Acta Protozoologica 2008 48/4
Jagiellonian University Kraków, Poland
© Jagiellonian University
Acta Protozool. (2008) 47: 287–291
Acta
Protozoologica
How Might Mixing Bias Protozoan-experiments that Use the Common
Micro-alga Isochrysis galbana?
Naomi Downes-Tettmar and David J.S. Montagnes
School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street Liverpool, UK
Summary. Although there are strong recommendations to mix micoalgal cultures, to improve productivity, there is only anecdotal evidence
to encourage protozoologists to mix cultures, to maintain constant quality of microalgae. Isochrysis galbana is extensively used in laboratory
experiments associated with applied and pure protozoan studies; thus, there is a need to assess how routine mixing may bias experiments. Although, mixing of cultures harms some microalgae, there is no indication of how mixing affects I. galbana. We address this problem by assessing
mixing effects on key experimental variables: specific growth rate, cell volume, nutritional quality (as carbon and nitrogen content and ratio),
and production (the product of growth rate and carbon or nitrogen content). Treatments, quantified as dissipation (ε) m3s-1 were: mixed once per
day (ε = 0), rotated on a plankton wheel (ε = 5.3 × 10-5), and shaken on agitating tables (ε = 8.2 × 10-3, 2.32 × 10-2). Mixing levels regularly used in
experimental work (no-mixing, plankton wheel, and low agitation) did not alter I. galbana specific growth rate and thus need not be carefully
controlled for, but exceptionally high-mixing did significantly reduce growth rate. Cell volume decreased at exceptionally high-mixing, but otherwise there was no size-bias caused by mixing. Carbon and nitrogen levels were highest at low-mixing but were otherwise generally unaffected
by mixing. The nutritional quality of I. galbana (i.e. carbon:nitrogen) remained unaltered by mixing. Production was doubled at low-mixing,
compared to all other treatments (which did not differ). Overall, I. galbana is a relatively robust species that withstands mixing levels well within
those experienced in the laboratory and still grows at exceptionally high-mixing levels. Thus, within reason, protozoologists using I. galbana do
not need to be rigorous in their monitor of mixing. Furthermore, meta-analyses that examine this species need not be too concerned with variation
in mixing methods as a confounding factor. However, the data suggest that gentle mixing, using a plankton wheel, is preferable to no-mixing.
Key words: Biochemical composition, carbon, experimental design, growth, nitrogen, phytoplankton, production, nutritional quality.
INTRODUCTION
Although there are strong recommendations to mix
micoalgal prey cultures to maintain their constant quality (Stein 1973), there is only anecdotal evidence to
encourage protozoologists to mix their prey. In fact,
key texts that outline routine maintenance methods
Address for correspondence: D. Montagnes, School of Biological
Sciences, University of Liverpool, Biosciences Building, Crown
Street Liverpool, UK, L69 7ZB
Telephone number: (44) 151 794 7926; E -mail: [email protected].
(e.g. Fogg 1965, Stein 1973, Boney 1975, Becker 1994,
Andersen 2005) fail to provide guidance regarding the
application, extent, or consequences of mixing. There
is, admittedly, a belief by many protozoologists that
mixing of cultures is important, but to what extent?
Here we address this question, focusing on an extensively used experimental species in marine protozoology, to provide guidance for future work.
Since its description ~60 years ago (Parke 1949),
Isochrysis galbana has been extensively and routinely
used in laboratory experiments associated with applied
and pure marine sciences; every year 100s of studies
Acta Protozoologica 2008 48/4
Jagiellonian University Kraków, Poland
© Jagiellonian University
288 N. Downes-Tettmar and D.J.S. Montagnes
examine this species. In these works, I. galbana is often employed as a model to investigate various algal
processes (e.g. light responses, Thompson et al. 1990;
temperature responses, Montagnes and Franklin 2001;
clonal differences Sayegh et al. 2007). Furthermore, for
the last 35 years I. galbana has routinely been used as
a prey for a wide range of marine protozoa (e.g. Gold
1973, Stoecker et al. 1988, Bernard and Rassoulzadegan 1990, Verity 1991, Christaki et al. 1996, Montagnes 1996, Dolan and Šimek 1997, Jakobsen and
Hansen 1997, Strom and Morello 1998, Klein Breteler
et al. 1999, Johansson and Coats 2002, Kimmance
2006, Guermazi et al. 2008). The quality of I. galbana
grown in experimental culture is thus a focus of concern, and studies have examined the impact of several
key environmental variables (e.g. light, temperature,
salinity, nutrients) on a range of attributes, including
specific growth rate, composition (e.g. carbon and nitrogen content), predator-prey dynamics, and prey choice
(e.g. Thompson et al. 1990, Montagnes and Franklin
2001, Montagnes et al. 2001).
Here we test the hypothesis that mixing will affect
the outcome of experimental studies that use I. galbana, as mixing microalgal cultures, in general, can alter
their quality by reducing nutritional gradients, improving gas transfer, preventing sedimentation, and ensuring equal light exposure (Jiménez et al. 1996), but excessive mixing can be detrimental to some microalgae,
disrupting nutrient uptake, causing cell wall damage,
inducing filament loss, and increasing mortality (Thomas and Gibson 1990, Borowitzka 1997). If I. galbana
responds like other microalgae, then this is a concern
for experimental protozoologists, and if it is not affected, then this observation will provide extremely useful
time-saving guidance for researchers.
Surprisingly, virtually nothing is known regarding
mixing effects on I. galbana, except that it is not affected
by low-level turbulence (i.e. a dissipation rate of 10-4 m2 s3
; Havskum 2003). A review of the literature also reveals
that a wide range of mixing levels are used in experimental work, and these are rarely quantified: some studies mix
cultures once a day, others continuously mix them, and in
many studies mixing it is not mentioned (e.g. Davidson et
al. 1992, Kleppel and Burkart 1998, Martel 2006). Thus,
we test if variation in mixing could alter the outcome of
experiments that use I. galbana, and determine if future
experiments should carefully consider mixing. To this
end, we have assessed the impact of mixing I. galbana
cultures over a range of levels that cover, and extend beyond, the spectrum potentially used in laboratory incubations. We have focused on examining mixing effects on
key experimental variables examined by others and applicable to protozoan-related experiments: growth rate, cell
volume, carbon and nitrogen content of cells (as proxies
of nutritional quality), and production (the product of
growth rate and carbon or nitrogen content).
Methods
Isochrysis galbana Parke 1949, CCAP 927/1 was maintained
in artificial seawater, enriched with f/2 (Guillard 1972) at 16 ± 1°C
under a constant irradiance of 85 µmol photons m-2s-1. Cultures
(sample volume 175ml) were grown in 250 ml plastic, rectangular
tissue culture flasks. Four replicated treatments were applied (n = 3):
non-mixing, where flasks were only mixed (by gentle rotation) once
daily when sampled; low-mixing, where flasks were rotated on
a vertically oriented ‘plankton wheel’; medium and heavy mixing,
where flasks were placed on horizontal agitating tables that moved
back and forth, rather than rotating (Table 1).
For the agitated samples, mixing was characterised as dissipation (ε, m2 s-3); ε = U 3/L, where U, the velocity (m s-1), was determined from the speed of agitation (Table 1), and L, length (m), was
determined as the cube-root of the sample volume (0.056 m). For the
rotated samples (low-mixing), turbulence was also assessed as dissipation, but this was a more complex calculation. In this case U was
determined from the kinetic energy of rotation of the fluid and the
bottle together, in the first instance assuming it was a ridged body;
then assuming that the fluid rotates with the bottle, kinetic energy
was converted to velocity by U = (0.5 Iω/Mw)0.5, where Mw is the mass
of the water, ω is the angular rotation (2π/rotation time), and I is
the moment of inertia, which is (Mw + Mf)r2 (where r is the radius of
rotation and Mf is the mass of the flask), but assuming Mw >> Mf this
approximates to Mwr2; U then simplifies to (0.5r2ω)0.5. Furthermore,
mixed samples are likely to experience shearing as well as a dissipation effect (e.g. Havskum 2003); thus we have made estimates
Table 1. Characteristics of the four mixing regimes that Isochrysis galbana was exposed to. Calculations are described in the text.
Regime
Method
Displacement (m)
Cycles
Velocity (U, m s–1)
Dissipation (ε, m2s–3)
Shear (S, s–1)
–
–
–
~0
~0
None
Mixed daily
Low
Rotated on wheel
Wheel radius = 0.115
Rotation time 202 s
0.014
5.3 × 10–5
0.25
Medium
Shaker table
Shaking distance = 0.06
77 cycles min
0.077
8.2 × 10
1.38
High
Shaker table
Shaking distance = 0.06
109 cycles min–1
0.109
2.32 × 10–2
–1
–3
1.95
Acta Protozoologica 2008 48/4
Jagiellonian University Kraków, Poland
© Jagiellonian University
Mixing and Isochrysis galbana 289
of shear rate (S, s-1), using the above parameters (i.e. S = U/L, see
Table 1). However, for the purpose of this study we have focused on
dissipation (ε) as a parameter to quantify mixing. See Tennekes and
Lumley (1972) for a more detailed description of these estimates.
Specific growth rates (µ, d-1) were determined by regressing ln
cell numbers vs. time, over the exponential growth phase. Cell volumes were determined from linear dimensions of >30 live cells, harvested from exponentially growing cultures, using an image analysis
system (Scion image for Windows, Scion Corp., MD, USA); cells
were assumed to be prolate spheroids. Carbon and nitrogen content
(pg cell-1) was determined from duplicate samples, from each replicate, by elemental analysis (NC 2500, CE Instruments). The C:N
ratio, C and N per unit volume, and C and N production (i.e. the
product of specific growth rate and biomass) were then determined.
Treatment effects on the above measurements were compared
using one way ANOVA, followed by pairwise comparisons (Holmsidak method, α = 0.05, n = 3). All datasets were normally distributed and homoscadastic; Kolmogorov-Smirnov test).
We have addressed this issue by assessing growth rate,
volume, nutritional quality, and production at a range of
mixing levels that cover and extend above those typically
used in experimental laboratory practices (Table 1, Richmond 1987); each of these is, in turn, addressed below.
Growth rate is undoubtedly the most measured parameter associated with examining microalgae; for
I. galbana it is used as an index of physiological response (e.g. to temperature Montagnes and Franklin
2001), as a component of population dynamic studies
and ecosystem models (e.g. Kimmance et al. 2006), and
as an integral part of other measurements (e.g. determination of grazing on microalgae, Kimmance et al. 2006).
We found that I. galbana growth rate decreased due to
heavy mixing (a level far beyond that typically applied
to laboratory cultures) but remained unaffected by the
Results
There was a significant decrease in specific growth
rate of I. galbana between the lowest and highest mixing
levels (Fig. 1a), but non-mixing was not significantly
different from low-mixing or medium-mixing. Cell volume was significantly higher in low-mixing compared
with high-mixing, and there was an apparent decrease in
volume with mixing, although there was no significant
difference between all the other treatments (Fig. 1b).
The carbon and nitrogen analysis demonstrated that at
low-mixing C and N cell quota was significantly higher
than all other treatments, and there were no significant
differences across the other mixing treatments (Fig. 1c).
There was no significant effect of mixing on C:N ratio
(Fig. 1d). The estimates of C and N per unit volume were
significantly higher at high-mixing, and there were no significant differences between the other mixing treatments
(Fig. 1e). Production (in terms of both C and N) was significantly higher at low-mixing compared with the other
mixing treatments, and there were no differences between
the other mixing treatments with the exception of N production where there was a small but significant difference
between medium-mixing and high-mixing (Fig. 1f).
Discussion
Given the extensive use of Isochrysis galbana in
experimental protozoological works (see Introduction),
there is a need to assess if routine maintenance will alter culture parameters and potentially bias experiments.
Fig. 1. A comparison of Isochrysis galbana responses to mixing.
a – comparison of the specific growth rates (µ, d-1); b – comparison of­ cell volume (µm3); c – comparison of the total carbon and
nitrogen contents (pg cell-1); d – comparison of C:N ratios; e – comparison of the carbon and nitrogen content per unit volume (C, N pg
µm-3); f – comparison of production C, N pg d-1). Error bars are one
standard error. See Table 1 and Methods for a detailed explanation
of treatments. In Figs 1c, e, and f, carbon is represented by (●), and
nitrogen is represented by (▲).
Acta Protozoologica 2008 48/4
Jagiellonian University Kraków, Poland
© Jagiellonian University
290 N. Downes-Tettmar and D.J.S. Montagnes
other mixing treatments. This decrease in growth rate at
an excessively high level could result from increased cell
lyses and damage at the air surface interface (Havskum
2003) or hydrodynamic stress acting on the cells causing
a physiological disruption (Thomas and Gibson 1990).
This might be relevant if studies were interested in the
impact of breaking waves on shallow-water, coastal
populations, but otherwise mixing would have little ecological or laboratory significance. Clearly, I. galbana is
a robust species, compared to other more sensitive microalgae (Thomas and Gibson 1990, Borowitzka 1997).
Small changes in prey cell size will also alter experiments with protozoa; e.g. prey choice and grazing
experiments may be influenced by prey size (e.g. Jonsson 1986) and changes in volume may alter the cell nutritional quality (see below). However, I. galbana cell
volume remains unaffected by lower mixing levels and
only decreases with the influence of heavy mixing. Possibly heavy mixing inhibits nutrient uptake at the cell
surface thus selecting for smaller cells, or possibly the
small cell size is an adaptive mechanism to reduce cell
lyses (see above). Regardless, of the cause, there does
appear to be a potential size-bias caused by mixing.
Protozoologists must also recognise the potential
impact of mixing, on the nutritional quality of I. galbana; this is often determined by examining total carbon
(C) and nitrogen (N) cell content (e.g. Mitra and Flynn
2005). We found that C and N quotas per cell reflect the
overall trend observed in cell volume, with large cells
at low-mixing having the highest C and N levels and
small cells at high-mixing having the lowest C and N
quotas. However, in terms of C and N per unit volume
the relative densities of C and N were the same between
non- and medium mixed treatments, but at high-mixing the smaller cells were more densely packed with
C and N. We also found that there was no significant
stoichiometric change in C:N ratio of cells; this ratio
can be used as an index of prey quality, and our data
thus suggests that, in this respect, mixing will have little
influence on predator responses to I. galbana. Consequently, while mixing may affect growth rate and cell
volume at high-mixing, the overall nutritional quality
of I. galbana remains unaltered.
Finally, many experimental measurements (e.g. gra­
zing rates, population dynamics) require estimates of
production, rather than simply growth rate, as external
stimuli may influence both growth rate and nutritional
quality of cells (e.g. Montagnes and Franklin 2001).
Furthermore, there are many cases where production
needs to be maximised, such as when rearing batch cul-
tures of algae as food in grazing and prey choice experiments (Montagnes et al. 2001). We found a clear trend
across mixing treatments: as mixing is increased there
is a decrease in production, although unmixed cultures
had a significantly lower production than gently mixed
ones. Note that production, being the derived product
of growth rate and carbon or nitrogen content of cells
accentuates the trends exhibited by these independently. Our results illustrate that low-mixing approximately
doubles the productivity of cultures, supporting the case
to use plankton wheels to optimise production.
In conclusion, mixing effects on I. galbana quality
are contrary to those expected from past studies, which
indicate that microalgae can be highly sensitive to mixing (see Introduction). In contrast, I. galbana is a robust
species that withstands low to medium-mixing and is
not prevented from growth even at unrealistically highmixing levels. This suggests that within reason, protozoologists, and other researchers, using I. galbana,
do not require rigorous monitoring of mixing and that
meta-analyses that compare the many studies on this
species (e.g. Sayegh et al. 2007) need not be concerned
with variation in mixing methods.
Even though we suggest mixing variations are not
important, we still found effects of extreme mixing
levels and, therefore, can provide some guidance. For
instance, I. galbana was deleteriously affected by our
high-mixing treatment; fortunately, this level is rarely, if
ever, used in the laboratory and would even be beyond
levels experienced when samples are shipped in the
post. Furthermore, we found that low-mixing, which reflects most laboratory conditions, is the most favourable
treatment in terms of maximising cell size, production,
and C and N content. Finally, there was a clear indications that continuous low-mixing, provided more productive cultures with cells containing more carbon and
nitrogen than non-mixed (shaken once per day) cultures.
Many researchers do not continuously mix their cultures, and this may result in some bias. Thus, we recommend that although I. galbana is relatively insensitive
to mixing, experimental work should probably be conducted on gently mixed cultures, using a plankton wheel
(e.g. Crocker and Gotschalk 1997), which provides homogeneous mixing at extremely low levels; i.e. lower
than those achievable on a horizontal mixing plate.
Acknowledgements. We thank Jason Dzikowski, Jan Bissinger,
Sam Fielding, David Lewis, and Fabienne Marret for comments
and assistance. We also thank the Esmée Fairbairn Foundation, the
British Ecological Society, and the University of Liverpool Resarch
Development Fund for financial assistance.
Acta Protozoologica 2008 48/4
Jagiellonian University Kraków, Poland
© Jagiellonian University
Mixing and Isochrysis galbana 291
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Received on 20th June, 2008; revised version on 23rd September,
2008; accepted on 25th September, 2008