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: hyhu@tsinghua.edu.cn

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

238 Hydrobiologia (2010) 652:237–246

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.

Hydrobiologia (2010) 652:237–246 239

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)]

240 Hydrobiologia (2010) 652:237–246

123

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)]

242 Hydrobiologia (2010) 652:237–246

123

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,

Hydrobiologia (2010) 652:237–246 243

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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