The effect of Poterioochromonas abundance on production of intra- and extracellular microcystin-LR...
Transcript of The effect of Poterioochromonas abundance on production of intra- and extracellular microcystin-LR...
PRIMARY RESEARCH PAPER
The effect of Poterioochromonas abundance on productionof intra- and extracellular microcystin-LR concentration
Xue Zhang • Hongying Hu • Yujie Men •
Kirsten Seestern Christoffersen
Received: 23 August 2009 / Revised: 29 May 2010 / Accepted: 14 June 2010 / Published online: 26 June 2010
� Springer Science+Business Media B.V. 2010
Abstract Due to its capability for producing vari-
ous microcystins, Microcystis aeruginosa is recog-
nized as one of the most toxic, bloom-forming
cyanobacteria. In this study, the fates of intra- and
extracellular microcystin-LR (MC-LR) were investi-
gated when the mixotrophic golden alga Poterioo-
chromonas sp. (ZX1) was grazing on M. aeruginosa
cells. In the control groups, the total MC-LR
concentration increased with the growth of M. aeru-
ginosa with an MC-LR content per cell of 0.5–1.5 9
10-8 lg cell-1. In the treatment with ZX1, the total
MC-LR decreased linearly throughout the incubation
period. In particular, intracellular MC-LR disap-
peared with a loss of M. aeruginosa cells in the first
few days. Part of the intracellular MC-LR was
released to the medium under the grazing stress,
resulting in an increase of extracellular MC-LR. The
degradation rate of MC-LR was positively related to
the initial abundance of ZX1 and negatively related to
that of M. aeruginosa. The inhibition ratio of MC-LR
production dropped sharply from 98 to 67% when the
initial abundance of M. aeruginosa increased from
106 to 107 cells ml-1. However, it increased from 84
to 99% when the initial ZX1 abundance increased
from 104 to 105 cells ml-1. The effective removal of
both M. aeruginosa cells and MC-LR was observed
under lower M. aeruginosa abundance (\106 cells
ml-1) and higher ZX1 abundance ([1% of M. aeru-
ginosa abundance). Light had little impact on MC-LR
degradation, but MC-LR degradation decreased due
to the loss of ZX1 after 10 days of darkness. This
study showed that the interactions between M. aeru-
ginosa and ZX1 were strongly influenced by their
initial abundances.
Keywords Golden alga � Grazing �Microcystis aeruginosa �Microcystin-LR degradation �Poterioochromonas sp. � Cell abundance
Handling editor: D. P. Hamilton
X. Zhang
Laboratory of Environmental Technology, Institute of
Nuclear and New Energy Technology, Tsinghua
University, Beijing 100084, People’s Republic of China
X. Zhang � H. Hu (&)
Environmental Simulation and Pollution Control State
Key Joint Laboratory, Department of Environmental
Science and Engineering, Tsinghua University, Beijing
100084, People’s Republic of China
e-mail: [email protected]
Y. Men
Department of Civil and Environmental Engineering,
University of California, Berkeley, CA 94720, USA
K. S. Christoffersen
Freshwater Biological Laboratory, University of
Copenhagen, Helsingørsgade 51, 3400 Hillerød, Denmark
123
Hydrobiologia (2010) 652:237–246
DOI 10.1007/s10750-010-0335-3
Introduction
The occurrence of harmful algal blooms is a world-
wide phenomenon. This has caused numerous
adverse effects on water quality and lake ecology.
Many cyanobacterial genera (e.g., Microcystis, Ana-
baena) are even more problematic because they
produce microcystins, a family of cyclic hepatotoxins
(Bourne et al., 2006). These hepatotoxins are respon-
sible for human or wildlife illness or even death
(Christoffersen, 1996; Fitzgerald et al., 1999; Svrcek
& Smith, 2004; Song et al., 2007; Kagalou et al.,
2008). For example, intake of water contaminated by
microcystins correlated with a high incidence of liver
cancer in China (Ueno et al., 1996). A total of 76
people died at a hemodialysis unit after exposure to
microcystin-contaminated water in Brazil (Carmi-
chael et al., 2001). The risks increase when rivers/
lakes containing algal blooms and toxins are utilized
as drinking water sources. The World Health Orga-
nization (WHO) has published a provisional guide-
line value of 1 lg l-1 microcystin-LR (MC-LR) in
drinking water (WHO, 1998) and 20 lg l-1 micro-
cystin for a moderate health alert in recreational
water (WHO, 2003). Therefore, investigations that
lead to the prevention of massive algal blooms and
eliminating their secondary metabolites (e.g., cyano-
toxins, taste and odor compounds) have become
increasingly important (Kotak et al., 1993; Eynard
et al., 2000; Falconer, 2001). Among the known
bloom-forming cyanobacteria, Microcystis aerugin-
osa is considered the most common cyanobacterium
found worldwide. Almost 60 variants of microcystins
(e.g., MC-LR, one of the most toxic and universal
variants) have been identified in this species (Park
et al., 2001).
Various microorganisms, such as viruses, bacteria,
and protozoans can cause mortality of cyanobacteria
and degradation of microcystins in harmful blooms
(Sigee et al., 1999). However, studies have mostly
focused on the role of bacteria in the degradation of
microcystins. Several species of bacteria, capable of
degrading microcystins, have been isolated, such as
Sphingomonas sp. (Jones et al., 1994; Saitou et al.,
2003) and Pseudomonas aeruginosa (Takenaka &
Watanabe, 1997). Studies have generally focused on
one of these two processes: either the feeding
behavior in relation to cyanobacteria or the degrada-
tion of the dissolved MC-LR compound. Most
microorganisms reported cannot perform two
processes simultaneously. For example, microcy-
stin-degrading bacteria are able to degrade the
microcystin when it was released from Microcystis
cells. There are also other bacteria capable of
degrading the Microcystis cells (Maruyama et al.,
2003). Some Daphnia can feed on M. aeruginosa, but
they accumulate microcystins in their bodies (Thost-
rup & Christoffersen, 1999; Mohamed, 2001) and
suffer from poisoning (Rohrlack et al., 2001). Some
amoebae can be effective grazers of colonial cyano-
bacteria like Microcystis, but the degradation of
microcystins remains largely unknown in this group
(Nishibe et al., 2004).
Some species of mixotrophic golden algae (Pote-
rioochromonas sp.) are able to graze on a wide range
of bloom-forming algae including cyanobacteria
(e.g., M. aeruginosa) and degrade dissolved MC-LR
(e.g., Zhang et al., 1996; Ou et al., 2005). We
hypothesized that Poterioochromonas may be able to
both graze Microcystis as well as degrade MC-LR.
However, the fate of intra- and extracellular MC-LR
produced by M. aeruginosa when the cells are grazed
by Poterioochromonas has not yet been studied. Such
results will help in understanding the interactions
between a mixotrophic alga and M. aeruginosa.
In our previous study (Zhang et al., 2008), a
mixotrophic golden alga (Poterioochromonas sp.
strain ZX1) was isolated and confirmed to be able
to degrade MC-LR while grazing on M. aeruginosa
cells which were at concentrations of 7.3 9 105–
4.3 9 106 cells ml-1. However, grazing was signif-
icantly influenced by the initial abundances of both
M. aeruginosa and Poterioochromonas ZX1 (Zhang
et al., 2009). Hence, the abundances of the two
cultures were assumed to be important factors
influencing the degradation of MC-LR by Poterioo-
chromonas ZX1. The aim of this study was to
investigate the fate of both intracellular MC-LR (cell-
bound MC-LR) and extracellular MC-LR (dissolved
MC-LR), while ZX1 was grazing on M. aeruginosa
under different conditions (i.e., light intensity, initial
abundance of both prey and predator). Experiments
with different abundances of M. aeruginosa
(1.8 9 106–1.2 9 107 cells ml-1) and ZX1, as well
as different light conditions, were designed. The
MC-LR production characteristics of M. aeruginosa
cultured in BG11 medium were simultaneously
investigated. Based on these results, the influence of
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123
cell abundance on the interactions between Poterioo-
chromonas and M. aeruginosa is elucidated.
Materials and methods
Cultures
Microcystis aeruginosa FACHB915 was purchased
from the Freshwater Algae Culture collection of the
Institute of Hydrobiology (FACHB) Wuhan, Hubei
Province, China. The axenic culture was grown in
200 ml sterilized BG11 medium in a 500 ml flask
(Rippka et al., 1979). The culture was grown under
standard conditions (white light of 40 lmol pho-
tons m-2 s-1, light: dark = 14 h: 10 h, 25�C) and
used as inoculants after reaching the log growth
phase. Cultures were shaken at least once a day.
M. aeruginosa appeared as single cells in this study.
The mixotrophic golden alga, Poterioochromonas
(strain ZX1), was isolated in our previous study
(Zhang et al., 2008). A single cell was picked using a
micropipette and cultured in 10 ml sterilized BG11
medium under standard conditions. These procedures
were repeated more than three times to get an axenic
culture of ZX1. The golden alga was fed with
M. aeruginosa cells (105–106 cells ml-1) in BG11
medium and cultured under standard conditions.
After M. aeruginosa cells were grazed down (under
the detection limit of 104 cells ml-1), Poterioochro-
monas cells were harvested by centrifugation at
1,500g for 10 min at 25�C, and then washed (resus-
pended in sterilized BG11 medium and then centri-
fuged at 1,500g for 10 min) twice before use in the
following experiments.
Analytical methods for assessing cell abundance
and MC-LR concentration
Subsamples were taken out of each group periodi-
cally. For daily cell abundance count, 100 ll was
taken as the subsample, while for intra- and extra-
cellular MC-LR concentration tests every 4–6 days, a
30 ml subsample was obtained. The flasks were
shaken to make the suspension uniform before
sampling. All operations were performed under
sterilized conditions.
Subsamples were fixed with buffered glutaralde-
hyde (1% final concentration) and the cell abundances
of both cultures (M. aeruginosa and ZX1) were
determined using a hemocytometer (YA-XQ100,
Improved Neubauer Counting Chamber, China) using
light microscopy (COICTM, XSZ-HS3, magnification:
9400).
The intra- and extracellular MC-LR were deter-
mined by solid-phase extraction with high perfor-
mance liquid chromatography (SPE-HPLC), which
has previously been described by Men & Hu (2007).
The subsamples (30 mL for MC-LR test) were
centrifuged at 10,000g for 30 min. MC-LR in the
supernatant (i.e., extracellular MC-LR) was extracted
by solid phase columns (SPE) and then determined by
HPLC. The MC-LR in the sediment (i.e., intracellular
MC-LR) was extracted by acetic acid (5%). Further-
more, the extraction was done by SPE. The MC-LR
concentrations in the samples were then determined
by HPLC. All chemicals used for MC-LR detection
were of analytical reagent grade. The concentration
of MC-LR was determined from a calibration curve
generated using commercially available MC-LR
(Alexis Corporation). In the treatment groups, M.
aeruginosa and ZX1 cells were mixed together, and
the intracellular MC-LR concentrations in these
groups included the MC-LR content in both M.
aeruginosa and ZX1 cells. The total MC-LR con-
centration was calculated as the sum of intra- and
extracellular MC-LR concentrations.
Grazing on M. aeruginosa and degradation
of MC-LR by ZX1
Experiments were carried out in 500 ml flasks with
different volumes of the two inoculants (M. aerugin-
osa and ZX1) and sterilized BG11 medium. The total
volume was 200 ml. Subsamples were periodically
tested to determine cell abundances and intra- and
extracellular MC-LR concentrations. All treatments
were done in triplicate. The details of the experiments
are as follows.
(1) Effects of initial ZX1 abundance on MC-LR
degradation: Four groups were prepared with
the same initial abundance of M. aeruginosa
(6 9 106 cells ml-1) and four different initial
abundances of ZX1 (0 (control), 1 9 104,
5 9 104 and 10 9 104 cells ml-1). All treat-
ments were cultured under standard conditions
for 18 days.
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123
(2) Effects of initial M. aeruginosa abundance on
MC-LR degradation: Three treatment groups
with different initial abundances of M. aeru-
ginosa (1.8 9 106, 6.5 9 106 and 12 9 106
cells ml-1) and the same initial abundance of
ZX1 (3.5 9 104 cells ml-1) were prepared.
One ZX1-free control group was also prepared
for each treatment group. All six treatments
were cultured under standard conditions for 18
days.
(3) Effects of light on MC-LR degradation: Three
light treatments were tested, including continu-
ous darkness, intermediate light exposure
(40 lmol photons m-2 s-1, light: dark = 14 h:
10 h) and high light exposure (90 lmol pho-
tons m-2 s-1, light:dark = 14 h:10 h). The
temperatures were all set to the incubation
temperatures (25�C). Under each light condi-
tion, one treatment group with M. aeruginosa
(3.0 9 106 cells ml-1) and ZX1 (1.0 9 105
cells ml-1), as well as one ZX1-free control
group, was prepared. The incubation lasted for
16 days.
Data analysis
The inhibition ratio of M. aeruginosa growth (gg, %)
was calculated as: gg = (1 - Ntt/Nct) 9 100%, where
Ntt and Nct (cells ml-1) are the abundances of
M. aeruginosa in the treatment group and in the
control at time t, respectively.
The inhibition ratio of MC-LR production (gp, %)
was calculated as: gp = (1 - Ctt/Cct) 9 100%, where
Ctt and Cct (lg l-1) are the concentrations of total
MC-LR in the treatment group and in the control at
time t, respectively.
The degradation rate of total MC-LR (m,
lg l-1 d-1) was calculated as: m = (C0 - CT)/T,
where C0 and CT (lg l-1) are the concentrations of
the total MC-LR at the beginning and the end of
the experiment, respectively, and T (days) is the
time.
Statistical analysis of data was performed with
SPSS for Windows (SPSS Inc., V13.0). A paired t
test was used to compare significant differences of
M. aeruginosa abundance curves, and MC-LR
degradation rates among the treatment and control
groups.
Results
Effects of ZX1 abundance on MC-LR degradation
Four M. aeruginosa treatments were incubated start-
ing with different initial abundances of ZX1 (0,
1 9 104, 5 9 104 and 10 9 104 cells ml-1). Signif-
icant differences in the abundance curves of M.
aeruginosa were determined between the control and
treatment groups (P \ 0.05, t test) (Fig. 1). In the
control, M. aeruginosa grew well and the abundance
increased from 6 9 106 cells ml-1 to 20 9 106
cells ml-1 after 18 days. In contrast, in the treatment
groups, M. aeruginosa decreased sharply due to ZX1
grazing. The inhibition ratios of M. aeruginosa
growth were higher than 99% after 4 days. No
significant differences in the abundance curves of
M. aeruginosa were observed among the three treat-
ment groups (P [ 0.05, t test). Furthermore, ZX1
grew rapidly and achieved a maximum density of
4–6 9 105 cells ml-1 after 4 days in all the three
treatment groups (data not shown).
Similarly, significant differences in MC-LR
concentrations were observed between the control
and treatment groups during the incubation periods
(P \ 0.05, t test) (Fig. 2). In the control group, the
total MC-LR concentration increased proportionately
to the M. aeruginosa abundance, resulting in a
1.3-fold increase from the initial level after 18 days.
Fig. 1 Change in M. aeruginosa abundance with different
initial abundances of ZX1 [Values are averages ± standard
deviations (n = 3)]
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In contrast, the total MC-LR concentration decreased
linearly throughout the incubation period in the three
treatment groups and dropped to 55.3, 5.4 and
2.5 lg l-1 after 18 days, respectively. The inhibition
ratios of MC-LR production ranged from 84–99%.
The degradation rates of total MC-LR were not
significantly different between the two groups with
initial ZX1 abundances of 5 9 104 and 1 9 105
cells ml-1 (13–14 lg l-1 d-1) (P [ 0.05, t test).
However, they were significantly lower in the other
group (10 lg l-1 d-1) (P \ 0.05, t test).
In the treatment groups, the intra- and extracellular
MC-LR decreased in different patterns. The intracel-
lular MC-LR decreased steeply with a decrease of
M. aeruginosa abundance, and no MC-LR was
accumulated in the ZX1 cells. The extracellular
MC-LR, however, increased to a peak value (around
85% of the initial total MC-LR) on the fourth day and
then declined gradually (Fig. 3).
Effects of initial M. aeruginosa abundance
on MC-LR degradation
The effects of initial M. aeruginosa abundance on
MC-LR degradation were examined using three
different initial M. aeruginosa abundances (1.8 9
106, 6.5 9 106 and 12 9 106 cells ml-1). After
18 days, M. aeruginosa grew to 4.5–6.3 9 107
cells ml-1 in the control groups, but declined to
below 105 cells ml-1 in all the treatment groups (data
not shown). The periods needed to achieve 99%
inhibition of M. aeruginosa growth were 4, 6 and 12
days for the three treatment groups, respectively. This
showed a positive correlation with the initial abun-
dance of M. aeruginosa (r2 = 0.95, P [ 0.05). After
18 days, the inhibition ratios of M. aeruginosa growth
all increased above 99% in the treatments groups.
Significant differences in the concentrations of
intra- and extracellular MC-LR between the control
and the treatment groups were observed after 18 days.
The same observations were also found among the
three treatment groups with different initial abun-
dances of M. aeruginosa (P \ 0.05, t test) (Table 1).
In the three control groups, the total concentration of
MC-LR increased linearly with the abundance of
M. aeruginosa, and the MC-LR content per cell was
calculated to be 0.9–1.5 9 10-8 lg cell-1. More than
96% of the total MC-LR remained within the
M. aeruginosa cells.
In the treatment groups, the total MC-LR decreased
significantly compared with that of the control groups
(P \ 0.05, t test). The inhibition ratios of MC-LR
production ranged from 67–99%, with a significant
decrease in the group with an initial M. aeruginosa
abundance of 1.2 9 107 cells ml-1. In particular, the
intracellular MC-LR dropped steeply. The concentra-
tions were under the detection limit in the two groups
with lower abundances of M. aeruginosa, while
concentration was very low (4.5 lg l-1) in the third
group. The concentrations of extracellular MC-LR on
Fig. 2 Change in total MC-LR with different initial abun-
dances of ZX1 [values are averages ± standard deviations
(n = 3)]
Fig. 3 Change in extracellular MC-LR with different initial
abundances of ZX1 [values are averages ± standard deviations
(n = 3)]
Hydrobiologia (2010) 652:237–246 241
123
the 18th day increased with an increase of the initial
M. aeruginosa abundance. A significant increase was
observed in the groups with the highest initial M.
aeruginosa abundance (12 9 106 cells ml-1). When
the initial abundance of M. aeruginosa was lower,
ZX1 easily removed both the cells and MC-LR.
Effects of light condition on MC-LR degradation
In the control groups with no Poterioochromonas, no
obvious growth of M. aeruginosa was found in
continuous darkness, and the abundances remained
at around 3-4 9 106 cells ml-1 throughout the incu-
bation period. In contrast, the abundance of M.
aeruginosa increased 10-fold under intermediate
(40 lmol photons m-2 s-1) and high (90 lmol pho-
tons m-2 s-1) light exposure after 18 days. In the
treatment groups, the M. aeruginosa abundances all
decreased sharply to below the detection limit of 104
cells ml-1 after 5 days under all three light conditions
(data not shown). The abundances of ZX1 were of the
same level (3–4 9 105 cells ml-1) during the first 2
days. In subsequent days, the ZX1 abundance quickly
dropped in continuous darkness. After 10 days, the
ZX1 abundance stayed at around 3 9 105 cells ml-1
under intermediate and high light exposure, but it
dropped below 104 cells ml-1 in continuous darkness.
Significant differences in the concentration of total
MC-LR were observed between the control and test
groups under the three light conditions (P \ 0.05, t
test) (Fig. 4). In the control groups, the total MC-LR
increased quickly from approximately 50 lg l-1 to
300–500 lg l-1 under intermediate and high light
exposure after 18 days, while it stayed at the initial
level of 50 lg l-1 in continuous darkness. The
changes in the MC-LR concentration were consistent
with the changes in M. aeruginosa abundance, and
the MC-LR content was calculated to be 0.5–
1.1 9 10-8 lg cell-1. After 18 days, a maximum
total MC-LR content of 560.5 lg l-1 was detected in
the control group under intermediate light exposure,
while a maximum extracellular MC-LR content of
199.5 lg l-1 was detected in the control group under
high light exposure. Therefore, the light condition
influenced both the production and release of
MC-LR. In the treatment groups, total MC-LR
decreased to similar levels under the three light
conditions by the fifth day. MC-LR content then
remained steady in the continuous darkness
Table 1 Microcystis aeruginosa abundance and MC-LR concentration in the control and treatment groups with different initial
abundances of M. aeruginosa after 18 days
Initial abundance
of M. aeruginosa(cells ml-1)
Inhibition ratio
of M. aeruginosagrowth (%)
Concentration of intracellular
MC-LR (lg l-1)
Concentration
of extracellular
MC-LR (lg l-1)
Inhibition ratio
of MC-LR
production (%)
Control Treatmenta Control Treatment
1.8 9 106 99.9 746.3 ± 30.1 ND 6.6 ± 2.0 8.0 ± 1.9 98
6.5 9 106 99.9 982.9 ± 40.4 ND 45.1 ± 10.5 26.8 ± 4.8 97
12.0 9 106 99.6 1090.7 ± 52.3 4.5 ± 1.5 47.2 ± 7.5 369.7 ± 56.2 67
Initial ZX1 abundances were 0 and 3.5 9 104 cells ml-1 in the control and treatment groups, respectively. Values are means of three
parallel samples ± standard deviations after 18 days culture (n = 3)a Intracellular MC-LR included both MC-LR in cells of M. aeruginosa and ZX1 in the treatment group
ND not detected
Fig. 4 Change in total MC-LR under different light treatments
[solid symbols controls, blank symbols treatment groups with
ZX1; values are averages ± standard deviations (n = 3)]
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treatment, while it continued to decrease in the other
two treatments.
Discussion
MC-LR production characteristics
of M. aeruginosa under different conditions
The toxicity and growth characteristics of M. aeru-
ginosa have been investigated by many researchers.
Light, temperature, nutrients, iron, inorganic carbon,
and culture age have all been found to influence these
markers (Watanabe & Oishi, 1985; Westhuizen &
Eloff, 1985; Ame & Wunderlin, 2005; Rohrlack &
Hyenstrand, 2007; Jahnichen et al., 2007; Pyo & Jin,
2007). Among these factors, light has been studied in
the most detail. In this study, the results in the control
groups without Poterioochromonas represented the
growth and MC-LR production characteristics of
M. aeruginosa in BG11 medium under lab conditions.
Overall, the total MC-LR concentration was linearly
related to the M. aeruginosa abundance. However, the
MC-LR content per cell varied under different
conditions: the level of 0.9–1.5 9 10-8 lg cell-1
was maintained under intermediate light exposure and
continuous darkness throughout the incubation peri-
ods, but the level decreased from 1.1 9 10-8
lg cell-1 (4th day) to 0.5 9 10-8 lg cell-1 (17th
day) under high light exposure. Nevertheless, no
significant difference was observed in M. aeruginosa
growth, when it was cultured under intermediate and
high light exposure (P [ 0.05, t test). Different
findings have been reported with regards to the effect
of light. Wiedner et al. (2003) found that the total
microcystin content per cell in M. aeruginosa
PCC7806 increased to a maximum value at 126 lmol
photons m-2 s-1 and then decreased at higher irradi-
ation. Similar results were also reported by Utkilen &
Gjølme (1992). In contrast, Bottcher et al. (2001)
found that the microcystin content per cell of
M. aeruginosa HUB 5-2-4 was relatively constant
when it was cultured at growth-limiting irradiance
levels from 5 to 75 lmol photons m-2 s-1. The
differences among these results are likely to be due
to the differences in culture conditions and different
strains of M. aeruginosa used.
Intracellular MC-LR contributed more than 96%
of the total MC-LR in most samples under
intermediate light exposure, while it only contributed
75% in continuous darkness and 34% under high light
exposure after 17 days. Wiedner et al. (2003) also
found that the concentration of extracellular micro-
cystin was 20 times higher at 40 lmol pho-
tons m-2 s-1 than at 10 lmol photons m-2 s-1.
While these results indicate that light is an important
factor influencing the content and release of micro-
cystins, further studies are still needed to find out the
mechanisms for the changes in MC-LR.
Degradation of MC-LR by Poterioochromonas
ZX1
Microcystins are monocyclic heptapeptides that con-
tain seven variable amino acids. They are relatively
stable over a range of pH and temperature values and
resistant to the enzymatic hydrolysis of some com-
mon enzymes (e.g., pepsin, trypsin and chymotryp-
sin) (Svrcek & Smith, 2004). Until recently, only a
few species of bacteria (e.g., Sphingomonas, Pseu-
domonas) were found to be able to degrade micro-
cystins (Jones et al., 1994; Saitou et al., 2003;
Takenaka & Watanabe, 1997). In this study, the total
MC-LR decreased during ZX1 grazing on toxic M.
aeruginosa. That is, Poterioochromonas ZX1 can
degrade both M. aeruginosa cells and MC-LR. ZX1
can inhibit MC-LR production in two ways: degra-
dation of the initial MC-LR (the parts below the level
of initial MC-LR concentration in Fig. 4) and inhi-
bition of new MC-LR production by inhibiting the
growth of M. aeruginosa (the parts above the initial
MC-LR level in Fig. 4). Note that the contribution of
the latter mechanism gradually increased over time.
Both intra- and extracellular MC-LR could be
degraded by Poterioochromonas ZX1, but in quite
different patterns. The intracellular MC-LR decreased
steeply and disappeared with the M. aeruginosa cells
in the first few days. In contrast, the extracellular
MC-LR increased during the first few days and then
gradually decreased. These results indicate that
during the first few days, part of the cell-bound
MC-LR was degraded by ZX1, while M. aeruginosa
cells were digested (judging from the decreasing
total MC-LR content). The rest was released to the
medium under grazing stress. In the treatment
groups, above 98% of the total MC-LR dissolved
in the medium, which was quite different from the
case of the control groups. On the other hand,
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123
considering that the intracellular MC-LR included
MC-LR in the cells of M. aeruginosa and ZX1, no
MC-LR accumulated in ZX1 cells since intracellular
MC-LR disappeared with the loss of M. aeruginosa
cells.
Similar results were also reported by Watanabe
et al. (1996). In their study, no microcystins accu-
mulated in Poterioochromonas malhamensis after it
ingested and digested cells of Microcystis viridis
(A. Brown) Lemmermann (Chroococcales, Cyano-
bacteria), and part of the microcystins were released
to the culture medium during the grazing. However,
they did not find the gradual degradation of the
extracellular MC-LR during the latter part of the
incubation period. In this study, the degradation of
MC-LR was found to be significantly influenced by
the initial abundances of M. aeruginosa and ZX1.
One example is the cessation of MC-LR degradation
after ZX1 died out in continuous darkness.
Effects of cell abundance on the degradation
of MC-LR
The degradation of MC-LR was significantly influ-
enced by the initial abundance of both M. aeruginosa
and Poterioochromonas ZX1. With lower initial
abundance of M. aeruginosa, ZX1 more rapidly
degraded both M. aeruginosa cells and the toxin MC-
LR. In our previous study, with an initial M.
aeruginosa abundance of 7.3 9 105 cells ml-1, all
MC-LR was degraded in 40 h when M. aeruginosa
cells were grazed down (Zhang et al., 2008). The
inhibition ratios of MC-LR production were above
90% when the initial abundances of M. aeruginosa
were below 106 cells ml-1. However, the inhibition
ratio of MC-LR production decreased sharply to
67%, when the initial M. aeruginosa abundance
increased to 107 cells ml-1. Therefore, to remove
both M. aeruginosa cells and MC-LR, it is better to
use ZX1 under the condition that the initial M. aeru-
ginosa abundance is lower than 106 cells ml-1.
The initial abundance of ZX1 also plays an
important role in both total MC-LR degradation and
the release of intracellular MC-LR. More than 80% of
the intracellular MC-LR was released to the medium
in the treatment with initial ZX1 abundance of 104
cells ml-1, while 58% was released with an initial
ZX1 abundance of 105 cells ml-1. That is, less MC-
LR was released to the medium when there was a
higher initial abundance of ZX1. For the total MC-LR
degradation, the degradation rates of total MC-LR
(10–14 lg ml-1 day-1) were positively correlated
with the initial abundance of ZX1, although no
significant differences were observed in the abun-
dance of M. aeruginosa among the three treatments
(Fig. 5). These results show that MC-LR degradation
is more sensitive to the initial abundance of ZX1 than
the degradation of M. aeruginosa cells. Based on
these results, an initial ZX1 abundance that is higher
than 1% of the initial M. aeruginosa abundance is
suggested to effectively remove both M. aeruginosa
cells and MC-LR.
Under the three light treatments, no significant
differences in the removal of cells and MC-LR were
observed during the first 5 days. This means light has
minimal effect on the grazing and MC-LR degrada-
tion function of ZX1. However, the abundance of
ZX1 decreased sharply to zero when cells were
subjected to 10 days in continuous darkness, which is
consistent with the report by Zhang & Watanabe
(2001). The loss of ZX1 resulted in the termination of
the degradation of MC-LR in continuous darkness.
This indicates that the abundance and performance of
ZX1 played a key role in the degradation of MC-LR,
and light may be an indirect factor.
Fig. 5 The relationship between the total MC-LR concentration
and the corresponding M. aeruginosa abundance in groups with
different initial abundances of ZX1 [values are averages ±
standard deviations (n = 3)]
244 Hydrobiologia (2010) 652:237–246
123
Ecological implications
This study shows that Poterioochromonas ZX1 is an
effective grazer on M. aeruginosa. It can also degrade
MC-LR. However, these processes are significantly
affected by the abundance of both M. aeruginosa and
Poterioochromonas. M. aeruginosa and its microcys-
tins could be removed much slower when its grazers
(i.e., Poterioochromonas) were very few, especially
in the case of microcystin degradation. Considering
that the abundance of mixotrophic golden alga is
usually less than several thousand individuals per ml
under natural conditions (Boenigk & Stadler, 2004)
and the cyanobacteria are in much higher abundance
during blooms, it is easy to understand why cyano-
bacterial blooms occur even in the presence of a
grazer (e.g., Poterioochromonas) under natural con-
ditions. Intracellular MC-LR was released to the
surrounding water under the grazing stress, and more
was released under conditions of higher abundance of
M. aeruginosa and less abundance of ZX1. Aside
from the cell lysis by bacteria, the grazing stress may
be another important reason why the toxins are
released to the water during blooms.
Conclusions
The growth and MC-LR production of M. aeruginosa
in BG11 medium showed that total MC-LR concen-
tration increased with the growth of M. aeruginosa
with constant MC-LR content per cell of 0.9–
1.5 9 10-8 lg cell-1. Nevertheless, a reduction in
MC-LR content per cell was observed under high light
exposure after 17 days. The fate of intra- and
extracellular MC-LR was measured, while the living
cyanobacterium M. aeruginosa was grazed by ZX1.
Both intra- and extracellular MC-LR can be degraded
by ZX1, but in different modes: the intracellular MC-
LR decreased abruptly in the first few days; but the
extracellular MC-LR initially increased in the first
few days and gradually decreased thereafter. The
removal of MC-LR and M. aeruginosa cells were
strongly influenced by the initial abundance of M.
aeruginosa and ZX1. Less MC-LR was released to the
medium and faster degradation of MC-LR occurred in
groups with fewer M. aeruginosa and more ZX1. The
effective removal of both M. aeruginosa cells and
MC-LR was observed when the initial abundance of
M. aeruginosa was lower than 106 cells ml-1 and the
initial abundance of ZX1 was higher than 1% of the
M. aeruginosa abundance. This study demonstrated
that the initial abundance of cyanobacteria and their
grazers plays an important role in their interactions,
and helps in understanding such interactions under
natural conditions.
Acknowledgments This study was funded by China National
Science Fund for Distinguished Young Scholars (No.50825801)
and NSFC-JST joint-project (No. 50721140017). We wish to
thank Prof. Lirong Song for his kind help with MC-LR
detection method, to Trine Perlt Warming for her comments in
paper writing.
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