Aluwihare Lab
  Chemistry and Biology of Oceanic Organic Matter
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Ongoing Research Areas

Organic Nitrogen Cycling in Surface Waters of the Oligotrophic Ocean
In many areas of the World’s ocean, dissolved organic nitrogen is the most abundant form of nitrogen (excluding N2) and may be an important N source for both primary and secondary producers. This is particularly true in the oligotrophic ocean where the supply rate of inorganic nitrogen from deeper waters to the surface ocean is low. I am interested in identifying the biological and physical processes that control the chemical structure and inventory of dissolved organic nitrogen in these surface waters.

Travis Meador (graduate student) and I are currently studying how N2-fixers such as Trichodesmium sp., influence the composition and cycling of the DON pool. Using a combination of chemical (identifying and isolating proteins and amino sugars) and isotopic (measuring the δ15N signature of various organic pools) characterization methods, we have been tracking the dissolved nitrogen produced by Trichodesmium in the field. Thanks to Doug Capone (USC; who was funded by NSF’s Biocomplexity program) we were able to collect a large data set from the Atlantic and Pacific Ocean. Our preliminary data suggest that large amounts of Trichodesmium derived dissolved nitrogen do not accumulate in the surface ocean; but appears to be transferred to other organisms in the surface ocean through a tightly coupled uptake mechanism. We! have been focusing on the heterotrophic bacterial uptake of Trichodesmium derived N. Now, using a variety of biomarkers, we are beginning to investigate the transfer of fixed-N2 into other autotrophic organisms such as Prochlorococcus and Synechococcus.

These preliminary results have led to many other interesting avenues of research such as delineating the pathways of nitrogen isotope fractionation during biosynthesis in prokaryotes and eukaryotes, factors controlling bacterial and phytoplankton community structure in the oligotrophic ocean, factors controlling the nitrogen isotope composition of DON, the composition and sources of proteins in DON, DON turnover rates in the oligotrophic ocean and relationships between increased upper ocean stratification and DON/DOC composition and cycling.

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Chemical Structure-Residence time Relationships of Marine DOM Dissolved organic matter has several intriguing qualities. One that I find particularly fascinating is its old radiocarbon "age" or long residence time in seawater. DOC spends, on average, 6000 years in the ocean before being removed. Due to its long residence time relative to ocean circulation, this old DOC component is well mixed through out the water column and so, exists in surface waters as well. Although the age of DOC in the deep ocean is constrained (Willams and Druffel, 1987; Druffel et al., 1992), we don’t know the identity of these old (or new, for that matter) components within DOC, or how they were produced and where they go. The production of these "old" compounds represents a potentially important pathway for biological carbon sequestration and so the mechanisms of formation need to be identified.

We are working on many aspects of this problem, but we are currently focusing on addressing the relationship between the chemical structure of organic compounds and their residence time in seawater. For example, do certain organic compounds accumulate in the ocean because their chemical structure makes them particularly unattractive to heterotrophic organisms? Roman De Jesus (graduate student) and I are working on developing new methods to isolate chemically distinct fractions of DOM for radiocarbon measurements. We have successfully measured the 14C content of over 70 samples (at LLNL-CAMS, in collaboration with Michaele Kashgarian) that can be classified into 5 distinct chemical classes. We are also working on isolating individual compounds (from these 5 classes) for radiocarbon measurements. Our preliminary results suggest that there is a distinct relationship between chemical composition and residence time in seawater. These results compliment some earlier work that I did with Dan Repeta (WHOI) (submitted) where we measured the radiocarbon content of sugars isolated from DOM.

This research has raised several interesting questions regarding the source and cycling of "old" compounds and has identified some unique chemical compounds within surface and deep ocean DOM. (This work was funded by ONR and LLNL).

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Carbon Sources Fueling Prokaryotic Production in Subsurface Waters The old radiocarbon ages for DOC discussed in the previous section also raises some interesting questions regarding the various sinks for DOC in the ocean. For example, which mechanisms are ultimately responsible for removing "old" compounds from seawater? In surface waters, a large fraction of the prokaryotes are heterotrophic and DOC likely fuels their growth and activity. In deeper waters, DOC concentrations are low and the average age of the DOC is remarkably old, yet free-living prokaryotes are ubiquitous (~104 cells/mL). How do these organisms eek out a living in the dark ocean? Some preliminary data suggest that "recently" produced (in surface waters) DOC compounds are present even in deep waters (Aluwihare et al., 2002; Repeta and Aluwihare, submitted) perhaps released from sinking particles. Others have suggested that Archaea, which can make up as much as 60% of all DAPI staining cells in the meso- and bathypelagic ocean, may be chemoautotrophic (Pearson et al., 2001; Wuchter et al., 2003).

We have recently developed a method to extract large quantities of DNA from high-capacity filters and Roberta Hansman (graduate student) and I have used this method to isolate and radiocarbon date the DNA from bacteria living both in surface and subsurface waters. Radiocarbon dating is particularly useful because many of the carbon sources in the deep ocean have distinct residence times and therefore, radiocarbon ages. Since the radiocarbon content of bacterial DNA directly reflects the Δ14C signature of the carbon source utilized by prokaryotes, this measurement allows us to identify the primary carbon source to prokaryotes in the deep ocean. Our preliminary results are very intriguing and suggest that fresh carbon (recently synthesized in surface waters) plays only a small role in sustaining these deep-ocean prokaryotic populations. We are currently in the process of establishing the importance of chemoautotrophy (in collaboration with Ann Pearson, Harvard) and the role of "old" DOC compounds in sustaining heterotrophic production.

As part of this research we have begun to delve into the dark depths of molecular biology and microbial ecology. In addition to quantifying both the Archaeal and bacterial populations with methods such as FISH, we have also been looking at community composition and diversity. We are planning to develop some functional gene based PCR methods to probe nitrogen and carbon metabolism in these prokaryotes. In addition to studying the prokaryotic metabolism in the deep, open ocean, we are also beginning to look at community composition and carbon sources that fuel prokaryotic production in areas of the water column that overly methane seeps/vents. Our radiocarbon measurements are made in conjunction with the very generous Ellen Druffel at the UCI, Keck AMS Center. (Part of this work was funded by NSF-OCE)

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Chemical Composition of Unreactive Dissolved Organic Nitrogen Although the C/N ratio of DOM (between 14-22) is lower than that of POC (between 7-10), the ocean contains a large reservoir of organic nitrogen. Over 90% of this DON is stored in the deep ocean. The fact that a fraction of the biologically produced organic nitrogen escapes degradation and accumulates in the well oxygenated deep ocean, on long timescales, is intriguing; particularly because we expect N and P to be recycled efficiently within the water column. The composition of DON in the deep ocean has remained fairly elusive but new evidence suggests that this DON is distinct from much of the DON that is added in the surface ocean (Aluwihare et al., 2005). We are currently in the process of developing new methods to characterize and quantify DON compounds in the deep ocean. For many years, we have relied on hydrolysis methods to generate monomers such as amino acids and monosaccharides from DOM, which we can then quantify to mass-balance the marine organic c! arbon and nitrogen inventory. However, we have good evidence suggesting that acid hydrolysis is not very efficient at de-polymerizing DOM. Analytical techniques that can quantify proteins and polysaccharides directly, are likely to be more successful. These techniques include gel electrophoresis, immunochemical methods, fluorescent probes, and MALDI based mass spectrometry methods. Developing such analytical techniques is something of a holy grail for me and I work on these techniques in my very abundant spare time.

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Organic-Inorganic Associations Within Marine Gels and Particulate Organic Matter (POM) Recently, several studies have demonstrated the importance of the phase transition between DOM to POM (Chin et al., (1998); Kerner et al., (2003); Engel et al., (2004)) as a sink for DOM. These studies identify particular types of organic compounds that are involved in the phase transitions, and the Chin study in particular, implicates the importance of inorganic ions in mediating this phase transition. In unrelated work, Hedges et al., (2001), among others, have alluded to the importance of mineral-organic associations in protecting organic compounds in POM, from bacterial attack. This work has led me to ponder about the importance of interactions between certain types of organic and inorganic moieties in seawater.

I have been lucky to work with Hiroshi Furutani and Kim Prather (UCSD) using their innovative mass spectrometer - a single particle, laser desorption/ionization (LDI) time of flight (TOF) mass spectrometer (ATOFMS). This instrument allows one to measure the inorganic and basic organic composition of individual particles, and at the same time, obtain a general idea of the aerodynamic size of these individual particles. Using this technique, Hiroshi and I have been looking at the inorganic-organic associations between individual particles in seawater and gels formed from phytoplankton cultures. We have been able to identify the general characteristics of gels formed from the DOM produced in diatom cultures. In addition, Hiroshi has obtained some interesting data on the composition of various phytoplankton and we have been using this to see whether we can differentiate certain types of organisms in a natural assemblage.

As part of this collaboration, we are also studying the importance of marine particles emitted from the ocean into the atmosphere, the factors that control the amount and composition of these emissions and also, correlations between atmospheric "particle events" (such dust depositions) and marine biological responses. Through this collaboration, Hiroshi and Kim launched the first ever TOF-MS CalCOFI voyage in November, 2004.

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Chemical Composition and Photoreactivity of colored DOM (CDOM) In collaboration with Kathy Barbeau (SIO), I have also been studying the importance of photodegradation as a sink for DOM in surface waters. In addition, we have also been investigating the role of iron in the photochemistry of particular fractions of DOM. Of particular interest to me is the importance of photodegradation as a sink for refractory DOC. The mass balance of DOC radiocarbon in the ocean requires that a small fraction of "old" DOC is removed from the upper ocean during ocean-mixing, and Mopper et al., (1991) have demonstrated the importance of photodegradation as a sink for a significant fraction of DOC. My interest lies in identifying the fraction (and in particular, which compounds) of DOM that is most susceptible to photodegradation. In addition, we would like to identify the mechanisms by which this DOM photoreacts and how these organic compounds are first introduced into the marine system. To this end, we ha! ve been investigating the photoreactivity of both size- and chemically- fractionated marine DOM from various sites in the Pacific Ocean. We find a distinct relationship between size, chemistry and changes in UV-Vis absorbance as a result of irradiation. Our data also suggests that binding of iron by certain fractions of DOM might control the extent of photodegradation. We are currently in the process of identifying irradiation-induced structural changes in the DOC pool and the role of iron-binding organic compounds. Julian Herszage, who was a joint post-doc in the Barbeau and Aluwihare labs, performed much of the preliminary research on this project. (This work was funded by ONR).

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DOC and DON Cycling in the Southern California Bight (SCB) Recently, many studies have suggested that the warming of surface North Pacific Ocean waters and increased upper ocean stratification may be responsible for observed changes in community composition (e.g. Venrick et al., 1987; Fields et al., submitted), organic nutrient inventories (e.g. Karl et al., 2002; Church et al., 2002) and carbon export to the deep ocean (Ruhl and Smith, 2004). The region of the California Current that is surveyed by CalCOFI offers an excellent context in which to study the response of ecosystems and upper ocean nutrient inventories to climate forcing on several timescales (e.g. El Nino, PDO etc). As a result, the southern section of the California Current was recently made an LTER site (CCE-LTER). As part of this larger study, I am interested in assessing the inventory of DOC and DON within the Southern California Bight. In addition to quantifying the inventory, Roman DeJesus and I are investigating the mechanisms that control temporal (seasonal) and spatial (horizontal and vertical) variations in both DOC and DON concentration in this region of the Pacific Ocean.

In addition, together with Ralf Goericke, we are hoping to assess the relationship between in-situ CDOM fluorescence and DOC/DON concentrations in these waters. Our ultimate goal is to determine whether features in both the DOC and fluorescence profile are controlled by common mechanisms.

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Nitrate availability and Nitrate Utilization Within the California Current based on nitrogen isotopes in suspended POM The wind driven upwelling of subsurface waters in the northeastern section (off Point Conception) of the CalCOFI grid is a major source of nitrate to the CalCOFI system. However, even in the spring, surface nitrate concentrations (along the northern most transect) rarely exceed 5μM N. Over the last several years we have been collecting suspended POM samples from stations throughout the CalCOFI grid for stable carbon and nitrogen isotope measurements (at the SIO ULF facility) in the hopes of identifying nitrogen sources to, and extent of nitrate utilization by phytoplankton in this region. We have recently begun to analyze some of these samples and we find a wide range of δ15N values, some significantly higher than the deep ocean average. We are hoping to use these data to generate a spatial distribution of the extent of nitrate utilization in the SCB.

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