isolation of a poterioochromonas capable of feeding on microcystis aeruginosa and degrading...

R E S E A R C H L E T T E R

IsolationofaPoterioochromonas capable offeeding onMicrocystisaeruginosa anddegradingmicrocystin-LRXue Zhang, Hong-Ying Hu, Yu Hong & Jia Yang

Environmental Simulation and Pollution Control State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University,

Beijing, China

Correspondence: Hong-Ying Hu,

Environmental Simulation and Pollution

Control State Key Joint Laboratory,

Department of Environmental Science and

Engineering, Tsinghua University, Beijing

100084, China. Tel.: 186 10 6279 4005;

fax: 186 10 6279 7265; e-mail:

[email protected]

Received 20 May 2008; accepted 28 August

2008.

First published online 22 September 2008.

DOI:10.1111/j.1574-6968.2008.01355.x

Editor: Hans-Peter Kohler

Keywords

Poterioochromonas sp.; Microcystis

aeruginosa; microcystin-LR degradation;

ingestion effect; mixotrophy.

Abstract

Algal blooms have become a worldwide issue recently, especially those comprised

of toxic cyanobacteria. Grazers’ predation of bloom-forming algae plays an

important role in water clearing. In this study, a species of golden alga (strain

ZX1), capable of feeding on the toxic cyanobacteria Microcystis aeruginosa, was

isolated and identified as Poterioochromonas sp. (GenBank accession: EU586184)

on the basis of morphological characteristics and 18s rRNA gene sequencing.

Feeding experiments showed that ZX1 could clear high densities of M. aeruginosa

(7.3� 105–4.3� 106 cells mL�1) in a short time (40 h), with inhibition ratios

higher than 99.9%. ZX1 grew during the feeding processes and achieved a

maximum density of 10–20% of the initial M. aeruginosa density. Furthermore,

this study is the first to report that ZX1 was able to degrade microcystin-LR

(MC-LR) in cells of M. aeruginosa while digesting the whole cells, and that the

degradation process was determined to be carried out inside the ZX1 cell. For a

total MC-LR (intra- and extracellular) concentration of up to 114 mg L�1, 82.7%

was removed in 40 h. This study sheds light on the importance of golden alga in

aquatic microbial ecosystems and in the natural transportation/transformation of

MC-LR.

Introduction

Recently, blue algal blooms have broken out frequently in

lakes and reservoirs around the world, causing great damage

to the biodiversity and to the equilibrium of aquatic

ecosystems. Several species of blue algae (e.g. Microcystis

and Anabaena) are more harmful than others as they release

toxins into water bodies, causing illness or the death of

wildlife and humans. Millions of dollars will be spent to deal

with the problems (e.g. off-flavor, drinking water, and health

care) caused by harmful algal bloom, and 4 2000 cases of

human poisoning are caused globally by cyanotoxins each

year (Men et al., 2007). Microcystis aeruginosa is the most

common toxic cyanobacterium found worldwide, and pro-

duces potent cyclic peptide hepatotoxins called microcys-

tins. So far, almost 60 variants of microcystins have been

isolated; microcystin-LR (MC-LR) is regarded as the most

common and most toxic variant of microcystins (Vasconce-

los et al., 1996; Park et al., 2001). A provisional guideline

value of 1 mg L�1 has been issued for MC-LR in drinking

water by the World Health Organization (WHO, 1998).

Therefore, M. aeruginosa and MC-LR are the standard

species of bloom-forming algae and the standard microcys-

tin in studies, respectively.

Algal blooms en masse may be dispersed via physical

factors or removed from the water column due to sinking,

while the mortality of individual cells within blooms may be

affected by autolysis, viruses, predatory bacteria, or grazing

zooplankton (Rosetta & McManus, 2003). A number of

relevant microorganisms have already been isolated either

from bloom-forming water or during laboratory studies.

Cyanophage (a virus of cyanobacteria e.g. LPP-1), bacteria

(e.g. Bacillus sp., Flexibacter sp., Myxococcus sp.), fungi (e.g.

Rhizophidium planktonicum, Acremonium, Emericellopsis,

and Verticillium), and some actinomycetes (e.g. Strepto-

myces) are all potential biocontrol agents. These micro-

organisms may act in one of three major ways to control

the overgrowth of bloom-forming algae: production of

extracellular products, contact lysis, or entrapment lysis

(Sigee et al., 1999). Also, some kinds of protozoa [e.g.

FEMS Microbiol Lett 288 (2008) 241–246 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Tintinnids (Adrniraal & Venekamp, 1986), Strombidium

lingulum (Montagnes & Humphrey, 1998), Oxyrrhis marirta

(Jeong et al., 2003), Gyrodinium dominans (Nakamura et al.,

1995), and Naegleria (Liu et al., 2006)] and mixotrophs [e.g.

Ochromonas danica (Cole & Wynne, 1974)] may act as

alternative biocontrol agents that can graze on certain types

of bloom-forming algae; these species can thereby play an

important part in balancing the flow of matter in the case of

phytoplankton blooms (Tillmann, 2004).

Besides bacteria, viruses and protozoa, other kinds of

microorganisms may also influence bloom-forming algae,

including other species of algae. These species, especially

mixotrophic algae, which can graze on certain types of algae,

play an important part in forming the community structure

of the aquatic ecosystems (Boraas et al., 1988). Several

species of golden algae, such as Ochromonas, Poterioochro-

monas, and Chrysamoeba, are mixotrophic and can feed on

organic particles, bacteria, and certain species of green algae,

cyanobacteria, and diatoms (Daley et al., 1973; Zhang

et al., 1996; Holen, 1999; Kristiansen, 2005; Ou et al.,

2005). Ochromonas danica and Poterioochromonas malha-

mensis can feed on the toxic bloom-forming cyano-

bacterium M. aeruginosa (Daley et al., 1973; Zhang et al.,

1996), and a species of Poterioochromonas can degrade

MC-LR (Ou et al., 2005). However, only a few species of

golden alga capable of feeding on M. aeruginosa have

been reported, and there is a great dearth of data related to

the feeding characteristics and degradation processes of

microcystins.

In this study, a species of golden alga (strain ZX1) that

feeds on M. aeruginosa was isolated and identified. The

feeding characteristics of the golden alga predation on

M. aeruginosa were studied along with the degradation of

MC-LR during the feeding period. Based on these results,

the significance of golden algae in aquatic microbial ecosys-

tems was discussed.

Materials and methods

Materials

Microcystis aeruginosa PCC7806 was purchased from the

Freshwater Algae Culture collection of the Institute of

Hydrobiology (FACHB) (Wuhan, Hubei Province, China)

and cultured in sterilized BG11 medium (Rippka et al.,

1979). The algae were cultured under the standard condi-

tion (800–1100 lx white light; light : dark = 14 h : 10 h,

25 1C) for about 14 days to reach the log phase before being

used as inoculants. The flasks were shaken at least once

a day.

ZX1 was fed with M. aeruginosa cells in BG11 medium

and cultured under the standard condition. The culture

with only ZX1 was used as the grazer inoculants, after

M. aeruginosa cells were cleared out.

Golden alga identification

ZX1 was identified by both morphological characteristics

and 18s rRNA gene sequencing. The morphological char-

acteristics were observed via light microscopy (Leica

DM6000B) with an electronic flash device for photography

and transmission electron microscopy (TEM). The pro-

cesses of DNA analysis included PCR amplification of the

18s rRNA genes, BLAST analysis, and comparison with

sequences in the GenBank nucleotide database (http://www.

ncbi.nlm.nih.gov/BLAST/). The polygenetic tree was used to

describe the relationship between the golden alga and other

strains. The primers and PCR amplification processes were

according to Ou (2005).

Feeding experiments

Feeding experiments of ZX1 on M. aeruginosa were carried

out in 500-mL flasks, with different initial volumes of the

two algal inoculants and sterilized BG11 medium, reaching

200 mL in total. Three different initial densities of

M. aeruginosa were prepared: 7.3� 105, 2.4� 106, and

4.3�106 cells mL�1. In the test groups, 10 mL liquid inocu-

lants containing 8� 106 ZX1 cells were added to each flask,

while no ZX1 was added to the controls. Groups were tested

in triplicate and cultured under the standard condition.

Subcultures were taken periodically over a 40-h incubation

period, and cell densities of the two algae were determined

using a hemocytometer under light microscopy (magnifica-

tion: 10� 40). The feeding experiments were repeated

three times.

Analysis method for MC-LR

The concentrations of both extra- and intracellular MC-LR

were determined by the SEP-HPLC method, according to

Men & Hu (2007). The concentration of MC-LR was

determined by developing a calibration curve from com-

mercially available MC-LR. All chemicals used were of

HPLC or of analytical grade.

Data analysis

The inhibition ratio of M. aeruginosa (IR, %) was calculated

as IR = (1�Ntt/Nct)� 100, where Ntt and Nct are the

densities (cells mL�1) of M. aeruginosa in the test group and

in the control at time t, respectively.

The growth-curve parameters during the log phase of

M. aeruginosa were calculated following the formulas be-

low. Because the grazing period was short, the growth of

M. aeruginosa during this period was ignored. A linear

model was used to describe the reduction of M. aeruginosa

FEMS Microbiol Lett 288 (2008) 241–246c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

242 X. Zhang et al.

according to the grazing of the golden alga. The clearance

rate of each ZX1 (RC, nL per cell of golden alga h�1) was

calculated as

RC ¼2� 106 � ðN0 �NtÞ � ln ðMt=M0ÞðN0 þ NtÞ � ðMt �M0Þ � Dt

where N0 and Nt are the densities (cells mL�1) of M. aeruginosa

at the beginning and the ending points of the log phase,

respectively; M0 and Mt are the densities (cells mL�1) of ZX1

at the beginning and the ending points of the log phase of

M. aeruginosa, respectively; and Dt is time of the log

phase (h).

The specific growth rate of ZX1 (m, h�1) was calculated

as m= ln(Mt/M0)/Dt.

The specific attenuation rate of M. aeruginosa (n, h�1) was

calculated as n= ln(Nt/N0)/Dt.

The removal ratio of total MC-LR (Z, %) was calculated

as Z= (1�Ctt/Cct)� 100, where Ctt and Cct are the concen-

trations (mg L�1) of total MC-LR in the test group and in the

control at time t, respectively.

Results

Morphology and taxonomy of ZX1

ZX1 was yellow brown, transparent, free swimming, had no

cell walls, and presented in spherical, ovum, or amoeba form

with one or two flagella (Fig. 1). The cell lengths and

movement speeds were 5–15 mm and 0–5mm s�1, respec-

tively. The food storage products were oils, and the mode of

reproduction was cell division. The species proved to be

mixotrophic (autotrophic, osmotrophic, and phagotrophic)

and grew faster phagotrophically than autotrophically. ZX1

was identified as a species of golden alga on the basis of its

morphological characteristics, referring to the descriptions

of golden alga by Kristiansen (2005). After PCR amplification and 18s rRNA gene sequencing,

one sequence of 1477 bp was obtained. Alignments and

phylogenetic analysis (Fig. 2) demonstrated that the gene

sequences of ZX1 (GenBank accession: EU586184) had 99%

maximum identification with the P. malhamensis strain

SAG933.1c (GenBank accession: EF165114). Therefore,

ZX1 was identified as a species of the Poterioochromonas

genus.

Growth and grazing characteristics of ZX1 onM. aeruginosa

Three different initial densities of M. aeruginosa were fed to

ZX1, and the density changes of M. aeruginosa in the control

and the test groups are shown in Fig. 3. The densities of

M. aeruginosa kept growing slowly in the controls but

declined steeply in the test groups. These results showed that

ZX1 could feed on the cyanobacterium M. aeruginosa andFig. 1. (a, b) TEM photos of ZX1 in different forms (magnification:

� 5000); one flagellum of ZX1 can be seen in (b) (arrow).

Fig. 3. Density change of Microcystis aeruginosa with three different

initial densities: 7.3� 105(’, &), 2.4� 106(�, �), and 4.3�106(m,

n) cells mL�1; solid symbols represent controls and blank symbols repre-

sent test groups with ZX1. Values are means� SDs (n = 3).

Fig. 2. Phylogenetic tree based on 18S rRNA gene sequences of ZX1.

Species from this study are shown in bold.


243A microcystis-grazing and MC-LR-degrading Poterioochromonas

clear M. aeruginosa of high density (4.3� 106 cells mL�1) in

a short time (40 h). The inhibition ratios of M. aeruginosa

were higher and the medium was apparently clearer in the

lower initial M. aeruginosa density groups during the first

20 h. Therefore, we recommend that ZX1 be placed in the

water body during the early periods of blue algal blooms if it

is to be applied in controlling future blooms.

In all three test groups, there were lag phases during the

first 20 h, which might have been due to low ZX1 densities.

As ZX1 density increased up to around 105 cells mL�1,

M. aeruginosa density decreased quickly and the green color

of the medium faded noticeably. After 34.5 h of incuba-

tion, M. aeruginosa densities in all the test groups were

o 104 cells mL�1 (the detection limit), with inhibition ratios

around 99.9%. The medium plateaued at this stage for

several days.

The growth curve of ZX1 followed the ‘S’ pattern, and

there were three phases as shown in Fig. 4, including the lag,

log, and stationary phases. The maximum densities of ZX1

(among 1–5� 105 cells mL�1) increased linearly with the

initial densities of M. aeruginosa, ranging from 5� 105 to

5.1� 106 cells mL�1 (R2 = 0.80), where these maximums

represented 10–20% of the initial M. aeruginosa densities.

The decreasing density of the grazer met the pyramid of

energy flow in the food chain principle. The stationary

phase lasted for 10–20 days, at which point ZX1 declined in

density and slowed down in movement, approaching the

declining phase (data not shown). Based on these results, we

can deduce that ZX1 will not form blooms.

Growth and grazing characteristic parameters of ZX1

feeding on M. aeruginosa were calculated during the log

phase of M. aeruginosa (Table 1). The specific growth rates

of ZX1 and the specific attenuation rates of M. aeruginosa

increased with the initial densities of M. aeruginosa.

Degradation of microcystins (MC-LR) by ZX1

As shown in Table 2, MC-LR was degraded by ZX1. The

concentrations of MC-LR in controls increased with the

initial density of M. aeruginosa, and were mostly contained

within cells. However, the concentrations of intracellular

and total MC-LR decreased dramatically in the test groups,

with removal ratios of total MC-LR 4 82.7% in 40 h.

Intracellular MC-LR in the test groups included MC-LR in

cells of both M. aeruginosa and ZX1, and its disappearance

indicated both that MC-LR within cells of M. aeruginosa was

degraded by ZX1 and that there was no MC-LR accumula-

tion in ZX1 cells. Furthermore, the degradation processes

were carried out inside ZX1 cells while ZX1 was digesting

M. aeruginosa cells. However, the concentrations of extra-

cellular MC-LR in the test groups were higher than those of

the controls, which may have been due to an increased

release of microcystin by M. aeruginosa under the grazing

pressure of ZX1. More studies should be carried out to verify

the assumption and to clarify the degradation pathway of

MC-LR by ZX1.

Fig. 4. Growth curve of ZX1 in the test groups with three different initial

densities of Microcystis aeruginosa: 7.3� 105(&), 2.4�106(�), and

4.3� 106(n) cells mL�1. Values are means� SDs (n = 3).

Table 1. Growth characteristic parameters of ZX1 and Microcystis

aeruginosa

Initial density of

M. aeruginosa

(cells mL�1)

Specific

attenuation rate

of M. aeruginosa

(h�1)

Clearance rate

of each ZX1

(nL per cell of

golden alga h�1)

Specific growth

rate of ZX1 (h�1)

7.3� 105 0.233� 0.070 1.874� 0.198 0.034� 0.011

2.4� 106 0.339� 0.011 0.815� 0.074 0.076� 0.011

4.3� 106 0.334� 0.055 0.612� 0.102 0.129� 0.020

Table 2. Concentrations and the removal ratios of MC-LR in different groups

Initial density of

M. aeruginosa (cells mL�1)

Intracellular MC-LR (mg L�1) Extracellular MC-LR (mg L�1)Removal ratio of total

MC-LR (%)Control group Test group� Control group Test group

7.3� 105 13.5 0 0 0 100

2.4� 106 69.5 0 0 1.7 97.6

4.3� 106 114.0 2.7 0 17.0 82.7

�Intracellular MC-LR included MC-LR in cells of both M. aeruginosa and ZX1 in the test groups.


244 X. Zhang et al.

Discussion

Golden algae and mixotrophy

Golden algae (Chrysophytes) constitute a group of micro-

scopic algae that are primarily characterized by their golden

color (Kristiansen, 2005). The chrysophytes show an unu-

sual and remarkable range of different nutrition strategies,

including phototrophy, osmotrophy, or phagotrophy. Some

species have a combination of phototrophy with any or all of

these methods, called mixotrophy. The golden alga ZX1

isolated in this study is mixotrophic (phototrophic, osmo-

trophic, and phagotrophic).

The mixotrophs are widespread in aquatic habitats and

represent an important step of eukaryote evolution; they

possess advantages that facilitate their survival under parti-

cular conditions, such as low light and scarce mineral

nutrients. In the illuminated surface strata of a lake,

mixotrophs steeply reduce prey abundance and escape

competition with, or losses to, higher grazers that cannot

persist. Furthermore, the mixotrophs structure prey abun-

dance along the vertical light gradient (Martin & Hofle,

2001; Tittel et al., 2003). They can even invade established

plankton communities depending on the trophic status of

the system, affecting the food web structure, species diver-

sity, nutrient dynamics, and the flux of material through

planktonic food webs (Jones, 2000; Domaizon et al., 2003;

Katechakis & Stibor, 2006). However, until now, quantifying

the role of mixotrophs in plankton communities has proven

extremely difficult, especially in a field study.

As a mixotroph, ZX1 success is favored in shaded, slightly

humic, mesotrophic ponds with some organic nutrients.

Mixotrophic chrysophytes may constitute 4 50% of the

total phytoplankton biomass in some lakes (Kristiansen,

2005). Mass developments, or blooms, of chrysophytes (e.g.

Dinobryon, Synura, Chrysococcus, and Uroglena, not includ-

ing Poterioochromonas sp.) may sometimes occur in ponds

or lakes, but in smaller quantities than for inducing cyano-

bacterial blooms. The study of golden algae feeding on

bloom-forming cyanobacteria is valuable in the study of

mixotrophs and bloom-forming algae.

Feeding on toxic M. aeruginosa

Several protozoa, both in freshwater and in the sea, capable

of grazing on algae, have been reported (Tillmann, 2004),

but few feed on the toxic cyanobacteria M. aeruginosa.

Daphnia can feed on M. aeruginosa, but accumulate micro-

cystins in their bodies at a level of 1.78 mg toxin per

25 daphnids (Mohamed, 2001). Until now, only a few

species of golden alga (Poterioochromonas sp. and O. danica)

have been reported to be capable of grazing on M. aerugino-

sa (Cole & Wynne, 1974; Zhang et al., 1996; Ou et al., 2005).

The ingestion and digestion processes of grazing by

P. malhamensis and O. danica on M. aeruginosa and other

organic particles were observed using light and electron

microscopy by Cole & Wynne (1974) and Zhang et al.

(1996). The processes were as follows: (1) the membrane

that was derived from the plasma membrane and that

surrounded the prey disappeared sometime after ingestion,

(2) the food vacuole was then formed by successive fusion of

numerous homogeneous vesicles accumulated around the

prey, and (3) the prey was enclosed in a single membrane-

bound food vacuole and then digested. Ou et al. (2005)

reported that Poterioochromonas sp. could degrade MC-LR.

We thus believed that MC-LR was degraded in ZX1 cells

while ZX1 was digesting M. aeruginosa cells.

In conclusion, the golden alga Poterioochromonas sp. is

widespread in freshwater environments and is capable of

feeding on toxic cyanobacterium M. aeruginosa and biode-

grading MC-LR. Based on these results, Poterioochromonas

sp. is deduced to play an important part in the flux of

material through planktonic food webs and the community

structure of the aquatic ecosystems. However, this only

heralds the very beginning of studies regarding interactions

between golden algae and M. aeruginosa and the degrada-

tion of microcystins by golden algae. This leaves a great deal

of work to be carried out in determining the roles of golden

algae in the aquatic microbial food chains and in natural

transformation/transportation of microcystins.

Acknowledgements

This study was funded by the NSFC-JST joint project

(No.50721140017).

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