Download Exploring Ocean Biogeochemistry by Single

J. Eukaryot. Microbiol., 55(3), 2008 pp. 151–162
r 2008 The Author(s)
Journal compilation r 2008 by the International Society of Protistologists
DOI: 10.1111/j.1550-7408.2008.00320.x
Exploring Ocean Biogeochemistry by Single-Cell Microprobe Analysis of
Protist Elemental Composition1
BENJAMIN S. TWINING,a STEPHEN B. BAINES,b STEFAN VOGTc and MARTIN D. de JONGEc
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina, and
b
Department of Ecology and Evolution, Stony Brook University, Stony Brook, New York, and
c
X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois
a
ABSTRACT. The biogeochemical cycles of many elements in the ocean are linked by their simultaneous incorporation into protists. In
order to understand these elemental interactions and their implications for global biogeochemical cycles, accurate measures of cellular
element stoichiometries are needed. Bulk analysis of size-fractionated particulate material obscures the unique biogeochemical roles of
different functional groups such as diatoms, calcifying protists, and diazotrophs. Elemental analysis of individual protist cells can be
performed using electron, proton, and synchrotron X-ray microprobes. Here we review the capabilities and limitations of each approach
and the application of these advanced techniques to cells collected from natural communities. Particular attention is paid to recent studies
of plankton biogeochemistry in low-iron waters of the Southern Ocean. Single-cell analyses have revealed significant inter-taxa differences in phosphorus, iron, and nickel quotas. Differences in the response of autotrophs and heterotrophs to iron fertilization were also
observed. Two-dimensional sub-cellular mapping indicates the importance of iron to photosynthetic machinery and of zinc to nuclear
organelles. Observed changes in diatom silicification and cytoplasm content following iron fertilization modify our understanding of the
relationship between iron availability and silicification. These examples demonstrate the advantages of studying ocean biogeochemistry at
the level of individual cells.
Key Words. Iron fertilization, phytoplankton, plankton, Southern Ocean, synchrotron X-ray fluorescence.
THE BIOGEOCHEMISTRY OF PLANKTONIC PROTISTS
T
HE field of biogeochemistry concerns, in part, the impact of
biological processes on the chemical composition of the environment. Many critical transformations of biologically active
elements in natural ecosystems are mediated by protists.
Autotrophic protists are often responsible for entry of elements
into food webs. As consumers, mixotrophic and heterotrophic
protists play a key role in returning elements to the abiotic world.
Our knowledge of the mechanisms driving these transformations
is poised to rapidly expand in the near future as a molecular level
understanding of cellular physiology, biochemical composition,
and gene expression develops. Such an understanding will be
needed if we are to anticipate the consequences of anthropogenic
alterations to global geochemical cycles. Many of the changes
already underway will in turn impact protist biology and ecology
in the future in ways that may depend on their elemental
requirements.
The importance of protists to global biogeochemistry is perhaps
most obvious in the sea. Covering 70% of the Earth’s surface, the
oceans play numerous critical roles in global biogeochemistry and
climate. The oceans buffer temperature fluctuations, provide an
important food supply for maritime countries, and mediate changes in the global C cycle. The oceans have absorbed approximately
120 petagrams (1 Pg 5 1015 g) of anthropogenic carbon dioxide in
the past two centuries, about half of the total amount released by
human activities during this time (Sabine et al. 2004). Carbon in
surface waters is ‘‘pumped’’ to deep waters and the sea floor via
the sinking of planktonic organisms. Through this ‘‘biological
pump’’, phytoplankton play a major role in the global C cycle
(Longhurst 1991; Sarmiento and Gruber 2006). Calcifying protists
Corresponding Author: B. S. Twining, Department of Chemistry and
Biochemistry, University of South Carolina, Columbia, South Carolina—Telephone number: 11 803 777 7765; FAX number: 11 803 777
9521; e-mail: [email protected]
1
Invited presentation delivered during the joint annual meeting of the
Phycological Society of America and the International Society of Protistologists, Providence, Rhode Island, August 5–9, 2007.
such as coccolithophores and foraminifera further impact the
ocean C cycle through the formation and export of external shells
composed of CaCO3 (Langer, this issue). Ocean sediments in
some regions are described as calcareous ‘‘oozes’’ because of the
abundance of calcareous shells of protists. The growth and metabolism of marine phytoplankton may also impact global climate
through the production of dimethylsulphide (DMS). Certain
phytoplankton species produce DMS and its precursor dimethylsulphoniopropionate (DMSP) for osmonic regulation and antioxidant protection in the cell (Sunda et al. 2002). Phytoplankton
DMS can diffuse into the overlying atmosphere and may serve as
a significant source of non-sea salt sulfate aerosols and cloud
condensation nuclei (Charlson et al. 1987). This process may impact global climate through the enhancement of cloud albedo
(Bates, Charlson, and Gammon 1987).
Marine protists also control the biogeochemical cycling of
many elements in the ocean besides C. The nutrient elements N,
P, Si, Fe, Mn, Ni, Cu, and Zn are accumulated by phytoplankton in
surface waters and exported to depth as sinking biogenic particles.
This process results in the surface-depleted ‘‘nutrient profile’’
characteristic of many bioactive elements (Donat and Bruland
1995). Protistan metabolic activities can also alter the chemical
form of many elements. Eukaryotic phytoplankton reduce nitrate
to ammonia upon uptake, convert silicic acid to opaline shells, and
modify the chemical speciation and reactivity of dissolved metals
(e.g. Coale and Bruland 1988; Rue and Bruland 1995). Grazing
processes can further change the physico-chemical speciation of
metals and non-metals (Hutchins and Bruland 1995; Sato, Takeda,
and Furuya 2007).
Numerous approaches have been taken to study the biogeochemical interactions between ocean protists and their marine environment. Incubations with isotopically labeled compounds have
revealed differences in the relative availability of different nutrient substrates to ambient phytoplankton populations (Dugdale and
Goering 1967; Hutchins et al. 1999; McCarthy 1972), enzyme
activities have been measured as markers of nutrient utilization
and limitation (Glibert et al. 2004; Webb, Moffett, and Waterbury
2001), and gene expression has been used to determine nutrient
cycling by specific phenotypes in field assemblages (Church et al.
2005). Comparisons of nutrient stoichiometries have also been
critical to our understanding of ocean biogeochemistry. Alfred
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J. EUKARYOT. MICROBIOL., 55, NO. 3, MAY–JUNE 2008
Redfield famously noted similarities in the stoichiometries of N
and P in bulk surface plankton and dissolved nutrients at depth
(Redfield 1934; Redfield 1958), and this approach has since been
applied to a number of other bioactive elements, including trace
metals (e.g. Löscher 1999; Sunda 1997).
THE NEED FOR A SINGLE-CELL APPROACH
The biogeochemical cycles of bioactive elements are linked by
their simultaneous incorporation into protists. Therefore, in order
to understand these elemental interactions, accurate measures of
cellular element stoichiometries are needed. These are typically
obtained for field communities using bulk size-fractionation (Collier and Edmond 1984; Copin-Montegut and Copin-Montegut
1983; Martin and Knauer 1973). However, this macroscopic approach obscures significant internal processes and limits our ability to link ecology and biogeochemistry. Protists play unique
ecological roles in pelagic plankton communities. A simplified
model of such a system is shown in Fig. 1. Photoautotrophs form
the base of the food web and provide organic substrates to heterotrophic bacteria, protists, and mesozooplankton, as well as
mixotrophic protists (Caron 2000). Photoautotrophs occur in each
of the traditional plankton size classes (0.2–2, 2–20, 20–200 mm).
Heterotrophic nanoflagellates, dinoflagellates, and ciliates consume autotrophs and bacteria, moving carbon through the food
web and also contributing dissolved organic compounds for use
by bacteria and dissolved inorganic and organic nutrients for use
by autotrophs. These trophic relationships are organized by size in
Fig. 1, although it is unlikely that predation and grazing occur
only between these clear size delineations. Each size class contains both autotrophs and heterotrophs (and likely mixotrophs),
and bulk elemental analyses performed on all cells collected on
0.2-, 2-, and 20-mm membranes would obscure differences between ecological functional groups in the same size range. Because of the presence of abiotic particulate material and the
sorption of dissolved and colloidal metals to filters, bulk chemi-
cal analyses often cannot be used to estimate the average cellular
elemental stoichiometries for the entire protistan community.
Even in open-ocean high-nutrient low chlorophyll (HNLC) areas,
biogenic iron may only account for a small fraction of total particulate iron (Strzepek et al. 2005). Further complicating matters,
delicate cells may burst upon contact with the filter membrane
under vacuum.
Diverse pelagic protists from the same size class can perform
unique biogeochemical roles. Eukaryotic autotrophs such as coccolithophores and diatoms produce mineral shells composed of
calcite and silicate, respectively, that provide relatively dense
‘‘ballast’’ to the cells. It is thought that these taxa are more likely to sink from surface waters and contribute to carbon export than
non-ballasted autotrophs (Armstrong et al. 2002; Klaas and Archer 2002). Coccolithophores and diatoms will also impact carbon
and silicon geochemical cycles differently than non-mineralized
taxa by removing silicon and additional carbon from surface waters and reducing alkalinity. Given their different biogeochemical
roles, it is desirable to separate ballasted and non-ballasted cells
for elemental analysis. Diazotrophic (N-fixing) organisms such as
Trichodesmium and Crocosphaera are also present in the same
size classes (and in the case of the endosymbiont Richellia, in the
same organism) as non-diazotrophs, but they clearly play a unique
and very important role in nitrogen biogeochemistry. Given the
high iron requirements for nitrogenase (Kustka et al. 2003),
diazotrophs are also likely to impact iron biogeochemistry differently than other cells types (Berman-Frank et al. 2001; Falkowski
1997). Specific taxa can also impact sulfur geochemistry, because
prymnesiophytes such as Emilinia huxleyii and Phaeocystis sp.
produce far more intracellular DMS/DMSP than other algal species (Matrai and Keller 1994; Stefels and Vanboekel 1993). To
advance our understanding of biogeochemistry, we must collect
elemental information at the level of ecological and biogeochemical functional groups. That is, since taxonomy influences biogeochemistry, biogeochemical measurements should reflect
taxonomic differences.
Fig. 1. Description of common particulate material in pelagic waters, including a hypothetical food web demonstrating the diversity of ecological
and biogeochemical functional groups contained within common size classes. Picoplankton (o2 mm) are typically composed of heterotrophic and autotrophic prokaryotes, including Synechococcus, Prochlorococcus, and some pico-eukaryotes. Nanoplankton (2–20 mm) generally comprise the bulk of
the protist biomass. This size class includes heterotrophs, mixotrophs, and ‘‘naked’’ and ‘‘ballasted’’ autotrophs. Microplankton (420 mm) can include
large diatoms, dinoflagellates, foraminifera, radiolarians, and zooplankton nauplii.
TWINING ET AL.—SINGLE-CELL ELEMENT ANALYSIS OF PROTISTS
Recent modeling efforts have begun to recognize these varied
biogeochemical roles and have incorporated multiple plankton
functional groups. Moore, Doney, and Lindsay (2004) and Moore
et al. (2002) include three phytoplankton groups—small phytoplankton, diatoms, and diazotrophs—and assign unique parameters to each, including different nutrient uptake kinetics, grazing
mortality, and minimum cell quotas. Salihoglu and Hofmann
(2007) further separate the picoautotrophs into low light-adapted
Prochlorococcus, high light-adapted Prochlorococcus, and Synechococcus, in addition to autotrophic eukaryotes and diatoms.
Others have chosen to separate the behavior of herbivorous microzooplankton grazers and bactivorous heterotrophic nanoflagellates (Lancelot et al. 2000). In order to accurately parameterize
these models and ground truth the output, it is necessary to make
elemental measurements of each relevant group. Given the broadly overlapping sizes of the autotrophic and heterotrophic functional groups, group quotas cannot be resolved with standard bulk
chemical analysis techniques.
There is substantial evidence from laboratory culture work that
element quotas are dynamic and can vary significantly in protists
as a function of taxonomy, trophic function, and nutrient substrate. Much of the work has focused on the micronutrient iron.
Brand (1991) found the iron requirements of eukaryotes to be
significantly lower than those of prokaryotes. Minimum iron quotas (often normalized to cellular carbon or phosphorus) required
for growth have been shown to be 2- to 4-fold lower in oceanic
diatoms compared to coastal diatoms (Maldonado and Price 1996;
Sunda, Swift, and Huntsman 1991). It also appears that mixotrophic and heterotrophic phagotrophs require 2- to 3-fold more iron
than autotrophic protists (Chase and Price 1997; Maranger, Bird,
and Price 1998; Sunda et al. 1991). The cell quotas of other bioactive metals also vary between taxa. Diatoms contain less iron,
cobalt, copper, and molybdenum (normalized to P) than dinoflagellates grown in the same media (Ho et al. 2003), and selenium
concentrations in phytoplankton can vary by more than four orders of magnitude among species at ambient levels of selenite
(Baines and Fisher 2001). Furthermore, phytoplankton metal quotas are sensitive to the ambient nutrient environment. The additional iron required for nitrate reductase raises iron quotas of
diatoms grown on nitrate compared to those grown on ammonium
(Maldonado and Price 1996). Trichodesmium iron requirements
increase 5-fold during nitrogen fixation as a result of nitrogenase
synthesis (Kustka et al. 2003). Other trace nutrients such as manganese, copper, and nickel may also vary in cells in response to
iron status and nutrient utilization patterns (Peers and Price 2004;
Peers, Quesnel, and Price 2005; Price and Morel 1991). Furthermore, phytoplankton have the ability to accumulate and store
considerable amounts of trace metals in response to environmental concentrations (Sunda and Huntsman 1998a).
Further complicating the extrapolation of culture studies to the
field is the uncertainty surrounding bioavailability of Fe and other
trace elements in the field. In the sea, the dissolved fraction of Fe
and many other trace elements exists predominantly as complexes
with dissolved organic ligands (Coale and Bruland 1988; Ellwood
and Van den Berg 2000; Rue and Bruland 1995; Saito, Rocap, and
Moffett 2005). In the free-ion model, the ligand-bound metal is
assumed to be unavailable to cells (Sunda and Huntsman 1998a).
This assumption allows modelers to calculate Fe quotas using
simple Michaelis–Menten uptake kinetics, measured Fe concentrations, and presumed concentrations of Fe-binding ligands (e.g,
Salihoglu and Hofmann 2007). However, at least some Fe bound
to organic ligands in nature may be directly accessible to protists
(Maldonado and Price 1999; Maldonado et al. 2005). Alternatively, phagotrophic protists may sidestep the dissolved pool by directly ingesting colloidal Fe or bacteria (Chase and Price 1997;
Maranger et al. 1998). Iron is not the only element for which bio-
153
availability of dissolved fractions is open to question. Both nitrogen and selenium are accumulated not only as inorganic ion, but
also in the dissolved organic form (Baines et al. 2001; Bronk et al.
2007).
Given the plasticity of protist metal quotas and the uncertainty
regarding bioavailability of dissolved trace elements, it is problematic to assume that field quotas match those measured in laboratory cultures. Perhaps more importantly, valuable information
regarding phytoplankton physiology, biogeochemical cycling,
and ambient nutrient bioavailability can be gained from measurements of in situ protist cell quotas. The elemental content of individual protists cells can be assayed through one of several
microscope-based technologies (Table 1). Light microscopy can
be used in conjunction with fluorescent dyes to detect various
metal ions within cells (Kikuchi, Komatsu, and Nagano 2004;
Thomas et al. 1999; Yang et al. 2005). Such dyes are commercially available, utilize relatively common light and confocal microscopes, and have become widely used. However, only labile
forms of the metals that are available to bind with the probes are
detected, so absolute quantification of metal quotas is difficult.
Fluorescent probes are also prone to interferences from competing
metal ions, further complicating quantitation. More promising for
analytical work are microscopy techniques which base detection
on the characteristic radiation absorption and fluorescence properties of individual elements.
A REVIEW OF APPLICABLE MICROPROBE APPROACHES
Fluorescence X-rays are emitted when atomic electrons transition from outer shells to lower energy inner shell vacancies created by incident ionizing radiation. The fluorescence energies
associated with these transitions correspond to the difference between inner and outer shell electron binding energies, and are thus
unique to each element, making it possible to deduce the cellular
elemental composition from the X-ray fluorescence spectrum. For
example, for iron the predominant X-ray fluorescence emission
has an energy of 6.404 keV, corresponding to the transition of an
L-shell electron into a K-shell vacancy. The number of detected
X-ray photons for each element generally scales directly as a
function of atomic abundance, thus elemental content can be
quantified in a straightforward manner. The yield of fluorescence
photons is lowest for the lighter elements since the excess energy
of the relaxed electron for such elements is more likely to be carried away through ejection of a secondary emitted electron (Auger
electron) rather than via a fluorescent X-ray photon.
Characteristic X-rays can be generated by one of several forms
of higher energy ionizing radiation, and each technique has advantages and disadvantages (Table 1). Electron microprobes are
commercially available and fairly common, and electron beams
can be easily focused, although the high flux requirements to detect trace elements in biological samples limit the achievable spatial resolution in analytical mode to 20 nm typically. Electrons are
rapidly absorbed by light elements (Zo10) in biological samples,
typically limiting the depth of penetration to several micrometers
at best. More importantly, multiple inelastic scattering will cause
the electron beam to broaden rapidly, thus increasing the excited
volume in the sample significantly and lowering achievable spatial resolution (Ingram et al. 1999). To circumvent this problem
thin sections (o100 nm) are typically used, and larger protist targets may need to be embedded and sectioned prior to analysis.
Lower ionization cross-sections for high mass (Z420) transition
metals and significant Bremsstrahlung background combine to
limit the sensitivity of electron microprobe X-ray microanalysis
(XRMA) for the heavier bioactive metals (e.g. Mn, Fe, Cu, Zn).
Cellular elemental quantification and sub-cellular mapping can
also be formed by measuring energy loss of electrons as they pass
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J. EUKARYOT. MICROBIOL., 55, NO. 3, MAY–JUNE 2008
Table 1. Comparison of some available techniques for trace element mapping in protists.
Typ.
spacial
resolution
(nm)
Typ.
sample
thickness
(mm)
Resolution
limitation
Light microscope
200
30
Wavelength Requires use of fluorescent dyes
Cells generally need to be
hydrated
Analytical electron
microprobe (XRMA,
EPXMA)
20
0.1
Sample
thickness
EELS/EFTEM
2
0.005–0.05
Radiation
damage
Proton microprobe
1,000
50
Radiation
damage;
Flux
Samples typically need to
be dried
Synchrotron X-ray
microprobe
30–200
10
Optics
(currently)
Samples typically need to
be dried, or imaged frozenhydrated
Sample preparation
Thick samples need to be
sectioned
Samples typically
need to be dried, or imaged
frozen-hydrated
Requires ultrathin sections
Advantages (1)/disadvantages (
)
1 Changes in living cells can be monitored
1/ Can only detect ‘‘labile’’ ions in
solution and not total element content
Absolution quantification is difficult
1 Simultaneously detect 410 elements
Analyses are slow
Significant radiation damage
Only some elements are readily accessible
(e.g. P, Fe)
Co-localization can be difficult (EFTEM)
or slow (EELS)
1 Simultaneously detect420 elements
1 high sensitivity
Analyses are slow
Significant radiation damage
1 Very high sensitivity, low background,
selective excitation of analytes
1 Simultaneously detect410 elements
1m-XANES for chemical state mapping
Analyses are slow
XRMA, X-ray microanalysis; EPXMA, electron probe X-ray microanalysis; EELS, electron energy-loss spectroscopy; EFTEM, energy-filtered transmission
electron microscopy; PIXE, proton-induced X-ray emission; SXRF, synchrotron X-ray fluorescence; m-XANES, micro X-ray absorption near-edge structure.
through thin biological samples. Electron energy loss spectroscopy (EELS) measures the energy loss of electrons at a single
focused spot while the energy of the incident electron beam is
systematically shifted across the absorption edge for the element
of interest (Table 1). A closely related technique, energy-filtered
transmission electron microscopy (EFTEM), involves the collection of a series of images of the whole specimen at specific
energies spanning the absorption edge. The ‘‘stack’’ of hyperspectral images can then be processed to produce element maps of
extremely high spatial resolution (Leapman 2004). These techniques are also capable of remarkable elemental sensitivity; for
example EELS has been used to detect single atoms of Ca and Fe
in biomolecules mounted directly onto TEM grids (Leapman
2003). These techniques require samples o100 nm in thickness
to avoid multiple scattering interactions between the electrons and
the sample matrix, so their applicability to natural protist samples
is limited.
Protons may also be used to generate characteristic X-ray fluorescence in protist cells, a technique termed proton-induced
X-ray emission (PIXE). Although less common than analytical electron microprobes, nuclear (proton) microprobes are more readily
available and less expensive than synchrotron facilities. A a result
of their higher momentum, protons generate less Bremsstrahlung
background than electrons and have higher sensitivity for transition metals (Garman 1999). Lower absorption coefficients also
eliminate the need for sample sectioning for most protist samples.
Protons can be focused to only 1 mm at the required fluxes, and
larger beam sizes are commonly used to analyze marine samples
(Iwata et al. 2005; Pallon et al. 1999). Proton-induced X-ray
emission is therefore generally inappropriate for studies of subcellular element distributions. Radiation damage can also be significant. Proton-induced X-ray emission analyses can be supplemented with direct carbon and nitrogen measurements via proton
backscattering, and sample thickness can be quantified by measurement of proton absorption by the sample (scanning transmission ion microscopy) (Pallon et al. 1999).
Synchrotron-based X-ray fluorescence (SXRF) microprobes are
rapidly becoming a powerful tool for single-cell element analysis.
With low Bremsstrahlung background and high ionization crosssections for heavier elements, SXRF has the highest sensitivity for
transition metals (Sparks 1980). Improvements in Fresnel zoneplate optics are allowing spatial resolution to approach that of
XRMA (Wang, Yun, and Jacobsen 2003; Yun et al. 1999). The
development of high brightness third-generation synchrotron
X-ray sources has provided the needed sensitivity to measure
transition metal stoichiometries in cells collected from the most
pristine regions of the ocean. Radiation damage is significantly
reduced in SXRF. Synchrotron-based X-ray fluorescence analyses
do not require high vacuum conditions and can be performed on
frozen-hydrated samples to reduce radiation damage and preserve
cell structure optimally. However, it is often most practical
to prepare dried protist samples in order to immobilize mobile
species and preserve elemental composition until analysis. Synchrotron-based X-ray fluorescence measurements can also be supplemented with microscale chemical speciation measurements
that provide redox and coordination environment information
(e.g. Bacquart et al. 2007).
A representative SXRF spectrum for a marine protist is shown in
Fig. 2. The spectrum shown is the sum of 156 individual spectra
collected during a raster scan of a small pennate diatom collected
from the equatorial Pacific Ocean. The Ka fluorescence peaks for Si,
P, S, Cl, Ar, K, Ca, Mn, Fe, Co, Ni, Cu, and Zn are clearly visible, as
well as Kb peaks (for Ca and Cu especially). Using custom designed
software (Vogt 2003), each peak in the spectrum is iteratively fit to
an exponentially modified Gaussian curve to find the peak areas. At
the same time background is estimated from the data using a peak
stripping algorithm (SNIP; Ryan et al. 1988). Fitting of a complex
series of peaks to data can be time consuming and prone to artifacts
due to the number of free parameters. To make the fitting procedure
tractable and efficient, ratios of the Ka and Kb peak areas for an element are tightly constrained according to the ratios observed in the
thin-film X-ray fluorescence standards from NIST. Also, relative
TWINING ET AL.—SINGLE-CELL ELEMENT ANALYSIS OF PROTISTS
Fig. 2. Synchrotron-based X-ray fluorescence spectrum for a pennate
diatom collected from the surface waters of the equatorial Pacific Ocean.
Fluorescence data are plotted in gray, and model functions are shown as
black lines of varying style: fitted model (solid), Ka peaks (dashed), Kb
peaks (dotted) and background (dash-dot) features unique to the detector.
The elements corresponding to the Ka peaks—which are used for quantification—are listed at the top of the graph.
positions of the peaks are fixed in accordance with the known X-ray
emission lines for each element. Quantitation is achieved by comparing the areas under the curves with corresponding peaks from the
NIST standard.
Synchrotron-based X-ray fluorescence also provides twodimensional maps of element distribution in target cells. Given
a typical focused beam spot size of 200–300 nm for 10 keV hard
X-ray microprobes, informative maps of sub-cellular element
localization can be generated for larger protists. An example is
shown in Fig. 3. This silicoflagellate was collected from the
Southern Ocean during the Southern Ocean Iron Experiment (SOFeX) project. The edge of a neighboring pennate diatom can be
seen to the right of the cell. The chloroplasts are clustered in the
middle of the cell. Silicon maps onto the silicate test, while S is
broadly indicative of the internal cytoplasm components. In this
cell Fe and Zn are most highly localized in the chloroplasts, although Zn distributions are often found to correlate with P in other
cells. In addition to providing information about the biological
functions of the elements in target cells, these maps are extremely
useful for identifying extra-cellular abiotic particles that can easily confound bulk analyses (Twining et al. 2003b).
SINGLE-CELL ANALYSIS OF PLANKTON BY ELECTRON
MICROPROBE
Electron microprobe X-ray fluorescence analysis, often termed
XRMA, energy-dispersive X-ray microanalysis or electron probe
X-ray microanalysis has been used to study the elemental composition of eukaryotic algae and cyanobacteria collected from
freshwater systems. Sigee, Levado, and Dodwell (1999) analyzed
the dinoflagellate Ceratium hirundinella sampled from several
depths in a stratified lake and consistently detected Mg, Si, P, Cl,
K, and Ca in the cells. Sodium, Al and Fe were occasionally detected, as well, but transition metals generally occur below the
detection limits of XRMA. Minor decreases in some elements
were observed below the epilimnion, but elemental ratios were
consistent throughout the water column. Concentrations of individual elements varied significantly between cells from the same
population (location and depth), but concentrations were normally
distributed. Other researchers have also noted significant intrapopulation variability in elemental composition (Fagerbakke,
Heldal, and Norland 1991; Villareal and Lipschultz 1995).
155
A draw-back of XRMA is the limited depth of penetration of
electrons into target cells. As mentioned above, impinging electrons are strongly absorbed by biological materials. Beam penetration will vary as a function of the accelerating voltage,
specimen density, and the critical excitation energies of elements
in the specimen. Typically, XRMA measurements are limited to
the upper 1 mm of the target cell (Krivtsov, Bellinger, and Sigee
2000; Reed 1975). Therefore, while XRMA is useful for studying
the surface composition of eukayotic cells (Krivtsov et al. 2000;
Sigee and Levado 2000), interpretation of XRMA data as wholecell concentrations can be misleading. Diatoms are more problematic than other cell types as a result of their dense silicate test.
Tien (2004), for example, analyzed planktonic and epilithic diatoms from a polluted river with XRMA, and presented the resulting concentrations as ‘‘intracellular chemical levels’’. However,
given the limited beam penetration into the 10–15 mm diam.
diatoms, the characteristic X-rays were likely not representative
of the intracellular matrix. A comparison of the reported element
concentrations for planktonic diatoms collected from the same site
showed Hg and Pb concentrations (4.4 and 2.7 mg/g, respectively)
to actually be higher than P concentrations (1.7 mg/g) in the same
cells. Iron concentrations in epilithic diatoms were comparable to
P concentrations. These findings depart significantly from our understanding of metal stoichiometries in organisms (Outten and
Twining 2007), and such intracellular concentrations are biologically implausible. It is likely that the measured metals are instead
sorbed to the outer surfaces of the cells, and the resulting stoichiometries may not be representative of the rest of the cell.
Because of these limitations, marine scientists have used electron microprobes primarily to analyze prokaryotes (Fagerbakke,
Heldal, and Norland 1996; Gundersen et al. 2002; Heldal, Norland, and Tumyr 1985; Vrede et al. 2002). Heldal et al. (2003)
measured C, N, P, and S in single cells from several strains
of cultured Prochlorococcus and Synechococcus. The range of
C:N:P stoichiometries spanned the Redfield ratio, but interpopulation variability was observed. Carbon:phosphorous ratios
ranged from 156 6 to 215 9 and 73 8 to 350 30 for
Prochlorococcus and Synechococcus, respectively. Sodium, Mg,
Cl, K, and Ca were also detected in the Prochlorococcus cells, but
the bioactive transition metals were not detectable via XRMA.
Reliable XRMA measurements of trace metals such as Mn and Fe
appear limited to metal-sequestering bacteria which contain
1,000- to 10,000-fold more metal in extracellular appendages
than typical microorganisms (Heldal et al. 1996).
PLANKTON ELEMENTAL ANALYSIS WITH PIXE
Within the marine sciences, PIXE has primarily been used to
study the elemental composition of inorganic particulate material
collected on aerosol filters (e.g. Reis et al. 2006), but several
groups have applied PIXE for element analysis of algae. Zhang
et al. (1997) investigated the binding of Ag, Ba, Cd, Cu, Hg, and
Pb to the cell wall of Chlorella vulgaris under controlled laboratory conditions. The metals were easily detected in the samples,
which were composed not of individual cells but of a layer of
freeze-dried cells collected on a membrane filter. A similar approach was used by Iwata and colleagues to measure metal bioaccumulation by actively growing phytoplankton cultures (Iwata
2001; Iwata et al. 2005). These researchers used a 2-mm proton
beam to assay mats of cells collected on filters. More recently,
Gisselson, Graneli, and Pallon (2001) mapped C, N, P, S, Cl, K,
and Ca concentrations in individual cells of Dinophysis norvegica
collected from the Baltic Sea. Carbon and N were quantified by
proton backscattering, and the heavier elements were assayed with
PIXE. Absolute cellular quotas (mol/cell) of C, N, and P were
lower when measured with the nuclear microprobe than with
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J. EUKARYOT. MICROBIOL., 55, NO. 3, MAY–JUNE 2008
Fig. 3. Light, epifluorescence, and false-color SXRF element maps of a silicoflagellate cell collected from the Southern Ocean. Warmer colors
(yellow and red) indicate higher concentrations of elements. The color scales are different in each element map, so a red pixel in the Si map does not
necessarily indicate the same concentration as a red pixel in the Fe map. Scale bar 5 20 mm.
traditional bulk techniques, but C:P and N:P ratios were similar.
More intra-population variability was observed for N quotas (6.4fold variation) than C or P quotas (2.2- and 3.4-fold, respectively),
which the authors suggested might indicate mixotrophic or
heterotrophic ingestion of prey by some of the D. norvegica cells
to obtain N.
SINGLE-CELL SXRF ANALYSIS OF MARINE PROTISTS
Synchrotron X-ray fluorescence was first used to analyze planktonic protists in culture and collected from the coastal Atlantic
(Twining et al. 2003a). Following method development (Twining
et al. 2003b), the first large-scale field sampling program came
during SOFeX (Coale et al. 2004). Single autotrophic and
heterotrophic protist cells were collected within and outside the
fertilized patch and analyzed with SXRF. Here we will summarize
and expand upon the results of this study, which demonstrates the
power of the single-cell approach to studying ocean biogeochemistry.
The importance of Fe to the functioning of HNLC regions was
not apparent until the adoption of stringent trace metal ‘‘clean’’
techniques to oceanographic sampling and analysis protocols
TWINING ET AL.—SINGLE-CELL ELEMENT ANALYSIS OF PROTISTS
Fig. 4. Comparison of P, Mn, Fe, Ni, and Zn quotas (normalized to C)
in diatoms and flagellated cells collected from the Southern Ocean. For
each element the bar represents the difference between the logged mean
quotas of diatoms and flagellated cells (all quotas were log-transformed to
stabilize variance). Quotients greater than 0 indicate higher quotas in diatoms, while negative quotients indicate higher quotas in flagellated cells.
Statistical significance is denoted with asterisks (Po0.05, Po0.01).
Modified from Twining et al. (2004a, 2004b).
(Martin and Fitzwater 1988; Martin, Gordon, and Fitzwater 1990;
Martin, Gordon, and Fitzwater 1991). The resulting observations
of low dissolved Fe led Martin (1990) to hypothesize that variations in delivery of airborne Fe-rich dust to the Southern Ocean
over time may have caused past glacial periods by generating
large blooms of phytoplankton. This hypothesis has stimulated a
series of large-scale Fe addition experiments, modeling exercises,
and even proposals to geoengineer atmospheric CO2 by adding Fe
to stimulate phytoplankton growth and reduce atmospheric CO2
(Kintisch 2007). Cellular Fe:C ratios in the plankton are of particular interest because they affect the relationship between C sequestration and Fe addition, and therefore the economic viability
of attempts to bioengineer atmospheric carbon through Fe fertilization. Quotas of other nutrient elements such as P, Mn, Ni, and
Zn are also important for their potential to limit cellular growth.
Cellular quotas of these elements were measured with SXRF on
single diatom, autotrophic flagellate and heterotrophic flagellate
cells.
Iron quotas were detectable even under HNLC low Fe conditions, and community wide averages of Fe:C agreed well with
radioisotopic measurements of C and Fe uptake (Twining et al.
2004b). However several interesting differences between cell
types emerged. First, diatoms collected from unfertilized waters
had lower cellular Fe:C (with carbon estimated from biovolume)
than did flagellates (Fig. 4). While cell volume-normalized elemental contents might be expected to be lowest for diatoms with
large vacuoles, the differences in Fe:C ratios may reflect more
interesting ecological or biological distinctions. For example, diatoms are constrained to obtain Fe from the dissolved fraction and
may thus be more susceptible to limitation than flagellates, which
in the open ocean are widely believed to feed upon other cells
which have already done the work of concentrating Fe from the
environment (Maranger et al. 1998). Indeed, the Fe:C ratios of
heterotrophic flagellates more closely resembled the uptake ratio
of Fe and C into the bacterial size fraction as determined by
radioisotopes. Alternatively, the higher Fe content in heterotrophs
may reflect a greater requirement. Respiratory enzymes that make
up the electron transport system have a higher Fe content than
does the photosynthetic apparatus that makes up much of autotrophic cell mass (Chase and Price 1997; Raven 1988).
157
Cell types differed in a number of other important respects as
well. For example, estimated P:C and P:S ratios were lower for
diatoms than for flagellated cells (Fig. 4), a finding in opposition
to culture studies indicating that diatoms as a group have high P
contents (Ho et al. 2003). Diatoms also exhibited much higher Ni
and Zn quotas. Elemental fluorescence maps indicate that most,
but not all, of the Ni in diatoms was associated with the frustule,
and in particular with the densely silicified girdle region (Twining
et al. 2003b). The only known Ni enzyme in eukaryotes is urease,
which can be used to assimilate reduced urea nitrogen from the
environment. Although nitrate was present at non-limiting concentrations (Coale et al. 2004), urease expression in cultured
phytoplankton is not generally suppressed in the presence of nitrate (Lomas 2004; Peers, Milligan, and Harrison 2000). Furthermore, diatoms in the Southern Ocean may favor the use of reduced
N when cellular energy is limited by light and Fe availability
(Price, Ahner, and Morel 1994). In contrast with Ni, Zn seemed to
be associated mostly with the nucleus (Twining, Baines, and Fisher 2004a), perhaps reflecting the importance of Zn-containing finger proteins in the overall Zn budget of the cell. This observation
is interesting because most discussion of Zn in phytoplankton revolves around carbonic anhydrase, a well described enzyme
known to contain Zn, and does not consider the role of Zn finger
proteins (Morel et al. 1994; Saito, Goepfert, and Ritt 2008).
Measured elemental ratios differed from expected values in
several important respects. Cellular Fe:C quotas ranged between
6 and 14 mmol mol 1, 2–4 times higher than the 3–5 mmol 1
Fe mol 1 C estimated by Moore et al. (2004) for Southern Ocean
plankton. This discrepancy may reflect inter-specific differences
in the Fe quotas of Southern Ocean phytoplankton compared to
the cultured species used to parameterize most models. It also remains unclear how to extend culture work performed in EDTAbuffered media to natural systems where the bioavailability of
dissolved Fe is largely unknown. Furthermore, higher Fe quotas
might result from physiological adjustments to low-light conditions in the Southern Ocean (Sunda and Huntsman 1997). As
might be predicted for a region with excess dissolved phosphate,
cellular P was also unusually high in Southern Ocean cells, being
42-fold more abundant in flagellates than predicted by Redfield.
Diatom Zn:P ratios were elevated relative to bulk plankton from
the Pacific Ocean but similar to bulk plankton from Southern
Ocean (Twining et al. 2004a). This suggests there may be unique
ecological or biogeochemical aspects of Southern Ocean taxa that
are reflected in the elemental composition. Measured diatom Zn
quotas were more than an order of magnitude higher than those
reported for cultured diatoms by Ho et al. (2003). This may indicate greater bioavailability of Zn bound to natural organic ligands
than typically assumed. Diatom Zn quotas in culture may also
have been lowered by relatively high Mn concentrations in the
media, as Mn can inhibit Zn uptake in marine phytoplankton
(Sunda and Huntsman 1998b). These comparisons highlight the
uncertainties introduced when culture data is applied to natural
systems and the benefit of field measurements.
The cellular concentrations of several elements changed markedly in response to Fe addition. Iron cellular concentrations
changed most, increasing in all cell types by a factor of 3–6 over
the course of 6 days and two additions. While the Fe content of
diatoms and autotrophic flagellates increased after both Fe additions, the Fe:C ratio in heterotrophic flagellates exhibited a delayed response which probably reflected a particulate food supply
that was continuously changing in Fe content (Fig. 5). Such metal
cycling within plankton communities is extremely difficult to
study without single-cell analytical tools, because the dominant
autotrophs and heterotrophs have similar sizes. Interestingly, P
and S content of diatoms increased markedly after Fe addition,
perhaps reflecting an increase in cytoplasm within the frustule. It
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J. EUKARYOT. MICROBIOL., 55, NO. 3, MAY–JUNE 2008
Fe, lack of an effect of Fe availability on cellular silicification, an
increase in cellular S and P content when Fe limitation is relieved,
and high cellular Ni and Zn. Large differences among cell types
were also noted, although not all patterns conformed to those observed in the Southern Ocean. In addition, recent improvements in
analytical sensitivity of the SXRF microprobe have allowed trace
element contents to be measured in prokaryotes for the first time,
and these have proven to be distinct from those of eukaryotic cells.
FUTURE METHODOLOGICAL DEVELOPMENTS
Fig. 5. Carbon-normalized Fe quotas for diatoms (white circles), autotrophic flagellated cells (white triangles), heterotrophic flagellated cells
(gray triangles) and the mean of all three cell types (black circles) following fertilization of Southern Ocean waters. Cellular Fe was measured
directly with SXRF. Cellular C was calculated from cellular S using a C:S
stoichiometry of 100. Points are geometric means ( SE). Modified from
Twining et al. (2004b).
may be that reduction of the cytoplasmic mass (or cell volume) is
a general response to Fe limitation. In addition to these changes,
cellular concentrations of the bioactive metals Mn, Ni, and Zn also
increased 1.5- to 4-fold after Fe addition. In general, these results
suggest that relaxation of Fe limitation caused a physiological
cascade in protist cells leading to a high growth, high metabolic
rate condition.
Some of the most interesting findings regarded cellular silica in
diatoms. Diatoms in the Southern Ocean were heavily silicified
prior to Fe fertilization. The average diatoms had Si:C ratios of
0.5 mol mol 1, which is nearly 4 times the typical value for diatoms in culture (Brzezinski 1985). These measurements imply
that SiO2 constitutes 20% of wet mass and 50% of the dry
mass of the diatoms in the Southern Ocean. The high degree of
silicification may explain why the Southern Ocean plays such an
important role in the global C cycle since dense silica ballast enhances sinking rates of aggregates and fecal material produced in
these regions. It is widely believed that high silicification is typical of Fe-limited regions because silica incorporation is inexpensive in energy terms and tends to continue even as cellular growth
rates slow in response to limitation by other nutrients (Franck
et al. 2000). However, Fe addition in the Southern Ocean produced only a transient 40% decline in Si per cell, after which cellular Si contents rebounded to match those of cells collected
before fertilization and from a control patch after the experiment.
There was a 2-fold decline in Si:P and Si:S ratios, but this reflected the increase in cellular P and S rather than a decrease in Si.
Clearly, the maintenance of high cellular silicification in the
Southern Ocean requires an explanation other than Fe limitation.
The SXRF data collected during the SOFeX project demonstrate the power of single-cell element measurements for studying
the role of protists in the biogeochemistry of natural systems. This
project has been recently followed up by SXRF studies in the
Eastern Equatorial Pacific, which is also believed to be Fe-limited,
and in the Sargasso Sea, which is nutrient poor but generally considered Fe-replete. These analyses are ongoing and as yet unpublished, but they generally confirm many trends noted for diatoms
in the Southern Ocean, most notably higher than expected cellular
Synchrotron X-ray microprobes offer arguably the best combination of analytical sensitivity, analytical breadth and spatial resolution while imparting the least radiation damage to protist
samples. There are several limitations to their usefulness for environmental and other microbiologists, however. First, SXRF typically cannot measure the major components of organic matter (C,
N, and O), which complicates interpretation of cellular concentrations and two-dimensional element maps. Second, localization
of elements within the cell is currently only possible in two dimensions, which makes it difficult to determine how much of an
element is associated with, for example, the cell surface or cellular
organelles embedded in protoplasm. Third, routine spatial resolutions are currently too low to resolve cell structures smaller than
the nucleus or chloroplast.
Estimation of cellular mass simultaneous with SXRF analyses
is possible in one of two ways. Just like visible light passing
through water or air, X-rays passing through biological samples
are scattered in proportion to the X-ray density of the substance.
These scattered X-rays are registered by the detector and the area
under the Compton scattering peak can be used as a proxy for cell
mass at a particular pixel. Without a vacuum chamber, the method
is fairly insensitive, because background scattering peaks due to
He atmosphere are large, resulting in a high ratio of noise to signal
and high error. Also, the XRF detector is typically positioned to
minimize detection of scattered radiation (and thus improve sensitivity), though use has been made of a second detector at a position optimized for detection of increased scatter (Golosio et al.
2003). A much more sensitive method that can be used for thin
biological samples without a vacuum is X-ray phase contrast microscopy (Hornberger, Feser, and Jacobsen 2007). X-rays are subject to phase shift as they pass through biological samples to a
degree dependent on the thickness and composition of the sample.
Using a specially designed and fabricated segmented detector positioned downstream of the sample source, differences in phase
can be recorded to produce a differential phase contrast image
(Feser et al. 2006). Given assumptions about the C, O, and N
composition of the sample, and direct measurements of other elements available from the fluorescence spectra, the phase shift can
be reconstructed (de Jonge et al. submitted; Hornberger et al.
2007) and used to estimate cellular mass at each pixel. Because
the method uses the same X-ray beam to construct a map of fluorescence and phase contrast (Hornberger et al. 2006), the two
maps coincide perfectly (Fig. 6). This method has been calibrated
on latex beads, and a comparison with standard chemical determinations of elemental composition of algal cells is underway
(Hornberger et al. 2007).
X-ray fluorescence computed tomography (XFCT) offers the
possibility of visualizing both elemental distributions and phase
contrast maps in three dimensions (Boisseau and Grodzins 1987;
Golosio et al. 2003; La Riviere and Vargas 2006; La Riviere et al.
2006). In XFCT the target is repeatedly imaged in two dimensions
under different angular positions. Once the desired angular range
is covered, the three-dimensional elemental or mass distribution
that best reconciles all of the images is determined. This process
can be complicated for larger cells due to the absorption of lower
TWINING ET AL.—SINGLE-CELL ELEMENT ANALYSIS OF PROTISTS
159
available and are useful when studying major elements in small
prokaryotes. Proton microprobes can detect a wide range of elements but at limited spatial resolution. Synchrotron-based X-ray
fluorescence combines unmatched sensitivity for transition metals
and spatial resolution that approaches XRMA, at the expense of
light element quantification and instrument availability. Each of
these microprobe techniques can be used to help open the ‘‘black
box’’ of plankton elemental composition with single-cell measurements. Only by making geochemical (elemental) measurements of individual functional groups will we advance our
understanding of the relationships between protist biology, ecology, and biogeochemistry.
ACKNOWLEDGMENTS
We would like to thank Ian McNulty for collaboration on
differential phase contrast experiments at beamline 2-ID-B. This
work was supported by grants from NSF to BST (OCE 0527062)
and SBB (OCE 0527059). Use of the Advanced Photon Source
was supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No.
DE-AC02-06CH11357.
LITERATURE CITED
Fig. 6. Shown in the upper panels are a light (differential interference
contrast) micrograph (1), Si SXRF map (2), and X-ray differential phase
contrast (3) image of a cultured pennate diatom (Pseudonitzschia multiseries, CCMP 2708). The scale bar 5 10 mm. The lower panel (4) shows a
higher resolution X-ray differential phase contrast image of two small
pennate diatoms collected from the equatorial Pacific Ocean. The data
presented in panels 2 and 3 were collected with the hard X-ray fluorescence microprobe at beamline 2-ID-E of the APS, and the data in panel 4
were acquired at the intermediate-energy scanning X-ray microscope at
beamline 2-ID-B of the APS.
energy fluorescent X-ray photons by cellular organic matter (Golosio et al. 2003). However, recent developments in statistical fitting techniques that also estimate this self absorption using
penalized-maximum likelihood have been able to correctly reconstruct the three dimensional test structures for which self absorption is significant, making quantitative tomography possible (La
Riviere et al. 2007). This approach effectively increases the size of
targets that can be imaged. Anticipated improvements in terms of
X-ray source brightness should improve the speed of such techniques in the future.
The focused spot sizes (i.e. spatial resolution) of hard X-ray
microprobes continue to improve with advances in X-ray optics
(Hignette et al. 2005; Kang et al. 2006). Current instrumentation
at third-generation synchrotron facilities is capable ofo200 nm
spatial resolution with 10 keV X-rays, with ongoing efforts to
reduce this to 30 nm. Beamlines utilizing intermediate energy
X-rays (2–3 keV), such as 2-ID-B at the Advanced Photon Source,
have already achievedo100 nm resolution (Fig. 6D) (McNulty
et al. 2003). This level of focus will allow elemental measurements in viruses and the smallest prokaryotes, which can be
important food resources for protists. It will also enable more
precise sub-cellular element mapping in eukaryotes, expanding
our ability to study the biological roles of metals in protists.
CONCLUSIONS
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Received: 02/01/08; accepted: 02/02/08