Coccolithophores play a key role in the marine carbon cycle and ecosystem.
The carbonate shells produced by coccolithophore, named as coccolith, could
be well preserved in the marine sediment for millions of years and become an
excellent archive for paleoclimate studies. The micro-filtering and
sinking–decanting methods have been successfully designed for coccolith
separation and promoted the development of geochemistry studies on
coccolith, such as the stable isotopes and Sr / Ca ratio. However, these two
methods are still not efficient enough for the sample-consuming methods. In
this study, the trajectory of coccolith movement during a centrifugation
process was calculated in theory and carefully tested by separations in
practice. We offer a MATLAB code to estimate the appropriate parameter,
angular velocity at a fixed centrifugation duration, for separating certain
coccolith size fractions from bulk sediment. This work could improve the
efficiency of coccolith separation, especially for the finest size fraction,
and make it possible to carry out the clumped isotope and radio carbon analyses
on coccoliths in sediment.
Introduction
Coccolithophores are a group of marine calcifying eukaryotic phytoplankton,
whose calcite exoskeletons (i.e., coccolith) contribute significantly to the
particulate inorganic carbon (PIC) export from the euphotic zone into the
deep ocean (Young and Ziveri, 2000). Coccoliths preserved in marine sediment
are also excellent archives for paleo-productivity reconstruction (Beaufort
et al., 1997). The element ratio Sr / Ca in coccoliths is correlated with the
growth rate of calcite crystals (Stoll et al., 2002), thereby becoming a
competitive candidate for coccolithophore growth rate, which is an essential
parameter in the paleo-CO2 reconstruction by alkenone carbon isotope.
However, the coccolith geochemical analyses are limited by the difficulty of
separating coccoliths from bulk sediment. To solve this problem, different
separating methods have been proposed in the past over a few decades (Paull and
Thierstein, 1987; Stoll and Ziveri, 2002; Minoletti et al., 2008).
Most of them, in general, could be categorized into two groups: the first
one is micro-filtering and the second is the sinking–decanting technique. The
micro-filtering method relies heavily on the specifications of micro-filter
membrane (such as 3, 5 and 8 µm pore sizes), which are
highly effective at separation of the larger-size coccoliths but useless
for coccoliths smaller than 2 µm. The sinking–decanting method, on the
other hand, could offer more freedom in coccolith size by adjusting the
sinking durations, thereby separating both small and large coccoliths.
However, because of the slow sinking speed, a single separation of particles
smaller than 2 µm may take more than 10 h in settling. Moreover, operations should be repeated about 6–8 times, which means a full
separation may take at most 1 week. Hence, it is necessary to improve
this method by reducing the time cost in coccolith separation.
Based on the Stokes sinking equation, the sinking rate of a certain particle
increases with the increase of density difference between particle and
liquid, decrease of the liquid viscosity, and the increase of gravity.
Changing the physical property of liquid often leads to an organic and
toxic solvent which could lead to potential contamination for further
geochemistry analyses. A better way to accelerate coccolith sinking speed is
changing the gravity or the acceleration speed of the reference system,
which can be easily achieved by centrifugation. One study has mentioned the
usage of centrifugation in coccolith separation, but only centrifugation
settings for a special case were provided (Hermoso et al., 2015). Here in
this study, the separation of coccoliths by centrifugation method is
introduced systemically. We first calculate the trajectory of coccolith
movement in centrifugation processes and show how to estimate the
centrifugation parameters in different situations. After that, two tests are
performed to confirm the robustness of our calculations. Ultimately, a
sample containing coccoliths ranging from 2 to 12 µm is
selected for a separation case in practice.
Trajectory of coccoliths during centrifugation
The movement of coccoliths under centrifugation is similar to that under the
gravity. Previously, we have calculated the separation ratio variation with
time during the settling (Zhang et al., 2018). All calculations in this
study are with an assumption that the coccolith is in force balance all
the time during both settling and centrifugation for a convenience of
calculation. Here we offer a brief proof for this assumption based on spherical
particles (the sink speeds of spherical particles are ∼ 30 %
higher than those of coccoliths of the same size) and give a quick review of
the derivation we did before.
Based on Newton's second law, the force balance of a spherical object during
sinking can be described by the following equation:
F=43πr3ρpg-43πr3ρlg-6πηrv=43πr3ρpdvdt,
where F is the joint force of particle, which is equal to zero in force
balance; r is the radius of sphere; ρp and ρl are the
density of particle and liquid, respectively; η is the velocity of
liquid; v is the particle sinking speed; and dv/dt is the particle acceleration
speed, which can be also denoted as a. On the right-hand side of the first equal to
sign, the first term is the gravity force, the second term is buoyancy and
the third term is the dragging force from liquid. By transforming Eq. (1), we can
obtain the expression of accelerated speed (a=F/m) of a sphere as Eq. (2):
a=dvdt=-9η2r2v+gρcalρp-ρl.
Given that the initial value of sinking speed is equal to zero at the initial
time (t=0), we can solve the differential equation (Eq. 2) and obtain the
variation of velocity with time as the following equation:
v=-e-9η2r2t+ln-gρcalρp-ρl+gρcalρp-ρl9η2r2.
When the value of t is large enough, the first term of the numerator in Eq. (3) is
close to zero, which means the sinking velocity is close to the
terminal sinking velocity described in the Stocks equation (Eq. 4).
v=2ρp-ρlgr29η
Given a spherical calcite carbonate particle with a 5 µm radius and density of 2.7 g cm-3, the density of water being equal to 1.0 g cm-3, and when t is equal to 10-7 s, then the first term of the numerator
is 3.7×10-44 m s-2 and small enough to be neglected
compared with the second term, which is 6.3 m s-2. The timescale in
coccolith separation is minutes for centrifugation and hours for settling;
Therefore, we suggest that it is reasonable to assume that the coccolith sinks
with the “terminal speed” from the very beginning.
The only difference between the terminal speed under centrifugation and under
gravity is the acceleration speed. If g in Eqs. (1)–(4) is adapted by a,
which is the acceleration speed of the coccolith during centrifugation, these
four equations above can also describe the sphere's movement in the
centrifugation if we adapt the gravity to centripetal acceleration (ca).
Here we define a new parameter named sinking parameter (sp):
sp=vg.
The physical meaning of sp is the influence of coccolith shape and liquid
property (density and viscosity) on sinking velocity without considering the
effect of gravity (or the acceleration rate of the reference system). The
sinking speed of the coccolith in water during a centrifugation (v′) can be
described as follows:
v′=sp×ca=sp×ω2×(L+D),
where the ca is centripetal acceleration during centrifugation, ω is
angular velocity of the centrifuge, (L+D) is the rotation radius as
illustrated in Fig. 1. Parameter L is a fixed value for a certain type of
centrifuge, and D depends on the position of the coccolith in the tube. Here
we should note two issues. The first one is that the rotation radius is
varying when coccolith is moving in the centrifuge tube; in other words, D
is always changing. This effect could be ignored when L is much larger
than D, but, unfortunately, most centrifuges employed in geochemistry
laboratories are not large enough. The second one is that angular velocity is
dynamic when the centrifuge is accelerating and decreasing. To solve
these two dynamic parameters, Eq. (6) was transformed into a form of
differential equation as Eq. (7) for the convenience of integration in the
next step.
dt=dDv=dDsp×ω2×L+D
For all centrifugation steps, there are three stages: the acceleration stage
(t1 to t2 in Fig. 1), the constant angular velocity stage
(t2 to t3 in Fig. 1) and the deceleration stage (t3 to
t4 in Fig. 1). The durations of the acceleration stage and deceleration
stage can usually be controlled and the angular velocity is changing with a
constant speed. For those machines where the angular velocity dynamic
(ω=f(t)) is unknown, we should measure it manually.
The position of coccolith and the variation of ω in the
three centrifuging stages: L represents the minimum rotation radius, and
V1 and V2 represent the volumes of the two parts; in the first stage,
the angular velocity increases from zero to ω1 (it could be linear
or cubic, which depends on the machine). Meanwhile, the coccolith moves a
distance of D2–D1; similarly, the coccolith moves a distance of
D3–D2 in the second stage, and it moves a distance of
D4–D3 in the last stage.
After knowing the angular velocity curve, integrate D over t in Eq. (7) by three steps from t1 to t4:
8sp×∫t1t2ω12dt=lnL+D2-lnL+D1,9sp×∫t2t3ω22dt=lnL+D3-lnL+D2,10sp×∫t3t4ω32dt=lnL+D4-lnL+D3.
Adding Eqs. (8)–(10) together gives
sp×∫t1t2ω12dt+∫t2t3ω22dt+∫t3t4ω32dt=lnL+D4-lnL+D1.
Set D4 equal to D, which represents the maximum distance that a
coccolith can move in the upper suspension V1. Now we can use the
coccolith sinking property, sp, and centrifugation settings to describe the
coccolith position after centrifugation D1:
D1=L+Desp×∫t1t2ω12dt+∫t2t3ω22dt+∫t3t4ω32dt-L.
The meaning of D1 is that all coccoliths with an initial position on the
right side of D1 in Fig. 1 will move to the right side of D4 and
then be kept in the suspension after pumping, while the coccoliths on the
left side of D1 will be removed by pumping.
In our previous publication (Zhang et al., 2018), we defined a parameter
named separation ratio (R), which represents the percentage of coccolith
removed in one separation if we pump the upper V1 volume suspension out
of (V1+V2) suspension in total.
R=V1×D1DV1+V2
Replacing the D1 in Eq. (15) with Eq. (12) gives the separation ratio (R)
as a function of centrifugation settings:
R=V1V1+V2×1D×L+Desp×∫t1t2ω12dt+∫t2t3ω22dt+∫t3t4ω32dt-L.
The R can be employed in estimating the centrifugation parameters for
separating one type of coccoliths from another. For example, if we want to
separate a group of coccolith (marked as CoccolithA, with sinking
parameter spA) from another group of coccolith (marked as
CoccolithB, with sinking parameter of spB and spA< spB), the R of CoccolithB should be set as zero, which means all
CoccolithB in the section V1 have sunk into V2 after
centrifugation; therefore, all coccolith pumped out should be
CoccolithA. To solve the angular velocity (ω2) and
centrifugation duration (t=t3–t2) in Eq. (14), we need to fix at
least one of them. Usually the duration could be safely set as 1 min or 2 min, and then we solve the suitable angular velocity with known parameters V1,
V2, D and L. The MATLAB code for the parameter estimation is in
the Supplement. After repeating these “centrifugation–pumping” routines several
times, CoccolithA could be fully separated from CoccolithB.
Test of the correctness of calculationsExperimental design
To test the robustness of our estimation in the last section, we performed
two groups of experiments comparing the observed with predicted separation
ratios. Here we select two different coccoliths, Florisphaera profunda and small Gephyrocapsa, with small size
and thereby slow sinking speed, sampled from ODP 807 and IODP U1304,
respectively. Most of the small Gephyrocapsa employed in this study are smaller than 3 µm with a mixture of G. muellerae less than 10 %. Two centrifuges from Anting Company,
TDL-40B and DL-5B, were selected to perform the tests. The angular
velocity of DL-5B can be set as linear increased or decreased with time in
the acceleration or deceleration stages, while the angular velocity of
TDL-40B was measured manually by reading the number on the instrument
panel. The centrifugation duration can only be adapted by a step of 1 min on both of these machines. The slowest angular velocities of
these two machines is 500 revolutions per minute (rpm). If we selected
water as dispersion agent, most of the coccoliths we used will sink to the
tube bottom after 2 min even with the slowest angular velocity. Hence,
to slow down the coccolith sinking speed in these tests, glycerol solution
was employed in this equation test, which can be dissolved with water in any
proportion and washed away from carbonate calcite particles conveniently.
The density and viscosity data can be found in Table 1.
All calculations above are for the situation that particles are sinking in
water or diluted solution, the physical property of which is close to
water. However, in this case, the property of glycerol is significantly
different from water. Here we define a new parameter, τ, to transform
the sinking speed in water to that in different liquid. The physical meaning
of τ is a ratio turning the sinking velocity in water (v) into the
velocity in any liquid with different density and viscosity (v′):
v′=v×τ.
Based on the definition of Stokes equation, the term τ can be
calculated as follows:
τ=ρp-ρlρp-ρw×ηwηl,
where the ρp, ρl and ρw are densities of
particle, liquid (in this study is glycerol solution) and water, respectively. ηl and ηw are the viscosity of liquid and water, respectively.
Combining Eqs. (14)–(16) forms the separation ratio as a function of
centrifugation settings in different liquid:
R=V1V1+V2×1D×(L+De[vg×ρp-ρlρp-ρw×ηwηl×∫t1t2ω12dt+∫t2t3ω22dt+∫t3t4ω32dt]-L).
In this test, the calculated R by Eq. (17) will be compared with the measured one.
To perform these tests, about 100 mg of bulk sediment was scattered into 30 mL 0.5 % ammonia, and after that, particles larger than 20 µm were removed by a mesh. In this test, we should obtain suspensions
with nearly monospecific coccoliths. To achieve this, in the test with F. profunda,
coccoliths larger than 3 µm were removed by the sinking method
described in Zhang et al. (2018), and coccoliths larger than 5 µm were
removed by the same method in the test with small Gephyrocapsa. Briefly, the suspension
was (1) set in a 100 mL reagent bottle, sinking freely for a few hours, and
then (2) pumped out of the upper 2 cm. These two steps were repeated 5–8 times
until coccoliths were purified. The sinking duration was 2 h for the F. profunda sample
and 1.25 h for the small Gephyrocapsa sample, respectively.
Then 50 mL tubes with 45 mL coccolith suspensions were mounted in the
centrifuge and run with the settings shown in Table 1. After centrifugation,
the upper 30 mL supernatant was pumped out by pipette and then filtered onto
a 0.4 µm polycarbonate membrane filter with a vacuum pump. The coccoliths on
the polycarbonate membrane were resuspended into 20 mL diluted ammonia again, and
coccolith number in the suspension was measured with the same method described in our previous work (Zhang et al., 2018). Finally, the separation
ratio, R, was calculated by the coccolith number in the upper 30 mL
suspension and divided by the total coccolith number. All the centrifuging
experiments were carried out in the laboratory with temperature controlled
to be around 20 (± 1) ∘C to avoid variation of physical
properties, especially the viscosity, with temperature.
The settings of two tests: the density and viscosity of glycerol at
20 ∘C; data are from Dorsey (1940); the parameters of the centrifuge
employed in this study are the following: Fp and G60 represent the experiments carried out with
F. profunda in 70 % glycerol and small Gephyrocapsa (< 3 µm) in 60 % glycerol,
respectively; L represents the minimum rotation radius of centrifugation,
which represents the distance between the shaft and top of suspension as
illustrated in Fig. 1. A, B and C are the terms on the left-hand side of the equal to
sign in Eqs. (8)–(10).
In the test, a 30 mL suspension was pumped out from a 45 mL suspension, leading
to the result that the initial R should be 60 %. However, the intercept of calculated R is
smaller than 60 % as the gravity settling in Zhang et al. (2018), because
the time in the x axis of Fig. 2 is the period in which angular velocity
remains constant. In other words, even though the time is set as zero, the
centrifuge will still do the acceleration and deceleration processes, and
coccoliths will move toward the bottom. The results of observed R (dots in
Fig. 2) are close to the theoretical values (dash lines in Fig. 2),
although a few measured results are lower than the prediction. We suggest that
this difference may be caused by coccolith loss during harvesting of the
coccolith from glycerol solutions into ammonia solution.
So far, we have obtained the coccolith movement equation in the
centrifugation and proved its correctness. In the next section, a case of
coccolith separation by the centrifuging method will be carried out giving an
example of separation.
The comparison of theoretical and measured separation ratio (R):
the dots represent the measured values, and dashed lines are theoretical
calculations. The error bars represent 95 % error based on the assumption
that the error of counting coccolith follows the Poisson distribution. The
orange dots represent the measured R in small Gephyrocapsa test with 60 % glycerol
(G60-M), and the blue ones represent the measured R in F. profunda text with 70 % glycerol (Fp-M). The dashed orange line is the theoretical values for small
Gephyrocapsa test with 60 % glycerol(G60-T), and the blue one is the theoretical
values for F. profunda test with 70 % glycerol (Fp-T). Raw pictures for coccolith
counting are shown in Figs. S1 and S2.
Separation of coccoliths in practiceSeparation steps
The aim of this section is to separate a
sample in practice using the centrifugation method. A sample form ODP 982B (56X Section 5 5–9 cm) dated
around mid-Miocene (nannofossil zone NN4) was selected in this test. The
coccolithophore Reticulofenestra spp. dominated in the assemblage, with long-axis length
ranging from 2 µm to more than 12 µm, offering an ideal sample to
test the coccolith separation method. Calcidiscus spp. (4–10 µm), Helicosphaera spp. (5–10 µm) and Coccolithus spp. (6–8 µm) were also found in this sample, which
contributed less than 10 % to all coccoliths together. The preservation of
fossil was moderate with many coccolith fragments but no evidence of
dissolution in the raw sample. The detailed operations are the following:
Step 1. Weigh about 40 mg bulk sediment, scatter with 45 mL 0.5 % ammonia
solution and transfer the suspension into a 50 mL centrifuging tube;
Step 2. Calculate the centrifugation parameters (angular velocity and
duration). Here we did not measure coccolith sinking velocities but employ
the length–velocity relationship in the previous study directly: sinking
rate at 25∘= 0.0982 × length2 (Zhang et al., 2018).
Based on this length-velocity equation and the centrifuge properties listed
in Table 1, we estimated that the angular velocity and duration for
separating coccolith with a length of 2, 3, 5, 8 and 10 µm should be 1850 rpm for 2 min, 2250, 1400,
1000 and 600 rpm for 1 min, respectively. The MATLAB code for
calculating the angular velocity at fixed centrifugation duration (1 or 2 min) is in the Supplement.
Step 3. Mount the tube into the centrifuge and balance weight; set the
angular velocity to 1850 rpm and the duration to 2 min and start the
machine;
Step 4. Pump out the upper 30 mL from each suspensions and release it into a beaker
(500 mL or larger beaker, depends on how many times this step is repeated) and
drop about 100 µL onto a glass coverslip. Dry the suspension on glass coverslip
and mount the coverslip on a slide. The details of this step follow Bordiga et
al. (2015);
Step 5. Repeat step 2–5 with different centrifugation parameters listed in
Table 2;
Step 6. Take pictures of coccoliths in each slide on the microscope and measure
the coccolith size on the computer with the method described by Fuertes et al. (2014).
Centrifugation parameters in the Miocene coccolith
separations.
<2µm2–3 µm3–5 µm5–8 µm8–10 µmAngular velocity1850225014001000600(ω2, rpm)Duration12060606060(t=t3–t2, s)Coccolith length in each fraction
The coccolith size distribution harvested from different centrifugation
settings are shown in Fig. 3 (the coccolith size was measured in a circular-polarizing microscope, and coccoliths under a cross-polarizing microscope are
shown in Figs. S3–S9 for species identification). The results show that the
separated coccolith size increased with the decrease of angular velocity, and
the differences of mean coccolith lengths are significant between each size
fraction. However, we should also note that there is still overlap of
coccolith sizes between two neighboring fractions. With the centrifugation
parameters set as 2250 rpm and 2 min, the coccoliths harvested have long-axis lengths around 2–4 µm, and when the centrifugation parameters were
varied to 1400 rpm and 1 min, the coccolith long-axis size ranges from 3 to 7 µm, which means coccoliths with a length between 3–4 µm appear in two fractions. Such situations may also happen in both settling
and micro-filtering methods, but the range of overlap seems to be larger for
the centrifugation method compared with the size fractions harvested by
other methods.
The coccolith size in different fractions after centrifuge
separation: the yellow, red, green and blue dots represent 2250 rpm for 2 min,
1400, 1000 and 600 rpm for 1 min, respectively.
Two methods to reduce the coccolith size overlapping. (a) Adaption
of pipette tip: the orange part on the tip is sealed by solidified glue,
and the gray parts mean that small holes should be drilled allowing the
suspension to flow in horizontally; (b) choose a proper pumping position
to avoid extracting the coccolith on the tube wall. The lightest gray part in
the tube represents the suspension in which the smaller coccolith floats;
most of the larger coccoliths are in the lower part of the suspension and
the tube bottom.
Troubleshooting
The first potential reason leading to overlap may be that the repeating times are
not enough. This could be the main problem for settling under gravity, since
the time costs for separation under gravity are much larger than the
centrifugation method. Bolton et al. (2012) suggested that
separations done 4–6 times are enough for fossil extraction, and in our separations we
repeated separations more than 8 times for a certain centrifugation setting. Considering
these facts, we suggest that this overlapping was not caused by the
separation times.
Another reason could be that larger coccoliths, which are supposed to sink
into the lower suspension, are pumped out after centrifugation. When the
upper suspension was pumped out, the pumping speed could be too fast, drawing
up larger coccoliths from the lower suspension. This problem could be solved
by reducing the pumping speed. Hoverer, in practice, the pumping speed of
a pipette is difficult to control. Here we recommend to modify the tips of
pipettes as follows: (1) suck a drop of glue into the top of pipette
tips (the Norland optical adhesive 74 was employed in this study), (2) solidify the glue with ultraviolet light to seal the top of tips, and (3) drill
holes above the glue horizontally. After this modification, the suspension
will go into tips horizontally instead of vertically (Fig. 4a) to avoid
mixing larger coccoliths with smaller ones.
The size overlapping could also be caused by the centrifugation tube not
remaining perfectly horizontal during centrifugation. In our calculations,
the tubes are assumed to be perfectly horizontal during all centrifugation
processes; thereby, it was assumed that there should be no collisions
between coccoliths or the tube wall. However, in practice,
the tubes in a centrifuge are not always horizontal and even a few degrees
slope of the tubes can lead some coccoliths will knock and stick on the tube
wall forming a significant coccolith layer on one side of the tube wall as
illustrated in Fig. 4b. These coccoliths on tube wall will be pumped out
after centrifugation, causing the coccolith length overlapping among two
fractions. To avoid this problem, before the step of pumping out suspension,
we should observe the tube carefully. If a coccolith layer can be found on
the tube wall, the pipette tip should be placed on the opposite of the
coccolith layer to reduce the size overlapping.
Summary
In this study, we described the method of separating coccolith from bulk
sediment by centrifuging. The rotation speed for separating coccoliths within
a certain range of length could be solved after measuring the rotations
radius (property of centrifuge) and fixing the centrifugation duration.
The centrifugation method is not perfectly accurate and could still mix
different species of coccolith as other traditional separating methods. The
size overlapping of this method could be reduced by adapting the pipette
tips and avoiding pumping out the coccolith on tube wall. However, this
method is more efficient in separating the finest particle (smaller than 3 µm) out of bulk sediment, which is always the time-consuming step in
micro-filtering and sinking methods. Thereby, this method can be widely used
in the sample preparation for analyses needing a large amount of material,
such as coccolith clumped isotope and radioactive carbon isotope
measurements. Moreover, the centrifugation method can be combined with other
separation steps, for example using the centrifugation method to remove the
finest particles followed by micro-filtering with different sizes of
membranes. This method could largely reduce the time cost in sample
preparation for coccolith geochemistry analyses and has the potential for
wide use in the future.
Code availability
The code for calculating centrifuging parameter is contained in the Supplement.
Data availability
All necessary data are included in Table 1 and in the Supplement.
The supplement related to this article is available online at: https://doi.org/10.5194/bg-18-1909-2021-supplement.
Author contributions
This study was conceived by HZ and CL Measurements and calculations were
conducted by HZ. HZ, HS and LMM wrote the paper.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
This study was funded by the National Science Foundation of China (41930536, to
Chuanlian Liu), ETH core funding (to Heather Stoll), European Union's Horizon 2020 research and
innovation program under the Marie Skłodowska-Curie grant agreement (795053
to Luz María Mejía) and the Chinese Scholarship Council (CSC) with a scholarship to Hongrui Zhang. We
thank the Integrated Ocean Drilling Program (IODP) for providing the samples. We thank Guodong Jia for providing two centrifuges to test our
work and Xinquan Zhou for identification the Miocene nannofossils.
Financial support
This research has been supported by the National Natural Science Foundation of China (grant no. 41930536), the ETH core funding, the Chinese Scholarship Council (grant no. 201706260032), and Marie Skłodowska-Curie (grant no. 795053).
Review statement
This paper was edited by Hiroshi Kitazato and reviewed by two anonymous referees.
ReferencesBeaufort, L., Lancelot, Y., Camberlin, P., Cayre, O., Vincent, E., Bassinot,
F., and Labeyrie, L.: Insolation cycles as a major control of equatorial
Indian Ocean primary production, Science 278, 1451–1454,
10.1126/science.278.5342.1451, 1997.Bolton, C. T., Stoll, H. M., and Mendez-Vicente, A.: Vital effects in coccolith calcite: Cenozoic climate-pCO2drove the diversity of carbon acquisition strategies in coccolithophores?, Paleoceanography, 27,
10.1029/2012pa002339, 2012.Bordiga, M., Bartol, M., and Henderiks, J.: Absolute nannofossil abundance
estimates: Quantifying the pros and cons of different techniques, Rev.
Micropaleontol., 58, 155–165, 10.1016/j.revmic.2015.05.002, 2015.
Dorsey, N. E.: Properties of ordinary water-substance, Reinhold
Publishing Corporation, New York, 1940.Fuertes, M.-Á., Flores, J.-A., and Sierro, F. J.: The use of circularly
polarized light for biometry, identification and estimation of mass of
coccoliths, Mar. Micropaleontol. 113, 44–55,
10.1016/j.marmicro.2014.08.007, 2014.Hermoso, M., Candelier, Y., Browning, T. J., and Minoletti, F.: Environmental
control of the isotopic composition of subfossil coccolith calcite: Are
laboratory culture data transferable to the natural environment?, GeoResJ, 7, 35–42, 10.1016/j.grj.2015.05.002, 2015.Minoletti, F., Hermoso, M. and Gressier, V. Separation of sedimentary
micron-sized particles for palaeoceanography and calcareous nannoplankton
biogeochemistry, Nat. Protoc., 4, 14–24,
10.1038/nprot.2008.200, 2009
Paull, C. K. and Thierstein, H. R.: Stable isotopic fractionation among
particles in Quaternary coccolith-sized deep-sea sediments, Paleoceanography
2, 423–429, 10.1029/PA002i004p00423, 1987.Stoll, H. M. and Ziveri, P.: Separation of monospecific and restricted
coccolith assemblages from sediments using differential settling velocity.
Mar. Micropaleontol., 46, 209–221, 10.1016/S0377-8398(02)00040-3, 2002.Stoll, H. M., Rosenthal, Y., and Falkowski, P.: Climate proxies from Sr / Ca of coccolith calcite: calibrations from continuous culture of Emiliania huxleyi, Geochim. Cosmochim. Ac., 66, 927–936,
10.1016/S0016-7037(01)00836-5, 2002.Young, J. R. and Ziveri, P.: Calculation of coccolith volume and it use in
calibration of carbonate flux estimates, Deep Sea Res. Pt. II, 47, 1679–1700, 10.1016/S0967-0645(00)00003-5, 2000.Zhang, H., Stoll, H., Bolton, C., Jin, X., and Liu, C.: Technical note: A refinement of coccolith separation methods: measuring the sinking characteristics of coccoliths, Biogeosciences, 15, 4759–4775, 10.5194/bg-15-4759-2018, 2018.