N 2 production rates limited by nitrite availability in the Bay of Bengal oxygen minimum zone

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1 In the format provided by the authors and unedited. 5 SUPPLEMENTARY INFORMATION DOI: /NGEO2847 N 2 production rates limited by nitrite availability in the Bay of Bengal oxygen minimum zone L.A. Bristow*, C.M. Callbeck*, M. Larsen*, M.A. Altabet, J. Dekaezemacker, M. Forth, M. Gauns, R.N. Glud, M.M.M. Kuypers, G. Lavik, J. Milucka, S.W.A. Naqvi, A. Pratihary, N.P. Revsbech, B. Thamdrup, A.H. Treusch, D.E. Canfield Figure S1. Depth profiles of salinity, temperature, sigma-t, chlorophyll and oxygen. The data presented are from up-casts only, sampled at 24 s -1, and subsequently low-pass filtered according to the manufacturer s recommendations. NATURE GEOSCIENCE Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 10 Figure S2. Comparison of in situ oxygen recordings. Measurements with the SBE 43 polarographic oxygen sensor compared to the STOX sensor, at station BoB OMZ sulfur cycle Gene abundance results suggest an active sulfur cycle as found in other OMZs supporting N 2 production. Thus, the abundance of 16S rrna genes affiliated to the sulfide-oxidizing SUP05 group peaked in the low-oxygen waters of the BoB, similar to the OMZs in the ETSP and ETNP 1,2. We also found high copy numbers of 16S rrna genes related to the 20 Gammaproteobacterial Sulfur Oxidizers (GSO) 3,4. The GSO group includes SUP05, but the much higher numbers for the GSO suggest the presence of additional taxa. We also found high numbers of the functional gene APS reductase of subunit A (apra) (Figure S3) used in sulfide oxidation, with similar distribution and numbers to the GSO group, suggesting that the GSO may house the apra. 2

3 Depth (m) SUP05 (copies ml -1 ) GSO (copies ml -1 ) apra (copies ml -1 ) A B C Stn 1 Stn 4 Stn 5 Figure S3. Abundance of 16S rrna genes for the SUP05 group and Gammaproteobacterial sulfur-oxidizing group (GSO) as well as the abundance of the functional gene APS reductase (apra). The dashed box outlines the approximate depth interval of the OMZ. Note variability in x axes scales Anammox (nm N d -1 ) Stn 6 Stn 7 Anammox (nm N d -1 ) Depth (m) NH NO 2-15 NO NH Figure S4: Depth profiles of anammox rates at stations 6 and 7. Error bars represent the standard error. 3

4 A role for nitrite reoxidation 35 Field and laboratory studies have highlighted that during dissimilatory nitrate reduction to nitrite, the ratio of kinetic isotope effects ( 15 ε: 18 ε) is approximately 1:1 5. Deviations from this expected 1:1 relationship in low oxygen systems, can be used to identify processes acting concurrently to nitrate reduction, this is described by the nitrate isotope anomaly, (15, 18) 6. This anomaly is expressed as ( 15, 18) = (δ 15 - NO 3 - δ 15 - NO 3 deep ) 15 ε / 18 ε x (δ 18 - NO 3 - δ 18 - NO 3 deep ), where 40 δ 15 NO 3 - and δ 18 NO 3 - are the values measured, δ 15 NO 3 - deep and δ 18 NO 3 - deep are the average values in the regional deep water (with values of 6.1 and 2.6 used for δ 15 - NO 3 and δ 18 - NO 3 respectively in the BoB) and 15 ε / 18 ε is the ratio of the N to O isotope effects of dissimilatory nitrate reduction. If nitrate reduction is the only process influencing the nitrate pool (15, 18) values should be close to zero. Negative (15, 18) values have been observed in low-oxygen 45 waters and attributed to the remineralisation of newly fixed N or the reoxidation of nitrite 6-9. The cycling of N between nitrate reduction and nitrite oxidation would have little net effect on the isotopic signature of δ 15 NO - 3, but would cause δ 18 - NO 3 values to increase as the nitrate reduced is initially lower in δ 18 O than the reoxidised nitrite, resulting in the negative anomaly. In the lowoxygen waters of the BoB (100 to 350 m), persistent negative (15, 18) values (Figure S5) 50 indicate a coupled anaerobic /aerobic N cycle, likely regulating N loss in this system. 4

5 !!!!!!!!!!!!!!!(15, 18) ( ) Depth (m) Stn 1 Stn 4 Stn 5 Stn 6 Figure S5: Depth profiles of the nitrate isotope anomaly; (15, 18) ( ). 5

6 Supplementary Information References Canfield, D. E. et al. A cryptic sulfur cycle in oxygen- minimum- zone waters off the Chilean Coast. Science 330, (2010). 2 Carolan, M. T. & Beman, J. M. Transcriptomic evidence for microbial sulfur cycling in the eastern tropical North Pacific oxygen minimum zone. Front. Microbiol. 6, doi: /fmicb (2015). 3 Glaubitz, S., Kiesslich, K., Meeske, C., Labrenz, M. & Jurgens, K. SUP05 Dominates the Gammaproteobacterial Sulfur Oxidizer Assemblages in Pelagic Redoxclines of the Central Baltic and Black Seas. Applied and environmental microbiology 79, , doi: /aem (2013). 4 Stewart, F. J., Ulloa, O. & DeLong, E. F. Microbial metatranscriptomics in a permanent marine oxygen minimum zone Environ Microbiol 14, (2012). 5 Granger, J., Sigman, D. M., Lehmann, M. F. & Tortell, P. D. Nitrogen and oxygen isotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria. Limnol. Oceanogr. 53, , doi: /lo (2008). 6 Sigman, D. M. et al. Coupled nitrogen and oxygen isotope measurements of nitrate along the eastern North Pacific margin. Glob. Biogeochem. Cycle 19, doi: /2005gb (2005). 7 Casciotti, K. L., Buchwald, C. & McIlvin, M. Implications of nitrate and nitrite isotope measurements for the mechanisms of nitrogen cycling in the Peru oxygen deficient zone. Deep- C Research I 80, (2013). 8 Casciotti, K. L., Bohlke, J. K., McIlvin, M. R., Mroczkowski, S. J. & Hannon, J. E. Oxygen isotopes in nitrite: Analysis, calibration, and equilibration. Anal. Chem. 79, , doi: /ac061598h (2007). 9 Gaye, B., Nagel, B., Dahnke, K., Rixen, T. & Emeis, K. C. Evidence of parallel denitrification and nitrite oxidation in the ODZ of the Arabian Sea from paired stable isotopes of nitrate and nitrite. Glob. Biogeochem. Cycle 27, , doi: /2011gb (2013). 10 Mason, O. U. et al. Prokaryotic diversity, distribution, and insights into their role in biogeochemical cycling in marine basalts. The ISME journal 3, , doi: /ismej (2009). 11 Ovreas, L., Forney, L., Daae, F. L. & Torsvik, V. Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR- amplified gene fragments coding for 16S rrna. Applied and environmental microbiology 63, (1997). 12 Zaikova, E. et al. Microbial community dynamics in a seasonally anoxic fjord: Saanich Inlet, British Columbia. Environ Microbiol 12, (2010). 13 Lavik, G. et al. Detoxification of sulphidic African shelf waters by blooming chemolithotrophs. Nature 457, , doi: /nature07588 (2009). 14 Wuchter, C. et al. Archaeal nitrification in the ocean. Proc. Natl. Acad. Sci. U. S. A. 103, (2006). 15 Throback, I. N., Enwall, K., Jarvis, A. & Hallin, S. Reassessing PCR primers targeting nirs, nirk and nosz genes for community surveys of denitrifying bacteria with DGGE. FEMS microbiology ecology 49, , doi: /j.femsec (2004). 16 Lam, P. et al. Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc. Natl. Acad. Sci. U. S. A. 106, , doi: /pnas (2009). 17 Pester, M. et al. NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite- oxidizing Nitrospira. Environ Microbiol 16, , doi: / (2014). 6

7 18 Blazejak, A., Kuever, J., Erseus, C., Amann, R. & Dubilier, N. Phylogeny of 16S rrna, ribulose 1,5- bisphosphate carboxylase/oxygenase, and adenosine 5 '- phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (Oligochaeta) from Bermuda and the Bahamas. Applied and environmental microbiology 72, (2006)

8 110 Table S1. STOX measurements. Values below the LOD of the sensor are indicated in red and by *. Only concentrations below 2x10 4 nm are shown. At Stn 5 and 6, STOX measurements were performed in two consecutive CTD casts. Stn 1 Stn 4 Stn 5#1 Stn 5#2 Stn 6#1 Stn 6#2 Stn 7 Depth (m) O 2 (nm) Depth (m) O 2 (nm) Depth (m) O 2 (nm) Depth (m) O 2 (nm) Depth (m) O 2 (nm) Depth (m) O 2 (nm) Depth (m) O 2 (nm) E E E E E E E E E E E E E E E E E E E E E E E E * E * E * * E * E * E E E E E E E E E E E E E E E E E

9 Table S2. Results from the oxygen regulation experiments. Site Depth Manipulated Oxygen Conc Anammox Denitrification Nitrite Oxidation m µm SD nm N d -1 SE nm N d -1 SE nm N d -1 SE Stn Stn Stn Stn Stn Stn Stn Stn Stn Stn Stn Stn

10 Table S3. qpcr primers Primer Name B27F U519R U519F 1048R SUP05 B27F GSO477- R AOAamoA-f AOAamoA-r cd3af R3cd Scnir372F Scnir845R nxrb169f nxrb638r aps1f aps4r Sequence 5' --> 3 ' AGR GTT YGA TYM TGG CTC AG TTA CCG CGG CKG CTG CAG CMG CCG CGG TAA CCA TCT CTG GAA AGT TCC GTS T AGR GTT YGA TYM TGG CTC AG CTA AAG TTA ACG TCA AGG CTG AYT GGG CYT GGA CAT C TTC TTC TTT GTT GCC CAG TA GTS AAC GTS AAG GAR ACS GG GAS TTC GGR TGS GTC TTG A TGT AGC CAG CAT TGT AGC GT TCA AGC CAG ACC CAT TTG CT TAC ATG TGG TGG AAC A CGG TTC TGG TCR ATC A TGG CAG ATC ATG ATY MAY GG GCG CCA ACY GGR CCR TA Annealing Temp o C Target Gene Target Organisms Efficiency of Standard 55 16S rrna Bacteria % 63 16S rrna SUP05 group 50 16S rrna Gammaprote o-bacterial Sulfur Oxidizers 59 Ammonia monooxygenase subunit A 58 Nitrite reductase subunit S 61 Nitrite reductase subunit S 57 Nitrite oxidoreductase subunit B 53 Adenylylsulfate reductase subunit A Archaeal nitrifiers (Th amoa) Denitrifiers (Den nirs) Scalindua (Sc nirs) Nitrospira/ Nitrospina (nxrb) % % % % 86% 88-90% diverse % Refs

11 Table S4. Nutrient and geochemical data from the Bay of Bengal Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) N- NO O- NO 3 N E m µm µm µm µm Where available duplicate samples were analyzed for excess N 2, indicated by columns labeled 1 and 2.

12 Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) 15 - N- NO O- NO 3 N E m µm µm µm µm

13 Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) 15 - N- NO O- NO 3 N E m µm µm µm µm

14 Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) 15 N- NO 3-18 O- NO 3 - N E m µm µm µm µm

15 Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) 15 N- NO 3-18 O- NO 3 - N E m µm µm µm µm

16 Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) 15 N- NO 3-18 O- NO 3 - N E m µm µm µm µm

17 Stn Latitude Longitude Depth Nitrate Nitrite Phosphate N Deficit Excess N 2 (µm) 15 N- NO 3-18 O- NO 3 - N E m µm µm µm µm Other Stations