International Journal of
Environmental Research
and Public Health
Article
Biomethylation and Volatilization of Arsenic by
Model Protozoan Tetrahymena pyriformis under
Different Phosphate Regimes
Xixiang Yin 1,2 , Lihong Wang 3 , Zhanchao Zhang 1 , Guolan Fan 1 , Jianjun Liu 1 , Kaizhen Sun 1
and Guo-Xin Sun 4, *
1
2
3
4
*
Jinan Research Academy of Environmental Sciences, Jinan 250014, China; [email protected] (X.Y.);
[email protected] (Z.Z.); [email protected] (G.F.); [email protected] (J.L.);
[email protected] (K.S.)
Key Laboratory of Urban Environment and Health, Institute of Urban Environment,
Chinese Academy of Sciences, Xiamen 361021, China
Shandong Analysis and Test Center, Shandong Academy of Sciences, Jinan 250014, China;
[email protected]
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences, Beijing 100085, China
Correspondence: [email protected]; Tel.: +86-10-6284-9328
Academic Editor: Helena Solo-Gabriele
Received: 10 November 2016; Accepted: 10 February 2017; Published: 14 February 2017
Abstract: Tetrahymena pyriformis, a freshwater protozoan, is common in aquatic systems. Arsenic
detoxification through biotransformation by T. pyriformis is important but poorly understood. Arsenic
metabolic pathways (including cellular accumulation, effluxion, biomethylation, and volatilization)
of T. pyriformis were investigated at various phosphate concentrations. The total intracellular
As concentration increased markedly as the external phosphate concentration decreased. The highest
concentration was 168.8 mg·kg−1 dry weight, after exposure to As(V) for 20 h. Inorganic As was
dominant at low phosphate concentrations (3, 6, and 15 mg·L−1 ), but the concentration was much
lower at 30 mg·L−1 phosphate, and As(V) contributed only ~7% of total cellular As. Methylated
As contributed 84% of total As at 30 mg·L−1 phosphate, and dimethylarsenate (DMAs(V)) was
dominant, contributing up to 48% of total As. Cellular As effluxion was detected, including inorganic
As(III), methylarsenate (MAs(V)) and DMAs(V). Volatile As was determined at various phosphate
concentrations in the medium. All methylated As concentrations (intracellular, extracellular,
and volatilized) had significant linear positive relationships with the initial phosphate concentration.
To the best of our knowledge, this is the first study of As biotransformation by protozoa at different
phosphate concentrations.
Keywords: arsenic; accumulation; biotransformation; phosphate; protozoan; Tetrahymena pyriformis
1. Introduction
Arsenic is a carcinogen that is widely found in aquatic environments such as rivers and seas.
Drinking water contaminated with As is a major health problem that is causing concern in a number
of places. Arsenic concentrations in drinking water in many parts of the world, such as parts of
Bangladesh, China, India, and Vietnam, are higher than the maximum concentrations of 10 µg·L−1
specified by the World Health Organization [1–3]. Millions of people in these areas suffer from
excessive exposure to As. Arsenic can enter the food chain in crops irrigated with contaminated
groundwater [4].
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www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2017, 14, 188
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The environmental fate of As and toxicity of As to organisms depend on the speciation of
the As present. Inorganic As is mainly present in the pentavalent and trivalent forms, As(V) and
As(III), respectively, in aquatic ecosystems. Many studies of the biotransformation of inorganic As by
microorganisms and plants have been performed [5]. The reduction of arsenate, i.e., the reduction of
As(V) to As(III), within cells is a crucial step in the biotransformation of inorganic As. The reduced
product As(III) is involved in various bio-metabolic pathways and can be oxidized to As(V) or
transformed into methylated arsenic species. Methylated As species are less toxic than inorganic As,
so the generation of the organic As species methylarsenate (MAs(V)), dimethylarsenate (DMAs(V)) and
trimethylarsine oxide (TMAs(V)O) as well as trimethylarsine (TMAs) is considered to be an effective
pathway through which organisms detoxify As [6]. The biovolatilization of inorganic As is seen as
an important metabolic approach used by organisms [7,8]. It has been found in previous studies that
some green microalgae and bacteria have strong capacities to volatilize As [6–9].
Phosphorus is an essential nutrient that is involved in the growth and development of cells,
accounting for about 2%–4% of the dry weight of cells. Increasing P levels in the soil due of human
actions, such as fertilizers and animal feeds, elevate the P runoff to aquatic ecosystems and cause
P eutrophication [10]. Phosphorus concentration in aquatic environments could reach µM levels.
Phosphate and arsenate are chemically similar, so they are absorbed competitively by microorganisms.
The P concentration in the environment of a microorganism can affect the accumulation and
transformation of As by the microorganism and the toxicity of As to the microorganism [11,12].
There have been many reports of As(V) being taken up by eukaryotic plants through the phosphate
pathway, and the processes involved are relatively well understood. The mechanism through which
inorganic As(V) is taken up by aquatic microorganisms in environments containing different P
concentrations has also been studied [13]. The results of a previous study indicated that As(V) may
activate phosphate sensors and disturb the phosphate signal pathway, which could cause an organism
to display phosphate starvation responses through repressing genes [14].
Tetrahymena pyriformis is a freshwater protozoan that is found in various aquatic systems in which P
and As are both commonly present. The sensitivity of T. pyriformis to environmental contamination has
led to it being used widely as a model organism to monitor aquatic toxicity and assess environmental
risks [15]. It has been found that T. pyriformis can survive in the presence of 40 µM arsenate, which is far
higher than realistic environmental concentrations of arsenate [16]. In some lakes such as Mono Lake
and Searles Lake in California, the concentrations of As are up to 200 µM and 3000 µM, respectively [17].
The aim of the study described here was to investigate the metabolism of As in T. pyriformis cells under
different phosphate regimes because of the importance of P to cell growth, the pervasiveness of As in the
environment, and the crucial aquatic ecological niches protozoa occupy. Competition between phosphate
and As(V) accumulation and transport in bacteria and plants has been demonstrated in numerous studies,
but the effect of phosphate on the biotransformation of As(V) by protozoa has been little studied.
2. Materials and Methods
2.1. Culturing T. pyriformis
A culture of T. pyriformis was acquired from the Institute of Urban Environment, Chinese Academic
of Sciences. The culture was kept at 30 ◦ C, without shaking, in a modified Neff medium consisting
of 0.25% proteose peptone (BD, Franklin Lakes, NJ, USA), 0.5% glucose (Sinopharm, Beijing, China),
0.25% Difco yeast extract (Thermo Fisher Scientific, Waltham, MA, USA), and 3.33 µM FeCl3 .
2.2. Growth of T. pyriformis Exposed to As at Different Phosphate Concentrations
Cells were harvested in the early exponential phase (21 h post-inoculation), and washed with
sterile Milli-Q water. Pellets of the cells were inoculated into 250 mL flasks containing Neff medium
with phosphate added. Na3 AsO4 was added to give a concentration of 40 µM, and the phosphate
concentrations in the different flasks were 3, 6, 15, and 30 mg·L−1 . This allowed the effects of As
toxicity on the cells in media containing different phosphate concentrations to be investigated. Growth
Int. J. Environ. Res. Public Health 2017, 14, 188
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was determined by measuring the optical density at 600 nm (OD600 ) after the cells had been exposed
to As(V) for 20 h. Three replicate experiments were performed at each phosphate concentration.
2.3. Total As Concentrations and Speciation in the Cells and Media
The cultures were incubated for 20 h, then each culture was centrifuged at 4000× g and 0 ◦ C for
5 min and samples of the cells and supernatant were collected. The cells were washed with sterile
Milli-Q water, then stored at −80 ◦ C.
The total cellular As and P concentrations in each cell sample were determined. Approximately
0.01 g dry weight (DW) of a cell sample was weighed and transferred into a 50 mL polypropylene
Int. J. Environ. Res. Public Health 2017, 14, 188
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digestion tube, then 2 mL of concentrated nitric acid was added and the mixture left overnight. Quality
Total As Concentrations
andthree
Speciation
in the Cells
and Media
control was2.3.
achieved
by analyzing
standard
reference
material GBW10010 (rice flour) samples and
The
cultures
were
incubated
for
20
h,
then
each
culture
was
centrifugedthe
at 4000×
g and
0 °C mixtures
three reagent blanks in the same way as the samples. The tubes
containing
partly
digested
for
5
min
and
samples
of
the
cells
and
supernatant
were
collected.
The
cells
were
washed
with
sterile
were heated in a microwave-accelerated reaction system (CEM Microwave Technology, Buckingham, UK)
Milli-Q water, then stored at −80 °C.
using a three-stage
temperature
programinthat
previously
[18]. The samples
The total
cellular As ramping
and P concentrations
eachhas
cell been
sampledescribed
were determined.
Approximately
were then cooled,
and
each
digest
wassample
diluted
50 mLand
with
Milli-Q
water.
The
solutions were then
0.01 g dry
weight
(DW)
of a cell
was to
weighed
transferred
into
a 50 mL
polypropylene
◦ C until
digestion
tube,they
then 2were
mL of analyzed
concentrated[16].
nitricThe
acid was
added
mixture
left overnight. Quality
stored at −20
total
As and
andthe
total
P concentrations
in the digests
control was achieved by analyzing three standard reference material GBW10010 (rice flour) samples
were determined using an inductively-coupled plasma mass spectrometer (Agilent 7500 ICP-MS;
and three reagent blanks in the same way as the samples. The tubes containing the partly digested
Agilent Technologies,
CA, USA) [16]. reaction
The total
As (CEM
in each
medium
(i.e., supernatant)
mixtures were Santa
heated Clara,
in a microwave-accelerated
system
Microwave
Technology,
UK) using
three-stage
temperature
ramping program
that has through
been described
sample wasBuckingham,
also determined.
A a5 mL
aliquot
of the supernatant
was passed
a 0.45-µm filter,
previously
[18].
The
samples
were
then
cooled,
and
each
digest
was
diluted
to
50
mL
with
Milli-Q
◦
acidified by adding 50 µL concentrated nitric acid, then stored at −4 C until analysis.
water. The solutions were then stored at −20 °C until they were analyzed [16]. The total As and total P
The speciation
of inthe
in were
eachdetermined
cell sample
investigated using
the following
concentrations
the As
digests
using was
an inductively-coupled
plasma mass
spectrometer procedure.
Approximately
0.01
g
DW
of
a
cell
sample
was
weighed
and
transferred
to
a
50-mL
polypropylene
(Agilent 7500 ICP-MS; Agilent Technologies, Santa Clara, CA, USA) [16]. The total As in each medium
(i.e.,
supernatant)
sample
was
also
determined.
A
5
mL
aliquot
of
the
supernatant
was
passed
through
digestion tube, then 8 mL of 1% nitric acid was added and the mixture was left for 10 h. A 5-mL
a 0.45-μm filter, acidified by adding 50 μL concentrated nitric acid, then stored at −4 °C until analysis.
aliquot of the supernatant was passed through a 0.45-µm filter, then the extract was analyzed by high
The speciation of the As in each cell sample was investigated using the following procedure.
performance
liquid chromatography
with
inductively-coupled
plasma
mass spectrometer
Approximately
0.01 g DW of a cellcoupled
sample was
weighed
and transferred to a 50-mL
polypropylene
digestionTechnologies).
tube, then 8 mL ofThe
1% nitric
acid waswere
added separated
and the mixture
wasaleft
for 10 h. Aanion
5-mL exchange
(ICP-MS, Agilent
As species
using
PRP-X100
aliquot of the
supernatant
was passed
a 0.45-μm
filter,
then the
extract was6.66
analyzed
by ·high
column (Hamilton,
Reno,
NV, USA).
Thethrough
aqueous
mobile
phase
contained
mmol
L−1 NH4 NO3
performance
liquid
chromatography
coupled
with
inductively-coupled
plasma
mass
spectrometer
and 6.66 mmol
·L−1 (NH )2 HPO4 , andThe
theAspH
was adjusted to 6.2 by adding ammonia solution.
(ICP-MS, Agilent4 Technologies).
species were separated using a PRP-X100 anion exchange
The total
As and
total PReno,
limits
ofUSA).
detection
(LOD)mobile
were phase
0.013contained
and 0.009
µgmmol·
·L−1L, −1respectively.
The As
column
(Hamilton,
NV,
The aqueous
6.66
NH4NO3
−1
and 6.66for
mmol·
(NH4)2HPO
4, and the pH
was adjusted
to 107%
6.2 by adding
ammonia
solution.
and P recoveries
theLstandard
reference
materials
were
and 103%,
respectively.
The limits of
total As and total P limits of detection (LOD) were 0.013 and 0.009 μg·L−1, respectively. The As
detection for theThe
different
arsenic species (As(III), As(V), DMAs(V), and MMAs(V)) were all 70 ng·As·L−1 .
and P recoveries for the standard reference materials were 107% and 103%, respectively. The limits of
detection for the different arsenic species (As(III), As(V), DMAs(V), and MMAs(V)) were all 70 ng·As·L−1.
2.4. Chemotrapping of Volatile Arsenic
2.4. Chemotrapping of Volatile Arsenic
The volatile As species produced by T. pyriformis were trapped in our previous study [16]. The trap
The volatile As species produced by T. pyriformis were trapped in our previous study [16]. The
device used in the
study described here is shown in Figure 1. The volatile As concentration in a trapped
trap device used in the study described here is shown in Figure 1. The volatile As concentration in a
sample wastrapped
determined
using
the method
wemethod
have we
described
previously
[16].
sample was
determined
using the
have described
previously
[16].
Figure 1. The chemo-trapping for volatile arsenicals. Silica gel (0.4-mm diameter) was steeped with
Figure 1. The
chemo-trapping for volatile arsenicals. Silica gel (0.4-mm diameter) was steeped with
10% AgNO3 solution (w/v) overnight, and dried at 70 °C; oxygen was supplied with an adjustable air
10% AgNO3pump
solution
overnight, and dried at 70 ◦ C; oxygen was supplied with an adjustable air
for the(w/v)
cell growth.
pump for the cell growth.
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2.5. Scanning Electron Microscopy (SEM) Analysis
Int. J. Environ.
Health 2017,
188
4 of 9 cells
The
effectsRes.
ofPublic
different
As14,
species
on the morphological characteristics of T. pyriformis
were identified by performing scanning electron microscopy on cells cultured in 40 µM Na3 AsO4
2.5. Scanning Electron Microscopy (SEM) Analysis
in media containing P concentrations at 3 or 30 mg·L−1 . As in the experiment described above,
The effects
of different
Ash,species
on the morphological
T. pyriformis
cells
the cells were
incubated
for 21
then arsenate
was addedcharacteristics
and the cellsofwere
cultured
at were
25 ◦ C for
by performing
electron
microscopy
on cells cultured
in 40The
μM Na
3AsO
4 in media
20 h. identified
Each culture
was thenscanning
centrifuged
and
the supernatant
discarded.
cells
were
fixed with
−1. As in the experiment described above, the cells were
containing
P
concentrations
at
3
or
30
mg·
L
2.5% glutaric dialdehyde overnight, then washed with 0.1 M phosphate buffer and dehydrated using
incubated for 21 h, then arsenate was added and the cells were cultured at 25 °C for 20 h. Each culture
ethanol. Each cell sample was then dried and coated with metal using a Polaron SC7620 sputter
was then centrifuged and the supernatant discarded. The cells were fixed with 2.5% glutaric
coater (Quorum Technologies, Ashford, UK), then observed using a Hitachi S-2000N scanning electron
dialdehyde overnight, then washed with 0.1 M phosphate buffer and dehydrated using ethanol. Each
microscope
(SEM)
High-Technologies,
Tokyo,
Japan).
cell sample
was(Hitachi
then dried
and coated with metal
using
a Polaron SC7620 sputter coater (Quorum
Technologies, Ashford, UK), then observed using a Hitachi S-2000N scanning electron microscope
(SEM) (Hitachi High-Technologies, Tokyo, Japan).
3. Results and Discussion
3.1. Toxic Effects of As in T. pyriformis at Different Phosphate Concentrations
3. Results and Discussion
Similar trends of As transformation by T. pyriformis at high (40 µM) and low (2 µM)
3.1. Toxic Effects of As in T. pyriformis at Different Phosphate Concentrations
As concentrations
were observed in our previous study [16], and the flux of volatilized As
Similar trends of
As ng
transformation
T. pyriformis
at high than
(40 μM)
μM) As
at high concentration
(9.3
·mg−1 ·day−1by
) was
much higher
thatand
at low
low(2concentration
concentrations
observed in
our previous(40
study
the flux
of volatilized
Asinvestigation
at high
(1.3 ng
·mg−1 ·day−1were
). A high-level
concentration
µM)[16],
wasand
therefore
chosen
for further
−1·day−1) was much higher than that at low concentration (1.3 ng·mg−1·day−1).
concentration
(9.3
ng·
mg
in this study. The morphologies of T. pyriformis cells that had been exposed to 40 µM arsenate and two
A high-level concentration (40 μM) was therefore chosen for further investigation in this study. The
different phosphate concentrations were investigated by scanning electron microscopy to allow us
morphologies of T. pyriformis cells that had been exposed to 40 μM arsenate and two different
to gain an understanding of the toxic effects caused by As(V). The microscopy images are shown in
phosphate concentrations were investigated by scanning electron microscopy to allow us to gain an
Figure
2A. No significant
differences
wereby
observed
in the
morphological
characteristics
of 2A.
the cells
understanding
of the toxic
effects caused
As(V). The
microscopy
images are
shown in Figure
−1 ), and no obvious differences
exposed
at
the
two
different
phosphate
concentrations
(3
and
30
mg
·
L
No significant differences were observed in the morphological characteristics of the cells exposed at
were the
found
the cellphosphate
membranes,
pores, and(3flagella.
WeL−1also
investigated
the growthwere
of T.found
pyriformis
two in
different
concentrations
and 30 mg·
), and
no obvious differences
incubated
h in 40 µMpores,
arsenate
and different
phosphate
concentrations.
Cell
growthincubated
was retarded
in the for
cell20
membranes,
and flagella.
We also
investigated
the growth of T.
pyriformis
−1 ) compared
for 20 h in 40
and different
phosphate concentrations.
Cell·Lgrowth
was retarded
by 14.6%–30.3%
atμM
the arsenate
lower phosphate
concentrations
(3, 6, and 15 mg
with by
the cell
−1) compared with the cell
−
1
14.6%–30.3%
at
the
lower
phosphate
concentrations
(3,
6,
and
15
mg·
L
growth rate at 30 mg·L phosphate (Figure 2B). It has been found in previous studies that As(V) can
growth
rate at 30
L−1 bacterium
phosphate (Figure
2B). cells
It hasatbeen
in previous
studies that [12,19,20].
As(V) can It is
inhibit
the growth
of mg·
some
and plant
lowfound
phosphate
concentrations
inhibit the growth of some bacterium and plant cells at low phosphate concentrations [12,19,20]. It is
possible that excessive intracellular As(V) decreases the effectiveness of the cellular energy source,
possible that excessive intracellular As(V) decreases the effectiveness of the cellular energy source,
causing cell death. Competitive absorption between As and P means that higher external phosphate
causing cell death. Competitive absorption between As and P means that higher external phosphate
concentrations
can effectively
decrease
As(V)
toxicity
by inhibiting
intracellular
accumulation
concentrations
can effectively
decrease
As(V)
toxicity
by inhibiting
intracellular
accumulationofofAs(V),
meaning
that
the cells
better
than
at lower
concentrations
(Figure
2B).2B).
As(V),
meaning
thatcan
thegrow
cells can
grow
better
than atphosphate
lower phosphate
concentrations
(Figure
1.5
B
A
OD600
1.0
0.5
0.0
P3
P6
P15
P30
Medium phosphate (mg L-1)
Figure
2. Growth
of Tetrahymena
pyriformis
exposed
to various
P concentrations
h.Scanning
(A)
Figure
2. Growth
of Tetrahymena
pyriformis
exposed
to various
P concentrations
afterafter
20 h.20(A)
Scanning electron microscopic photographs of T. pyriformis. −1Left, 3 mg·L−1 P treatment; −1
electron microscopic photographs of T. pyriformis. Left, 3 mg·L P treatment; Right, 30 mg·L
Right, 30 mg·L−1 P treatment; (B) Growth of T. pyriformis cell. All data are means ± SE (n = 4). OD:
P treatment; (B) Growth of T. pyriformis cell. All data are means ± SE (n = 4). OD: optical density.
optical density.
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Int. J. Environ. Res. Public Health 2017, 14, 188
3.2. Effects of Phosphate on As Accumulation and Biotransformation in T. pyriformis
5 of 9
Total arsenic in the cells
(mg kg-1 dry weight)
Total phosphate in the cells
(mg g-1 dry weight)
3.2.
Effects
Phosphate on As Accumulation
and Biotransformation
pyriformis
The
total
Asofconcentrations
in the cells increased
markedlyinasT.the
external phosphate concentration
−
1
The total 168.84
As concentrations
in the
cells
increased
markedly
as the external
phosphate
decreased, reaching
mg·kg DW
at the
lowest
phosphate
concentration,
3 mg
·L−1 (Figure 3).
−1
concentration
reaching
168.84 mg·
kgP DW
at the lowestwhich
phosphate
concentration,
3 mg·
L−1
It has been
reporteddecreased,
that As(V)
is absorbed
via
transporters,
would
explain the
competitive
(Figure 3). It has been reported that As(V) is absorbed via P transporters, which would explain the
effect between
As(V) and P uptake [12,20]. The cellular P concentrations tended to increase as the
competitive effect between As(V) and P uptake [12,20]. The cellular P concentrations tended to
phosphate
concentration
in the medium increased. Similar results have been reported for some
increase as the phosphate concentration in the medium increased. Similar results have been reported
photosynthetic
algae and higher
[11,21].
for some photosynthetic
algaeplants
and higher
plants [11,21].
P3
P6
P15
P30
Medium phosphate (mg L-1)
3. Total
concentrations
Asand
and P,
to 40toμM
under different
P levels forP levels
Figure Figure
3. Total
concentrations
ofofAs
P,when
whenexposed
exposed
40As(V)
µM As(V)
under different
20 h incubation. White columns: phosphate levels; gray columns: arsenic levels. All data are the means
for 20 h incubation. White columns: phosphate levels; gray columns: arsenic levels. All data are the
± SE (n = 4).
means ± SE (n = 4).
The concentrations of different As species in the cells were also determined (Figure 4). Inorganic
As
(As(III)
and As(V))
and two organic
arsenic
(MMAs(V)
anddetermined
DMAs(V)) were
detected
in
The concentrations
of different
As species
inspecies
the cells
were also
(Figure
4). Inorganic
the cells that had been exposed to different phosphate concentrations. The methylation (and therefore
As (As(III) and As(V)) and two organic arsenic species (MMAs(V) and DMAs(V)) were detected in the
detoxification) of accumulated As by T. pyriformis may have a number of steps. As(V), which is the
cells that
had beenAs
exposed
toaerobic
different
phosphate
concentrations.
methylation
(and to
therefore
predominant
species in
environments,
may
first be absorbed The
by the
cells, then reduced
detoxification)
of accumulated
As byAtT.phosphate
pyriformisconcentrations
may have aofnumber
which
As(III), which
is then methylated.
3, 6, andof
15steps.
mg·L−1,As(V),
inorganic
As is the
species As
were
the dominant
forms
within the cells,
for 67%–85%
of cells,
the total
predominant
species
in aerobic
environments,
mayaccounting
first be absorbed
by the
thenAsreduced
concentrations.
As(V)
accounted
for
39%–59%
of
the
As
concentrations.
The
organic
As
species
to As(III), which is then methylated. At phosphate concentrations of 3, 6, and 15 mg·L−1 ,
MMAs(V) and DMAs(V) accounted for 8%–17% and 6%–16%, respectively, of the total As
inorganic
As species were the dominant forms within the cells, accounting for 67%–85% of the
concentrations in the cells. The percentage of intracellular As(V) concentrations was around 7% of
total As concentrations. As(V) accounted for 39%–59% of the As concentrations.
The organic As
the total As concentrations at the high phosphate concentration of 30 mg·L−1, much lower than at the
speciesrelatively
MMAs(V)
and
DMAs(V)
accounted
for
8%–17%
and
6%–16%,
respectively,
of the
−1
low phosphate concentrations (3, 6, and 15 mg·L ). However, the methylated
Astotal As
concentrations
in
the
cells.
The
percentage
of
intracellular
As(V)
concentrations
was
around
concentrations in the cells treated at the high phosphate concentration accounted for 84% of the total 7% of
concentrations,
and DMAs(V)
accounted
for 48%
of the total As concentrations
There than at
the totalAsAs
concentrations
at the high
phosphate
concentration
of 30 mg·L−1(Figure
, much4).lower
−
1
are
two
crucial
As
detoxification
pathways
in
aquatic
organisms,
the
reduction
of
As(V)
and
the
the relatively low phosphate concentrations (3, 6, and 15 mg·L ). However, the methylated
As
methylation of the As(III) produced [22–24]. The results indicated that more cellular arsenate was
concentrations in the cells treated at the high phosphate concentration accounted for 84% of the total
transformed into arsenite and then methylated to give less toxic organic pentavalent arsenic species
As concentrations,
and DMAs(V)
accounted
ofphosphate
the total concentrations.
As concentrations
(Figure 4). There
at the high phosphate
concentration
than atfor
the48%
lower
The intracellular
are twoorganic
crucial
As
detoxification
pathways
in
aquatic
organisms,
the
reduction
of
As(V) and the
As (MMAs(V) and DMAs(V)) concentrations positively correlated with the initial phosphate
methylation
of the 3As(III)
produced
Thebyresults
indicated
that more
cellular
arsenate
was
level (Figures
and 4B).
This could [22–24].
be explained
more inorganic
As being
methylated
in the
cells
in the into
presence
of high
phosphate
concentrations
than toxic
in the
presence
of low phosphate
transformed
arsenite
and then
methylated
to give less
organic
pentavalent
arsenic species
concentrations.
Anconcentration
adequate cellular
P at
concentration
contributed
to the formation
of
at the high
phosphate
than
the lower therefore
phosphate
concentrations.
The intracellular
methylated As, and low cellular P concentrations may have decreased cell vitality, impeding the
organic As (MMAs(V) and DMAs(V)) concentrations positively correlated with the initial phosphate
biomethylation process. Similar results have been found for the freshwater alga Chlorella sp. and the
level (Figures
3 andOryza
4B). sativa
This [12,25].
could be explained by more inorganic As being methylated in the cells in
higher plant
the presence of high phosphate concentrations than in the presence of low phosphate concentrations.
An adequate cellular P concentration therefore contributed to the formation of methylated As, and low
cellular P concentrations may have decreased cell vitality, impeding the biomethylation process. Similar
results have been found for the freshwater alga Chlorella sp. and the higher plant Oryza sativa [12,25].
The effluxion of cellular As has been found to be an important detoxification pathway, in addition
to reduction and methylation, in many organisms [26]. In our study, methylated As was strongly
Int. J. Environ. Res. Public Health 2017, 14, 188
6 of 9
Arsenic species in the cells
(mg kg-1 dry weight)
The concentrations of cellular
organic arsenic (ng mL-1 )
effluxed by T. pyriformis into the culture media at different phosphate concentrations (Figure 5A).
We found inorganic As(III) and the two organic arsenic species MMAs(V) and DMAs(V) in the
culture media.
In the
range 6–30 mg·L−1 (i.e., not at the lowest
Int. J. Environ.
Res. phosphate
Public Health 2017,concentration
14, 188
6 of 9phosphate
−
1
concentration, 3 mg·L ), the predominant form of As found in the medium was DMAs(V), and As(III)
and MMAs(V) accounted for 3%–24%
and 4%–15%, respectively, of the total As concentrations.
A
B
The DMAs(V) concentrations in the media after the cells had
been cultured for 20 h clearly increased as
the phosphate concentration increased (Figure 5A). There was a significant positive linear correlation
between the extracellular organic As concentration and the initial
phosphate concentration (Figure 5B).
Medium phosphate (mg L )
A ready supply of phosphate may accelerate the As(V) reduction and methylation processes and result
in the effluxion of methylated As accumulated in the cells. Little As(III) effluxion occurred. Other
organisms, such as some eukaryotic algae and bacteria, behave quite differently from T. pyriformis,
and mainly efflux inorganic As [27,28]. The release of intracellular organic As allows T. pyriformis to
self-detoxify, and also causes relatively little environmental pressure because DMAs(V) is less toxic
than inorganic As species.
120
90
60
y = 2.6744x + 10.651
R2 = 0.9026
30
0
0
5
10
15
20
25
30
35
-1
Int. J. Environ. Res. Public Health 2017, 14, 188
6 of 9
P3
P6
P15
P30
The concentrations of cellular
organic arsenic (ng mL-1 )
Medium phosphate (mg L-1)
A
120
Arsenic species in the cells
(mg kg-1 dry weight)
B
Figure 4. Distribution of As species in the cell after 20 h As(V)
exposure under various P levels. (A)
90
60
Concentrations of four arsenic species. Black: As(III); dark gray: dimethylarsenate
(DMAs(V)); gray:
y = 2.6744x + 10.651
30
R = 0.9026
monomethylarsenate (MMAs(V)); white: As(V); (B) The relationship
between intracellular organic As
0
0
5
10
15
20
25
30
35
concentrations and the initial P levels. All data are the means ± SE
(nphosphate
= 4). (mg L-1 )
Medium
2
The effluxion of cellular As has been found to be an important detoxification pathway, in
addition to reduction and methylation, in many organisms [26]. In our study, methylated As was
strongly effluxed by T. pyriformis into the culture media at different phosphate concentrations (Figure
5A). We found inorganic As(III) and the two organic arsenic species MMAs(V) and DMAs(V) in the
culture media. In the phosphate concentration range 6–30 mg·L−1 (i.e., not at the lowest phosphate
concentration, 3 mg·L−1), the predominant form of As found in the medium was DMAs(V), and As(III)
and MMAs(V) accounted for 3%–24% and 4%–15%, respectively, of the total As concentrations. The
DMAs(V) concentrations in the media
after the P6
cells had been
P3
P15 cultured for
P30 20 h clearly increased as
Medium5A).
phosphate
the phosphate concentration increased (Figure
There (mg
wasLa-1)significant positive linear correlation
between the extracellular organic As concentration and the initial phosphate concentration (Figure
Figure 4. Distribution of As species in the cell after 20 h As(V) exposure under various P levels. (A)
Figure5B).
4. ADistribution
of phosphate
As species
inaccelerate
the cell the
after
20 h
As(V) exposure
underprocesses
variousand
P levels.
ready supply of
may
As(V)
reduction
and methylation
Concentrations of four arsenic species. Black: As(III); dark gray: dimethylarsenate (DMAs(V)); gray:
result in the effluxion
ofarsenic
methylated
As accumulated
in thedark
cells.gray:
Little dimethylarsenate
As(III) effluxion occurred.
(A) Concentrations
of
four
species.
Black:
As(III);
(DMAs(V));
monomethylarsenate (MMAs(V)); white: As(V); (B) The relationship between intracellular organic As
organisms, such as(MMAs(V));
some
eukaryotic
algaethe
and
bacteria,
behave
quite differently
T.
gray: Other
monomethylarsenate
white:
(B)± SE
The
between from
intracellular
concentrations and the initial
P levels. All
data areAs(V);
means
(n =relationship
4).
pyriformis, and mainly efflux inorganic As [27,28]. The release of intracellular organic As allows T.
organic As concentrations and the initial P levels. All data are the means ± SE (n = 4).
pyriformis
to self-detoxify,
and also
causes
relatively
little
because
DMAs(V)
The effluxion
of cellular
As has
been
found to
beenvironmental
an important pressure
detoxification
pathway,
in
is
less toxic
inorganic
species. in many organisms [26]. In our study, methylated As was
addition
to than
reduction
and As
methylation,
The concentrations of extracellular
organic arsenic (ng mL-1)
Efflux of arsenic species in the
medium (ng mL-1)
strongly effluxed by T. pyriformis into the culture media at different phosphate concentrations (Figure
5A). We found inorganic As(III) and the two organic arsenic species MMAs(V) and DMAs(V) in the
A
culture media. In the phosphate 800
concentration range 6–30 mg·L−1 (i.e., not at the lowest phosphate
−1
B
concentration, 3 mg·L ), the predominant
form of As found in the medium was DMAs(V), and As(III)
600
and MMAs(V) accounted for 3%–24%
and 4%–15%, respectively, of the total As concentrations. The
400
y = 20.348x
- 25.05cultured for 20 h clearly increased as
DMAs(V) concentrations in the media
after the cells had
been
200
R = 0.9206
the phosphate concentration increased
(Figure 5A). There was a significant positive linear correlation
0
0
5
10
15
20
35
between the extracellular organic As
concentration
and25the 30initial
phosphate concentration (Figure
Medium phosphate (mg L-1 )
5B). A ready supply of phosphate may accelerate the As(V) reduction and methylation processes and
result in the effluxion of methylated As accumulated in the cells. Little As(III) effluxion occurred.
Other organisms, such as some eukaryotic algae and bacteria, behave quite differently from T.
pyriformis, and mainly efflux inorganic As [27,28]. The release of intracellular organic As allows T.
pyriformis to self-detoxify, and also causes relatively little environmental pressure because DMAs(V)
is less toxic than inorganic As species.
2
The concentrations of extracellular
organic arsenic (ng mL-1)
Efflux of arsenic species in the
medium (ng mL-1)
P3
P6
P15
P30
Medium phosphate (mg L-1)
A
800
Figure 5. Efflux of As speciation
in the medium after cultures were grown for 20 h by
B
600
T. pyriformis. (A) Concentrations of As species. Black: As(III); gray: DMAs(V); dark gray: MMAs(V);
400
= 20.348x - 25.05
(B) The relationship between extracellular
organic As yconcentrations
and the initial P levels. All data
200
R = 0.9206
are the means ± SE (n = 4).
0
2
0
5
10
15
20
25
Medium phosphate (mg L-1 )
30
35
Figure 5. Efflux of As speciation in the medium after cultures were grown for 20 h by T. pyriformis.
(A) Concentrations of As species. Black: As(III); gray: DMAs(V); dark gray: MMAs(V); (B) The
relationship between extracellular organic As concentrations and the initial P levels. All data are the
means ±Res.
SE Public
(n = 4).
Int. J. Environ.
Health 2017, 14, 188
7 of 9
Volatile arsenic (ng)
3.3. Volatilization of As by T. pyriformis at Different Phosphate Concentrations
3.3. Volatilization of As by T. pyriformis at Different Phosphate Concentrations
Volatile As generated by microorganisms has been found to play a crucial role in the
Volatile Ascycle
generated
by Itmicroorganisms
been bacteria
found to
play
a crucial role
in alga
the
biogeochemical
of As [23].
has been found has
that some
and
photosynthetic
green
biogeochemical
cycle of As
It has been
found that
some
bacteria
andasphotosynthetic
greenpathway
alga can
can
volatilize arsenicals
by [23].
methylating
inorganic
As and
that
this acts
a biodetoxification
volatilize
arsenicals
by methylating
inorganic
As andon
that
this
acts as a biodetoxification
[6,29].
[6,29]. Previous
studies
have mainly
been focused
the
production
of volatile As bypathway
organisms
at
Previous studies
have mainlyand
been
focused
on the
production
of volatile
organisms
at potential
different
different
As concentrations
after
different
exposure
periods
[15,30].As
Inby
our
study, the
As
and after by
different
exposure
periods
[15,30].atIndifferent
our study,
the potential
for As to be
for concentrations
As to be biovolatilized
T. pyriformis
was
investigated
phosphate
concentrations,
biovolatilized
pyriformis
was investigated
different was
phosphate
concentrations,
thefor
results
and
the resultsby
areT.shown
in Figure
6A. When T.atpyriformis
incubated
with 40 μM and
As(V)
20 h,
are shown
in Figure
6A. When
pyriformis as
wasthe
incubated
with 40 µMconcentration
As(V) for 20 h,inthe
production
the
production
of volatile
AsT.increased
initial phosphate
the
medium
of volatile As
increased asconcentrations
the initial phosphate
in and
the medium
At phosphate
increased.
At phosphate
of 3 andconcentration
6 mg·L−1, 26.69
29.24 ng,increased.
respectively,
of volatile
−
1
concentrations
of 3 and Much
6 mg·L more
, 26.69volatile
and 29.24
respectively,
of volatile
As were
produced.
Much
As
were produced.
Asng,was
produced
at higher
external
phosphate
more volatile Aswith
was49.58
produced
at higher
concentrations,
with
49.58 and 64.71
concentrations,
and 64.71
ng ofexternal
volatile phosphate
As being found
at phosphate
concentrations
of ng
15
−
1
−1
of volatile
AsL being
found at phosphate
concentrations
of 15
30supply
mg·L of
, respectively.
and
30 mg·
, respectively.
The results
indicated that
a and
good
phosphate The
mayresults
have
indicated that
a good supply
of phosphate may
have
increased
the methylated
As concentrations
increased
the methylated
As concentrations
in the
cells,
accelerating
the generation
of volatile As
in the cells,
accelerating
generation
of volatile
(Figures supply
4 and 6A).
This may be
explainedthe
by
(Figures
4 and
6A). Thisthemay
be explained
by aAs
sufficient
of phosphate
decreasing
a sufficient supply
of phosphate
decreasing
the cytotoxicity
of inorganic
As, allowing
normal cellular
cytotoxicity
of inorganic
As, allowing
normal
cellular metabolic
viability
to be maintained
and
metabolic viability
to be maintained
the
of gaseous
Asstudies
by the cells.
has
facilitating
the production
of gaseousand
Asfacilitating
by the cells.
It production
has been found
in some
that aIthigh
been
found
in
some
studies
that
a
high
intracellular
methylated
As
concentration
is
a
key
factor
in
the
intracellular methylated As concentration is a key factor in the production of volatile arsenicals [16].
production
arsenicals
Taking intoofconsideration
the importance
of the
Taking intoof volatile
consideration
the[16].
importance
the biovolatilization
of As
in biovolatilization
microorganism
of As in microorganism
detoxification
and environmental
the removal of As
fromT.environmental
media,
detoxification
and the removal
of As from
media,
pyriformis seems
to beT.apyriformis
potential
seems to be
a potential
candidate forof
use
in bioremediation
of As-polluted aquatic environments.
candidate
for
use in bioremediation
As-polluted
aquatic environments.
A
80
B
60
40
y = 1.457x + 22.885
R2 = 0.966
20
0
5
10
15
20
25
30
35
Medium phosphate (mg L-1)
P3
P6
P15
P30
Medium phosphate (mg L-1)
Figure
6. Volatile
pyriformis. (A)
Figure 6.
Volatile As
As level
level produced
produced by
by T.
T. pyriformis.
(A) Exposure
Exposure to
to 40
40 μM
µM As(V)
As(V) for
for 20
20 h
h under
under
different
concentrations;(B)
(B)The
Therelationship
relationshipbetween
between
volatile
and
initial
P levels.
different PP concentrations;
volatile
AsAs
and
thethe
initial
P levels.
All All
datadata
are
are
mean
± SE
= 4).
mean
± SE
(n =(n4).
4.
4. Conclusions
Conclusions
T.
pyriformis had
had aa strong
strong capacity
capacity to
to methylate
methylate As
As and
and excrete
excrete the
the products
products to
to the
the external
external
T. pyriformis
environment.
Thephosphate
phosphateconcentration
concentration
played
a critical
the metabolism
As by T.
environment. The
played
a critical
role inrole
the in
metabolism
of As byof
T. pyriformis.
Arsenic methylation and volatilization by protozoan may be influenced in the presence of phosphate.
Acknowledgments: This study was supported by the Natural Science Foundation of China (No. 41671485),
Opening Fund of State Key Laboratory of Agricultural Microbiology (AMLKF201409), Doctor Foundation of
Shandong (No. BS2013HZ009), Jinan Innovation Plan (No. 201302123) and Key Research Program of Shandong
(2015GSF120010).
Int. J. Environ. Res. Public Health 2017, 14, 188
8 of 9
Author Contributions: Xixiang Yin and Guo-Xin Sun conceived and designed the experiments; Xixiang Yin,
Lihong Wang and Zhanchao Zhang performed the experiments; Guolan Fan, Jianjun Liu and Kaizhen Sun
analyzed the data; Xixiang Yin and Guo-Xin Sun wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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