Description of Research:
This research cruise, initiated in San Diego, California on August 21st, 2000 and ending in Arica, Chile on September 29, 2000 will focus on the effect of bioactive trace metals (iron, zinc, manganese, cobalt, copper and cadmium) on phytoplankton and bacterioplankton dynamics. The main emphasis will be on the coastal upwelling regime off Peru, with a secondary emphasis on the equatorial Pacific upwelling regime near the Galapagos Islands. We will also run a major transect connecting the equatorial Pacific upwelling system with the Peru coastal upwelling regime. Finally, we plan on spending one and a half days examining a coastal upwelling area off the southwest coast of Baja California, Mexico. The major working hypothesis is that we will observe a mosaic of conditions with high concentrations of macro nutrients such as nitrate and phosphate, but with highly variable iron concentrations ranging from extremely low resulting in severe iron limitation, to high concentrations where iron is not biolimiting. We also predict a range of silicic acid concentrations driven by different assimilation ratios of silicic acid to nitrate dependent upon the ambient iron concentrations. Zinc, cadmium and cobalt may all play a significant role in carbon assimilation. We propose that the trace metal chemistry will be a factor determining whether or not extensive blooms of large diatoms develop in the macro-nutrient rich upwelled waters.
Factors responsible for variations in trace metal concentrations have to do with both the magnitude of external sources and the assimilation rates of the bioactive trace metals relative to the macronutrients. The riverine inputs of trace metals to the Peru coastal waters during our investigation are predicted to be minimal. The existence of a wide continental shelf, however, can trap the iron-rich suspendend particles delivered during the previous flood seasons and previous years. The continental shelf can then act as an external source of iron (and zinc and manganese) to be entrained with the macronutrient rich upwelled waters. Extensive regions of the Peru coast have only a minimal shelf. We predict that upwelled waters in these regions will have extremely low iron concentrations and will tend to exhibit iron limitation. A wide variety of biological parameters will be examined; ranging from growth rates of phytoplankton, to various physiological indicators, to molecular biological indexes. It is argued that many of these parameters will prove to be sensitive diagnostic indicators of the extent to which the biological communities might be limited by a particular trace or macro-nutrient. There will be a multitude of on-deck incubations occurring throughout the cruise. These incubations will have different amounts of trace metals/organics added in order to examine the response of the phytoplankton and bacteria communities. There will also be a contingent of marine chemists aboard making near real time measurements of the concentrations and speciation of the various trace metals.
The bulk of the sampling will be of near surface water during the major surface transects. The surface transects will be carried out at speeds of approximately 4 to 8 knots and will have our clean surface pumping system deployed. We will periodically stop during the surface transects for a quick CTD cast down to a depth of 300 to 500 meters. We will take advantage of the latest satellite and remote sensing data to help guide our transects. We will also take advantage of what we learn during the surface transects in order to reoccupy a station and sample more intensively at appropriate locations.
Latest Personnel List:
1. Ken Bruland, Prof., UCSC - Chief Scientist - Proposal title - "The interdependence of the chemistry of Fe and Zn and phytoplankton dynamics in coastal upwelling regimes." This is a major field study of upwelling regimes (with Bruland and Hutchins as the two core PI's) to assess the interaction between bioactive trace metals and phytoplankton. The emphasis of this field effort is on the Peru coastal upwelling system. We will investigate the extent to which elevated levels of iron (and Zn and Mn) are necessary for the development of extensive blooms of large diatoms in upwelling systems and how these trace metals can be important factors regulating bloom development. Recent results have shown that upwelling regimes off central California can range from severe iron depletion to iron replete and lead to a mosaic of Fe limitation conditions. For reasons similar to the Central California upwelling systems, we hypothesize that large areas of the Peru coastal upwelling system are Fe-limited. Factors suggestive of Fe-limitation off Peru are a very narrow shelf, low riverine inputs (temporally out of phase with the upwelling), and historical evidence for HNLC conditions. The Peru upwelling system is adjacent to the eastern equatorial Pacific upwelling system and we will also examine the gradation between these two regimes as well as the "island effect" of the Galapagos Islands. The core funding of this major field effort also allows for a variety of complementary biological and chemical studies to be carried out by additional collaborating scientists.
2. Dave Hutchins, Asst. Prof., U. Delaware - Co-Chief Scientist - Proposal title - "Iron limitation and the silicate pump in the California and Peru upwelling systems." Dave will coordinate his group's study of the effects of Fe and Zn on biological parameters and silicon and nitrogen biogeochemistry. Numerous small "survey" shipboard incubations (see Giselle Firme's proposed efforts) will be coupled with fewer large volume (20L), in-depth more "process-oriented" experiments. Dave will have the primary responsibility for the latter. Process experiments will be carried out at selected sites and will involve a comprehensive examination of nutrient biogeochemistry and effects of metal additions on productivity and diversity of important plankton groups, including phytoplankton, bacteria and grazers. This dual approach of numerous smaller survey and select larger incubations is designed to give us both a broad spatial coverage of the relationship between metal availability and changes in Si:N utilization ratios, as well as a detailed look at iron's influence on community structure and nutrient biogeochemistry in areas of special interest. Dave will also attempt an experimental 'continuous culture' systems with natural communities to look at the effects of variations in iron supply rates on community dynamics and nutrient drawdown ratios; the intent is to mimic different rates of upwelling in a shipboard setting. Planned collaborations (in addition to the core collaborations with Bruland and coworkers) include studies with Kudela (N uptake kinetics), Franck (Si uptake kinetics), Wilhelm (genetic regulation of Fe uptake), and Kirchman/Cottrell (Fe and C limitation of bacterial growth).
3. Geoffrey Smith, Senior Technician (Bruland), UCSC - Responsible for clean seawater sampling systems for all clean water collections - both chemical and biological (treat him well, he is crucial to your success). In addition, Geo is responsible for the collection of samples to return to UCSC where he will perform shore-based analyses for a suite of trace metals, including Fe.
4. Eden Rue, postdoc (Bruland), UCSC - Shipboard iron (and Cu) chemistry via cathodic stripping voltammetric methods. Edie will be involved in near-real time determinations of dissolved Fe distributions as well as iron speciation studies. The mapping of the Fe distributions will be essential for interpreting much of the other spatial data. She is also interested in collaborative studies with other participants doing incubation experiments.
5. Unnamed, (Bruland) Shipboard iron chemistry via our FIA catalytic spectrophotometric method. Involved with mapping of dissolved iron distributions.
6. Fabiana Corami, grad student (Bruland) UCSC (Italian national) - Zn chemistry via voltammetric methods. Mapping of dissoved Zn (and Cd) distributions and speciation studies.
7. Jenn Conn, technician (Bruland), UCSC (UK national) - Nutrient analysis and mapping
8. Heather Macrellis, grad student (Bruland), UCSC - Nutrient analysis and mapping together with Jenn Conn.
9. Laura Lessin, grad student (Bruland), UCSC - Chlorophyll or Mn mapping??
10. Bettina Loscher, postdoc (Hutchins) U. Delaware (German national) - Bettina will examine diel rhythms in size-fractionated iron (and possibly Zn) uptake using radiotracers in deckboard incubation experiments. Their preliminary culture work suggests that prokaryotes, in particular, have a pronounced diel rhythm in iron uptake tied to light/dark cycles and possibly to the cell division cycle. Bettina will also carry out a number of experiments to examine effects of Fe and Zn availability on sinking rates of phytoplankton assemblages both from the deckboard incubation experiments, and from high- and low-metal regimes along the cruise track. Finally, she will assist with the large-volume '"process" Fe- and Zn-addition experiments.
11. Rumi Fukuda, postdoc (Hutchins), U. Delaware (Japanese national) - studies of bacterial metalloenzymes. The goal of this study is to understand how trace metals affect DOC utilization and carbon export in marine environments through control of bacterial enzymatic activities. Although a few recent studies have examined how iron deficiency affects bacterial production, little attention has been paid to the role of other metals in bacterial DOC hydrolysis activities. Many strains of marine bacterial isolates have extracellular aminopeptidases that have zinc cofactors, so low Zn concentrations in open ocean seawater could potentially limit protein hydrolysis, with large consequences for DOC cycling. The purpose of this study is to test for the inhibition of protein hydrolysis by zinc deficiency in ocean surface water, as well as to examine the potential for metal limitation of other important bacterial ectoenzymes. Since it is possible that bacteria can shift their utilization of specific limiting metals to others that are more abundant, it is important to understand the effects of multiple trace metals on bacterial ectoenzyme activities. Seawater incubation experiments will be used to monitor the influence of metal availability (zinc, iron, cadmium and manganese) on bacterial ectoenzymes. Enzyme activities will be monitored in metal addition bottles and in unamended controls, including aminopeptidase activity (a spectrofluorophotometric assay), alkaline phosphatase, and b-glucosidase. Samples will also be taken for later laboratory analyses of bacterial productivity and abundance, and dissolved combined amino acids and sugars. Incubations will also be conducted with additions of RI-labeled proteins (extracted from labeled phytoplankton) in order to examine how trace metal limitation affects DOC cycling in metal-deficient regimes. This work will be coordinated with the Fe/DOC bacterial work to be carried out by Kirchman and Cottrell.
12. Giselle Firme, grad student (Hutchins), U. Delaware (Brazilian national - 'Survey' of Fe and Zn limitation with shipboard incubations and growout experiments. These survey experiments are small-volume (1 L) Fe and Zn addition experiments designed to map spatial and temporal variability in metal limitation and the effects on Si:N utilization ratios. They involve starting a large number of addition experiments (4 or more a day) involving only relatively simple size-fractionated chl a and dissolved and particulate Si and N measurements. This approach has successfully been used in their last cruise to gauge the areal extent of Fe limitation along the California coast, where they carried out almost 50 individual experiments throughout the upwelling zone. This mapping approach will be highly complimentary with the planned measurements of dissolved iron, phytoplankton photosynthetic efficiency, and flavodoxin/ferredoxin ratios along the cruise track by DiTullio's group.
13. Jack DiTullio, Assoc. Prof., U. Charleston - Proposal title "Ferredoxin: Flavodoxin ratios as indicators of Fe-limitation". The method uses high performance capillary electrophoresis (HPCE) that allows separation and quantification of these two proteins. It also resolves isoforms of the proteins that are produced by different species. In conjunction with the Fe numbers and some physiological measurements this will be a good test for using this ratio in the field. Samples can also be size fractionated to estimate diatom and prokaryotic algal Fe stress from the fd:flv ratio. C-14 pigment labeling will be used to get at class-specific algal growth rates. HPLC pigment profiles will be mapped. This method also allows us to measure algal C/chl ratios. Jack will also be responsible for underway Chl mapping on the surface transects as well as frrf measurements. Fe-light experiments.
14. Jen Maucher, grad student (DiTullio), U. Charleston - Flavodoxin/Ferredoxin ratios as indicators of Fe-limitation. Jen will carry out a broad survey to compare with Fe distributions and examine the shipboard incubation experiments.
15. Unnamed, grad student (DiTullio) - assist with the projects of Jack DiTullio.
16. Charlie Trick, Prof, (Canadian national) - The composition and physiological status of phytoplankton populations undergoing the changing nutrient chemistry of upwelled waters provides a unique opportunity to study the dynamic relationship between nutrient competition and the resulting community. C.G. Trick will examine how communities are formed as the cells compete for iron in waters of different iron status. Trick will provide a flow cytometer with cell sorting capabilities and a series of natural and artificial iron chelators. In one series of experiments the flow cytometer will be routinely used to characterize the structure of the different communities. Periodically the cells of the population will be sorted into the specific fluorescent/size groups and then examined for biochemical and physiological parameters (ferridoxin/flavodoxin, iron-inducible membrane proteins, photosynthetic efficiencies (Fv/Fm)). These will be collaborative studies with individuals such as Jack DiTullio and Jen Maucher. The purpose of this study would be to relate the biochemical parameters measured in the mixed community to the physiological activities of each of the distinct phytoplankton groupings. In a second series of experiments, natural and synthetic iron chelators will be provided along with radioactive Fe to ascertain the routing of iron through the competing cells of the population. After exposure to various iron/ligand compositions the cells will be fixed and sorted using the sorting cell cytometer and the distribution of the iron considered in relation to the biomass of the collected cell fractions. The purpose of this study is to ascertain how the ligand-iron combination influences the competitive ability of the natural cells.
17. Raphe Kudela, Asst. Prof., UCSC - Because iron is a key requirement for many different enzymatic processes, iron limitation and subsequent release from limitation in natural assemblages or grow-outs manifests itself in several different metabolic pathways. These changes do not necessarily occur simultaneously. For example, Fe additions typically result in an almost instantaneous change in the quantum efficiency of PSII (measured by FRR). Changes in carbon uptake versus irradiance patterns take >24 h, while changes to nitrogen metabolism (as either a change in preference, or a change in N uptake vs. irradiance) typically take > 36-48 h. I am interested in following the time-dependent changes in availability of reducing power once Fe limitation is released. Specifically, I propose to measure 15N-NO3, 15N-NH4, and 14C kinetics (uptake vs. irradiance) in comparison with the FRR data from DiTullio. The working hypothesis is that modest Fe additions stimulate the recovery of PSII (and therefore increase Fv/Fm), but may not be sufficient to stimulate both C and N fixation. This could potentially result in imbalances in the Redfield assimilation ratio. By comparing specific uptake rates and uptake vs. irradiance parameters with the variable fluorescence data, it will be possible to determine the [Fe] thresholds for full recovery and the time-dependent processes that lead to this state.
18. Jim Moffett, Assoc. Res. Scientist, WHOI - ?????
19. Mak Saito, postdoc (Moffett), WHOI - Cobalt Chemistry and its interaction with plankton dynamics. Mak will determine the distribution of cobalt and its speciation. They have evidence that there are Co-binding ligands, cobalophores, that act analogous to siderophores. Mak would also like to collaborate on growouts for cobalt limitation.
20. Phoebe Lam, grad student (Morel), Princeton - Effect of metal additions on the CaCO3/organic carbon ratio of the plankton community.
21. Phillipe Tortell, postdoc (Morel), Princeton - Mechanisms of inorganic carbon uptake and the potential role of metals in the carbonic anhydrase system.
22. Valerie Franck, grad student (Brzezenski), UCSB - I will investigate changes in diatom biomass and species composition, Si and NO3 uptake rates and Si:NO3 uptake ratios along some of the major onshore-offshore transects. Transect sampling will be coordinated with Ken Bruland's group in the hope of correlating changes in these parameters with changes in trace-metal availablity. In order to understand the mechanism behind changes in Si:NO3 uptake rates due to trace-metal availability, I will look at Fe and Zn effects on Si and NO3 uptake kinetics (uptake rate vs substrate concentration) in 10L growouts. I will also participate in Dave Hutchins' larger 20L growout experiments, measuring size-fractionated Si and NO3 uptake rates, Si and NO3 kinetic parameters and diatom abundance and species composition, and will try to use various methods (32Si autoradiography or live-cell staining) to get at species-specific changes in Si uptake.
23. Dave Kirchman, Prof., U. Delaware - Proposal title "Metal/bacteria interactions - in particular, carbon versus iron limitation of heterotrophic bacteria". Influence of low iron on bacterial growth efficiency. Variation in carbon mineralization rates and respiratory electron transport in seawater incubations with carbon and iron additions. Various radiotracers will be used including 3H-leucine and 3H-thymidine for bacterial production, as well as 14C-glucose and 14-C amino acids for bacterial growth efficiency.
24. Matt Cottrell, postdoc (Kirchman), U. Delaware - Metal/bacteria bacteria interactions - in particular, carbon versus iron limitation of heterotrophic bacteria. Composition of bacterial assemblages in iron deplete versus iron replete waters. Assessment of the abundance of specific phylogenetic groups of in contrasting iron regimes assessed using fluorescence in situ hybridization (FISH) of rRNA-directed oligonucleotide probes. Influence of iron and carbon supply on the composition of bacterial assemblages assessed using seawater incubations with iron and carbon additions. Microautoradiography using tritiated compounds including protein, amino acids, chitin and N-acetyl glucosamine will be used in combination with 16S rRNA directed fluorescent probes (MICRO-FISH) to assess uptake of organic matter by different phylogenetic groups of bacteria. MICRO-FISH may also be used with different 55Fe-ligand complexes to assess iron uptake by various phylogenetic groups of bacteria.
25. Steven Wilhelm, Asst. Prof., U. Tennessee (Canadian National). Proposal title - "Influence of viruses on Fe and C cycling". As major mechanisms of microbial mortality, viruses act to recycling organically complexed Fe and other DOMs in the water column. In conjunction with Dave Hutchins, we will use 55Fe labeled cells and viral enrichment assays to look at the effect of viruses on the recycling and transfer of Fe amongst marine microbes. As well, we will continue investigations of the fungal siderophore DFB as an inducing agent for lysogenized bacterial cells in marine systems. A second virus project (carried out with Charlie Trick) will build on studies from the Sargasso Sea to look for evidence of virus-infected Synechococcus and Prochlorophytes. Synechococcus and Prochlorophytes will be collected by flow cytometry at various stations during the cruise, and samples returned to the UTK labs for analysis. The final project is to field test of Fe-responsive biosensor in natural and amended samples. We have placed the fepA-fes promoter (from a Fur-regulated siderophore biosynthesis gene) in front of a luxCDABE cassette in a mini-TN5 transposon, and inserted this construct into several bacteria. The resulting organism(s) produce light in response to bioavailable Fe in their environment. This will involve a series of small (1 L) incubations and addition assays (titrated Fe, DFB, ferrichrome) to determine the bioavailability of Fe to prokaryotes in waters collected from various stations. We will work closely with Rue/Bruland to determine the sensitivity/response of this system relative to the [Fe].
26. Melanie Eldridge grad student (Wilhelm), U. Tennessee. "Identifying Fe-dependent responses at the genetic regulatory level". Research is directed at identifying changes at the genetic level in response to Fe availability. Two systems are currently being focussed on. The ferric uptake repressor (fur) gene encodes a 17 kDa protein product that acts as the internal sensor for Fe concentration in prokaryotes. We have been studying the phylogenetics and regulation of this gene in the lab, and will undertake amendment studies to look at the regulation of this gene product in the field (in collaboration with Hutchins). As well, we have developed PCR primers specific to cyanobacterial flavodoxin, and will look at the regulation of this bioindicator of cellular iron status in these same samples. This work will be paralleled with 16s rDNA/rRNA studies to look at changes in this phylogenetic indicator (with Hutchins). These studies will involve relatively small (1-2 L) incubations of amended and non-amended cultures over 24-72 h time frames, as well as direct filtration of surface water.
27. Martina A. Doblin, Post-Doc (Greg Cutter), Old Dominion University (Australian National) - Will conduct some Se enrichment experiments involving incubation of natural phytoplankton communities with different chemical forms of Se. In addition, will collect seawater samples for Se determinations, involving 2-3 vertical profiles (sampling at approximately 8 depths in the upper 500m), and 8-10 surface grab samples along the inshore-offshore transects.
28-32. Foreign observers