre Kamdem Toham{, Annelisa M. Kilbournk, Sally A. Lahmq, Stefanie Latourk, ` Fiona Maiselsk**, Christian Mbinak, Yves Mihindouk, Sosthene Ndong Obiang#, Ernestine Ntsame Effa#, Malcolm P. Starkeyk??, Paul Telfer???, Marc Thibault{, Caroline E. G. Tutin??, Lee J. T. Whitek & David S. Wilkiek
* Department of Ecology and Evolutionary Biology, Guyot Hall, Princeton, New Jersey 08540, USA ? ? Centre International de Recherches Medicales, BP 769, Franceville, Gabon ? Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, UK ? ? § Departamento Biolog?a Animal (Vertebrados), Facultad de Biolog?a, Universidad de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain k Wildlife Conservation Society, Bronx, New York, New York 10460-1099, USA { WWF Central Africa Regional Program Of?ce, BP 9144, Libreville, Gabon ` ` ? ? # Ministere de l’Economie Forestiere, des Eaux, de la Peche charge de l’Environnement et de la Protection de la Nature, Direction de la Faune et de la Chasse, BP 1128, Libreville, Gabon q Institut de Recherche en Ecologie Tropicale, BP 13354, Libreville, Gabon ** Institute of Cell, Animal and Population Biology, Edinburgh University, Edinburgh EH9 3JT, UK ?? Department of Geography, University of Cambridge, Downing Place, Cambridge CB2 3EN, UK ?? New York University, Department of Anthropology, 25 Waverly Place, New York, New York 10003, USA
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Because rapidly expanding human populations have devastated gorilla (Gorilla gorilla) and common chimpanzee (Pan troglodytes) habitats in East and West Africa, the relatively intact forests of western equatorial Africa have been viewed as the last stronghold of African apes1. Gabon and the Republic of Congo alone are thought to hold roughly 80% of the world’s gorillas2 and most of the common chimpanzees1. Here we present survey results conservatively indicating that ape populations in Gabon declined by more than half between 1983 and 2000. The primary cause of the decline in ape numbers during this period was commercial hunting, facilitated by the rapid expansion of
611

NATURE | VOL 422 | 10 APRIL 2003 | www.nature.com/nature

? 2003 Nature Publishing Group


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