PROPOSAL FOR AN INTERNATIONAL RESEARCH CRUISE IN THE ARABIAN SEA (19 September 23 October 2007) Bess Ward Princeton University 3 May 2007

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1 PROPOSAL FOR AN INTERNATIONAL RESEARCH CRUISE IN THE ARABIAN SEA (19 September 23 October 2007) Bess Ward Princeton University 3 May Background The Arabian Sea is a unique oceanographic province, due to its geography, climate and oceanographic circulation. Of particular interest to biogeochemists is the strong permanent oxygen minimum zone that occurs in the central region of the Sea. Due to limited circulation (lack of deep water formation in the north) and relatively high seasonal productivity, the mid waters of the central region are permanently depleted in oxygen. This leads to some very interesting and globally significant chemistry, including the world record for oceanic emissions of the climatically important greenhouse gas, nitrous oxide (N 2 O). Nitrous oxide is an intermediate in the processes of nitrification and denitrification, microbially mediated biochemical pathways that determine the availability of fixed nitrogen to the ecosystem. Net nitrous oxide production appears to be favored by low oxygen and the extent to which these two processes contribute to the flux is not well understood. To make matters more complicated, another process that could be of great significance to nitrogen cycling in oxygen minimum zones was discovered about 10 years ago. Anammox refers to the anaerobic oxidation of ammonium, and it results in the production of dinitrogen gas, as does denitrification. Previously, it was thought that denitrification was the most important process leading to loss of fixed nitrogen and N2, and the discovery of anammox raises very important questions about the N budget and its regulation. The microbes that perform nitrification, denitrification and anammox differ in some important ways, and we suspect therefore that they are regulated in different ways by environmental factors. Their different impacts on the carbon and nitrogen cycles of oxygen minimum zones are focus of our project. 2. Objectives of the Cruise 1. What is the relative importance of heterotrophic denitrification and anaerobic ammonium oxidation (anammox) in molecular nitrogen (N 2 ) production? The Arabian Sea is one of the three major oceanic sites where an acute oxygen deficiency within a large body of intermediate waters causes large-scale microbially-mediated production of N 2. Heterotrohic denitrification has long been believed to be the primary process responsible for the combined nitrogen loss. However, the excess N 2 concentrations computed from the measured N 2 /Ar ratio in the suboxic water column seem to far exceed nitrate deficits derived from the Redfield stoichiometry and the observed nutrient data. At least a part of this discrepancy may be caused by the recently discovered anammox process. In fact, in most of the studies undertaken in the other oceanic suboxic zones (e.g. off Namibia, Kuypers et al., PNAS, 102, , 2005) anammox has been found to be the dominant pathway of N 2 formation. There is as yet no published data on anammox activity in the Arabian Sea.

2 We have developed and proposed a suite of new measurement methods to evaluate the rates of anammox and denitrification under conditions that are realistic in the ocean. These will enable us to evaluate the contribution of anammox and denitrification to N 2 production, the contribution of nitrification and denitrification to N 2 O production, and the degradation of organic carbon and N mineralization by denitrification. 2. How diverse is the bacterial community involved in denitrification/anammox? Although the Arabian Sea has known to be an active site of water column denitrification, very little is known about the bacteria that carry out denitrification. Previous observations including those made on the SK209 cruise have shown that the suboxic layer is associated with a bacterial biomass maximum and a high abundance of nitrite reductase genes (the signature gene for the process of denitrification) implying that in some samples essentially all of the bacteria present possessed the genetic capability for denitrification. The relative abundance of the gene (normalized to total DNA present) decreased with increasing distance from shore along an offshore transect, and as expected, was generally higher in the oxygen minimum zone than in shallower or deeper regions surrounding the zone. Subsequent detailed investigations of nitrite reductase gene sequences provided evidence of distinct bloom dynamics in denitrifying assemblages as denitrification intensifies, both in the open sea and in the coastal region. Anammox organisms, in contrast, do not show significant changes in cell number, implying a quite different dynamic in response to environmental variables. These dynamics have important implications for the rates and temporal variability in denitrification and anammox, and the resulting N 2 and N 2 O formation. 3. Cruise Track The US National Science Foundation has supplied funding for a US research vessel (Roger Revelle, operated by the Scripps Institution of Oceanography; please visit for details of the vessel) to support the proposed research. Our cruise represents the second leg in a collaborative effort with Dr. Wajih Naqvi of the National Institute of Oceanography, who has submitted a separate application. We will meet the ship in Oman at the end of Dr. Naqvi s cruise and embark from there on 22 September. We are in the process of identifying Omani scientists who might like to participate and welcome their involvement in the work. 4. Work Plan 4.1. Sampling and in-situ observations Water: Water samples for routine chemical measurements will be collected using a CTD fitted with Niskin samplers. Sampling for trace-metal work will be performed using 30 L Go-flo bottles on a Kevlar rope. A submersible pump will be used for continuous profiling at selected stations and an in situ pump will be used to collect the particulate matter from the particle maximum layer.

3 Phyto- and Bacterio-plankton: Phytoplankton and bacterioplankton sub-samples will be taken from the Niskin bottles Sediment Traps: Floating 0.5 m net traps will be deployed at each station for a period of 4 days. Trapped material will be retrieved for analysis of chemical and microbial parameters on board and for use in incubations (see below) On-board processing/analyses Water Routine measurements: Water samples at all stations and sampling depths will be analysed on board ship for routine hydrographic and chemical parameters [salinity, dissolved oxygen and nutrients (nitrate, nitrite, ammonia, phosphate and silicate)] following standard procedures: Oxygen will be estimated titrimetrically using a automated system while a Skalar analyser will be used for nutrient measurements Dissolved gases other than oxygen: Subsamples from most stations will be analysed on board ship for the climatically important gas [nitrous oxide (N 2 O) using gas chromatographic procedures Dissolved/particulate organic carbon (DOC/POC): Samples from selected stations and depths will be filtered for the analysis of POC concentration as well as its isotopic (carbon and nitrogen) composition. Unfiltered samples from same sampling depths will be preserved for analysis of DOC in the shore laboratory using a Shimadzu TOC 5000 analyzer Natural abundances of stable nitrogen isotopes: Samples for studying the natural abundance of nitrogen isotopes in nitrate, and both nitrogen and oxygen isotopes in N 2 O will be taken from selected depths at selcted stations. While the nitrate samples will be from the main CTD cast, those for N 2 O isotopes will be from another cast using specially-modified samplers. The former will be poisoned with mercuric chloride while the latter will be purged with argon and the dissolved N 2 O will be concentrated on MS 5A columns. In the shore laboratory N 2 O will be thermally desorbed, purified and analysed for determining its dual isotopic composition Incubation experiments to determine water-column nitrogen transformation rates: Experiments will be carried out using 15 N-labelled substrates as substrate to quantify the rates of water column denitrification and anammox. Organic material retrieved in the sediment traps will be added to bag incubations as a source of nutrient stimulation Characterization of denitrifying and anammox bacteria: Water samples from selected depths within the denitrifying zone will be collected by Niskin bottle sampling from the CTD rosette. Approximately four litres of seawater from each depth will be concentrated by filtration onto Sterivex filter capsules aboard ship, and the filter capsules will stored frozen. Upon return to the shore Laboratory (Princeton University) the DNA

4 filters will be extracted using standard protocols. Using either clone libraries or DNA microarrays, we will characterize the diversity of genes representing denitrification and anammox. Using quantitative PCR, we will determine the distribution and abundance of both groups Trace metals Iron speciation will be determined on board. For total dissolved Fe, samples will be preserved for analysis in the shore laboratory. 5. Utilization of Results The results of the study will be published in the form of joint research papers in highimpact scientific journals. As required, all data from the Omani waters will be made available to the Omani scientists for their use. 6. Potential Benefits of Collaboration Besides making available data needed to address issues listed in Section 2, the proposed cruise will provide an impetus for collaboration between India and Oman in ocean sciences that will help in capacity building in the region. The Omani scientists will gain more experience in field studies through participation in this multi-disciplinary oceanographic cruise. Incidentally, the season chosen for the cruise is when fish mortality often occurs along the Omani coast, but so far very little information is available from the near shore region to understand the causative processes. The data collected during this cruise are expected to provide crucial information on the hydrographic conditions, the associated nutrient and oxygen fields and productivity levels - factors that control the living resources of the region. 7. Tentative Composition of Scientific Team Princeton University, USA 3 University of Washington, USA 4 National Institute of Oceanography, Goa 3 Woods Hole Oceanographic Institution, USA 4 University of Southern California, USA 2 Sultan Qaboos University, Al Khod, Oman - 3 Old Dominion University, USA - 3

5 Contact Person (Chief Scientist): Professor Bess B. Ward Princeton University Princeton, New Jersey USA Tel: Fax: