w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Evaluation of the mechanisms of the effect of ultrasound on

Microcystis aeruginosa at different ultrasonic frequencies

Xiaoge Wu, Eadaoin M. Joyce*, Timothy J. Mason

Sonochemistry Centre, Faculty of Health and Life Science, Coventry University, CV1 5FB, UK

article info abstract

Article history: Blooms of cyanobacteria are now considered to be a common environmental issue. They
Received 29 September 2011 are hazardous to both domestic and wild animals and humans. Current treatments are
Received in revised form unable to effectively control such blooms as they become tolerant to biocides and it is
8 February 2012 difficult to degrade cyanobacterial toxins in water. Alternative methods for control are
Accepted 13 February 2012 currently under investigation. One potential effective method is ultrasonic irradiation.
Available online 28 February 2012 Ultrasound inactivates algal and cyanobacteria cells through cavitation by generating
extreme conditions, resulting in a number of physical, mechanical and chemical effects.
Keywords: The aim of this study was to investigate the effect of ultrasound at different frequencies on
Cyanobacteria Microcystis aeruginosa. Flow cytometry was used to measure cyanobacterial metabolic cell
Microcystis aeruginosa viability in addition to the more commonly used haemocytometry, optical density and
Ultrasound fluorimetry. Results indicate low frequency 20 kHz ultrasound with high intensity
Mechanism (0.0403 W cm
3) is effective for the inactivation of cyanobacterial cells. Higher frequencies
of 580 kHz (0.0041 W cm
3) also resulted in an inactivation effect, but 1146 kHz
(0.0018 W cm
3) showed a declumping effect as evidenced by flow cytometry. Ultrasonic
treatment over time under different sonication conditions demonstrates the following:
1. Acoustic cavitation via mechanical effects can induce sufficient shear forces to
directly rupture cyanobacteria cells.
2. At higher ultrasonic frequencies the mechanical energy of cavitation is less but
a larger proportion of free radicals are produced from the ultrasonic degradation of water,
which chemically attacks and weakens the cyanobacteria cell walls.
3. At higher frequencies free radicals also damage chlorophyll a leading to a loss in
photosynthetic cell viability.
4. At low powers ultrasonic energy results in declumping of cyanobacteria.
ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction activated carbon (National Rivers Authority, 1996 and WHO,

The inactivation and control of cyanobacteria blooms in water
bodies is an increasing world-wide problem. A number of
approaches to control are currently available or are under
investigation and these include minimizing nutrient loading,
addition of algaecides and the use of filters embedded with
2003). The use of ultrasound for environmental remediation
is attractive because it is considered to be a ‘green’ technology
in that it involves sound energy and requires either no addi-
tional chemicals or a much reduced quantity (Adewuyi, 2001).
In the particular case of the treatment of cyanobacteria
blooms it has been proven to be effective in laboratory trials

* Corresponding author.
E-mail addresses: e.joyce@coventry.ac.uk, aa3276@coventry.ac.uk, ejoyce2@hotmail.com (E.M. Joyce).
0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2012.02.019
2852 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8
and has the potential for becoming one of a suite of tools
available for large scale treatment. For many years ultrasound
has been proposed for the protection of the environment from
biological contamination due to its inactivation effect on
microorganisms (Mason, 2007). The inactivation effects arise
from the collapse of acoustic cavitation bubbles which
generates both physical and chemical effects (Joyce et al., 2003
and Koda et al., 2009).
 The physical effects involve intense shock waves and shear
forces produced by the bubble collapse and acoustic
streaming. Mechanical effects of this type are known to
break down biological cell membranes and for many years
these have been used in microbiology laboratories for the
release of cell contents (Frizzell, 1988 and Cameron et al.,
2008).
 The chemical effects are due to the formation of free radi-
cals, HO and H, from the decomposition of water vapour
within the collapsing bubble. Each cavitation bubble
collapse will result in extreme conditions involving high
temperatures (>5000 K) and high pressures (several thou-
sand atmospheres), this combination is easily capable of
fragmenting water into radicals. These radicals can attack
cell membranes leading to lysis of the cell walls (Mason and
Peters, 2002 and Koda et al., 2009).
We have reported previously on the effects of different
ultrasonic frequencies and acoustic intensities on Microcystis
aeruginosa based on optical density and haemocytometry
measurements (Joyce et al., 2010). Ultrasonic irradiation was
found to have two effects on this cyanobacteria, declumping
and inactivation. The former was observed using a 40 kHz
bath (0.0213 W cm
3) however at other frequencies up
to1146 kHz the concentration of cyanobacteria cells decreased
with the optimum effect occurring at a frequency of
580 kHz(0.0490 W cm
3) (Joyce et al., 2010). These results were
in accord with previous observations that ultrasonic treat-
ment could be more effective at higher frequencies (Tang
et al., 2004, Zhang et al., 2006a). However as the ultrasonic
frequency is increased there will be more power required to
achieve the same degree of cavitation obtained at lower
frequencies because of the shortening in rarefaction phase for
the formation of cavitation bubbles (Mason and Lorimer,
2002). Thus in order to achieve large scale cyanobacterial
bloom control using ultrasound a balance must be struck
between the frequency employed and power consumption.
One way of approaching an answer to this problem is to gather
more information on the actual mechanism of ultrasonic
inactivation of cyanobacteria because this will allow a more
informed selection of effective and economic parameters for
treatment.
Information available to date on the mechanism of cya-
nobacteria inactivation by ultrasound indicates that the
action of ultrasound is dependent upon the frequency used
but can be summarized in terms of mainly mechanical at low
frequencies, mainly chemical at high frequencies or a mixture
of these. This is a well-known effect in sonochemistry and has
been highlighted recently in terms of the change in the
surface effects of ultrasound with frequency (Mason et al.,
2011).
Sonication can collapse gas vacuoles (Walsby, 1992) and
this may cause the cyanobacterial cells to sink. However,
sinking may not lead to cell death. The hypothesis is that
using sufficient high power ultrasound; acoustic cavitation
can directly rupture whole cells or gas vacuoles within the
cells. TEM evidence has shown this effect on a single M. aer-
uginosa cells following ultrasonic treatment at 28 kHz (inten-
sity 0.12 W cm
3) for 30 s (Lee et al., 2001). Using relatively
lower power ultrasound, cell death may not always occur as
the gas vacuoles may regenerate as Hayes and Walsby (1984)
found the collapsed gas vacuole proteins may be recycled
(Walsby, 1994). In nature, during the period of gas vacuoles
reformation other phytoplankton species may temporarily
dominate the ecosystem (Walsby, 1992).
Another possible route to inactivation is ultrasonic damage
to chlorophyll a which will result in a reduction in photo-
synthetic ability. When M. aeruginosa was sonicated using
25 kHz ultrasound (intensity of 0.32 W cm
3) for 5 min the
absorbance of both chlorophyll a and phycocyanin (PC)
(photosynthetic pigment) were reduced (Zhang et al., 2006b).
This research concluded that ultrasound damaged the
photosynthetic function in cyanobacteria inhibiting photo-
synthesis thus impairing cyanobacterial growth.
At higher ultrasonic frequencies the mechanical energy of
cavitation is less but a larger proportion of free radicals are
produced from the ultrasonic degradation of water. This may
well help to explain why such higher frequencies achieve high
inactivation (Zhang et al., 2006a). At higher frequencies, free
radicals can also damage chlorophyll a which would also lead
to a loss in photosynthetic cell activity. However, the actual
mechanisms involved in the inactivation of cyanobacteria
with ultrasound have not yet been fully resolved and this
current study is designed to provide further information
through the use of flow cytometry as an analytical tool for cell
viability studies. The technique evaluates live and/or dead cell
numbers, based on a total cell sample by measuring the
fluorescence and light scattering of individual cyanobacteria
cells in large populations (Marie et al., 2005).
M. aeruginosa was chosen as a cyanobacteria species since
it is found in many cyanobacteria blooms producing micro-
cystin toxins (WHO, 2004).
2. Experimental
2.1. Materials
M. aeruginosa was purchased from the Culture Collection of
Algae and Protozoa (CCAP
strain number 1450/15) and
cultured using blue-green medium (BG11
CCAP). Cyano-
bacterial suspensions were placed in a plant growth room and
incubated at 25  C along with exposure to 12-hour incandes-
cent light and 12-hours darkness to reproduce natural day and
night cycles.
Before sonication the cyanobacterial cell concentration
was adjusted to an OD of 0.2 at 680 nm (6  106 cells per mL) by
either diluting or centrifuging the suspensions.
The effect of ultrasound on cyanobacteria was assessed
using 20, 580 and 1146 kHz ultrasound as low, medium and
high frequency sonication.
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8 2853

2.2. Ultrasonic irradiation carried out in triplicate, with the average recorded and used to
calculate ultrasonic power. Power is calculated using the
2.2.1. Low frequency ultrasound equations below:
200 mL standard suspensions of M. aeruginosa was placed in
Power ¼ ðdT=dtÞCp ðH2 OÞM
a 250 mL flask and sonicated for 30 min using a 20 kHz probe
(intensity 0.0403 W cm
3, Serial 23188E, model VCX600, Sonics
& Materials). The flask was immersed in an ice bath to Intensity ¼ Power=Area
maintain the temperature at 25  C. Samples were taken at 0, 5, T indicates temperature ( C) and t is time (seconds). Cp
10, 20 and 30 min sonication. The cell number was calculated relates to heat capacity of water at 25  C (J Kg
1 K
1) and M is
using a haemocytometer (HAE, Weber, BS748) and spectro- molar mass of H2O (kg).
photometer at 680 nm (optical density (OD), Corning) as two
common methodologies used (Baptista et al., 2009; Rajaskehar
et al., 2012; Ma et al., 2012). All experiments were performed in
3. Results and discussion
triplicate and results were reported as the average reduction
percentage, calculated using the following equation:
3.1. Effect of sonication on M. aeruginosa at three
% cell reduction ¼ ð1
C30 =C0 Þ  100% different frequencies monitored by haemocytometer and
optical density
In the equation, C30 indicates cell number after 30 min
sonication. C0 is cell number before sonication. The results from Table 1 for the sonication of 200 mL cyano-
bacteria suspension under different ultrasonic conditions
2.2.2. High frequency ultrasound show that for each individual frequency the reduction in
200 mL standard suspensions of M. aeruginosa was sonicated concentration was related directly to the power applied.
for 30 min using a multi-frequency bath (Serial 00002405, However the ultrasonic effect leading to the maximum
model M11-010, Meinhardt) at 580 and 1146 kHz using three reduction appears to be dependent on the method of
different intensities: 40%, 80% and maximum with a circu- measurement thus by haemocytometry it was 1146 kHz at
lating cooling system (Julabo, FL 300) to control the tempera- power setting, 0.0248 W cm
3 but by OD it was 580 kHz at
ture below 25  C. Samples were taken at 0, 5, 10, 20 and 30 min. 0.0493 W cm
3. This difference is probably due to the fact that
The cell numbers were calculated using a haemocytometer haemocytometry records only whole cell numbers remaining
(HAE, Weber, BS748) and spectrophotometer at 680 nm whereas the latter is an overall measure of cyanobacteria
(optical density (OD), Corning). All experiments were per- concentration (ruptured cells will still register, because their
formed in triplicate and results were reported as the average contents remain in suspension).
reduction percentage, calculated using the equation above. The differences that occur as a result of increases in the
applied frequency reflect the change in balance of cavitation
2.3. Cell integrity and viability tests effects from mainly mechanical (low frequency) to mainly
radical (high frequency). Petrier has demonstrated that there
200 mL standard suspensions of M. aeruginosa with an OD of will be an increase in radical production through the decom-
0.2 at 680 nm (6  106 cells per mL) were sonicated for 30 min at position of water as the frequency is increased while the
various frequencies and powers. Samples were taken after 0, power remains constant (Petrier et al., 1992) and Mason has
5, 10, 20 and 30 min and the cell numbers were calculated shown that there is a change in balance from mainly
using a haemocytometer. The in vivo fluorescence intensity at mechanical to chemical effects with rise in frequency (Mason
25  C was measured using a fluorospectrometer (Shimadzu, et al., 2011).
RF5301) which determined the phycobiliproteins (photosyn- The results that were obtained at higher power and low
thetic pigment) of a sample. Cyanobacterial cell viability was frequency are in accord with those of Graham-Rowe who
also analyzed with a BD FACS Calibur flow cytometer using an reported that high intensity ultrasound may control cyano-
argon laser (excitation at 488 nm). 1 mL M. aeruginosa was bacterial blooms through the disruption of whole cells or of
stained with 1.0 mL SYTO-9 and 1.0 mL Propidium Iodide (PI) the gas vacuoles within the cells. When gas vacuoles burst the
using a LIVE/DEAD Baclight bacterial viability kit (Invitrogen,
L10316). The auto fluorescence of the standard settings
used for flow cytometry analysis were: FSC ¼ E00, SSC ¼ 242, Table 1 e Effect of ultrasound on 200 mL Microcystis
green fluorescence (FL1) ¼ 550 nm and red fluorescence aeruginosa suspensions.
(FL3) ¼ 610 nm. Freq. Power Intensity % Reduction % Reduction
(kHz) setting (W cm
3) (HAE) (OD)
2.4. Measurement of acoustic power by calorimetry 20 e 0.0403 39.25 49.18
580 40% 0.0041 24.55 22.13
Acoustic power entering each ultrasonic system was deter- 580 80% 0.0216 59.33 36.84
mined by calorimetry (Mason and Peters, 2002). The temper- 580 Maximum 0.0493 44.11 47.37
1146 40% 0.0018 14.77 8.33
ature of 200 mL water (H2O) was recorded every 10 s over a set
1146 80% 0.0124 66.19 23.89
period of time (180 s) starting at ambient using continuous
1146 Maximum 0.0248 91.54 44.63
sonication at frequencies of 20, 580 and 1146 kHz. This was
2854 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8

cell will sink and then during the period of gas vacuoles
reformation other phytoplankton species may temporarily
dominate the ecosystem (Graham-Rowe, 2009; Walsby, 1992).
The effect of higher frequencies in terms of radical production
and attack has been reported by Koda who concluded free
radicals attack the cell surface breaking cell walls resulting in
leakage of cell contents (Koda et al., 2009).
The results of sonicating 200 mL M. aeruginosa suspensions
at three different frequencies over 30 min using haemocy-
tometry (HAE) are shown in Fig. 1. Following ultrasonic
treatments with the 20 kHz probe (0.0403 W cm
3) the
concentration of cyanobacteria decreased to 39.25%. The
effects of higher frequencies were studied at markedly lower Fig. 2 e Inactivation of 200 mL Microcystis aeruginosa using
powers 580 (0.0041 W cm
3) and 1146 kHz (0.0018 W cm
3). 20, 580 and 1146 kHz for 30 min sonication (optical
The results appeared to follow the order of frequency i.e. density).
20 > 580 > 1146 kHz and the same order is found using OD
(Fig. 2). However these results are somewhat misleading
because the acoustic power entering the system has not been
taken into account. This can be done using the concept of
Table 2 e Effect of ultrasound on 200 mL Microcystis
ultrasonic efficiency (Joyce et al., 2010). Ultrasonic efficiency
aeruginosa suspensions at 20, 580 and 1146 kHz.
can be determined by dividing the reduction in cyanobacteria
Frequency (kHz) 20 580 1146
by the acoustic power entering the system (Table 2). It then

3
becomes clear that 20 kHz ultrasound performs the lowest Power (W cm ) 0.0403 0.0041 0.0018
efficiency by either of these monitoring techniques and that Volume (mL) 200 200 200
the choice of optimum frequency is 580 by optical density and Sonication time 30 30 30
(minutes)
1146 by haemocytometry. The difference in the direct results
%HAE 39.25 24.55 14.77
obtained from these two measurement techniques has Efficiency (HAE) 973.94 5987.80 8205.56
already been explained above. %OD at 680 nm 49.18 22.13 8.33
These results suggest that the chemical (i.e. radical) effects Efficiency 1220.35 5397.56 4627.78
of ultrasound may be more significant than mechanical (OD at 680 nm)
effects and so higher frequencies are better than lower in
terms of energy consumption. However the time taken to
deactivate the cyanobacteria is rather longer at higher activity. The technique is based on the sensitive detection of
frequencies and lower powers and so this should be taken into the in vivo fluorescence of the phycobiliproteins peak at
account when considering larger scale treatment. 665 nm and this signal comes only from metabolically active
cells. Thus changes in the signal reflect changes in the
3.2. Effect of sonication on M. aeruginosa at three metabolic activity of a suspension of cyanobacteria. Fluorim-
different frequencies monitored by fluorimetry etry assesses ultrasonic effects on cyanobacterial photosyn-
thetic functions. Damage to photosynthetic function may lead
When measuring the effects of ultrasound on cyanobacteria to subsequent death of cyanobacterial cells.
the most commonly used methods are OD and HAE as
described above. Another technique which has been used is
fluorimetry which provides a different measure of metabolic

Fig. 1 e Inactivation of 200 mL Microcystis aeruginosa using

20, 580 and 1146 kHz for 30 min sonication Fig. 3 e Inactivation of 200 mL Microcystis aeruginosa using
(haemocytometry). 20, 580 and 1146 kHz for 30 min sonication (fluorimetry).
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8
Fig. 4 e Live and dead cyanobacteria control sub-
populations analyzed by a forward scatter histogram plot.
Fig. 5 shows the effect of 20, 580 and 1146 kHz ultrasound at
the powers shown in Table 2 after 30 min sonication.
The effect of ultrasound at three frequencies over 30 min
were also recorded using fluorimetry using the phycobilipro-
teins peak at 665 nm which reflects metabolic activity (Fig. 3).
Sonication with a 20 kHz probe (0.0403 W cm
3) resulted in
fluorimetry peaks at 665 nm decreasing with ultrasonic
treatment. The peak at 665 nm reduced after sonication
indicating the photosynthetic system of cyanobacteria
cells was damaged. Sonication with the 580 kHz bath
(0.0041 W cm
3) also resulted in a slight decrease in the peaks
at 665 nm, indicating that the metabolic activity of cyano-
bacteria cells was damaged. Sonication with the 1146 kHz bath
(0.0018 W cm
3) showed no significant reductions of the
fluorimetry peak during treatment suggesting that sonication
did not affect cyanobacterial metabolic activities.
Fig. 5 e Live and dead cyanobacteria control sub-populations analyzed by a “dot plot”.
2855
Fig. 6 e Effect of sonication on Microcystis aeruginosa using
20, 580, 1146 kHz and control (no sonication) cell counts vs.
forward side scatter histogram plot.
3.3. Effect of sonication on M. aeruginosa at three
different frequencies monitored by flow cytometry
Flow cytometry (FCM) provides different and complementary
information relating to cell viability than can be obtained from
the methods referred to above. In particular it is able to
discriminate and enumerate between and live and dead cells
plus cell fragments. The method involves passing an aqueous
suspension of the cyanobacteria in a narrow tube through the
path of a laser beam. The laser beam is scattered by the
suspension and the scattered light is collected by sensors and
processed to provide information on the characteristics of the
suspended material. Cyanobacteria have intrinsic fluores-
cence properties (see Section 3.2 above) but the fluorescence
intensity may be interfered by side scatter sensitivity from gas
vacuoles (Brookes et al., 2000) so that FCM cell vitality analysis
2856 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8

is commonly augmented by specific staining, in this case
SYTO-9 for live cells and PI for dead (Lee et al., 2000 and
Surono et al., 2008). Forward scatter is used to analyse the
material in terms of size from larger whole cells down to small
cell fragments recording some 10,000 events in seconds.
In order to calibrate the flow cytometry data in terms of live
and dead cyanobacteria cells two control samples were
prepared and stained. A fresh cyanobacteria suspension
(7 day culture) and a dead control suspension prepared by
killing the cyanobacteria by boiling for 30 min. The results of
FCM analysis of these samples are presented as histogram
plots using forward side scatter (Fig. 4). In this plot the hori-
zontal axis of the graph essentially shows the size of particle
and the vertical axis indicates intensity i.e. the number of cells
giving rise to each signal (cell counts). Fig. 5 clearly shows that
the loss in cell viability through boiling results in a loss of the
live sub-population which then shifts from right (live) to left
(which presented lower cell viability levels).
An alternative representation of this same data is the so-
called “dot plot” (Fig. 5). Each dot represents a single event

Fig. 7 e Effect of sonication on Microcystis aeruginosa using 20, 580 and 1146 kHz displayed as dot plots.
 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8
and the data is grouped into four segments which relate to cell
counts in different regions of the histogram. The quadrants are
arranged in a clockwise order with upper left (UL) being live cells
through upper right (UR) and lower right (LR) which contain
cells in different stages of inactivation with the lower left (LL)
containing dead cells and cell fragments. It should be noted that
the dot plot obtained from the live cell culture does show the
presence of some dead or fragmented cells but these would be
expected in any normal cell culture. On the other hand the dead
cell control shows almost entirely cells in LL quadrant.
When FCM analysis is applied to sonication over
30 min results under the conditions above the results dis-
played as a histogram (Fig. 6) indicate that cyanobacterial
cell viability levels are reduced in the following order:
20 < 580 < 1146 kHz < Control (no sonication).
These results are more revealing when displayed as dot
plots (Fig. 7). For each frequency the control FCM is shown and
these are slightly different because no two cultures taken on
different days can be exactly the same. Nevertheless each of
the controls shows the expected predominance of live cells in
the upper left (UL) quadrant. The effects of sonication at
different frequencies do however reveal significant differences.
The numbers of dots in each quadrant are displayed in Fig. 7
Fig. 7 (a2) shows that using 20 kHz (0.0403 W cm
3) results
in major inactivation with the population moving almost
entirely into the lower left (LL) quadrant. In Fig. 8 the actual
numbers of counts are displayed showing some 85% of the
counts in this “dead” quadrant. Sonication with the 580 kHz
bath (0.0041 W cm
3) for 30 min treatment illustrates a large
sub-population positioned in the lower right (LR) quadrant,
which represented cells which were between live and dead
sub-populations; thus sonication did not result in complete
inactivation. This indicates some cyanobacteria were between
live and dead states (live but not fully metabolically active).
This may be due to low intensities not generating sufficient
power to disrupt cyanobacterial cells. This is a significantly
different result from that obtained using the 20 kHz probe. We
believe that this may due to the difference in the cavitational
effects of 20 vs. 580 kHz. In the former, effects are mainly
mechanical and almost instantaneous whereas at 580 kHz
lower mechanical effects are accompanied by the production
of a greater amount of free radicals. Ultrasonically generated
Fig. 8 e % Microcystis aeruginosa cells remaining in UL, UR,
LL and LR quadrants before and after 30 min sonication.
2857
free radicals will not directly disrupt cell walls but will react
chemically with membranes eventually disrupting cell walls
but this may require long reaction times (Frizzell, 1988).
Sonication with the 1146 kHz bath (40% intensity,
0.0018 W cm
3) for 30 min resulted in the majority of sub-
populations remained in upper left (UL) and upper right (UR)
quadrants. Sub-populations in the lower left (LL) quadrant
increased following 30 min treatment. Sonication did not
inactivate cyanobacterial cells and a declumping effect
occurred. Joyce et al. (2010) demonstrated sonication at
low powers resulted in a declumping effect at 40 kHz
(0.0213 W cm
3) since ultrasound can deaggregate cyano-
bacterial clumps producing more individual cells. In this case,
although 1146 kHz belongs to high frequency range, low
intensities may also induce declumping effects. It is inter-
esting to note that haemocytometer and optical density
results did not illustrate declumping effects but rather low
reductions in cell numbers, which may due to higher sensi-
tivity of flow cytometry analysis.
4. Conclusions
Flow cytometry results allow us to draw the following
conclusions about the mechanism involved in the effects of
sonication on cyanobacteria. At low frequencies but high
powers (20 kHz), acoustic cavitation leads mainly to
mechanical effects i.e. high shear forces generated can lead
to the direct rupture of cells. At low ultrasonic frequencies
less radicals are produced than at higher frequencies and so
chemical damage to cells is small. At high frequencies
(580 kHz) with medium powers, the cavitation collapse
energy is smaller than at low frequencies leading to less
direct mechanical damage through sheer forces. On the other
hand more radicals are produced leading to inactivation but
not necessarily cell rupture i.e. cells are not necessarily
metabolically active. At high frequencies but low powers
(1146 kHz) the energy input is sufficient to deaggregate cya-
nobacteria clumps into individual cells resulting in
a declumping effect.
references
Adewuyi, Y.G., 2001. Sonochemistry: environmental science and
engineering applications. Industrial & Engineering Chemistry
Research 40 (22), 4681e4715.
Baptista, M., Stocher, T., Vasconcelos, V.M., Vasconcelos, M.,
2009. Fate and effects of octylphenol in a Microcystis aeruginosa
culture medium. Aquatic Toxicology 92 (2), 59e64.
Brookes, J.D., Canf, G.G., Oliver, R.L., 2000. Heterogeneity of
cyanobacterial gas vesicle volume and metabolic activity.
Journal of Plankton Research 22, 1579e1589.
Cameron, M., McMaster, L.D., Britiz, T.J., 2008. Electron
microscopic analysis of dairy microbes inactivated by
ultrasound. Ultrasonics Sonochemistry 15 (6), 960e964.
Frizzell, L.A., 1988. In: Suslick, K.S. (Ed.), Biological Effect of
Acoustic Cavitation, Ultrasound: Its Chemical, Physical and
Biological Effects. VCH, New York, pp. 287e290.
Graham-Rowe, D., 2009. Killer algal blooms won’t like the sound
of this. New Scientist 202, 22.
2858 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 8 5 1 e2 8 5 8
Hayes, P.K., Walsby, A.E., 1984. An investigation into the recycling
of gas vesicle protein derived from collapsed gas vesicles.
Journal of General Microbiology 130, 1591e1596.
Joyce, E., Phull, S.S., Lorimer, J.P., Mason, T.J., 2003. The
development and evaluation of ultrasound for the treatment
of bacterial suspensions. A study of frequency, power and
sonication time on cultured bacillus species. Ultrasonics
Sonochemistry 10, 315e318.
Joyce, E., Wu, X., Mason, T.J., 2010. Effect of ultrasonic frequency
and power on algae suspensions. Journal of Environmental
Science and Health, Part A Toxic/Hazardous Substances and
Environmental Engineering 45 (7), 863e866.
Koda, S., Miyamoto, M., Toma, M., Matsuoka, T., Maebayashi, M.,
2009. Inactivation of Escherichia coli and Streptococcus mutans by
ultrasound at 500 kHz. Ultrasonics Sonochemistry 16, 655e659.
Lee, T., Nakano, K., Matsumara, M., 2001. Ultrasonic irradiation
for blue-green algae bloom control. Environmental
Technology 22, 383e390.
Lee, T., Nakano, K., Matsumura, M., 2000. A new method for the
rapid evaluation of gas vacuoles regeneration and viability of
cyanobacteria by flow cytometry. Biotechnology Letters 22,
1833e1838.
Ma, M., Liu, R., Liu, H., Qu, J., 2012. Effect of moderate pre-
oxidation on the removal of Microcystis aeruginosa by
KMnO4eFe(II) process: significance of the in-situ formed
Fe(III). Water Research 46 (1), 73e81.
Marie, D., Simon, N., Vaulot, D., 2005. In: Anderson, R.A. (Ed.),
Phytoplankton Cell Counting by Flow Cytometry, Algal
Culturing Techniques. Academic Press.
Mason, T.J., 2007. Sonochemistry and the environment e
providing a “green” link between chemistry, physics and
engineering. Ultrasonics Sonochemistry 14 (4), 476e483.
Mason, T.J., Cobley, A.J., Graves, J.E., 2011. New evidence for the
inverse dependence of mechanical and chemical effects on
the frequency of ultrasound. Ultrasonics Sonochemistry 18,
226e230.
Mason, T.J., Lorimer, J.P., 2002. Applied Sonochemistry: The Use of
Power Ultrasound in Chemistry and Processing. Wiley-Vch,
Weinheim.
Mason, T.J., Peters, D., 2002. Practical Sonochemistry Power
Ultrasound Uses and Applications. Horwood Publishing,
Chichester.
National Rivers Authority (NRA), 1996. The Occurrence and Fate
of Blue-green Algal Toxins in Freshwaters. National Rivers
Authority, London.
Petrier, C., Micolle, M., Merlin, G., Luche, J., Reverdy, G., 1992.
Characteristics of pentachlorophenate degradation in
aqueous solution by means of ultrasound. Environmental
Science Technology 26, 1639e1642.
Rajaskehar, P., Fan, L., Nguyen, T., Roddick, F.A., 2012. Impact of
sonication at 20 kHz on Microcystis aeruginosa, Anabaena
circinalisand ChlorellaSp. Water Research 46 (5), 1473e1481.
Surono, I.S., Collado, M.C., Salminen, S., Merluto, J., 2008. Effect
of glucose and incubation temperature on metabolically
active Lactobacillus plantarum from dadih in removing
microcystin-LR. Food and Chemical Toxicology 46 (2),
502e507.
Tang, J., Wu, Q., Hao, H., Chen, Y., Wu, M., 2004. Effect of 1.7 MHz
ultrasound on a gas-vacuolated cyanobacterium and a gas-
vacuole negative cyanobacterium. Colloids and Surface B:
Biointerfaces 36, 115e121.
Walsby, A.E., 1992. A control of gas-vacuolate cyanobacteria. In:
Sutcliffe, D.W., Jones, J.G. (Eds.), Eutrophication: Research and
Application to Water Supply. Freshwater Biological
Association, Ambleside, UK, pp. 150e162.
Walsby, A.E., 1994. Gas vesicles. Microbiological Reviews 58 (1),
94e144.
WHO, 2003. In: WHO (Ed.), Algae and Cyanobacteria in Fresh
Water Guidelines for Safe Recreational Water Environments,
Coastal and Fresh Waters. WHO, Geneva.
WHO, 2004. In: Guidelines for Drinking-water Quality, third ed.
World Health Organization, Geneva.
Zhang, G., Zhang, P., Wang, B., Liu, H., 2006a. Ultrasonic
frequency effects on the removal of Microcystis aeruginosa.
Ultrasonics Sonochemistry 13, 446e450.
Zhang, G., Zhang, P., Liu, H., Wang, B., 2006b. Ultrasonic damages
on cyanobacterial photosynthesis. Ultrasonics Sonochemistry
13, 501e505.