CSIRO PUBLISHING
Research Paper
Environ. Chem. 2013, 10, 91–101
http://dx.doi.org/10.1071/EN12191
Influence of culture regime on arsenic cycling
by the marine phytoplankton Dunaliella tertiolecta
and Thalassiosira pseudonana
Elliott G. Duncan,A,B William A. Maher,A Simon D. FosterA and Frank KrikowaA
A
Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra,
University Drive, Bruce, ACT 2601, Australia.
B
Corresponding author. Email: [email protected]
Environmental context. Phytoplankton form the base of marine food-webs, and hence they have been
proposed as the likely source of many arsenic compounds found in marine animals. Because of the difficulties
associated with field experiments with phytoplankton, attempts to test this hypothesis have relied mainly on
laboratory experiments. This study assesses the environmental validity of this research approach by investigating
the influence of the culturing experimental protocol on the uptake, accumulation and biotransformation of
arsenic by marine phytoplankton.
Abstract. Arsenic cycling by the marine phytoplankton Dunaliella tertiolecta and the marine diatom Thalassiosira
pseudonana was influenced by culture regime. Arsenic was associated with the residue cell fractions of batch cultured
phytoplankton (D. tertiolecta and T. pseudonana), due to the accumulation of dead cells within batch cultures. Greater
arsenic concentrations were associated with water-soluble and lipid-soluble cell fractions of continuously cultured
phytoplankton. Arsenoribosides (as glycerol (Gly-), phosphate (PO4-) and sulfate (OSO3-)) were ubiquitous in
D. tertiolecta (Gly- and PO4- only) and T. pseudonana (all three species). Additionally, arsenobetaine (AB) was not
detected in any phytoplankton tissues, illustrating that marine phytoplankton themselves are not an alternate source of AB.
Arsenic species formation was influenced by culture regime, with PO4-riboside produced under nutrient rich conditions,
whereas Dimethylarsenoacetate (DMAA) was found in old (.42 days old) batch cultures, with this arsenic species
possibly produced by the degradation of arsenoribosides-arsenolipids from decomposing cells rather than by biosynthesis.
Nutrient availability, hence culture regime was thus influential in directly and indirectly influencing arsenic cycling and
the arsenic species produced by D. tertiolecta and T. pseudonana. Future research should thus utilise continuous culture
regimes to study arsenic cycling as these are far more analogous to environmental processes.
Additional keywords: arsenic species, arsenoribosides, batch culture, continuous culture, lipid-soluble arsenic.
Received 6 December 2012, accepted 9 February 2013, published online 18 April 2013
Introduction
Chemostats, also known as continuous phytoplankton cultures, maintain ‘steady-state’ phytoplankton growth by the
constant replenishment of nutrient resources coupled with the
subsequent removal of algal culture.[14,15] This process is more
analogous to natural processes[12] and as a result chemostats
have been used to investigate the cycling of essential nutrients
such as silicon, phosphorus and nitrogen by both marine and
freshwater phytoplankton.[11,14,16–19] Chemostats have, however,
never been used to investigate arsenic cycling.
This study investigates how arsenic uptake, accumulation
and the formation of arsenic species in the marine phytoplankton
Dunaliella tertiolecta and marine diatom Thalassiosira pseudonana vary when cultured under different batch and continuous culture regimes. This study specifically aims to illustrate if
arsenic uptake, accumulation and species formation by marine
phytoplankton can be influenced by culture regime, thus creating a platform to assess the environmental validity of different
Phytoplankton form the base of most marine pelagic foodwebs[1] and consequently are believed to be the source of many
of the precursors of the arsenic species (e.g. arsenoribosides)
found in higher marine organisms such as arsenobetaine
(AB)[2–4] (Fig. 1).
Arsenic cycling by marine phytoplankton is, however, difficult to study in situ and as a result all existing information has
been generated by laboratory-based batch culture experiments.[2,5–10] Batch phytoplankton cultures are an effective
experimental approach in the investigation of arsenic uptake,
accumulation and the formation of arsenic species in short-term
experiments as all arsenic and nutrient inputs can be easily
traced from media to phytoplankton.[11] Batch cultures, however,
represent a closed system[12] which does not adequately mimic
the cycling of elements (i.e. arsenic) and nutrients between the
atmosphere, water-column, sediments and biota.[12,13]
Journal compilation Ó CSIRO 2013
91
www.publish.csiro.au/journals/env
E. G. Duncan et al.
O
As
HO
OH
As
HO
OH
As
As
HO
CH3
CH3
OH
OH
OH
CH3
AsV
MA
DMA
Arsenite
Arsenate
Methylarsonate
Dimethylarsinate
CH3
As
H3C
CH3
O
O
OH
OH
As⫹
CH3
H3C
H3C
As
CH3
CH3
TMAO
TETRA
Tetramethylarsonium ion
DMAA
Dimethylarsinoylaceticacid
Dimethylarsinoylethanol
CH3
OH
H3C
O
CH3
CH3
OH
As⫹
H 3C
TMAP
OH
As⫹
O
CH3
CH3
Trimethylarsoniopropionate
O
CH3
DMAE
CH3
As⫹
As
CH3
Trimethylarsine oxide
H3C
HO
O
AsIII
O
H3C
O
AC
AB
Arsenocholine
Arsenobetaine
Dimethylated arsenoribosides:
O
H3C
O
As
OR
R⫽
Glycerol
OH
OH
CH3
O⫺
O
HO
OH
Phosphate
R⫽
Dimethylated thio-arsenoribosides:
O
As
O
OH
S
H3C
P
OR
Sulfonate
R⫽
O
OH
OH
SO3OH
CH3
HO
OH
Sulfate
Trimethylated arsenoribosides:
R⫽
OSO3OH
CH3
H3C
As⫹
O
OR
CH3
HO
OH
Fig. 1. Structures of common arsenic species.
experimental approaches used to investigate arsenic cycling by
marine phytoplankton.
regimes as listed in Table 1. Extended details on the rationale
behind the culture regimes and phytoplankton species used in
this study can be found in the Supplementary material.
Methods
Experimental design
The marine phytoplankton Dunaliella tertiolecta and Thalassiosira pseudonana were cultured under five different culture
Phytoplankton culture maintenance
Dunaliella tertiolecta cultures were obtained from the CSIRO
Centre for Analytical Chemistry (CSIRO, Lucas Heights
92
Arsenic cycling in marine phytoplankton cultures
Table 1. Culture regimes, nutrient and arsenic treatments used to culture D. tertiolecta and T. pseudonana within this study
Culture type
Batch
Continuous þ nutrients þ AsV
Continuous þ nutrients
Continuous þ AsV
Heat-treated batch
Nutrient treatment
f/2
f/2
f/2
None added
f/2
Arsenic treatment
1
2–5 mg L As
2–5 mg L1 AsV
None added
2–5 mg L1 AsV
2 mg L1 AsV
V
Science and Technology Centre, NSW, Australia), whereas
Thalassiosira pseudonana cultures were obtained from CSIRO
Marine and Environmental Research (Hobart, Australia).
All phytoplankton stock cultures were operationally sterile
(defined in this context as containing no culturable bacteria on
marine PYEA, peptone yeast-extract agar) and were prepared
using autoclave-sterilised 0.3-mm filtered seawater f/2 culture
media[20] under aseptic conditions in a laminar flow hood
(Gelaire, BSB-12, Sydney, Australia). Cultures were incubated
in plant growth chambers (3504 process controller, Eurotherm,
Sydney) under a light intensity of ,110 mmol photons m1 s1,
over a 12-h light exposure period and a temperature regime which
increased from 20 to 25 8C during light periods and was held at
20 8C during dark periods, with salinity maintained at 35 %.
Cultures were shaken daily to ensure cell motility, with
T. pseudonana cultures grown on magnetic stirrer plates (Industrial Equipment and Control Pty Ltd, Melbourne) to ensure
culture mixing. All stock cultures plus all prepared media were
tested for the presence of culturable microorganisms using
marine PYEA marine agar plates.[6]
Incubation periods
Phytoplankton species studied
4, 7, 42 days
7 (T. pseudonana only), 42 days
42 days
7, 42 days
7 days
D. tertiolecta and T. pseudonana
D. tertiolecta and T. pseudonana
D. tertiolecta
T. pseudonana
D. tertiolecta
Flow direction
Air pump
Nutrient ⫹
reservoir
Peristaltic
pump
AsV
Culture vessel
Collection vessel
Fig. 2. Diagrammatic illustration of the continuous culture set up used in
this study. Black lines and arrows indicate flow directions.
At this point (Day 4) the remaining two D. tertiolecta
cultures were amended with 300 mL of 0.2-mm filtered,
20 mg L1 AsV solution, which equated to an arsenic concentration of 2 mg L1 in each culture. The remaining T. pseudonana
cultures were amended with 500 mL of 0.2-mm filtered, 20 mg L1
sterile AsV solution, which equated to an arsenic concentration
of 5 mg L1 in each culture.
After a further 3 days of incubation (Day 7) a randomly
selected culture was removed from the chamber and harvested
as per the non-arsenic dosed (Day 4) culture, with this process
repeated with the final culture after 42-days incubation.
In addition, two batch D. tertiolecta cultures, within two
replicate experiments (n ¼ 2), were heat treated to create cultures containing purely dead cell material. These cultures were
created as per experimental cultures and incubated for 4 days
under the same environmental conditions. After 4-days incubation, all cultures were autoclaved (Tangent Tiger, Atherton,
Melbourne, Australia) at 121 8C for 15 min to ensure all
D. tertiolecta cells had been lysed. Cultures were allowed to
cool and were then spiked with 300 mL of 0.2-mm filtered,
20 mg L1 AsV solution which equated to an arsenic concentration of 2 mg L1 in each culture. Cultures were then re-incubated
for a further 3 days, with the dead cells harvested as per live
D. tertiolecta cells.
Batch culture preparation
In total 100 mL of D. tertiolecta and T. pseudonana culture was
transferred aseptically to sterile 5- and 2-L Erlenmeyer flasks
respectively containing 3 and 1 L of autoclaved sterilised 0.3-mm
filtered, f/2 seawater nutrient media.[20] All experimental cultures were operationally sterile upon the commencement of
experiments as identified through the use of PYEA marine agar
plates.[6] Cultures were also tested at the conclusion of the
incubation process for the presence of culturable bacteria;
however, as per the paper by Levy et al.,[21] cultures were likely
to have contained bacteria throughout the incubation process.
All cultures were incubated in an environmental chamber (3504
process controller, Eurotherm) under the same environmental
conditions described previously (see Phytoplankton culture
maintenance).
Three cultures were prepared per experiment (n ¼ 2) and
were randomly placed and moved throughout the chamber to
avoid the effects of any potential light and temperature gradients. To ensure culture mixing both D. tertiolecta cultures were
shaken daily by hand, whilst T. pseudonana cultures were placed
on magnetic spinner plates (Industrial Equipment and Control
Pty Ltd, Australia).
After 4 days incubation one culture was randomly selected
and removed from the chamber, with cells and media harvested
by centrifuge at 4500g at room temperature (20–21 8C) for
10 min (Eppendorf centrifuge 5804, Hamburg, Germany). Cells
and culture media were frozen, with cell tissue subsequently
freeze-dried (Labconco, Kansas City, KS, USA) and stored for
analysis. This culture acted as a non arsenic-dosed sample to
establish initial arsenic concentrations and species present in
D. tertiolecta and T. pseudonana.
Continuous culture preparation
Operationally sterile D. tertiolecta and T. pseudonana culture
(200 mL) was transferred aseptically to sterile 2-L Schott
bottles, containing 2 L of 0.3-mm filtered, autoclaved, sterilised
f/2 seawater nutrient media (Figs 2, S2).[20] Two cultures were
prepared per replicate experiment (n ¼ 2) and were incubated as
per batch cultures for 4 days in order to generate sufficient cell
biomass to avoid culture washout. These cultures were grown
under the same environmental conditions and tested for the
presence of culturable bacteria as described previously for batch
cultures.
93
E. G. Duncan et al.
Foster et al.,[26,28] and Kirby et al.[29] (Table S1). Individual
arsenic species standards used in analysis were synthesised as
per the literature procedures.[30–37]
Quality assurance and quality control (QAQC) values for
total arsenic, total phosphorus and arsenic species analysis are
also presented in the Supplementary material, with HPLC
column recoveries in Table S2.
After 4-days incubation both D. tertiolecta cultures were
constantly supplied with sterile f/2 seawater nutrient media by a
peristaltic pump (RP-150 series, Lachat, Loveland, USA) at a
flow rate of ,1 L day1 (Figs 2, S2). One of the two cultures was
also supplied with sterile AsV equating to a concentration of
2 mg L1 AsV within the culture, whilst the other culture
received no additional AsV, with the arsenic present in this
culture present in the seawater (,0.3 mg L1) used as the base
for the f/2 nutrient media.
For T. pseudonana, both cultures were continually supplied
with sterile AsV at a concentration of 5 mg L1 with one culture
constantly supplied with sterile f/2 seawater nutrient media by a
peristaltic pump (RP-150 series, Lachat, Loveland, USA) at a
flow rate of ,1 L day1 (Figs 2, S2). The other culture was
supplied with sterile seawater containing no additional nutrients
also at a flow rate of 1 L day1. T. pseudonana cultures were
placed on magnetic stirrer plates (Industrial Equipment and
Control Pty Ltd) to ensure mixing of cultures.
All continuous cultures were aerated using 0.2-mm filtered
air by an air pump (Resun LP-60, Shenzen, China) (Figs 2, S2),
which also aided in mixing, and fulfilling oxygen requirements
(Fig. 2). All cultures were grown for 42 days, with algal cells and
media harvested daily using the same technique described for
batch cultures.
Statistical analyses
All graphs were generated using Microsoft Excel software
(Microsoft, Redmond, CA, USA).
Results and discussion
Behaviour and growth of D. tertiolecta and
T. pseudonana in batch and continuous cultures
The growth of D. tertiolecta was similar across the different
culture regimes, with viable cell numbers in all three treatments
of ,9 105 cell mL1 after 42-days incubation (Fig. 3).
The growth of T. pseudonana under different culture
regimes, however, differed considerably (Fig. 3). Viable cell
populations in nutrient-supplied continuous cultures peaked at
,1 106 cells mL1 throughout the first 21 days of incubation,
with cell numbers gradually falling to ,3 105 cells mL1 by
the conclusion of the experiment (Day 42). Viable cell numbers
in T. pseudonana continuous cultures supplied with AsV but no
nutrients also peaked at ,1 106 cells mL1, however, in this
case viable cell numbers rapidly decreased over time, with
viable cell numbers of 4 104 cells mL1 present in cultures
from Day 21 onwards (Fig. 3). Viable cell numbers in batch
T. pseudonana cultures were typically lower (7 105 cells mL1)
than peak populations in continuous cultures (Fig. 3). Unlike in
continuous cultures, however, viable cell numbers in batch
Growth determination
The growth of both D. tertiolecta and T. pseudonana cultures
was measured by cell counts using a haemocytometer slide as
described in Foster et al.[6] As well as measuring cell numbers,
cells of both D. tertiolecta and T. pseudonana were also analysed for viability (i.e. in-tact cells), with motile and actively
dividing cells present throughout the 42-day experimental
period. In addition autoclaved-treated cultures were also
examined to ensure that no live cells were present in these
cultures.
1000.0
Analytical methods
Full details of the various analytical methods used in this study
can be found in the Supplementary material.
In brief, the digestion of phytoplankton tissue for cellular
total arsenic and phosphorus determination was performed
using concentrated nitric acid and microwave heating as
described by Baldwin et al.[22] Cellular total arsenic and phosphorus concentrations were measured by inductively coupled
plasma mass spectroscopy (ICP-MS) as per Maher et al.[23]
Total phosphorus (TP) and total nitrogen (TN) concentrations in microbial culture media were determined by alkaline
peroxodisulfate digestion and analysed by flow injection analysis as per Maher et al.[24]
Arsenic within the different biochemical fractions of
D. tertiolecta and T. pseudonana cells were separated using
2 : 1 (v/v) chloroform/methanol to separate lipid-soluble and
water-soluble arsenic as per Folch et al.[25] Arsenic associated
with residual cell fractions was extracted with 2 % HNO3 at
95 8C as per Foster et al.[26]
Total arsenic concentrations within lipid-soluble, watersoluble and residue cell fractions of phytoplankton tissue was
measured by electrothermal atomic absorption spectroscopy as
per Deaker and Maher.[27]
The arsenic species within lipid-soluble, water-soluble and
residue cell fractions of phytoplankton tissue were measured by
high pressure liquid chromatography (HPLC) ICP-MS as per
100.0
10.0
Cells ⫻ 104 mL⫺1
1.0
0
7
14
21
28
35
42
0
7
14
21
28
35
42
0.1
1000
100
10
1
0.1
Culture age (days)
Fig. 3. Culture growth of D. tertiolecta (Top) and T. pseudonana (Bottom)
when grown under batch cultures (black-solid), continuous cultures constantly supplied with f/2 nutrient media and AsV (grey) and continuous
cultures constantly supplied with either supplied AsV or nutrients but not
both (black-broken). Values are means (n ¼ 2) represented on a logarithmic
scale standard deviation.
94
Arsenic cycling in marine phytoplankton cultures
freshwater chlorophyte Chlorella sp.[41] In both of these studies
total arsenic concentrations peaked during the middle of the
incubation period (Nostoc sp. – Day 12, Chlorella sp. – Day 8)
and then decreased steadily over the remainder of the 21-day
experimental period.[40,41]
The most probable explanation as to why this scenario
occurred involves the accumulation of dead cells in batch
cultures.[12] Viable cell numbers in batch D. tertiolecta cultures
remained largely constant over time (Fig. 3), whereas T. pseudonana cell numbers decreased slightly (Fig. 3), however, over
time it was observed that the amount of dead cell material in
batch cultures noticeably increased. Consequently, the decrease
in total cellular arsenic concentrations in batch cultures was
probably a dilution effect resulting from the accumulation of
dead cell material, which illustrates a limitation with the use of
batch cultures to investigate active arsenic cycling.
Total arsenic concentrations in heat-treated batch D. tertiolecta cultures were similar (,6 mg g1 dry mass) to those of live
D. tertiolecta batch cultures particularly after 42-days incubation (Fig. 4). This provides a degree of confirmation that arsenic
is associated with both live and dead cells in laboratory-based
cultures, as has been shown to occur for phosphorus.[38] This
notion is enhanced by the observation that most of the arsenic
(50–80 % of total arsenic) associated with batch cultured phytoplankton is associated with residue cell fractions, which is also
the case in heat-treated D. tertiolecta tissue (76 6 %) (Fig. 4).
T. pseudonana cultures peaked after 42 days rather than in the
first 21 days of incubation (Fig. 3).
With the exception of the continuous T. pseudonana cultures
supplied with AsV and no added nutrients (Fig. 3), viable cell
numbers for both phytoplankton species were similar after 42days incubation across the different culture regimes (Fig. 3).
This is despite TP/TN concentrations in the culture media falling
considerably over time in batch cultures, whereas concentrations unsurprisingly remained more stable in continuous cultures (Figs S2–S4). This illustrates that the behaviour of
D. tertiolecta and T. pseudonana varied between culture
regimes with growth in continuous cultures being regulated by
the nutrient replenishment or dilution rate as described in many
previous publications,[11,14,15,17,19] whereas in batch cultures
nutrient sequestration (Fig. S5) is the likely means by which cell
growth is maintained despite reductions in external nutrient
resources (Figs S2–S4), which also is in agreement with past
research.[17,19,38,39]
Arsenic uptake and accumulation by D. tertiolecta
and T. pseudonana in batch and continuous cultures
Total arsenic concentrations in batch cultured D. tertiolecta and
T. pseudonana were similar (,7–11 mg g1 dry mass), and also
followed a similar pattern over time (Fig. 4). This represents a
similar scenario to that described within long-term batch cultures of the freshwater cyanobacteria Nostoc sp.[40] and the
D. tertiolecta total cellular As
(µg g⫺1 dry mass)
16
14
12
10
8
6
4
2
0
T. pseudonana total cellular arsenic
(µg g⫺1 dry mass)
Batch 4
(pre-AsV)
Batch 7
Batch 42
Continuous
42
Continuous 42
(no AsV)
Dead cell
12
10
8
6
4
2
0
Batch 4
(pre-AsV)
Batch 7
Batch 42
Continuous Continuous Continuous Continuous
7
42
7
42
(no nutrient) (no nutrient)
Culture regime
Fig. 4. Total arsenic concentrations (mg g1 dry mass) associated with water-soluble (white), lipidsoluble (grey) and residue (black) cell fractions of D. tertiolecta (top) and T. pseudonana (bottom) grown
under batch and continuous culture regimes. Values are means standard deviations (n ¼ 2).
95
E. G. Duncan et al.
To complicate matters, however, T. pseudonana behaved in a
different manner to D. tertiolecta in terms of arsenic uptake and
accumulation (Fig. 4). Total arsenic concentrations in continuously cultured T. pseudonana were lower (3–8 mg g1 dry mass)
than was found in batch cultures (9–11 mg g1 dry mass) (Fig. 4).
In addition, arsenic concentrations in water-soluble cell fractions (,1–1.5 mg g1 dry mass) were similar across treatments
and over time (Fig. 4), with concentrations in the lipid-soluble
fractions (,2 mg g1 dry mass) also similar with the exception
of concentrations in 42-day nutrient-supplied continuous cultures (,4 mg g1 dry mass) (Fig. 4).
It is hard to explain why arsenic uptake and accumulation
differed so distinctly between T. pseudonana and D. tertiolecta
under similar culture conditions (Fig. 4). To speculate, a possible explanation could reside in ecological differences between
the two species in question. D. tertiolecta is known to typically
inhabit very hostile environments such as tidal pools and
lagoons,[8,46–48] in which nutrient concentrations are known to
fluctuate.[49] To survive in these ecosystems D. tertiolecta
would have needed to evolve efficient nutrient utilisation
processes, which subsequently explains why under periods of
nutrient availability D. tertiolecta did not accumulate arsenic in
lipid-soluble fractions.
As T. pseudonana is a more oceanic species[48] it may have
accumulated arsenic in the lipids as a result of the ‘boom–bust’
response to growth nutrients exhibited by many phytoplankton
species. Phytoplankton typically inhabit nutrient poor environments,[50] thus many species are programmed to accumulate and
sequester nutrients (which as per recent research[44] could
include arsenic) in internal pools at concentrations that regularly
exceed physiological requirements.[11,38,39] These sequestered
nutrients form the basis of how phytoplankton are able to survive
under conditions of nutrient depletion.[50]
Consequently, total arsenic concentrations in laboratorycultured phytoplankton almost certainly represent the flux of
arsenic from media to cell tissue (live or dead) and as a result
provide limited information on active arsenic cycling.
Total arsenic concentrations and the concentrations of arsenic within different biochemical cell fractions of both
D. tertiolecta and T. pseudonana were more variable under
continuous culture regimes (Fig. 4). Total arsenic concentrations were higher for continuously cultured D. tertiolecta
(,13 mg g1 dry mass) than were found in batch-cultured
D. tertiolecta (7–11 mg g1 dry mass) and in addition greater
arsenic concentrations were found in the water-soluble cell
fractions (,5 mg g1 dry mass) than was the case in batch
cultures (,1.5 mg g1 dry mass) (Fig. 4). Conversely, far greater
arsenic concentrations were found in the lipid-soluble cell
fractions of D. tertiolecta when cultured under batch culture
conditions (,1–3 mg g1 dry mass) than under continuous
culture conditions (,0.5 mg g1 dry mass) (Fig. 4).
The high concentrations of arsenic in the water-soluble cell
fractions of continuously cultured D. tertiolecta can theoretically be explained by the high degree of nutrient availability in
these cultures. The essential growth nutrient phosphate (PO3
4 ),
[42]
and it has been
is structurally similar to AsV (i.e. AsO3
4 )
proposed in previous research that phytoplankton and other
marine primary producers incorporate AsV as they are unable
[43]
If this postulate is true
to discriminate between it and PO3
4 .
3
then the high PO4 concentrations (mg L1) within the culture
media used here[20] may have facilitated arsenic uptake in
D. tertiolecta. Batch cultures usually commence as a nutrientrich system, however, will ultimately become nutrient-depleted
due to their nature as a closed system. Consequently, if PO3
4
availability does indeed facilitate AsV uptake then it is not
surprising that less arsenic was present in the water-soluble cell
fractions of batch-cultured D. tertiolecta.
availability indirectly,
It is, however, possible that PO3
4
rather than directly, facilitates AsV uptake in D. tertiolecta.
Under the continuous culture system used here the proportion of
dead cells was observed to be considerably lower than in batch
cultures after 42-days incubation. The concentration of PO3
4
and other growth nutrients will dictate the ratio of live to dead
cells within the culture. As dead cells (by the use of heat-treated
D. tertiolecta cultures) were observed to remove arsenic from
solution, it is possible that dead cells compete for ‘available’
arsenic with live cells, and thus under batch culture conditions
D. tertiolecta may have incorporated low arsenic concentrations
in water-soluble cell fractions (Fig. 4) as it was all bound to
residual cell material instead.
The high nutrient availability in continuous D. tertiolecta
cultures may also provide an explanation as to why little arsenic
was accumulated in lipid-soluble cell fractions (Fig. 4). Recent
research[44] has illustrated that arsenolipids form analogues of
phospholipids, which are important components of phytoplankton cellular membranes.[45] Based on this research it now
appears possible that arsenic has a functional role in phytoplankton and algal cells, which has relevance to what was observed
here. If AsV is accumulated in lipid-soluble cell fractions as a
means to replace PO3
4 then under a nutrient rich continuous
culture system little arsenic may be accumulated because PO3
4
requirements are being met. Conversely, under batch culture
conditions nutrient depletion is more prominent and thus the
observation of greater lipid-soluble arsenic concentrations in
batch cultured D. tertiolecta provides some support to the idea
that arsenic may be accumulated to fulfil PO3
4 requirements.
Arsenic species produced by D. tertiolecta and
T. pseudonana in batch and continuous cultures
Lipid-soluble arsenic species
Arsenoribosides were the dominant arsenic species found in
hydrolysed lipid-soluble extracts of all D. tertiolecta and
T. pseudonana cultures (Figs 5, 6). For D. tertiolecta, glycerol
arsenoriboside (Gly-riboside) (Fig. 1) was the major arsenoriboside species present in all cultures accounting for ,98 % of the
total arsenic within the fraction in batch cultures, and ,76 % in
continuous cultures (Fig. 5). Conversely, sulfate arsenoriboside
(OSO3-riboside), Gly-riboside and on occasions phosphate
arsenoriboside (PO4-riboside) (Fig. 1) were all present, accounting for between 50 and 80 % of the total arsenic in lipid-soluble
fractions of T. pseudonana (Fig. 6).
Arsenoribosides are widely considered to be the final arsenic species produced by marine primary producers, by an
extension of the Challenger biomethylation pathway.[51,52]
Recently[44] an arsenoriboside-containing phospholipid has
been identified, thus it is possible that arsenoribosides are
present within marine primary producers as arsenolipid degradation products. The arsenoribosides found here (Figs 5, 6)
could theoretically be the hydrolysed products of arsenolipid
species present in the lipid-soluble cell fractions of D. tertiolecta and T. pseudonana. Furthermore, the presence of the same
individual arsenoriboside species in water-soluble fractions of
both D. tertiolecta and T. pseudonana (Figs 7, 8) makes the idea
that arsenoribosides formed by arsenolipid degradation more
plausible.
96
Arsenic cycling in marine phytoplankton cultures
Batch 7
Batch 42
AsV
2⫾1%
AsV
2 ⫾ 0.3 %
Batch 4
AsV
1⫾1%
Gly Ribose
98⫾33 %
Unknown anion
4.2 min
Gly Ribose 16 ⫾ 1 %
Gly Ribose
98 ⫾ 46 %
Continuous 42 (no As)
Continuous 42
PO4 Ribose
3⫾1%
Unknown anion
4.2 min
AsV
8⫾7%
5⫾1%
99 ⫾ 20 %
PO4 Ribose
5⫾6%
AsV
9 ⫾ 13 %
Gly Ribose
78 ⫾ 10 %
Gly Ribose
76⫾ 25 %
Fig. 5. Proportions of arsenic species found in hydrolysed lipid extracts of D. tertiolecta cultures grown under batch and
continuous culture regimes. Values are mean proportions standard deviations (n ¼ 2). Patterns indicate individual arsenic
species.
Batch 7
III
Batch 42
As
5⫾1%
Gly Ribose
28 ⫾ 9 %
DMA
16 ⫾ 4 %
Gly Ribose
21⫾ 9 %
MA
0.5 ⫾ 0.3 %
PO4 Ribose
11 ⫾ 24 %
OSO3 Ribose
27 ⫾ 18 %
Batch 4
Continuous 7
III
As
2⫾2%
Gly Ribose
35 ⫾ 11 %
DMA
9 ⫾ 12 %
MA
0.5 ⫾ 1 %
PO4 Ribose
10 ⫾ 9 %
DMAA
13 %
AsV
20 ⫾ 18 %
AsIII
10 ⫾ 13 %
DMA
13 ⫾ 16 %
MA
1⫾1%
PO4 Ribose
4⫾1%
V
As
8⫾13%
OSO3 Ribose
23 ⫾ 5 %
Continuous 42
III
As
9⫾9%
III
As
16 ⫾ 5 %
Gly Ribose
DMA
18 ⫾ 2 % 29 ⫾ 3 %
Gly Ribose
20 ⫾ 7 %
V
As
3⫾2%
DMA
22 ⫾ 9 %
MA
1 ⫾ 0.3 %
PO4 Ribose
4⫾3%
OSO3 Ribose
V
As
16 ⫾ 5 %
18 ⫾ 21 %
OSO3 Ribose
41 ⫾ 10 % OSO3 Ribose
30 ⫾ 0.5 %
Continuous 7 (no nutrient)
Gly Ribose
17 ⫾ 9 %
MA
0.3 ⫾ 0.2 %
Continuous 42 (no nutrient)
III
As
6 ⫾ 11 %
DMAE
3⫾3%
V
As
PO4 Ribose
1⫾2%
16 ⫾ 5 %
DMA
14 ⫾ 8 %
Gly Ribose
25 %
PO4 Ribose
1⫾1%
III
As
25 %
OSO3 Ribose
V
7%
As
OSO3 Ribose
27⫾ 1 %
V
As
32 ⫾ 26 %
2%
PO4 Ribose
2%
DMA
39 %
Fig. 6. Proportions of arsenic species found in hydrolysed lipid extracts of T. pseudonana cultures grown under batch and continuous culture regimes. Values
are mean proportions standard deviations (n ¼ 2). Patterns indicate individual arsenic species.
concentrations
Under nutrient rich conditions (PO3
4
.1 mg L1) (Figs S2–S4) typical of continuous cultures and
‘young’ (,7 days) batch cultures PO4-riboside was always
present (Figs 5, 6) and at times accounted for between 10 and
16 % of the total arsenic within the fraction (Figs 5, 6, S1–S3).
Although arsenoribosides were the major arsenic species
present in the lipid-soluble fractions of both D. tertiolecta and
T. pseudonana, subtle differences in the proportions and presence of individual arsenic species differed with culture regime
(Figs 5, 6).
97
E. G. Duncan et al.
Batch 7
DMA
5⫾6%
Batch 42
MA
2 ⫾ 0.2 %
Gly Ribose
29 ⫾ 11 %
Gly Ribose
18 ⫾ 25 %
III
As
25 ⫾ 35 %
PO4 Ribose
18 ⫾ 23 %
Batch 4
Gly Ribose
14 ⫾ 3 %
DMA
8⫾4%
DMA
10 ⫾ 14 %
MA
5⫾2%
PO4 Ribose
4⫾2%
AsV
38 ⫾ 11 %
AsV
46 ⫾ 42 %
PO4 Ribose
9 ⫾ 13 %
Continuous 42
Continuous 42 (no As)
DMA
18 ⫾ 3 %
AsV
69 ⫾ 26 %
DMA
16 ⫾ 2 %
Gly Ribose
31 ⫾ 6 %
Gly Ribose
33 ⫾ 3 %
PO4 Ribose
21 ⫾ 1 %
AsV
6⫾1%
PO4 Ribose
47 ⫾ 5 %
AsV
28 ⫾ 1 %
Fig. 7. Proportions of arsenic species found in water-soluble extracts of D. tertiolecta cultures grown under batch and continuous culture regimes. Values are
mean proportions standard deviations (n ¼ 2). Patterns indicate individual arsenic species.
Batch 7
Batch 42
DMA
8⫾2%
Gly Ribose
12 ⫾ 6 %
DMA
11 ⫾ 3 % PO4 Ribose
2⫾4%
PO4 Ribose
12 ⫾ 9 %
Gly Ribose
22 ⫾ 4 %
V
V
As
11 ⫾ 6 %
As
9⫾1%
OSO3 Ribose
59 ⫾ 16 %
Batch 4
Gly Ribose
15 ⫾ 5 %
DMA
13 ⫾ 5 %
PO4 Ribose
14 ⫾ 4 %
OSO3 Ribose
54 ⫾ 9 %
Continuous 7
DMA
13 ⫾ 2 %
Gly Ribose
18 ⫾ 7 %
PO4 Ribose
4⫾5%
DMA
14 ⫾ 1 %
Gly Ribose
17 ⫾ 0.2 %
PO4 Ribose
12 ⫾ 3 %
V
OSO3 Ribose
52 ⫾ 7 %
Continuous 42
As
6⫾7%
V
As
30 ⫾ 17 %
OSO3 Ribose
35 ⫾ 6 %
V
As
18 ⫾ 9 %
OSO3 Ribose
39 ⫾ 13 %
Continuous 7 (no nutrients)
Continuous 42 (no nutrients)
DMA
11 ⫾ 4 %
Gly Ribose
12 ⫾ 4 %
Gly Ribose
14 %
V
As
22 ⫾ 6 %
OSO3 Ribose
55 ⫾ 15 %
OSO3 Ribose
86 %
Fig. 8. Proportions of arsenic species found in water-soluble extracts of T. pseudonana cultures grown under batch and continuous culture regimes. Values
are mean proportions standard deviations (n ¼ 2). Patterns indicate individual arsenic species.
Under nutrient poor conditions (PO3
concentrations
4
,0.5 mg L1) (Figs S2–S4), typical of ‘old’ batch cultures and
in continuous cultures supplied with no additional nutrients,
PO4-riboside was not present in either cell fraction (Figs 5, 6).
is required in the formation of PO4-riboside,
As PO3
4
the formation of PO4-riboside is likely to be influenced by
nutrient availability, which has not been previously considered. Consequently, culture regime will also influence
arsenic species formation in marine phytoplankton and
thus regimes that do not mimic natural processes such as
nutrient replenishment are unlikely to be environmentally
representative.
98
Arsenic cycling in marine phytoplankton cultures
whereas AsV, Gly-riboside, PO4-riboside and DMA were all
found in similar proportions in continuous cultures (Fig. 7).
T. pseudonana conversely contained OSO3-riboside, Gly-riboside
and AsV as the dominant arsenic species in water-soluble cell
fractions (Fig. 8), with OSO3-riboside dominant in batch and
nutrient-limited continuous cultures and AsV was more prominent
in nutrient-supplied continuous cultures (Fig. 8).
Arsenic within the water-soluble cell fractions of marine
phytoplankton are likely to be comprised of a mixture of actively
incorporated arsenic plus arsenoriboside–arsenolipid degradation products. In younger batch cultures (primarily D. tertiolecta) and in nutrient-supplied continuous cultures (both
species) AsV and PO4-riboside were prominent (Figs 7, 8). This
suggests, first, that within nutrient-supplied continuous cultures,
arsenic as AsV is constantly being incorporated into cells, which
reinforces the importance of nutrient availability (primarily
PO3
4 ) in facilitating arsenic uptake.
The presence of PO4-riboside in young batch cultures and
nutrient-supplied continuous cultures (Figs 7, 8) reinforces the
findings presented in the previous section, whereby PO4riboside and therefore its parent arsenolipid species is produced
under periods of PO3
4 availability, whereas this arsenic species
is absent under nutrient limited conditions (Figs 7, 8), as was the
case in hydrolysed lipid extracts (Figs 5, 6).
Older batch cultures (,42-days incubation) and nutrientlimited continuous cultures contained far greater proportions of
likely arsenolipid–arsenoriboside degradation products, which
included species such as DMA, methylarsonate (MA) and AsIII
(Figs 7, 8). Although these arsenic species can be produced as
biosynthesised arsenic species from incorporated AsV by the
Challenger[52] pathway, the loss of arsenic in the water-soluble
cell fractions of both D. tertiolecta and T. pseudonana (Fig. 4)
makes it more likely that these species are degradation products
of higher arsenic species, i.e. arsenolipids and arsenoribosides.
The replenishment of nutrients was of particular importance in
influencing the relative production of arsenic species, as illustrated by the observation of similar proportions of individual
arsenic species in the two D. tertiolecta continuous cultures,
despite only one of these cultures being supplied with AsV
(Fig. 5). Conversely, when T. pseudonana was cultured with
and without f/2 nutrient media the proportions of individual
arsenic species changed considerably (Fig. 6). Specifically, when
T. pseudonana cultures were continually supplied with f/2 nutrient media and AsV the proportions of all three arsenoriboside
species in combination (OSO3, Gly and PO4) after 42-days
incubation was higher (61 % of total arsenic in fraction) compared
to ,34 % in cultures continually supplied with AsV only (Fig. 6).
Furthermore, a similar scenario is evident in the proportions
of other arsenic species such as dimethylarsinate (DMA) and
AsIII (Fig. 6). DMA and AsIII have been described in previous
publications[35,53–55] as arsenoriboside degradation products.
T. pseudonana cultures supplied with AsV only contained
,54 % of the total arsenic within the fraction as DMA and
AsIII, which is higher than the 38 % found in cultures continually
supplied with f/2 nutrient media. As stated previously, viable
cell numbers within T. pseudonana cultures supplied with AsV
only were far lower than in cultures supplied with f/2 nutrient
media (Fig. 3) and in addition the proportion of dead cell
material was observed to be higher in T. pseudonana cultures
supplied with AsV only.
It is thus likely that the increase in amounts of DMA and AsIII
found in T. pseudonana cultures supplied with AsV may have
resulted from the increase in dead cells, with these arsenic
species present as arsenoribosides degradation products[35,53–55]
rather than actively biosynthesised arsenic species.
In addition to DMA and AsIII, dimethylarsenoacetate (DMAA)
(Fig. 1) was found within the hydrolysed lipid-extracts of batch
T. pseudonana cultures incubated for 42 days (Fig. 6). DMAA has
never been previously found in marine phytoplankton tissue[2,5–10]
and environmentally is rarely found with live organisms,[2,51,56,57]
with the reduced product of DMAA, dimethylarsenoethanol
(DMAE), a prominent arsenic species in decomposing macroalgal tissue.[35,53–55] DMAE was found as a major arsenic species
in hydrolysed lipid-soluble extracts of D. tertiolecta in a recent
study,[5] in which D. tertiolecta was cultured in batch cultures
under harsh environmental conditions (30–35 8C, 170 mmol
photons m1 s1; 16 h of illumination per day).[5]
Consequently, both DMAA and DMAE have only been found
in batch phytoplankton cultures that are either old (i.e. 42 days of
incubation) or cultured under harsh environmental conditions[5]
in which the proportion of dead cell tissue was high. Thus the
presence of DMAA and DMAE in batch cultured phytoplankton
is unlikely to have occurred as a result of active AsV metabolism,
which concurs with available information for marine macroalgae,[28,58,59] which at a cellular level are similar,[60] with DMAA
and DMAE likely to have been produced by the degradation of
arsenoribosides in decomposing D. tertiolecta and T. pseudonana
tissue. This reinforces a major issue with the use of batch cultures,
whereby dead cell tissue accumulation makes it difficult to
discriminate between active arsenic metabolism and arsenic
degradation associated with tissue decomposition.
Implications of findings for understanding
of arsenic cycling in marine systems
The results from this study illustrate that arsenic cycling in the
marine phytoplankton species D. tertiolecta and T. pseudonana
is heavily influenced by the culture regime used.
From a purely arsenic cycling perspective this study demonstrated that total arsenic concentrations are of minimal relevance
to studies investigating active arsenic cycling as these concentrations provide no information regarding arsenic uptake as
arsenic is adsorbed to dead cell material.
This study also demonstrated that arsenic uptake in
D. tertiolecta and T. pseudonana is linked to nutrient availability, however, whether this is due to nutrients directly facilitating
arsenic uptake (i.e. as a result of structural similarities between
V
PO3
4 and As ) or simply by limiting the accumulation of dead
cell material, which negates the effects of arsenic binding to
dead tissue, is uncertain.
Arsenic was also demonstrated to accumulate in the lipids of
D. tertiolecta and T. pseudonana, particularly within batch
cultures, which may suggest that arsenic is being accumulated
under batch culture conditions to fulfil a metabolic purpose
within the cell, possibly as a replacement for phospholipids
which may be unable to be formed as a result of nutrient
limitation.
Arsenoribosides were found to be the major group of arsenic
species in the hydrolysed lipid-extracts of both D. tertiolecta and
T. pseudonana irrespective of culture regime. In addition,
arsenoribosides were also widely found in water-soluble cell
Water-soluble arsenic species
The proportions of individual water-soluble arsenic species
present in D. tertiolecta and T. pseudonana differed considerably
(Figs 7, 8). AsV and Gly-riboside were the major arsenic species in
the water-soluble cell fractions of batch cultured D. tertiolecta,
99
E. G. Duncan et al.
fractions under all culture regimes. Consequently, it is likely that
arsenoribosides are a major arsenic component in marine
phytoplankton as is the case for marine macro-algae.[28,58,59]
Furthermore as arsenoribosides were ubiquitous within hydrolysed lipid-extracts of both phytoplankton species under all
culture regimes illustrates the possibility that arsenoribosides
are formed by the degradation of arsenolipids rather than formed
in situ by an extension of the Challenger[52] biomethylation
pathway.
In addition AB (Fig. 1) was not present in tissues of either
D. tertiolecta or T. pseudonana under any culture regime, which
provides evidence that phytoplankton do not directly form AB
in situ, with its formation in marine animals occurring by
associated animal epiphytes or the further metabolism of
ingested arsenoribosides.
This study also aimed to determine if culture regime influences arsenic cycling and the formation of arsenic species by
marine phytoplankton. It was evident that culture regime influenced arsenic uptake by both D. tertiolecta and T. pseudonana,
with greater intra-cellular arsenic concentrations present in
continuously cultured phytoplankton. This can be attributed to
the two major differences between batch and continuous cultures, which are that continuous cultures are constantly nutrient
rich and thus result in a greater proportion of live cells at any
given time, which facilitates arsenic uptake either directly (as a
and AsV) or
result of structural similarities between PO3
4
indirectly by minimisation of the presence of dead cell tissues,
which was shown to compete with live cells for available arsenic.
These two processes were also shown to influence the
production of arsenic species, with nutrient-supplied cultures
producing PO4-riboside, whereas it was absent from nutrient
poor, batch cultures. Furthermore, the inevitable accumulation
of dead cell material in batch cultures resulted in an increased
presence of arsenic species such as DMAA and DMAE
which are more regularly associated with decomposing algal
tissue.[35,53–55] This demonstrates that the accumulation of dead
cell material in batch cultures makes it impossible to determine
the arsenic species produced by active biosynthesis from those
produced as a result of phytoplankton decomposition, thus
making interpretation of arsenic biogeochemical cycling
difficult.
Consequently, this study has demonstrated that nutrient
availability plays a key role in regulating arsenic cycling in
marine phytoplankton and thus future research should endeavour to mimic the cycling of nutrients as occurs in nature. Batch
cultures do not encompass this. Continuous cultures are more
successful in encompassing nutrient cycling and limiting the
accumulation of dead cells and therefore these cultures should
be utilised for future research.
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Acknowledgements
The authors acknowledge the assistance of Damon Bryce who assisted with
the culturing of D. tertiolecta and running of incubation experiments.
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