Applications

Plankton

Plankton is commonly assumed to have a C:N:P stoichiometry of 106 C:16 N: 1 P - referred to as the 'Redfield Ratio'. This ratio is an emergent property of marine biogeochemistry that represents an integrated response of the ocean over time scales of thousands of years (Falkowski, 1997). It has long been recognised that marked deviations from this stoichiometry may be found between identifiable water masses (Copin-Montegut & Copin-Montegut, 1983), amongst individual plankton samples (Redfield, 1934), or between phytoplankton species grown in unialgal or axenic cultures (Geider & La Roche, 2002). Although developed for the plankton, the Redfield ratio has been applied to marine biogeochemistry in general, including microalgal growth in benthic systems. Although limnologists have long recognised the marked variability in the elemental stoichiometry of the biota and non-living particulate matter in lakes and rivers (Elser et al., 2000), oceanographers have been more conservative regarding the potential importance of variability in C:N:P stoichiometry. For the plankton, deviation of the C:N:P stoichiometry from the Redfield ratio has been proposed as an index of the degree of nutrient limitation of algal growth rate (Goldman et al., 1979).

Resource-ratio theory was developed to describe and explain how variability in nutrient supply influences the size and composition of ecological communities (Curtis et al., 2003). Recently, a change in C:N:P of particulate matter has been used to suggest an ecosystem shift from N-limitation to P-limitation in the subtropical North Pacific (Karl et al., 1995). However, bulk C:N:P of particulate matter is a very crude index of the elemental stoichiometry of the microbial community and more taxonomic resolution is necessary. For example, Trichodesmium, which can be physically isolated from the rest of the particulate matter, is characterised by N:P = 40; considerably higher than the N:P of particulate matter.
Recently, Quigg et al. (2003) concluded that the C:P, C:N and N:P ratios varied systematically between marine algal phyla. Mean C:P ranged from about 200 mol C:mol P in the prasinophytes and chlorophytes to about 70 in the diatoms and prymnesiophytes. Mean C:N ranged from about 11 mol C: mol N in the dinoflagellates to about 6 in the chlorophytes. Mean N:P ranged from about 30 mol N:mol P in the chlorophytes to about 10 in the diatoms and prymnesiophyes. To this data set for the eukaryotic phytoplankton can be added observations for the cyanobacteria, Synechococcus and Prochlorococcus (Bertilsson et al., 2003). Moreover, between nutrient-replete and nutrient-limiting conditions, phytoplankton and bacterioplankton have the potential to show up to 100 fold variability in C:P, and about 5 fold variability in N:P (Geider & La Roche, 2002; Makino et al., 2003). Zooplankton also show pronounced variability in C:P and N:P ratios (Pertola et al., 2002; Elser et al., 2003). In particular, C:P and N:P increase as growth rate declines both with age (developmental stage) within a taxon and with the intrinsic maximum growth rate between taxa (Elser et al., 2003).

Some groups of phytoplankton and zooplankton display characteristic mineral phases. These include the coccolithophores and foraminifera, which precipitate CaCO3, and the diatoms and radiolaria, which precipitate opal (SiO2). The ratios of these inorganic phases to organic carbon vary amongst species, with some species more heavily calcified or silicified than others. In addition, variability in the coupling of opal and CaCO3 precipitation to particulate organic matter production may occur in response to nutrient limitation (Hutchins et al., 1998; Paasche, 1998). These mineral phases also include 'impurities' such as cadmium, which carry information about the nutrient status of the water masses in which the plankton grow. For example, the Cd:Ca ratio of foraminifera has been used to reconstruct changes in phosphate levels since the last glacial maximum (Elderfield & Rickaby, 2000).

One of the fundamental problems facing application of resource-ratio theory to microbial communities is our limited ability to characterise the elemental composition of individual micro-organisms. X-ray transmission electron microscopy (TEM) microanalysis can be used to obtain the elemental composition of individual cells as small as the picoplankton Prochlorococcus and Synechococcus in laboratory cultures (Heldal et al. 2003). X-ray TEM microanalysis has also been applied to Emiliania huxleyi in natural assemblages (Fagerbakke et al., 1994). However, application of this technique to natural assemblages is limited by the ability to identify particular taxa using TEM. Differentiating heterotrophic from autotrophic bacteria is a particular problem, but there are also difficulties in differentiating between heterotrophic and autotrophic nanoflagellates. In addition, sample preparation for TEM may destroy spatial structure, which can be preserved in wet mounts. The development of a microscope-based LIBS for examining elemental composition of microalgal cells is a timely technological advance. It will have immediate application in addressing key questions regarding the role of phytoplankton and zooplankton taxa that play key functional roles (such as N₂ fixation and CaCO₃ precipitation) in upper ocean nutrient (N, P, Fe) cycling and biogeochemistry. In addition, by combining Chlorophyll a fluorescence measurements (F_v/F_m and F_q'/F_m') with LIBS, we will be able to examine how photosynthetic performance is related to elemental composition.

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Microphytobenthos

Microphytobenthic algae (MPB) live in sediments with steep, and temporally varying, gradients of dissolved inorganic and organic nutrients, pH and redox potential. There has been much discussion of the degree to which cells living within this matrix are nutrient limited, and nutrient limitation does have consequences to how MPB respond to environmental stresses, for example UVB (Wulff et al., 2000). There is almost no data on the elemental ratios of benthic diatoms, though other ratios, e.g. C: Chl a, show great variability (de Jonge 1980). This has prevented any rigorous understanding of nutrient limitation in situ. One hypothesis that has gained credibility in recent years is that MPB in muddy systems are not nutrient limited, and that cells micro-migrate within the surface layers of biofilms, obtaining nutrients (including CO2) at greater depths, enabling them to photosynthesise at their maximum rate at the sediments surface (Kromkamp et al., 1998, Underwood & Kromkamp, 1999). In contrast to this is the measurement of high rates of production of extracellular carbohydrates in biofilms, attributed to unbalanced growth and release of excess photoassimilates (Staats et al., 2000; de Brouwer & Stal, 2001, Underwood & Paterson 2003). It is known that benthic diatoms in culture produce extracellular carbohydrates of different composition depending on environmental conditions (Underwood & Paterson, 2003) and the production of extracellular carbohydrates plays an important role in the physical structuring and the carbon dynamics of these sediment microbial systems.  To fully understand these processes in natural system requires a technique to determine the nutrient status of cells.
We have already shown that high-resolution imaging can determine the photosynthetic efficiency of different taxa of microphytobenthos within intact biofilms (Oxborough et al. 2000, Perkins et al. 2002). With the development of this system, we aim to answer the following questions:

1. How does elemental composition vary between cells at depth, newly arriving at the sediments surface and present on the surface and what are the rates of change over diel exposure cycles?
2. Is there evidence of nutrient limitation at the micro level within these 3 dimensional habitats?
3. How variable is the elemental composition of EPS produced by diatoms under different conditions within biofilms and how does this relate to photosynthetic performance?

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