Farooq Azam - US grants

Research is proposed to continue the study of the mechanisms whereby heterotrophic bacterioplankton utilize particulate organic matter (POM) and dissolved organic matter (DOM) in the sea. The PI is also studying the magnitude and patterns of organic matter flux into bacteria in both the epipelagic and the mesopelagic zones to determine how heterotrophic bacteria may influence the vertical transport of biogenic production in the sea. The research will focus on studying the mechanisms and rates of bacterial utilization of small detritus (1-50 um). The PI will use laboratory produced and well-characterized "model detritus" consisting of killed phytoplankton, microflagellates, and chroococoid cyanobacteria. He will study colonization growth rates of bacteria on POM and will examine enzymic mechanisms whereby bacteria hydrolyze POM into utilizable DOM; determine shifts in bacterial cell-size, growth rate, and modes of organic matter hydrolysis as a consequence of attachment to, or release from, POM. The PI asks whether a colonized particle becomes a point-source of DOM as well as of bacteria ("baby-machine"). A hypothesis will be tested that firm attachment to POM is a survival/dispersal strategy of bacteria; they survive the passage through metazoan gut and thrive in the fecal pellets. In field work using uniformly ?14C! labeled detritus, the PI will measure the turnover times of detritus of a broad size range, biochemical composition, and origins. The research proposed will contribute towards developing a mechanistic framework for bacteria-organic matter interactions in an ecosytem context. This knowledge should be of value in elucidating the role of bacterioplankton in organic matter fluxes in the oceans.

It is important to understand how particles which are formed in the upper layers of the ocean are transformed as they sink out of those upper layers into the benthos. Bacteria play a major role in the transformation of the particles on their way to the bottom. Much of the organic matter associated with these particles is converted to the dissolved state, a form in which it can be utilized by phytoplankton and other bacteria for growth. Dr. Azam's previous results have led to the hypothesis that the attached bacteria, via secretion of certain enzymes (hydrolases) into the particle, are responsible for large scale solubilization and fragmentation of the particles. The result is that most of the dissolved organic material diffuses out of the particle and is utilized by free-living bacteria. In the present study, the magnitude and mechanisms of this pathway are explored. The research focuses on the regulation of several enzymes that control the transformation from the particulate to the dissolved state. The study contributes toward the understanding of the role of bacteria in biogeochemical cycling pathways of organic matter.

Coupling of carbon from primary producers to bacteria can significantly influence the patterns of vertical flux of organic matter in the ocean. Because bacteria must hydrolyze particles before uptake, bacterial exohydrolases must assume a central role in carbon flux from particulate organic matter into the microbial loop. The expression of bacterial exohydrolase is regulated by substrate concentration and therefore may cease in waters depleted of particles and polymers. In high latitude waters there is no phytoplankton production during the protracted polar night. The hypothesis to be tested is that during the Antarctica winter, the production of bacteria exohydrolase ceases. Therefore, particles and polymers produced during the early spring bloom cannot be utilized by bacteria. Exohydrolases are expressed when organic matter accumulates to a critical level; consequently, the coupling between particulate organic matter and bacteria becomes strong. Field and laboratory manipulations will examine environmental cues which regulate exohydrolase activities. This knowledge should contribute to the understanding of biological production mechanisms and variability in the pathways of organic matter cycling in the Southern Ocean.

This proposal is part of an integrated scientific investigation to study the properties of the Arctic ocean, atmosphere, and sea-ice on a transect between Murmansk, USSR and Nome, Alaska. There are few published measurements of biomass or production from most shelf and basin regions of the Arctic Ocean. This project is one of four that will evaluate biological processes in the Arctic Ocean during the Trans-Arctic cruise of the SOVETSKIY SOYUZ. It will investigate role of bacteria in the cycling of organic matter in the Arctic Ocean. The program will study the spatial variation in bacterial biomass and production and their significance in organic matter cycling and the sinking flux out of the region of light penetration and into deeper depths. Concentrations of various organic fractions in sea water will also be accomplished.

Dissimilation of sinking organic aggregates is a central issue in the regulation of carbon fluxes from the ocean's surface into its depths. The role of attached bacteria in particle decomposition has been unclear. High concentrations (108-109 ml-1) of slowly growing bacteria commonly found on aggregates create intense ectohydrolase activities which rapidly solubilize (0.2-2.1 d turnover time) particulate combined amino acids but allow most hydrolysate to be released into seawater. This "uncoupled solubilization", particularly if it applies generally to aggregate's carbon pool, could have profound implications for downward flux of carbon. Using SCUBA collected marine snow and laboratory-made 14C labeled phytodetritus aggregates studies will 1) quantify DOC release (measured by high temperature catalytic oxidation) and POC turnover rates and how they vary with variation in colonization and enzyme activities on aggregates. 2) characterize the released DOC in terms of molecular size distribution (with molecular sieves), whether it contains colloids and sub-micron particles (by transmission electron microscopy), and whether some of it is refractory to bacterial utilization (or has long turnover time); 3) test whether enzyme action on aggregates causes differential solubilization of C, N and P. This work will be done on aggregates collected in waters off La Jolla, Santa Barbara, and at several offshore stations in Southern California Bight during 4 cruises. The proposed study will contribute to an understanding of the role of attached bacteria in POC DOC transition and hence in carbon cycling in the ocean.

The quantitative role of bacteriophages in the biogeochemical cycles of the marine environment is a most urgent question. At present, there is no direct method to measure phage production, only indirect estimates of phage production based on measures of phage decay rates by Norwegians scientists. This project will develop urgently needed and innovative techniques for measuring phage production in seawater. Two radiotracer approaches will be attempted to specifically measure DNA and RNA phage production. The approach is risky because the strategy is to metabolically label bacteria and secondarily the phage DNA and RNA. The methods will be calibrated and field tested to assess whether or not phage proliferation is an important process in marine ecosystems.

9317958 Palenik The flow cytometer provides the unique capability of rapid analyzing the fluorescence, scattering, and several chemical and biochemical parameters of individual cells. This proposal requests funds to acquire a flow cytometer for the Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography (SIO) to be jointly used by several investigators working in diverse areas. The instrument will be used to provide samples. This information will be used to understand the morphological and genetic diversity of these organisms, their effects on the biooptical properties of the water column, and their responses to nutrient and other environmental stresses. In a novel application, the instrument will be used to characterize the bioactive secondary metabolites produced by currently unculturable organisms isolated from field samples by flow cytometric sorting. The flow cytometer will also be used on laboratory cultures to study physiological responses of particular species to various environmental stresses. This research is part of efforts to develop molecular probes as indicators for particular stresses and to parameterize optical models of oceanic photosynthesis as a function of particular stresses.

9312695 Azam This is a joint research proposal (F. Azam and H. Ducklow, co-PIs) to perform quantitative studies on the significance of bacteria in mediating and regulating biogeochemical fluxes of carbon and nitrogen in the Arabian Sea, as a component of the US JGOFS Arabian Sea Process Study. We propose a program of core measurements to quantify carbon and nitrogen pools and fluxes as well as mechanistic studies to understand the response of the bacteria-mediated processes to dramatic changes in many primary production that occur regularly due to monsoonal reversals in the Arabian Sea. The JGOFS core measurements will include bacterial abundance and biomass, bacterial production (by both 3H thymidine and 3H leucine incorporation methods), DOC, POC and PN. Our research is design, further, to address two key questions concerning microbial heterotrophic processes identified by US JGOFS: 1) How do the community structures which occur during the oligotrophic vs eutrophic periods differentially impact carbon storage and export fluxes? 2) Does the decomposition of sinking particles slow down in the suboxic zone and result in enhanced vertical flux though this layer? We hypothesize that sequential occurrence of highly eutrophic and oligotrophic productivity regimes creates a uniquely robust microbial loop during the oligotrophic period at the expense of slow-to-degrade DOM which accumulates during the eutrophic period. We predict that the strong dominance of the microbial loop renders the oligotrophic period net- heterotrphic and this has implications for spatial and temporal patterns of carbon oxidation and exchange. The 'stored' DOC may also support high bacterial carbon demand (higher than would be supported by particle flux) below the mixed layer following shoaling of the pycnocline. A second hyothesis we wish to test is that sinking particles in the surface waters are rapidly solubilized by hydrolytic ectoenzymes of colonizing bacteria but t he enzymatic solubilization is greatly reduced in the suboxic layer resulting in much slower depth-dissipation of POC.

95-30851 Azam This research project is part of the US Joint Global Ocean Flux Study (JGOFS) Southern Ocean Program aimed at (1) a better understanding of the fluxes of carbon, both organic and inorganic, in the Southern Ocean, (2) identifying the physical, ecological and biogeochemical factors and processes which regulate the magnitude and variability of these fluxes, and (3) placing these fluxes into the context of the contemporary global carbon cycle. This research will investigate the role and quantitative importance of bacteria in the transformation of organic matter as part of the JGOFS Southern Ocean Process Study. Researchers hypothesize that extreme seasonal and spatial variation in the Southern Ocean leads to transient uncoupling between bacteria and primary producers as mediated through the dissolved organic matter pool. The principal goal of the research will be comprehensive spatial and temporal coverage of core measurements (bacterial abundance, biomass, and production). This will allow an integrated quantitative assessment of the fraction of primary production consumed by bacteria. Equally important is the study of the mechanisms controlling the rate and time frame in which primary production is processed by bacteria. This is critical to complement the core measurements and address these issues. Specifically, total bacteria counts will be complemented with counts of nucleoid-containing cells and respiring cells to determine the fraction of the bacterial assemblage that is active. Incubation experiments in collaboration with other researchers will examine variation in DOM lability and the growth yield of bacteria. This project will provide a quantitative and mechanistic understanding of variability in a major pathway of carbon flow, the microbial loop, in the Southern Ocean.

9708153 Azam Bacterial production, via 3H-thymidine incorporation, is a "core" parameter in biological oceanography and marine biogeochemistry. No non-radio-isotopic method is currently available but one would be highly desirable for a variety of logistical, safety and scientific reasons. This project will develop a non-radioisotopic method based on the use of the thymidine analog, 5-bromo-deoxyuridine (BrdU), for measuring the rate of bacterial DNA synthesis in seawater samples. BrdU behaves as a thymidine analog and becomes incorporated into newly synthesized DNA in place of thymidine. Its incorporation into DNA is quantified by an immunochemical method. After BrdU incorporation, the cells are permeabilized and treated with an antibody which binds to BrdU in the DNA. A second antibody, conjugated with a fluorochrome or a dye, is then used to quantify BrdU incorporation. Many different procedures for quantifying BrdU incorporation into DNA have been employed. Current protocols will be tested and modified as necessary to provide the simplest reliable method for aquatic microbial assemblages. An additional benefit of this approach is the possibility to estimate growth rates of individual bacteria in a sample in situ hybridization. The fluorescent signal of individual bacteria could be determined by epifluorescence microscopy or by flow cytometry. In principle, the BrdU-labeling could even be combined with rRNA gene probing and, using flow cytometry, growth rates could then be estimated for any specific group of bacteria in a complex assemblage. Such a capacity would revolutionize the study of microbial ecology, providing information on growth rates in microbial populations at a level of detail not possible with the current radiolabeling procedures.

This project is designed to extend investigations of viral diversity and the magnitude and variability of bacterial and algal mortality due to viral attack in the Arctic Ocean. The PI proposes to extend these studies with emphasis on sea ice microbial communities for comparison with the underlying sea water. Collection, fixation, and concentration of melted sea ice samples will be accomplished at sampling sites by deployment from US Navy submarine. Subsequent analyses will be conducted in the laboratory. Total counts of viruses and the frequency of viral infection in phytoplankton and bacteria in sea ice and seawater will be determined by electron microscopy on fixed samples. To examine natural community diversity, concentrates of viruses will be purified and subjected to pulsed-field gel electrophoresis to separate viral DNA based on genome size. From the number, positions, and staining intensity of different bands, the PI will estimate the diversity, genome sizes, and abundance of viruses in each sample. Through coordination with other researchers, the PI will relate viral abundance, the frequency of viral infections, and viral community diversity to hydrographic and biological variables to determine those which may be important in regulating viral propagation in arctic sea ice and seawater.

ABSTRACT

OCE-9819603

A very large fraction of the downward fluxes of biogenic silica and organic matter in the ocean are derived from the productivity of diatoms in the surface ocean. Not surprisingly then, there is much interest in understanding what controls diatom productivity, including mechanisms for recycling silica from sinking, decomposing particles back to growing diatoms. In this project researchers from the Scripps Institution of Oceanography and the University of California at Santa Barbara will study contribution of bacteria-mediated silica dissolution to total biogenic silica dissolution by measuring Si dissolution under conditions where bacterial processes have been selectively inhibited. The contribution of bacteria-mediated dissolution to biogenic silica production would be determined at the same time. Radio- (32Si) and stable isotope techniques developed through recent NSF grants would be used to attain high sensitivity and precision.

9900301, Azam
A methodology is presented that, if successful, will simultaneously quantify and qualify the majority of microbes present in an environmental sample. Such a methodology is a prerequisite to studying the distribution and diversity ofmarine microbes on the ocean basin scale. The research method is based on the fact that a seven base pair DNA tag, if sufficiently divergent, can differentiate between thousands microbial species. To be useful this tag region must be invariably located within a microbe's genome and it must be associated with a conserved DNA region that can be used to isolate the tag A tag region within the RNase P locus, a ubiquitous nbozyme, that fulfills the criteria ofbemg highly divergent and located next to a conserved region is identified. Fortuitously, the RNase P locus is also always found as one copy per genome. Therefore, this tag can be used as a quantitative, as well as qualitative, indicator of the microbial species present in a sample.

DEB 9972054
Azam
This project is designed to survey the diversity of marine phages from several sites in the Pacific Ocean. Bacteriophage are ubiquitous components of the marine environment frequently reaching concentrations of tens to hundreds of millions of individuals per ml. The decay rates of the bacteriophage are rapid, suggesting that production must be high to maintain the observed standing stocks. While numerous attempts have been made to incorporate these observations and their implications-such as mortality of marine bacteria-into the marine microbial food web, most of these studies have ignored the specificity of host/virus interactions. In fact, the diversity of marine phage is essentially unknown. Characterization of the phage isolates will be accomplished by electron microscopy, Pulse-field Gel Electrophoresis, and sequencing. These tests are specifically designed to identify the phage and classify them within known groups and to identify phage that represent novel groups. This study will be the first to systematically address the genetic diversity of this uncharacterized biota. The protocols develop and utilized here will provide the important background work for studies of phage in other environments.

This award will enable the purchase of an instrument that combines a rapid thermocycler with a microspectrofluorimeter. This technology allows the quantification of a PCR reaction as it occurs in real time and thus can convert the qualitative results typical of PCR into quantitative ones using the sensitivity and precision of the fluorimeter. Quantitative PCR is emerging as a potentially crucial, but still "risky" technique in the study of marine organisms. The barrier of instrumentation cost has also limited its development as a tool in marine sciences. The researchers will initially use the instrument in the detection of specific microorganisms (free-living photoautotrophs, heterotrophs, symbionts, and pathogens) in the marine environment, with the goal of developing a fundamental understanding of the mechanisms controlling microbial biodiversity. The detection of the genes for various microbial activities such as toxic metal metabolism or nutrient stress responses is a second potential use. Lastly, population genetic studies of eukaryotic marine organisms such as copepods and sea urchins will benefit from the utilization of the proposed instrument. Three laboratories in the Marine Biology Research Division of Scripps Institution of Oceanography, University of California, San Diego, will begin as major users of the instrument (Palenik, Azam and Burton), but use of the instrument will likely extend to several other labs. In all cases the instrument will be valuable in generating results that will open new research directions in marine sciences and provide preliminary results for future NSF proposals.

The human pathogen Vibrio cholerae is widely distributed along many coastlines including those of the United States. Environmental factors play a major role in determining the distribution of toxigenic V. cholerae. For example, the introduction of the seventh pandemic of cholera to Latin America in 1991 has been suggested to have been correlated with marine plankton blooms triggered by a climate change event such as that initiated by El Nino. In a effort to better understand marine vectors and conditions influencing the spread of cholera, we propose to examine the association of a variety of clinical and environmental strains of V. cholerae with marine plankton. Marine mesocosm experiments will be performed in which V. cholerae will be identified by marking cells with a modified version of the green fluorescent protein gene. growth of V. cholerae in the mesocosm will be followed using the frequency of dividing cells technique coupled to an image analysis method. Using selected marine model systems V. cholerae plankton colonization will be further examined, and the possible role of chemotaxis in plankton or phytodetritus association will be assessed. We will also isolate and characterize, or obtain mutants deficient in plankton colonization. The genetic defects will be identified, and the relationship, if any, between colonization of plankton and of mammals will be ascertained. Finally, the effect of biological and physiochemical factors on V. cholerae growth and distribution in mesocosms will be determined and the possibility of cholera toxin phage production and toxin gene exchange among cells in the marine environment will be explored. By understanding how biological, chemical, and physical factors influence the distribution, abundance and virulence of V. cholerae, together with the elucidation of the genetic requirements for its persistence with plankton in coastal ecosystems, it will be possible to better predict when environmental change is likely to present a cholera public health risk.

Our understanding of the marine environment was fundamentally changed when it was shown that marine phage (viruses that infect bacteria) are present in extraordinary numbers (ca.1010 per liter). Since this observation, marine microbial ecologists have attempted to incorporate phage into the microbial food web concepts. This work has shown that under various conditions phage account for 12-600% of all bacterial production via lysis. The high number of observable phage and their high production rates have called into question concepts of energy and nutrient cycles in the world's oceans. The observation of large numbers of phage in the ocean also raised two other extremely exciting questions: 1) What type of phage inhabit the marine environment?, and 2) How might phage affect genetic transfer (e.g., transduction or lysogenic conversion) in the marine environment?. Available evidence suggests that marine phage diversity is ten times as great as prokaryotic diversity and like their prokaryotic counterparts, it is believed that each phage will have a unique strategy for surviving and multiplying in the marine environment and these strategies will be reflected in the genes that each phage encodes. The life strategies adopted by phage will in turn help shape the MMFW and the biogeochemical processes of the ocean.

Current methods of phage characterization rely on phage morphology, genome size, and host range. However, these approaches are extremely limited in their applicability to environmental samples, primarily because the majority of host are not culturable. Drs. Azam and Rohwer ill attempt to circumvent this problem by sequencing the genomes of uncultured phage. This will be accomplished in three steps. First, phage genomes will be separated from each other using pulse field gels. Second, randomly amplified shotgun libraries (RASLs) will be constructed from the bands isolated from the PF-gels. Finally, the shotgun libraries will be sequenced.

The proposed research is a component of CREICO (Cooperative Research on Ecological Interactions in the Coastal Oceans) program in the Adriatic Sea. This international initiative is intended to develop cooperation between Croatia, Slovenia, Italy and the US in environmental science in the Adriatic, in concert with Adriatic Observing System (CAOS) being developed with NOAA. CREICO is intended to increase understanding of the structure and function of ecological systems in the Adriatic, and the system response to perturbations. The focus of CREICO is on "bloom phenomena" and their causes and consequences. This proposal focuses on the mucilage phenomenon (mare sporco). Over the last 3 centuries, enormous accumulations of "mucilage" have occurred along the coasts of the northern Adriatic Sea, affecting the coasts of Italy, Slovenia and Croatia. The cause remains a mystery. Mucilage events damage local fisheries and tourism. Scientifically, the huge organic matter accumulation represents an opportunity to study the ecosystem level mechanisms of variability in the carbon biogeochemistry, and North Adriatic offers a natural "laboratory." Predicting the mucilage events would be an excellent test of models of oceanic carbon biogeochemistry.

While phytoplankton blooms produce the organic matter that ultimately forms mucilage Dr. Azam hypothesizes that bacterial processing of phytoplankton biomass, to produce slow?to-degrade polysaccharide plays a significant role. They will test hypotheses on bacterial production of slow?to-degrade dissolved and colloidal organic matter (including bacterial capsular material and exudates) and its aggregation to form mucilage under relevant ecosystem conditions. This work will be done in collaboration with Serena Fonda?Umani (Italy), Vera Zutic (Croatia) and Valentina Turk (Slovenia). They will use mesocosms (in Fonda?Umani lab in Trieste, Italy) to test hypotheses through perturbation experiments. Further, they will take advantage of the planned two 15?d cruises. Bacterial studies will use molecular approaches, to explore species?specific roles in mucilage production and decomposition. Polysaccharide structure analysis will seek relationships between bacterial biochemical profiles and polysaccharide utilizability. Dr. Azam and his lab have been informally collaborating with Italian, Slovenia and Croatian marine scientists and students on mucilage studies, in research and training. This research will increase this collaboration; it will address a long?standing oceanographic problem, with potential of discovering fundamental principles of carbon cycling of relevance to research on global change.

The Shackleton Fracture Zone (SFZ) in the Drake Passage defines a boundary between low and high phytoplankton waters. West of Drake Passage, Southern Ocean waters south of the Polar Front and north of the Antarctic continent shelf have very low satellite-derived surface chlorophyll concentrations. Chlorophyll and mesoscale eddy kinetic energy are higher east of SFZ compared to values west of the ridge. In situ data from a 10-year survey of the region as part of the National Marine Fisheries Service's Antarctic Marine Living Resources program confirm the existence of a strong hydrographic and chlorophyll gradient in the region. An interdisciplinary team of scientists hypothesizes that bathymetry, including the 2000 m deep SFZ, influences mesoscale circulation and transport of iron leading to the observed phytoplankton patterns. To address this
hypothesis, the team proposes to examine phytoplankton and bacterial physiological states (including responses to iron enrichment) and structure of the plankton communities from virus to zooplankton, the concentration and distribution of Fe, Mn, and Al, and mesoscale flow patterns near the SFZ. Relationships between iron concentrations and phytoplankton characteristics will be examined in the context of the mesoscale transport of trace nutrients to determine how much of the observed variability in phytoplankton biomass can be attributed to iron supply, and to determine the most important sources of iron to pelagic waters east of the Drake Passage. The goal is to better understand how plankton productivity and community structure in the Southern Ocean are affected by the coupling between bathymetry, mesoscale circulation, and limiting nutrient distributions.
The research program includes rapid surface surveys of chemical, plankton, and hydrographic properties complemented by a mesoscale station grid for vertical profiles, water sampling, and bottle incubation enrichment experiments. Distributions of manganese and aluminum will be determined to help distinguish aeolian, continental shelf and upwelling sources of iron. The physiological state of the phytoplankton will be monitored by active fluorescence methods sensitive to the effects of iron limitation. Mass concentrations of pigment, carbon and nitrogen will be obtained by analysis of filtered samples, cell size distributions by flow cytometry, and species identification by microscopy. Primary production and photosynthesis parameters (absorption, quantum yields, variable fluorescence) will be measured on depth profiles, during surface surveys and on bulk samples from enrichment experiments. Viruses and bacteria will be examined for abundances, and bacterial production will be assessed in terms of whether it is limited by either iron or organic carbon sources. The proposed work will improve our understanding of processes controlling distributions of iron and the response of plankton communities in the Southern Ocean. This proposal also includes an outreach component comprised of Research Experiences for Undergraduates (REU), Teachers Experiencing the Antarctic and Arctic (TEA), and the creation of an educational website and K-12 curricular modules based on the project.

P.I. Papen, George Proposal #: 0428900

Project Summary
Nanoparticles, or colloids in aquatic environments, have sizes ranging from 1 to 1000 nm and are at the boundary between soluble chemical species and sinking particles. They are the most abundant particles in the ocean and other aquatic environments and account for a significant portion of "dissolved" organic carbon. In situ characterization of the physical and biochemical properties is crucial for a wide range of fundamental applications including: 1) ocean biogeochemistry 2) ocean optics and 3) aquatic biological hazards.

While the characteristics of nanoparticles are important for a variety of applications, their complex heterogeneous nature and small size makes the in situ determination of their physical and chemical characteristics extremely challenging. To date, most characterization techniques are laboratory-based and are thus limited. This project will develop in situ sensor networks for aquatic nanoparticle characterization that can address a broader range of applications and test them in an ocean environment. The research program consists of the development of microfluidic-based techniques that can preprocess and help analyze heterogeneous assemblies of aquatic nanoparticles and bacteria and the development of a pipelined suite of advanced optical techniques using the amplitude and the phase of the optical fields, multiple wavelengths, multiple scattering angles, polarization properties, and parallel interrogation volumes, which sequentially classify particle characteristics over a wide range of variables. The program will culminate in the deployment of a prototype in situ sensor network node that can measure both the physical and biogeochemical properties of aquatic nanoparticles forming the basis of a complete sensor network for in situ spatial and temporal monitoring.

The Shackleton Fracture Zone (SFZ) in Drake Passage of the Southern Ocean defines a boundary between low and high phytoplankton waters. Low chlorophyll water flowing through the southern Drake Passage emerges as high chlorophyll water to the east, and recent evidence indicates that the Southern Antarctic Circumpolar Current Front (SACCF) is steered south of the SFZ onto the Antarctic Peninsula shelf where mixing between the water types occurs. The mixed water is then advected off-shelf with elevated iron and phytoplankton biomass. The SFZ is therefore an ideal natural laboratory to improve the understanding of plankton community responses to natural iron fertilization, and how these processes influence export of organic carbon to the ocean interior. The bathymetry of the region is hypothesized to influence mesoscale circulation and transport of iron, leading to the observed patterns in phytoplankton biomass. The position of the Antarctic Circumpolar Current (ACC) is further hypothesized to influence the magnitude of the flow of ACC water onto the peninsula shelf, mediating the amount of iron transported into the Scotia Sea. To address these hypotheses, a research cruise will be conducted near the SFZ and to the east in the southern Scotia Sea. A mesoscale station grid for vertical profiles, water sampling, and bottle incubation enrichment experiments will complement rapid surface surveys of chemical, plankton, and hydrographic properties. Distributions of manganese, aluminum and radium isotopes will be determined to trace iron sources and estimate mixing rates. Phytoplankton and bacterial physiological states (including responses to iron enrichment) and the structure of the plankton communities will be studied. The primary goal is to better understand how plankton productivity, community structure and export production in the Southern Ocean are affected by the coupling between bathymetry, mesoscale circulation, and distributions of limiting nutrients. The proposed work represents an interdisciplinary approach to address the fundamental physical, chemical and biological processes that contribute to the abrupt transition in chl-a which occurs near the SFZ. Given recent indications that the Southern Ocean is warming, it is important to advance the understanding of conditions that regulate the present ecosystem structure in order to predict the effects of climate variability. This project will promote training and learning across a broad spectrum of groups. Funds are included to support postdocs, graduate students, and undergraduates. In addition, this project will contribute to the development of content for the Polar Science Station website, which has been a resource since 2001 for instructors and students in adult education, home schooling, tribal schools, corrections education, family literacy programs, and the general public.

Corals are increasingly threatened by disease induced mortality, and the risk of infection may be increased by elevated sea surface temperature either by promoting pathogen growth or virulence, reducing host resistance to infection, or a combination of both. This project will investigate the hypothesis that coral-associated bacteria mediate disease resistance through bacteria-bacteria interactions and this resistance mechanism is altered by elevated temperatures. The project tests three hypotheses related to the bacteria mediated resistance hypothesis: (1) Antagonistic interactions among coral-associated bacteria limit pathogen colonization and proliferation within the mucus layer. Antagonistic interactions among bacteria affect the diversity and spatial distribution of bacteria in marine pelagic and epibiotic communities, in part by preventing some bacteria from entering the established community. Analogous processes in the coral surface mucus layer will be examined with laboratory assays and in situ experiments using cultivable bacteria, combined with fluorescent in situ hybridization, phylotype-specific PCR, and denaturing gradient gel electrophoresis to assess non-cultivable bacteria. (2) Elevated temperature alters bacterial growth rates and bacteria-bacteria antagonism, facilitating pathogen colonization and proliferation. Growth rates are a fundamental force influencing bacteria community dynamics and can influence the relative abundance of phylotypes within a community. Temperature can affect growth rate as well as bacterial antibiotic production, thus altering the outcome of bacteria-bacteria interactions. Culture-dependent and -independent techniques will be used to explore bacteria-bacteria interactions on corals exposed to various temperature treatments and temporal samples of permanently tagged corals on the reef. (3) Other members of the coral-associated microbial community (e.g., eukaryotic grazers, viruses) impact the abundance and dynamics of coral-associated bacteria, potentially altering disease resistance. The coral mucus habitat contains not only prokaryotes but eukaryotic grazers and viruses. These predators strongly influence bacteria communities in the water column and their role is proposed to be analogous in the coral mucus layer. The intellectual merit of this project lies in the integration of coral reef ecology and microbial ecology to address fundamental questions regarding coral health and disease. Both culture-dependent and-independent techniques will be utilized to examine the ecological interactions of cultivable bacteria and assess dynamics of the whole community. The work is timely and imperative given the declining status of coral reefs and predictions of future decline. The broader impacts of this project include integration of research and education by developing seminars for the Professional Development Series for Education Staff at the Birch Aquarium at Scripps (La Jolla, CA). Participation of under-represented groups will be encouraged through 1) international coral disease workshops in the Philippines and East Africa facilitated in conjunction with the World Bank / Global Environment Fund Coral Reef Targeted Disease Research Group, 2) Mentoring of undergraduate students from the University of Guam, and 3) Developing a curriculum to engage minority high school students with ocean sciences and mentoring a team of minority high school students for the National Ocean Sciences Bowl.

Azam EAGER 1036613

Intellectual Merit: Pelagic marine bacteria and Archaea ("bacteria") play major roles in the variability in the biogeochemical fate of carbon fixed by phytoplankton in the ocean. The coupling of bacteria with the primary producers therefore has implications for climate and ecosystem models. Since bacteria interact with organic matter, including phytoplankton cells, at the nanometer to micrometer scale, understanding the biogeochemical coupling of bacteria with primary producers requires knowledge of the nature and strength of interactions at the micrometer scale. Using Atomic Force Microscopy (AFM) the PI recently discovered that a substantial fraction of heterotrophic (non-photosynthetic) bacteria, including the natural assemblages of Synechococcus, previously considered free-living, appeared conjoint with other bacteria. In a preliminary experiment, pelagic bacteria also became associated with cultures of Prochlorococcus (we do not yet know whether natural assemblages of Prochlorococcus harbor conjoint bacteria). In view of the biogeochemical importance of Synechococcus and Prochlorococcus as major primary producers in the ocean, their symbioses with heterotrophic bacteria could have far-reaching consequences.

This EAGER study has the potential to change ideas on microbial carbon cycling and marine ecosystems functioning. It may reveal a novel microspatial context, e.g., carbon and nutrient cycling 'hot spots', particularly in oligotrophic waters where Synechococcus and Prochlorococcus are major primary producers. This potentially transformative, but high risk research, is an excellent fit to the EAGER model for funding. Cutting edge methodologies, including cryo-electron tomography, nanoSIMS and single cell phylogenetics will be used through multidisciplinary collaborations to test hypotheses on the ultrastructure and ecosystem function of the symbioses. The phylogenetic identity of the conjoint partners will be determined by direct micromanipulation of seawater samples to pick individual conjoint cells followed by analysis by MDA (Multiple Displacement Amplification). NanoSIMS analysis of intercellular elemental exchanges will characterize the nature and biogeochemical significance of the symbioses. Field studies will examine the distribution of the symbioses in relation to relevant environmental factors. Success in the goals detailed by the PI should yield mechanistic understanding and biogeochemical significance of the symbioses. We will rapidly communicate our findings in a wide circulation journal as well as through talks at appropriate conferences.

Broader Impacts: This research project is aimed at gaining insights on fundamental mechanisms underlying the roles of microbes in the functioning of marine ecosystems, biological carbon cycling and the ocean's role in global climate variability. Therefore, the result should also be relevant to pressing societal issues fisheries and climate change. The PI's lab is actively involved in public education from local to international levels, and the findings of the proposed research will be disseminated broadly to the public in appropriately accessible formats. One postdoctoral fellow and one graduate student will receive training during this research.

The ocean plays a critical role in sequestering CO2 by exporting fixed carbon to the deep ocean through the biological pump. There is a pressing need to understand the systematics of carbon export in the Southern Ocean in the context of global warming because of the sensitivity of this region to climate change, already manifested as significant temperature increases. Numerous studies have indicated that Fe supply is a primary control on phytoplankton biomass and productivity in the Southern Ocean. The results from previous cruises in Feb-Mar 2004 and Jul-Aug 2006 have revealed the major natural Fe fertilization from Fe-rich shelf waters to the Fe-limited high nutrient low chlorophyll (HNLC) Antarctic Circumpolar Current Surface Water (ASW) in the southern Drake Passage, producing a series of phytoplankton blooms. Remaining questions include: How is natural Fe transported to the euphotic zone through small-meso-large scale horizontal-vertical transport and mixing in different HNLC ACC areas? How does plankton community structure evolve in response to a natural Fe addition, how does Fe speciation respond to biogeochemical processes, and how is Fe recycled to determine the longevity of phytoplankton blooms? How does the export of POC evolve as a function of upwelling-mixing, Fe addition-recycling and bacteria-plankton structure? This synthesis proposal will address these fundamental questions using a unique dataset combining multiyear physical, Fe and biogeochemical data collected between 2004 and 2006 from 2 NSF-funded Fe fertilization experiment cruises and 3 Antarctic Marine Living Resource (AMLR) cruises in the southern Drake Passage and southwestern Scotia Sea through collaboration with scientists in the AMLR program and US Southern Ocean GLOBEC projects. All investigators involved in this study are engaged in graduate and undergraduate instruction, and mentoring of postdoctoral researchers. Each P.I. will incorporate key elements of the proposed syntheses in our lectures, problem sets and group projects. The project includes support to convene a 4-5 day international workshop on natural Fe fertilization at Woods Hole Oceanographic Institution. The workshop will include scientists from United Kingdom, France and Germany who have conducted natural Fe fertilization experiments, and Korea and China who are planning to conduct natural Fe fertilization experiments. The participation of graduate students and postdoctoral scholars will be especially encouraged. The results will be published in a Deep-Sea Research II special issue.

Intellectual Merit: An ever-growing variety of diseases, including some demonstrably caused by bacteria, are decimating reef-building corals in many places around the globe. Corals are open systems persistently exposed to a great diversity of exogenous bacteria in the overlying seawater and attached to sinking particles. Furthermore, as coastal development continues to expand, increasing eutrophication, there is increasing concern that endogenous bacteria among the normal coral associated community could proliferate to become opportunistic pathogens causing tissue damage and coral death. How corals resist bacterial invasions is a complex issue, one critical aspect being the ability of endogenous bacteria to resist colonization and proliferation of potential pathogens.

Despite great strides in recent years uncovering the remarkable genetic diversity of coral-associated bacteria, mitigation and management of coral diseases remains hampered by a lack of understanding of in situ ecological interactions within the microbial community and with exogenous bacteria that underlie coral health and disease. Bacterial attachment and proliferation are critical steps in the infection process, thus inhibiting or preventing colonization and proliferation of pathogens is fundamental to disease resistance.

Recent research by the investigator demonstrates that corals exposed to organic matter enrichment can become colonized by potential pathogens, but the communities can rebound from such perturbations. Conducting in vitro studies, the investigator found that bacteria-bacteria antagonism is common among coral isolates which suggests it may be a mechanism to resist community shifts and pathogen colonization. It could thus drive resilience within coral-associated microbial communities. In this project the investigator will test these interactions in an in situ context.

Microbes are critical for the functioning of coral ecosystems at the global scale and, therefore, it is essential that a mechanistic understanding of microbial interactions at the microscale be attained. The following hypotheses will be tested which are designed to elucidate the ecological mechanisms by which bacterial colonization and proliferation in the coral mucus layer (CML) are prevented by microbial interactions: 1) Bacterial community homeostasis in the CML is maintained through the exclusion of potential colonizers 2) Microspatial organic matter hotspots within the coral mucus layer, particularly surrounding zooxanthallae, create loci of intense growth and antagonism 3) Spatially dispersed organic matter inputs overwhelm microscale hotspots and enable pathogens to colonize the CML

Broader Impacts: This project simultaneously improves our fundamental understanding of coral associated microbial ecology and provides a necessary scientific basis for marine resource management decisions. Public education, at both the local and international levels, is a key component of the routine activities of all participating researchers, and these activities will prosper from inclusion of cutting-edge ecological findings generated by this research. Additionally, three postdoctoral researchers (two full time and one at two months per year) and one graduate student will receive training from the integrated research and education activities supported through this renewal.

It is critical that we develop tools for improving our understanding of the impacts of natural systems on the climate system as the Earth undergoes unprecedented change. It is difficult to determine the impact of the oceans on clouds and climate in field studies due to complexities added in by human pollution, even out over the oceans. The PIs will development of a state-of-the-art environmental simulator that will allow studies of ocean impacts on clouds and climate in the presence and absence of human based pollution. The Scripps Ocean-Atmosphere Research Simulator (SOARS), will provide wind, waves, atmospheric, biological, and thermal controls capable of simulating real-world conditions in a laboratory scale simulator. SOARS will simulate the complex and interacting physical, chemical and biological components of the marine atmosphere boundary layer (MABL) driven by wind, waves, and microbial processes under varying scenarios of temperature, from tropical to polar, atmospheric gas phase concentrations, and ocean pH. An integrated, temperature-controlled smog chamber will allow unique studies of ocean-atmosphere exchange and atmospheric reaction studies. SOARS will be the only instrument in the world capable of studying the current and future states of the ocean/atmosphere system thus uniquely capable of simulating Earth?s rapidly changing ocean-atmosphere system. The ability to simulate biological, physical, and photochemical processes in a controlled laboratory setting will enable interdisciplinary studies at an unprecedented level. SOARS will be housed at the Hydraulics Laboratory (HLab) at Scripps Institution of Oceanography (SIO). The HLab has been a focal point for oceanic and atmospheric research for more than 50 years, providing experimental facilities for national and international scientists, including UCSD researchers and students. SOARS will support new science by enabling interdisciplinary teams of scientists to collaborate on quantifying ocean-atmosphere exchange and reaction processes. It will serve as a test bed for the next generation of instrumentation thus improving at-sea measurement techniques to address significant unknowns relevant to global change. SOARS will serve as a training tool for the next generation of interdisciplinary scientists cross-trained in marine biology, climate, atmospheric sciences, oceanography, and engineering. In addition, SOARS will facilitate classical fluid dynamical and engineering studies, as well as STEM education through practical demonstrations for UCSD classes on fluid mechanics that the soon-to-be decommissioned Wind Wave channel in the Hydraulics Laboratory has traditionally supported. The HLab and its affiliated wave channels have become popular for outreach activities at SIO (including artists, film makers, news media and on-site educational visits) and supports UCSD?s commitment to diversity. SOARS will leverage diversity programs promoted at SIO and by the Center for Aerosol Impacts on Climate and the Environment Center (CAICE), a major future SOARS user. Both SIO and CAICE actively seek opportunities to engage diverse audiences in Earth systems science, and SIO has recently appointed two diversity officers to increase broader participation in the STEM fields by inclusiveness in research, education, and outreach.

The motivation for SOARS lies in the critical role the marine atmosphere boundary layer (MABL) plays in weather, atmospheric chemistry, climate change, national security interests, and offshore civil engineering. SOARS will play a critical role in basic and applied research in these arenas as well as in educating and training the next generation of scientists and engineers working in these fields. The presence of biological, chemical and physical feedbacks on exchanges through the MABL makes the creation of controllable laboratory simulators essential to the understanding of these complex processes. SOARS will enable study of the full complexity of ocean-atmosphere exchange processes through interdisciplinary studies by chemists, biologists and physical oceanographers. Not only is SOARS designed for experiments using wind, waves and biology in natural seawater, with a controllable atmosphere, simulating tropical through polar conditions, but the conditions will also be customizable allowing experiments to unravel natural impacts on clouds and climate, as well as futuristic simulations of increasing CO2 levels and changes in ecosystems. Such studies will allow one to unravel impacts of human versus natural processes on our climate at a unique level.