Revisiting the Northern Hood Canal Sill: Exploring dissimilatory nitrate reduction to ammonium at the sediment water interface Jeremy Hudson jhuds@u.washington.edu (425) 497-1232 School of Oceanography University of Washington Seattle, WA 98195 February 27, 2005 1
Acknowledgments I would like to thank the faculty and students from the University of Washington Oceanography 443 class. I would also like to recognize several individuals who have been instrumental in this proposal thus far. I greatly appreciate their time, patience, advice, and expertise: Professor Roy Carpenter, Bonnie Chang, Lauren Curry, Professor Al Devol, Clara Fuchsman, Professor Mark Holmes, Professor Gabrielle Rocap, Jamie Pierson, Tiffany Straza, and Dave Wilbur. 2
Project Summary The occurrence of a seasonal ammonium plume at the Northern Hood Canal sill was first noted in 1998. This plume has since reoccurred every summer since 1998 when data has been collected at the Northern Hood Canal sill. Although it has not been proved, a previous study found that the ammonium in the plume had originated from the sediment. What was not determined was the source of the ammonium in the sediment. My plan is to further investigate the Northern Hood Canal sill during the March 21-25, 2005 Oceanography 443/444 cruise aboard the R/V Thomas G. Thompson. I will be sampling both the water column and the sediment in the hopes of gathering supporting evidence for the previous investigation which concluded that ammonium in the plume is of sedimentary origin. I also plan to study a possible mechanism for ammonium production in the sediment. This biological mechanism, dissimilatory nitrate reduction to ammonium, is a process mediated by nitrate reducing bacteria. My hypothesis is that increasingly anoxic conditions within the Hood Canal basin favor nitrate reducing bacteria, including those bacteria that reduce nitrate to ammonium. The ammonium plume could potentially be traced back to DNRA in future studies if this process is shown to occur at the Northern Hood Canal sill. 3
Introduction Nitrogen is a rate-limiting nutrient for all biology. Thus, the fates of the many forms of nitrogen are important in order to better understand ecological dynamics for a given environment. One form of nitrogen, ammonium, is especially important in the coastal and estuarine environments, where the concentration can far exceed that which is typically found in the open ocean. This is due in part to land run-off and untreated waste water. Ammonium is a biologically preferred source of nitrogen to many organisms, so the fate of ammonium and the sources that contribute to ammonium concentrations are important to understand as well, as these are components of the nitrogen cycle. Over the last several years, the occurrence of an ammonium (NH + 4 ) plume has been measured at the Northern Hood Canal sill (Fig. 1). The plume was first measured during the 1998 Puget Sound Regional Synthesis Model (PRISM) cruise (Fig. 2). In subsequent years since then, it has been shown that this ammonium plume has seasonal variability. Concentrations will typically build to a peak of 4 mol x L -1 in the summer months and dissipate thereafter. In addition, this variability seems to reproduce on a predictable annual basis (Norbeck 2000; Warner et. al. 2002). Many possible mechanisms could produce the ammonium in the plume either in isolation or combination. It was previously reasoned that the contributory source of ammonium to the plume was natural due to the fact that there were no known anthropogenic inputs nearby with the capacity to produce the plume, such as a sewer outfall. Furthermore, the plume is a localized anomaly with greater concentrations found at depth near the sediment water interface. These facts led to an original hypothesis that the ammonium was of sedimentary origin (Norbeck 2000). 4
At least two mechanisms exist for producing sedimentary ammonium. The first mechanism, which was also the assumption made in the previous investigation, is that ammonium is released into the water column as organic material settles out of the water column and is subsequently degraded. This process is known as mineralization, and the hypothesis is valid that enough ammonium could be produced to supply the plume concentration via this mechanism. Two potential problems exist for this hypothesis, however. First, ammonium is the only chemical measured at the sill to exhibit such a phenomenon as the plume. If mineralization was the primary source of the ammonium plume, one might expect similar concentration profiles for other nutrients that are released into the water column upon degradation (Newton, pers. comm.). Second, the lag time between the first phytoplankton bloom (the source of organic material), and the plume formation, may be far greater than the time required for organic material to sink out of the water column and degrade. The second mechanism for sedimentary ammonium formation involves dissimilatory nitrate reduction to ammonium (DNRA). This potential source of sedimentary ammonium typically occurs in anoxic environments via nitrate reducing bacterial species although it has also been shown to occur in oxygenated environments as well (Soonmo and Gardner 2002). At least one potential problem exists with this hypothesis. If the plume is a remnant of DNRA, then one might expect to find similar plumes elsewhere in Puget Sound, or at least elsewhere in the Hood Canal basin, but this is not the case. Conversely, if DNRA is occurring elsewhere, then why are no other plumes similar to that at the Northern Hood Canal sill produced as a result? 5
In light of this question, it is my hypothesis that nitrate reducing bacteria become increasingly favored as overlying water conditions change throughout the season, and the Hood Canal basin becomes increasingly anoxic. As such, I predict that the ammonium producing the plume could be supplied via DNRA. Strong evidence already suggests that denitrification occurs within Hood Canal sediment (Kassakian 2004). And since denitrification represents one of two nitrate reducing pathways, (DNRA represents the second) it stands to reason that the possibility exists for DNRA to occur as well. The ecological significance between the two nitrate reducing pathways is that nitrogen is lost to the environment when nitrate is reduced via denitrification, whereas nitrogen is conserved when nitrate is reduced via DNRA (Cole and Brown 1980; Bonin et. al. 1998). Past studies elsewhere have shown that DNRA is of quantitative significance and should be included as a component of the nitrogen cycle. Rates of ammonium formation via DNRA are comparable and sometimes exceed rates of denitrification, especially in coastal and estuarine sediments (Sørensen 1978; Bonin et. al. 1998; Kelly-Gerreyn et. al. 2001). Although the previous investigation (Norbeck 2000) had concluded that the ammonium concentrations in the sediment could produce the plume, it still has yet to be proven. It seems reasonable that the ammonium plume could be produced from the ammonium concentrations in the sediment, but as mentioned earlier, an assumption was made in that investigation that the contributory source to the ammonium in the sediment was mineralization. The prior investigation established a diffusive flux as great as 0.756 pmol NH + 4 x cm -2 x s -1 at the sediment water interface (Fig. 3). From this, along with the residency 6
time, a conservative calculation estimated that this sedimentary ammonium flux could account for up to 23% of the plume ammonium concentration (92% with bioturbation and advection included as factors). In the context of this prior work, I have two goals for my project: 1) Lend supportive evidence to the prior work which concluded that the ammonium plume could be produced from the sedimentary ammonium concentration. 2) Explore a sedimentary source of ammonium that has yet to be examined DNRA. Although I will not be able to quantify the rate of DNRA, I plan to show whether or not it is occurring by measuring the product (ammonium). By using nitrogen-15 nitrate ( 15 NO - 3 ), the ammonium produced via DNRA can be measured separate from any ammonium that might occur via mineralization. Since, only 0.37% nitrogen-15 occurs in nature, any nitrogen-15 ammonium ( 15 NH + 4 ) measured will have been produced via the DNRA mechanism (Sørensen 1978; Jørgensen 1989). 7
Proposed Research In order to test my hypothesis that the ammonium plume could be produced via DNRA, I plan to investigate the Northern Hood Canal sill/prism station #10, where a previous investigation was conducted and years worth of data has been collected. My plan to substantiate that the plume ammonium could originate from the sediment, and to show whether or not DNRA is occurring within the sediment is a three part approach, with a fourth component under consideration. First, I plan to take a sediment core with the Spade Box Core, sub-sample at 2 cm intervals (up to 10 cm in depth) and run (triplicate) nutrient analyses on those five interval depths in order to produce a nutrient profile. My reason for this component of the project is to place a nutrient profile in a temporal frame of reference, and to measure initial concentrations so that an estimated rate for DNRA can be constrained, should the process be found to occur. Nutrient and dissolved oxygen analyses will also be taken at six depths within the water column. Second, I plan to take another sediment core, sub-sample the top 2 cm, and compare the natural abundances of ammonium species ( 14 NH + 4 / 15 NH + 4 ) with that in the overlying bottom water. This portion of my project should lend further evidence to hypothesis that the ammonium in the bottom water originated from the sediment. This portion of my project will also serve as my control for the third portion of my project. Third, I plan to take a third sediment core, and sub-sample from this core using three 4-inch PVC pipes. These sub-cores will be enriched with up to 100 mol x L -1 of 15 NO - 3 and incubated under in-situ conditions (as best as can be simulated) for up to one week. In addition to the intact cores incubated, a slurry consisting of the top 2 cm of 8
sediment may also be prepared, enriched, and incubated under the same conditions as the intact cores. The difference between the intact core incubations and the slurry incubations is that any potential anoxic condition that may exist will be preserved in the slurry. This portion of my project should determine whether or not DNRA can occur at the Northern Hood Canal sill under the given conditions. The fourth possible portion of my project is still under consideration. This portion would involve bacterial characterization before and after incubation by Tiffany Straza. This portion of my project would provide further evidence for DNRA, if it is found to occur, by providing some of the bacterial species involved in carrying out nitrate reduction to ammonium via DNRA. If DNRA is found to occur, I would expect the abundance of the nitrate reducing bacteria to increase after incubation of the sub cores enriched with nitrate ( 15 NO - 3 ). Methods for each portion of my project are as follows, respectively: Cruise Experiment 1. 2 cm cross-section slices of the entire Spade Box Core will be sampled up to 10 cm in depth. Each 2 cm slice will be centrifuged for pore water, with the pore water frozen in liquid nitrogen, and stored onboard the R/V Thomas G. Thompson freezer for later analysis on shore. Dissolved oxygen measurements may be made on board using the Dosimat and titration protocols in place. Specifics for centrifuging sediment will be worked out in the weeks ahead with the aid of Bonnie Chang. 2. The overlying water retrieved by from the Box Core will be siphoned off the top of the sediment and stored onboard the R/V Thomas G. Thompson in the cooler at 9
approximately 8 C for later analysis on shore. The top 2 cm cross-section slice of the Box Core will be sub sampled, centrifuged for pore water and stored onboard the R/V Thomas G. Thompson in the cooler at approximately 8 C for later analysis on shore. 3. Sub-cores will be sampled from a Spade Box Core using 4-inch PVC pipe and caps. Each sub-core will be taped, wrapped in tinfoil, and stored onboard the R/V Thomas G. Thompson in the cooler at approximately 8 C for later analysis on shore. 4. In development Laboratory Experiment 1. Frozen triplicates for each 2 cm sediment depth will be analyzed for nutrients by the Marine Chemistry Laboratory. 2. Pore water and overlying bottom water will be measured for natural ammonium abundances using the ammonium distillation method and using mass spectrometry to characterize isotopes. The ammonium distillation method involves boiling the water sample in the presence of a base (NaOH). This process separates ammonium from bound substances and converts ammonium to ammonia gas (NH 3 ). The ammonia gas is then condensed and bubbled out in a separate flask converting ammonia back to ammonium that is unbound. The next process is to filter the ammonium through a molecular sieve (Zeolite) that has been prepared (Zeolite preparation involves baking in an oven to drive off any bound nitrogen particles). Lastly, the Zeolite enriched with ammonium is scraped into a tin boat and analyzed with mass spectrometry (Clara Fuchsman, pers. comm). 10
3. The overlying water in the PVC sub-cores will be injected with approximately 100 mol x L -1 of the enriched nitrate ( 15 NO 3 ) and incubated in a cold room (to be determined) as close to in-situ temperature that the samples were initially taken at. Sub-cores will be incubated for as much as one week. Incubation will cease at that time and pore water will be centrifuged, and frozen until samples can be distilled and run by mass spectrometry. Slurry preparations involve most of the same steps, with one addition. A mixture of sediment and pore water will be bubbled through with nitrogen or helium gas to drive out any potential oxygen before incubation begins (Al Devol, pers. comm.). 11
Project Budget Item Cost Quantity Duration Sub Total Offset Total Platform costs R/V Thomas G. Thompson N/A $18,000.00 1 5 $90,000.00 $90,000.00 $0.00 Platform Equipment Seabird SBE-9 CTD P10 $135.00 1 5 $675.00 $675.00 $0.00 A-frame N/A N/A 1 5 $0.00 $0.00 $0.00 Trawl/coring wire N/A N/A 1 5 $0.00 $0.00 $0.00 Cooler (approx. 8 C) & Freezer N/A N/A 1 5 $0.00 $0.00 $0.00 Rosette w/ 10-L Niskin bottles N/A N/A 1 5 $0.00 $0.00 $0.00 Pooled Equipment Biological Sampling Equipment Filter rack & Vaccuum pump B03 $6.00 1 5 $30.00 $0.00 $30.00 Centrifuge B04 $6.00 1 5 $30.00 $0.00 $30.00 Bottom Sampling Equipment Box Core (Spade) F10 $45.00 1 5 $225.00 $0.00 $225.00 PVC pipe (4-inch) F21 $1.00 3 5 $15.00 $0.00 $15.00 Core caps (4-inch) F22 $0.50 6 5 $15.00 $0.00 $15.00 Routine Chemistry Lab Alpkem RFA/2 R01 $45.00 1 5 $225.00 $0.00 $225.00 Dosimat (oxygen) R02 $15.00 1 5 $75.00 $0.00 $75.00 Nutrient bottles (per case) R03 $3.00 1 5 $15.00 $0.00 $15.00 Water Sampling Equipment U of W btl, 10-L W03 $3.00 1 5 $15.00 $0.00 $15.00 Salinity btls, iodine (case, 24 btls) W08 $3.00 1 5 $15.00 $0.00 $15.00 Oxygen btls (case, 24 btls) W09 $6.00 1 5 $30.00 $0.00 $30.00 Other Equipment (duct?) Tape (for sealing sub-cores) N/A TBD 1 5 $0.00 $0.00 TBD Container (Ice chest?) for sub-core storag N/A TBD 1 5 $0.00 $0.00 TBD Lead bricks for sub-core stabilization N/A TBD 2 5 $0.00 $0.00 TBD Bungie Cords N/A TBD 2 5 $0.00 $0.00 TBD Containers for water sample storage N/A TBD 1 5 $0.00 $0.00 TBD Siphon (to remove bottom water from core N/A TBD 1 5 $0.00 $0.00 TBD Material Saftey data sheet/haz Mat Spill K N/A TBD 1 N/A $0.00 $0.00 $0.00 Laboratory Equipment Centrifuge equipment & supplies (Bonnie N/A TBD TBD N/A TBD TBD TBD Cold storage room (ashore) N/A TBD 1 TBD TBD TBD TBD Distillation equipment & supplies (Clara F N/A TBD TBD TBD TBD TBD TBD Nitrogen-15 nitrate (Bonnie Chang) N/A $100.00 1 1 $100.00 $100.00 $0.00 Zeolite (Al Devol) N/A TBD 1 1 TBD TBD TBD Glassware Round-bottom flask (500 ml?) N/A $100.00 1 1 $100.00 $0.00 $100.00 Condenser N/A $25.00 1 1 $25.00 $0.00 $25.00 Miscellaneous N/A TBD TBD 1 TBD $0.00 $0.00 Analyses Marine Chemistry Lab (Kathy Krogslund) Nutrients (sediment, dilluted) N/A $15.00 15 1 $225.00 $0.00 $225.00 Nutrients (water column) N/A $10.50 15 1 $157.50 $0.00 $157.50 Salinity (CTD calibration) N/A $9.50 3 1 $28.50 $0.00 $28.50 Oxygen (CTD calibration) N/A TBD 3 1 TBD $0.00 TBD Filters for nutrients N/A $2.25 30 1 $67.50 $0.00 $67.50 Mass Spectrometry (Dave Wilbur) Isotope characterization N/A TBD 18 1 TBD $0.00 TBD Sub Total > $92,068.50 Total< $1,293.50 12
References Bonin, P., P. Omnes, and A. Chalamet. 1998. Simultaneous occurrence of denitrification and nitrate ammonification in sediments of the French Mediterranean Coast. Hydrobiologia 389: 169-182. Cole, J. A. and C. M. Brown. 1980. Nitrite reduction to ammonia by fermentative bacteria: A short circuit in the biological nitrogen cycle. FEMS Microbiol. Lett. 7: 65-72. Jørgensen, K. S. 1989. Annual pattern of denitrification and nitrate ammonification in estuarine sediment. Appl. Environ. Microbiol. 55: 1841-1847. Kassakian, S. 2004. Nitrogen isotopic variations in nitrate in the Hood Canal, Puget Sound Washington: Confirmation of the kinetic isotope effect of sedimentary denitrification. B.S. thesis. University of Washington. Kelly-Gerreyn, B. A., M. Trimmer, and D. J. Hydes. 2001. A diagenetic model discriminating denitrification and dissimilatory nitrate reduction to ammonium in a temperate estuarine sediment. Mar. Ecol. Prog. Ser. 220: 33-46. Norbeck, A. 2000. Sediment fluxes as the main source of high ammonium concentrations in Puget Sound, Washington. B.S. thesis. University of Washington. Soonmo, A. and W. S. Gardner. 2002. Dissimilatory nitrate reduction to ammonium (DNRA) as a nitrogen link, versus denitrification as a sink in a shallow estuary (Laguna Madre/Baffin Bay, Texas). Mar. Ecol. Prog. Ser. 237: 41-50. Sørensen, J. 1978. Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbiol. 35: 301-305. 13
Warner, J., M. Kawase, and J. Newton. 2002. Recent studies of the overturning circulation in Hood Canal. In T. Droscher, ed. Puget Sound Water Quality Action Team, Proceedings of the 2001 Puget Sound Research Conference. 14
Figure Captions Figure 1: Location for the Northern Hood Canal sill (PRISM station #10): 47 48.00 N by 122 43.20 W (PRISM web site) Figure 2: Vertical sections of dissolved ammonium (mol x kg -1 ) in Hood Canal during the June 17, 1998 PRISM cruise. Sample locations and depths are indicated by dots (Warner et. al. 2002) Figure 3: Mock ammonium concentration profile similar to that found by Norbeck in 2000 at the Northern Hood Canal sill 15
Figures Fig. 1 16
Fig. 2 17
0 Ammonium Concentration (um) 0 50 100 150 2 4 Depth (cm) 6 8 10 12 Fig. 3 18