Ultrasonic damages on cyanobacterial photosynthesis

Abstract

Excessive cyanobacterial growth in eutrophic water sources has been a serious environmental problem, and both sight preservation and drinking water production demand control of cyanobacterial growth in water. Ultrasonic treatment was reported to effectively inhibit cyanobacterial growth through vesicle collapsing and cell fracturing, but little was known about the change of cyanobacterial photosynthesis during sonication. This paper examined the ultrasonic inhibition of Microcystis aeruginosa cell growth and extracellular microcystins release, and the instant ultrasonic decreases of antenna complexes like cyanobacterial chlorophyll a and phycocyanins (PC), and the oxygen evolution rate. The results showed that sonication effectively damaged antenna complexes, slowed down the photo-activity, which significantly inhibited the cell growth and microcystins formation and release.

Full text

Ultrasonic damages on cyanobacterial photosynthesis
Guangming Zhang
a,*
, Panyue Zhang
b
, Hong Liu
c
, Bo Wang
a
a
Shenzhen Graduate School, Tsinghua University, Shenzhen 518055, China
b
Department of Environmental Science Engineering, Hunan University, Changsha 410082, China
c
Department of Environmental Engineering, Beijing University of Aeronautics and Astronautics, Beijing 100083, China
Received 25 July 2005; received in revised form 10 October 2005; accepted 4 November 2005
Available online 4 January 2006
Abstract
Excessive cyanobacterial growth in eutrophic water sources has been a serious environmental problem, and both sight preservation
and drinking water production demand control of cyanobacterial growth in water. Ultrasonic treatment was reported to effectively inhi-
bit cyanobacterial growth through vesicle collapsing and cell fracturing, but little was known about the change of cyanobacterial pho-
tosynthesis during sonication. This paper examined the ultrasonic inhibition of Microcystis aeruginosa cell growth and extracellular
microcystins release, and the instant ultrasonic decreases of antenna complexes like cyanobacterial chlorophyll aand phycocyanins
(PC), and the oxygen evolution rate. The results showed that sonication effectively damaged antenna complexes, slowed down the
photo-activity, which significantly inhibited the cell growth and microcystins formation and release.
2005 Elsevier B.V. All rights reserved.
Keywords: Cyanobacteria; Sonication; Chlorophyll a; Phycocyanins; Microcystins
1. Introduction
Algae bloom has been recognized as an environmental
problem since it significantly increases the water turbidity
and causes serious taste and odor problems [1]. Moreover,
some bloom-forming algae produce toxic compounds that
might kill fish and domestic animals [2]. Field investigations
in China have reported that microcystins in drinking water
were one major factor that resulted in locally high incidence
of liver cancer [3]. When the eutrophic water is used t o produce
drinking water, algae cells consume high amount of coagu-
lants and disinfectant, jam filters, generate disinfection by-
products, and deteriorate the water quality. Despite all efforts
[4–6], algal bloom, especially cyanobacterial overgrowth,
remains a major problem in many lakes and reservoirs, and
new engineering methods are needed to counteract excessive
cyanobacterial growth in eutrophic waterbodies.
Ultrasound is known to have harmful effects on the
structure and functional state of organisms [7,8].When
applied to water, power ultrasound causes acoustic cavita-
tion, in which millions of small bubbles collapse rapidly to
reach temperatures as high as 5000 K and pressures as high
as 100 MPa, so called ‘hot spots’, generate highly active
hydroxyl free radical, and cause high speed micro-jets [9].
Such extreme conditions induce chemical reactions and
enforce mechanic damages on substances in water, which
has been widely employed to accelerate chemical reactions
and to degrade environmental pollutants. A few studies
have developed algae bloom control by ultrasound irradia-
tion. It was reported that ultrasound effectively reduced the
growth rate of algae by collapsing the gas vesicles that con-
trol the floating of the cells, fracturing the cells, and inhib-
iting cell division, and that the extent of algal growth
reduction was influenced by ultrasonic parameters like fre-
quency, intensity and time [10–15]. However, little is
known about the impacts of sonication on the cyanobacte-
rial photosynthesis process, which is the key for cyanobac-
terial growth. Therefore, this paper was to study the
1350-4177/$ - see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2005.11.001
*
Corresponding author. Tel.: +86 755 2603 6736; fax: +86 755 2603
6445.
E-mail address: zhanggm@sz.tsinghua.edu.cn (G. Zhang).
www.elsevier.com/locate/ultsonch
Ultrasonics Sonochemistry 13 (2006) 501–505

ultrasonic induced changes of key components for cyano-
bacterial photosynthesis and their behaviors.
Antenna complexes are the components in plants and
algae that capture photons for photosynthesis. There are
two major antenna complexes: chlorophyll–protein com-
plex inside the cell membrane and phycobilin-peptides
(PBS) outside the cell membrane. PBS absorb light in the
range of 470–650 nm and chlorophyll–protein complex
absorb light in the range of 430–440 nm and around
670 nm, the combination of these complexes thus utilize
solar lights effectively. There are four types of PBS, namely
phycoerythrins (PE), phycocyanins (PC), phycoerythrocya-
nins (PEC), and allophycocyanins (APC). For cyanobacte-
ria, the most important chlorophyll and PBS are
chlorophyll aand PC, respectively, which were chosen
for study in this paper [16]. The typical structure of PC sub-
unit is shown in Fig. 1. The photo-activity was evaluated
by the oxygen evolution rate since oxygen was the product
of cyanobacterial photosynthesis. Microcystis aeruginosa
was chosen as representative of glomerate cyanobacteria
because it was major bloom-forming and poisonous algae
specie and was widely found in natural waters.
2. Materials and Methods
Cells of M. aeruginosa were purchased from the Chinese
Academy of Science and cultured at 25 ± 0.5 C with
incandescent light of 2000 lx (12 h dark, 12 h with light)
in Erlenmeyer flasks with BG11 medium in a special algal
incubator (SW-CJ-113, Shanghai Yiheng Inc.). Table 1
reports the components of the BG11 culturing medium.
The microcystins standards were purchased from Sigma
Aldrich Company. All other chemicals were purchased
from Guangzhou Chemical Inc. and used as received. Solu-
tions were made with pure water generated from a SYNS
50001 generator (Millipore Inc.). M. aeruginosa growth fol-
lowed S shape curve; it reached the exponential growth
stage after 4 days, which lasted around 10 days. All exper-
iments were done with cells in the exponential growth
stage. After grown for 14 days, algal concentration in the
culturing solution reached almost 10
9
L
1
. The solution
pH was around 7.6.
The sonication which was performed in a probe system
(JY90-II, Xinzhi Inc.) that emitted 25 kHz ultrasound
through a tip which had a surface area of 2.12 cm
2
. For
each experiment, 250 mL cyanobacteria solution was filled
in a stainless steel beaker and the ultrasonic probe was
dipped 3 cm into the cyanobacteria solution; the beaker
was put in a water bath (THZ95, SBL Ltd., China) to keep
the temperature in the beaker at 25 ± 2 C. The measure-
ment of the ultrasonic field inside the beaker was carried
out with a standard calibration ultrasound-needle-hydro-
phone (CS-3, Acoustic Academy of China) connected with
a TDS 3000 oscillograph (Tektronix Inc.). The solution pH
was not adjusted. The ultrasound power density was
0.32 W/mL and the sonication time was 5 min, the sonica-
Fig. 1. Typical PC subunit structure [20].
Table 1
Contents of BG11 culturing solution
Panel A: Major components (based on 100 mL)
NaNO
3
K
2
HPO
4
MgSO
4
Æ7H
2
O CaCl
2
Æ2H
2
O Citric acid Ammonium ferric citrate green EDTANa
2
Na
2
CO
3
A1
150 mg 4 mg 7.5 mg 3.6 mg 0.6 mg 0.6 mg 0.1 mg 2 mg 0.1 mL
Panel B: Components for A1 solution (based on 1 L)
H
3
BO
3
MnCl
2
Æ4H
2
O NaMoO
4
Æ2H
2
O CuSO
4
Æ5H
2
O ZnSO
4
Æ7H
2
O Co(NO
3
)
2
Æ6H
2
O
2.860 g 1.810 g 0.390 g 79 mg 0.222 g 49 mg
502 G. Zhang et al. / Ultrasonics Sonochemistry 13 (2006) 501–505

tion conditions were chosen following the literature reports
[12–14]. After irradiation, 50 mL of the solution was
siphoned 1 cm below the water surface and taken for anal-
yses of the immediate influences of sonication. The remain-
ing 200 mL solution was then filled back to Erlenmeyer
flasks and cultured in the illumination incubator. Blank
samples without irradiation were cultured under exactly
the same conditions.
The cyanobacterial cell concentration was measured
immediately after sampling. The number of algae was
counted with the aid of an Olympus 18-ZSX microscope,
which was very time consuming. The alternative way was
to monitor the optical transparency of the water sample
at 684 nm using an Ultra-Spec 2000UV/Visible spectro-
photometer (Pharmacia Inc.) since the M. aeruginosa solu-
tion had very strong absorbance at 684 nm (OD
684
). The
OD
684
value was linear with the counted cell number with
R
2
of 0.99 within the tested cyanobacteria concentration
range. Therefore, OD
684
, instead of the cell number, was
reported in the study. Both methods were utilized simulta-
neously and frequently cross-checked. Reported data were
the average of three or more repeated experiments.
Chlorophyll awas determined following methods
described by MacKinney [17] and refined by Hao [14]. Oxy-
gen evolution rate was measured following the method of
Hao [15]. Microcystins was quantified using competitive
enzyme-linked immuno sorbent assay (ELISA) method
since it is quick, sensitive and can measure the total micro-
cystins concentration. An ELISA plate reader (DG5031,
Huadong, China) was used to measure the microcystins
concentrations colorimetrically at 450 nm. The method
has a low detection limit of 0.16 lgL
1
. To measure the
concentration of extracellular microcystins, water sample
was filtered through a 0.45 lm fiber glass filter to remove
cyanobacteria cells, the filtrate was then extracted using a
Waters C18 solid extracting column according to Zhang
[18] and then measured following the methods of Chorus
[19]. To extract PC, the cyanobacteria sample was centrifu-
gated at 12,000g
1
for 10 min (3K30 Sigma Laboratory
Centrifuger, Sigma Inc.), the supernatant was discarded,
then 10 mL KH
2
PO
4
–K
2
HPO
4
cushion solution (final
concentration of 0.001 mol L
1
, pH of 7.0, with 0.001
mol L
1
BME, 0.001 mol L
1
NaN
3
and 0.2 mol L
1
NaCl) was added to the remaining solids. The so-prepared
solution was then wrapped with black paper, frozen at
4C and then defrozen at 4 C, which was repeated three
times; the solution was then centrifugated at 15,000g
1
for 45 min at 4 C, the supernatant was then used for
the measurement of PC. PC absorbed strongly at 620
nm, the absorbance of the supernatant at 620 nm was
thus used to quantify the PC concentration in M. aeru-
ginosa.
3. Results and discussion
The pH of cyanobacterial sample dropped slightly from
7.6 to 7.3 after 5 min sonication.
3.1. Instant M. aeruginosa cell decrease and 14 days
growth inhibition
The M. aeruginosa solution was sonicated for 5 min and
the cell concentration decreased by 10.8%. The instant cell
removal might be achieved by vesicle collapsing which
caused the cells losing buoyancy and settling to the bottom.
The sonicated solution was then cultured and the growth
curve is shown in Fig. 2. The cell concentration of the
un-treated sample (blank) increased by 18.9 times while
that of the sonicated sample increased by only 1.92 times,
implying very effective M. aeruginosa growth inhibition
by sonication. The final cell concentration of the sonicated
sample was only 14.1% of that of the un-treated sample.
3.2. Instant ultrasonic impacts on photosynthesis
components and photo-activity
The instant reductions of M. aeruginosa chlorophyll a
concentration, PC absorbance and oxygen evolution rate
were reported in Table 2. Ultrasonic treatment for 5 min
reduced the M. aeruginosa cell concentration by 10.8%,
the chlorophyll aconcentration by 21.3%, the PC absor-
bance by 44.8%, and the photo-activity by 40.5%, implying
that sonication strongly damaged the antenna complexes
and inhibited the photosynthesis of M. aeruginosa, which
significantly slowed down the cyanobacterial growth. Son-
ication reduced PC more effectively than it reduced the
chlorophyll a, which might because that the ‘‘empty rod’’
structure of PC (Fig. 1) was prone to collapsing in cavita-
tion [20] or because that the cell membrane protected the
in-cell chlorophylls against cavitation attack.
3.3. Instant and 14 days reduction of extracellular
microcystins
Microcystins, products of M. aeruginosa, are of great
concerns since they are health hazards. World Health
Organization and many countries have strict regulations
on the maximum acceptable microcystins concentration
in drinking water. Fig. 3 shows the instant degradation
and long time inhibition of extracellular microcystins
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 5 10 15
Time (d)
OD684
Sonication
blank
Fig. 2. Growth inhibition of Microcystis aeruginosa by sonication,
25 kHz, 0.32 W/mL, 5 min.
G. Zhang et al. / Ultrasonics Sonochemistry 13 (2006) 501–505 503

release by sonication. The results showed that 5 min soni-
cation only slightly decreased the extracellular microcystins
by 7.2%, but effectively inhibited the release of microcystins
from M. aeruginosa cells to water within the following 14
days culturing. The final extracellular microcystins concen-
tration of the blank sample was 137.7 lgL
1
while that of
the sonicated sample was only 22.1 lgL
1
, only 16% of
that of the control. The ratio of extracellular microcystins
in sonicated sample and that of the control (16%) the ratio
of cell concentration in sonicated sample and that of the
control (14.1%) after 14 days culturing. The results indi-
cated that sonication for 5 min did not effectively degrade
the extracellular microcystins, but significantly control
the M. aeruginosa cell growth; and fewer cells formed
and released fewer toxins.
4. Conclusions
•Sonication reduced M. aeruginosa cell concentration
and extracellular microcystins concentration slightly,
but inhibited the cell growth and extracellular microcys-
tins release very effectively.
•Sonication for 5 min at 0.32 W/mL and 25 kHz
decreased the M. aeruginosa chlorophyll aconcentration
by 21.3%, the PC absorbance by 44.8% and the oxygen
evolution rate by 40.5%. These factors, combined with
cell kill, contributed to the growth control of M. aerugi-
nos by sonication.
•Sonication for 5 min at 0.32 W/mL and 25 kHz effec-
tively controlled the cell growth and microcystins release
of M. aeruginosa. After 14 days culturing, the final cell
and extracellular microcystins concentrations of the
treated sample were 14.1% and 16% of those of the con-
trol sample, respectively.
Acknowledgement
The authors thank the Ministry of Science and Technol-
ogy of China (2002AA601120) for financial support.
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0
20
40
60
80
100
120
140
160
010
Time (d)
Microcystins (μg/L)
15
blank
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5
Fig. 3. Reduction of extracellular microcystins release by sonication,
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Table 2
Instant damage on Microcystis aeruginosa photosynthesis by sonication, 25 kHz, 0.32 W/mL, 5 min
Chlorophyll a(mg L
1
) PC absorbance Oxygen evolution rate (lmol L
1
h) Extracellular microcystins (lgL
1
)
Blank 0.33 0.38 2088 18.7
Sonicated sample 0.26 0.21 1243 15.5
Reduction (%) 21.3 44.8 40.5 17.3
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