ARTICLE IN PRESS

Biosystems Engineering (2004) 89 (3), 297308

doi:10.1016/j.biosystemseng.2004.08.012
PHPostharvest Technology

Ultrasonic Cellular Disruption of Yeast in Water-based Suspensions

A. Lo+ rincz
Faculty of Agricultural and Food Sciences, University of West Hungary, Mosonmagyarovar, Institute of Agricultural, Food and Environmental
Engineering, Hungary; e-mail: lorinczattila1@freemail.hu
(Received 1 May 2003; accepted in revised form 5 August 2004; published online 2 November 2004)

This study examines the qualitative and quantitative properties of water-based suspensions and the effect on
the occurrence and threshold levels of ultrasonic phenomena, standing wave, acoustic streaming and especially
cavitation. Commercial pressed (hydrated) and lyophilised (dehydrated) bakers yeast (Saccharomyces
cerevisiae), and a dolomite suspension that has the same average particle size as that of the yeast were used.
The experiments were conducted in the specially designed and constructed ultrasonic treating vessel under
conditions of 1
117 MHz frequency and 30120 kW m2 ultrasound output power. The levels of particle
concentration, expressed in g l1, in the ultrasound eld that were needed to terminate cavitation (that is the
cavitation threshold concentration), and the length of time that passed from the start of the experiment until
the restart of cavitation, which is the time period required for the formation of cavitation, were examined. The
experiments aimed at determining the time period required for the formation of cavitation was carried out at
concentration levels that were 1
5 times higher than the cavitation threshold concentrations. The acoustic
phenomena taking place in the ultrasound eld, and through these, the effects of ultrasound can be
characterised by these two measures. Under the conditions of an output power of 90 kW m2, it was found
that a concentration of 3
2 g l1 lyophilised Saccharomyces cerevisiae bakers yeast stopped cavitation in the
ultrasound eld. Then, by using multiples of the aforementioned concentration, the acoustic phenomena
occurring in the ultrasound eld were monitored and, simultaneously, the survival dynamics of the yeast cells
were examined. Physical parameters of the ultrasound eld had an essential effect on the acoustic phenomena
formed in the sound eld and on the threshold levels of their formation.
r 2004 Silsoe Research Institute. All rights reserved
Published by Elsevier Ltd

1. Introduction
1.1. Ultrasound physics
Cavitation means formation of cavities in the liquid; it
is of two types: transient and stable cavitation (Frizzel,
1988). There is no acoustical cavitation in the ultrasound eld until the amplitude of the acoustic pressure
exceeds a certain level, the cavitation threshold (Fry,
1978). The cavitation threshold is proportional to the
frequency of ultrasound, the hydrostatic pressure in the
liquid, and the viscosity of the sample and it is inversely
proportional to the gas content and temperature of the
sample (ter Haar, 1988). Due to the absorption, the
intensity of ultrasound decreases exponentially with
distance and the absorption coefcient primarily de1537-5110/$30.00

pends on the speed of propagation of the ultrasound in

the subject media (Kuttruff, 1991). The speed of
ultrasound largely depends on the particle size and
suspension concentration (Sayan & Ulrich, 2002).
Acoustic streaming is the movement of liquid that is
caused by an intensive ultrasound (Mitome, 1998).
Watmough et al. (1990) concluded that acoustic
streaming produced visible mixing of liquid in the
ultrasound eld. An acoustic reector placed opposite to
the transducer causes a visible standing wave to be
formed. In a standing wave, the materials whose density
are lower and higher than that of the liquid drift to
propagation cluster planes, pressure nodes and antinodes, respectively (ter Haar, 1988). Yeast (Saccharomyces cerevisiae) particles were manipulated in a
standing wave ultrasound eld at frequencies of
297

r 2004 Silsoe Research Institute. All rights reserved

Published by Elsevier Ltd

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298

Notation
C
Ca
Cb
D
k
N
NC

suspension concentration, g l1

cavitation threshold concentration, from the
auxiliary determination method, g l1
cavitation threshold concentration, from the
basic determination method, g l1
decimation time, s
destruction rate, s1
total cell count, cell ml1
relative living cell count between the cavitation, %

1 MHz. The particles formed visible bands in pressure

nodes whose distance from each other was equal to half
of the wavelength. In the direction of radiation, the
bands formed column-like structures (Hawkes et al.,
1998). Church and Miller (1983) observed that as a
result of the standing wave the cells and the bubbles
were separated into different layers; thus, no interaction
can occur between them.

NS
n0
nt
p
t
t0
tt
TC
l

relative living cell count between the standing

wave, %
initial living cell count, ml1
actually living cell count, ml1
ultrasound output power, kW m2
time, s
initial time, s
nal time, s
time period for formation of cavitation, s
wavelength, mm

bacteria in milk in an ultrasound system. The Gramnegative Pseudomonas fluorescens was less resistant to
ultrasound than the Gram-positive Streptococcus thermophilus because of the differences in their cell
membrane structures. Irradiation of milk by ultrasound,
either as a stand-alone treatment or combined with
traditional heat treatment technologies, is a promising
method as homogenisation of milk is also accomplished
simultaneously; thus, the total energy required for these
operations is reduced.

1.2. Cell biology

The most useful approaches in analysing the biological effects of ultrasound are examining its mechanical
and physical effects. The most important phenomenon
of all the effects is cavitation. Both types of cavitation
(transient and stable) caused a wide range of biological
effects (Miller et al., 1996). The transient cavitation may
have led to the formation of free radicals (e.g. hydrogen
peroxide) and of other sonochemical compounds in an
indirect way (Riesz & Kondo, 1992). The formation of
hydrogen peroxide and other sonochemical compounds
in sufcient concentration causes biochemical changes
in living cells (Miller & Thomas, 1994). The effects of
ultrasound on surviving cells may include structural
changes and effects on deoxyribonucleic acid (DNA)
(Liebeskind et al., 1979). Biological effects observed in in
vitro systems include fragmentation of cell membranes
that is caused by the collapse of cavitation bubbles, by
microstreaming near the boundary layer and by the
formation of sonochemical compounds (Miller, 1987).
According to Carstensen et al. (1993), the effectiveness
of cell disruption was inversely proportional to the
applied cell concentration. Radel et al. (2000) explained
that, where propagating waves dominated, strong cell
destruction was observed while in the case of a standing
wave, the cell destruction was negligible. Villamiel and
Jong (2000) examined the opportunities for deactivating
Pseudomonas fluorescens and Streptococcus thermophilus

1.3. Applied microbiology

According to Walsch et al. (1999), the most signicant
effects of the propagating ultrasonic waves on yeast
(Saccharomyces cerevisiae) physiology are the decrease
in the number of living cells and in the capability for
cellular division. These authors did not record any
signicant cellular destruction effect in the standing
wave eld. They examined the changes in vitality of cells
exposed to propagating and plane ultrasound waves by
methylene blue staining. According to B ro (1976),
staining is suitable for determining the vitality of
microorganisms, primarily of yeasts. By adding methylene blue to a suspension that consists of both living and
dead cells, the dead cells become blue immediately, while
the living ones remain unaffected. According to Deak
(1997), the majority of the experiments indicate that the
interferences acting through environmental factors that
cause the destruction of the microorganisms show
exponential relationships. The specic destruction rate
k in s1 of cells from Eqn (1):
k 2
303=t  t0  log10 n0 =nt

(1)

where: n0 is the initial number of cells at time t0; nt is the

number of surviving cells at time t; and the time interval
in s. Plotting the logarithm of the number of surviving
cells as a function of time, a linear relationship is

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ULTRASONIC CELLULAR DISRUPTION OF YEAST

established. If the time tt0 that occurs in Eqn (2) is

dened as the period of time during which the number
of surviving cell decreases to one-tenth of its original
value, the concept of decimation time D in s is
established. If t  t0 D and nt 0:1n0 ; then the value
of D is given by
D 2
303=k

(2)

Decimation time is the measure of the resistance of the

microorganism population expressed in s.

2. Objective
It is a general problem in the literature that almost all
the publications dealing with the biological effects of
ultrasound specify only the applied output power of
ultrasound as a factor affecting the results. In most cases
it is not enough for the reader to know which acoustic
phenomenon was generated by this output power during
the examinations. Knowledge of the prevailing acoustic
phenomenon is essential because it is not the intensity
that has an effect on the material but the acoustic
phenomenon that is formed in the given material under
the conditions of the applied ultrasound output power.
Effects of the formed acoustic phenomenon remain the
same in a wide range of ultrasound intensities. Based on
the above, the objective of this work is to understand the
dynamics of the formation of the acoustic phenomena as
a function of applied suspension density and, simultaneously, to examine the cell-disrupting effects of the
acoustic phenomena. Based on this work, ultrasoundinduced cell decomposition and particle manipulation
can be methods that are available for everybody to carry
out and to design ultrasound systems having different
geometry.

3. Materials and methods

3.1. Control instrument
The specially designed and constructed ultrasound
signal generator generated a sinusoidal signal at a
frequency of 1
117 MHz. The ultrasound amplier could
produce an amplication of 040 W that could be
converted to W m2 in the knowledge of the radiation
surface. The ultrasound resonators that are suitable for
manipulating, handling and controlling small suspended
particles consist of at least four parts, namely: the
piezoelectric transducer, the carrier glass container, the
suspension and the acoustic reector. The ultrasound
laboratory is shown in Fig. 1, and the ultrasound system
is shown in Fig. 2.

Fig. 1. Ultrasound laboratory: A, ultrasound system; B,

cavitation detection system; C, machine vision system

Fig. 2. Ultrasound system: A, signal generator and ultrasound

amplifier; B, transducer; and C, treating vessel

The continuous wave treatment method was applied,

primarily with longitudinal waves. The shape of the
treatment vessel allowed the formation of both standing
and propagating planar waves. A planar PZT-4 ceramic
body having a diameter of 22 mm was the active
component of the resonator. The ultrasound eld was
formed by the treated material itself. The suspension
was poured in a double-walled cylindrical glass vessel
having internal and external diameters and a height of
30, 80 and 200 mm, respectively. The temperature of this
vessel was maintained at 20 1C. The quantity of the
treated suspension, 0
025 l, is in the near-eld range of
the sound eld. After pouring into the vessel, when kept
motionless, the height of the liquid is 0
0352 m, that is,
26
7 times the wavelength l of 1
31 mm. The air layer
above the liquid, which behaves as an ideal reector, has
a primary role in the formation of the standing waves.
The transducer was connected to the ultrasound
amplier through an electric connector and the transducer is suitable for generating both standing and
propagating longitudinal waves. The non-rigid reector
was the air layer situated above the suspension, facing
the piezoelectric ceramics. Weight was measured by an

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analytical balance of Precisa 505M2020C DR SCS

type. Its accuracy was 0
001 g.

concentration were applied by the auxiliary method at

90 kW m2.

3.2. Suspension

3.4. Detection

Water was used as a suspending agent. Its temperature was maintained at 20 1C in a water bath that was
not shaken and it was conditioned for 0
51 h before
use. The dissolved oxygen contents of the water samples,
as a factor responsible for the formation of cavitation,
were determined in the range of 1112% after conditioning.
Lyophilised dehydrated and pressed hydrated bakers
yeast Saccharomyces cerevisiae and dolomite particles
having the same particle size as of the yeast were used as
suspended material. The use of yeast in the experiments
was justied by the fact that this is the most frequently
used microorganism in the post-harvest and food
technology applications. By examining the different
forms of this organism, our objective was to specify the
best conditions for the ultrasound treatment. By
comparing the behaviour of ground dolomite and the
biological material in the ultrasound eld, our objective
was to determine whether the results observed in the
ultrasound eld are based on physical effects exclusively,
or some biological factor shall also be considered when
these results are evaluated.

According to Veit (1977), cavitation appears as

acoustical noise which can be recorded by a microphone
and analysed. Components of the applied cavitation
detection arrangement are the microphone, amplier
and oscilloscope. The observable acoustic phenomena,
standing wave and acoustic streaming, as a function of
the length of the treatment period, were visually
examined. Changes in yeast vitality were examined by
methylene blue vitality staining.
During the examination, the formation and duration
of the individual acoustic phenomena (acoustical
streaming, standing wave, cavitation) and, simultaneously, their effect on the vitality of yeast cells placed
in the ultrasound eld were examined.

3.3. Experimental plan

From the initial examinations at a frequency of
1
117 MHz, the cavitation threshold concentrations in
g l1 were determined for the different suspended sample
particles at 20 1C under the output conditions of 30, 60,
90 and 120 kW m2 by the basic and auxiliary methods.
When applying the basic method, particles were added
to the material in the ultrasound eld until the
cavitation sound stopped. The particle density at which
cavitation ceased to exist is the cavitation threshold
concentration Ca. The value for the cavitation threshold
concentration provided by the basic method was used as
input in the auxiliary approximation method that was
considered as a more accurate one. Examination of the
time period required for the formation of cavitation TC
was carried out for every material and every output level
at concentrations that were equal to 1
5 times the
cavitation threshold concentration was measured by the
auxiliary method.
When the standing wave was formed, the point of
time at which cavitation occurred and the biological
effects were examined simultaneously; multiple concentrations (1; 1
5; 1
7; 2
2; 3) of the cavitation threshold

3.5. Cavitation threshold concentration

3.5.1. Basic method
When applying the basic method for determining the
cavitation threshold concentration, detectable cavitation
occurred in the presence of 0
025 l conditioned water
(20 1C) in the resonator at an ultrasound output power
of 90 kW m2. The yeast sample was added to the sound
eld until cavitation ceased to exist. The quantity of
yeast added to the system until the cessation of
cavitation is the cavitation threshold concentration in
g l1. The basic method is an informative test measurement whose result provides the basis for the auxiliary
cavitation threshold concentration measurements of
approximation type.
3.5.2. Auxiliary method
The objective for determining the cavitation threshold
concentration by an auxiliary method was to improve
the accuracy of the results compared to the results given
by the basic method. In the auxiliary method presuspended samples having known initial cell concentration are examined and the cavitation threshold concentration is the concentration of the sample where
cavitation is detected within 1 s. In the biological
examinations, the results of the auxiliary method were
used.
3.6. Time period required for the formation of cavitation
While examining the time period required for the
formation of cavitation TC, 0
025 l of conditioned tap

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ULTRASONIC CELLULAR DISRUPTION OF YEAST

3.7. Vital cell number and acoustic wave phenomena

Cell-disrupting effects of the ultrasound occur immediately in the cells. A pre-determined quantity of
biological material, as determined in the experimental
plan, was suspended into the sound eld by articial
agitation, and then the mixer was removed. From the
moment when the ultrasound was switched on, measurement of the elapsed time was started with a stopwatch. Samples of 0
05 ml were taken from the treated
suspension every 15 s, and the vital cell numbers of these
samples were determined by methylene blue staining.
Sampling was carried out until the steady state of the
acoustic wave phenomena was achieved. The time
elapsed from starting the experiment was measured with
a stop-watch until the formation of visible standing
wave or of cavitation (until cavitation sound was
detected). The decimation time D and the specic
destruction rates k were determined by the method of
Deak (1997) for different phases of the acoustic
phenomena and survival curves plotted.

Cavitation threshold, g l1

5
4
3
2
1
0
20

40
60
80
100
120
Ultrasound output power, kW m2

140

Fig. 3. Results of cavitation threshold concentration measurements for dehydrated yeast and ground dolomite: 3, dehydrated
yeast, basic examination; d, dehydrated yeast, auxiliary
examination; W, dolomite, basic examination; m, dolomite,
auxiliary examination

13.0
12.5
Cavitation threshold, g l1

water was poured into the ultrasound eld, while the

temperature of the ultrasound eld was maintained at
20 1C by a thermostat. In the case of the different types
of particles, the time period required for the formation
of cavitation was examined by adding particles into the
ultrasound eld until its concentrations were equal to
1
5 times the cavitation threshold concentration measured by the auxiliary method. The particles whose
quantity was equal to 1
5 times the cavitation threshold
concentration measured by the auxiliary method were
placed in a suspension and a sonic output equal to the
output power of the subject cavitation threshold
concentration was switched to the resonator. The
duration between switching on the ultrasound device
and initiation of cavitation is the time period for the
formation of cavitation. The experiments were repeated
four times.

301

12.0
11.5
11.0
10.5
10.0
9.5
9.0

20

40
60
80
100
120
Ultrasound output power, kW m2

140

Fig. 4. Cavitation threshold concentrations for pressed yeast:

}, basic examination; ~, auxiliary examination

4. Results and discussion

4.1. Cavitation threshold concentration
The results of determining the cavitation threshold
concentration for lyophilised yeast and ground dolomite, and pressed yeast as functions of different
ultrasound outputs are shown in Figs 3 and 4,
respectively. Standard deviations from the average
cavitation threshold concentration measured by the
basic method for ground dolomite and for lyophilised
yeast, as shown in Fig. 3, were 3
54 and 3
66
8%,

respectively. From the data shown in Fig. 3, the

functional relationships [Eqns (3) and (4)] between the
cavitation threshold concentration Cb and Ca in g l1
measured for dolomite by the basic and the auxiliary
methods, respectively, and the ultrasound output power
p in kW m2 are
C b 0
6431e0
01782p

(3)

C a 0
5218e0
01922p

(4)

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C b 1
6637e0
00812p

(5)

C a 1
543e0
00824p

(6)

with values for R of 0
9965 and 0
9981, respectively.

The standard deviation from the average cavitation
threshold concentration measured for pressed yeast by
the basic method, as shown in Fig. 4, was in the range of
1
953
4%. From the data shown in Fig. 4, the
functional relationships [Eqns (7) and (8)] between the
cavitation threshold concentration and the ultrasound
output measured for pressed yeast by the basic and
auxiliary methods are
C b 8
3934e0
00314p

(7)

C a 8
2478e0
0031p

(8)

with values for R of 0
968 and 0
992, respectively.

Interpreting the results, it was observed that the
cavitation threshold concentrations were proportional
to the output of the treating ultrasound for each
material examined, that is, if the output of the
ultrasound increased, the cavitation threshold concentration also increased and vice versa. Cavitation threshold concentration levels measured by the auxiliary
method were lower than the respective levels measured
by the basic method for all the suspensions at each
ultrasound output power. The reason for this is that no
preliminary suspension test was carried out by the basic
method; hence, the suspending power of the acoustic
streaming caused by the cavitation determined the
dispersion of the particles in the suspension. Due to
the surface tension, some particles remained at the
surface of the liquid; thus, the quantity of the particles
needed to be added to the suspension for achieving the
same suspension concentrationcavitation threshold
concentrationin the liquid was higher than in the
auxiliary experiment performed under the same treatment conditions.
4.2. Time period for the formation of cavitation
When the experiments were carried out at higher
suspension concentrations, three different acoustic
phenomena were observed in the ultrasound eld. They
always followed each other in the following sequence:
acoustic streaming, standing wave, cavitation. Figures 5
and 6 show the time period for the formation of

48
Time period for formation of cavitation, s

with values for the coefcient of determination R2 of

0
9998 and 0
9936, respectively.
Relationships [Eqns (5) and (6)] between the cavitation threshold concentration and ultrasound output
power measured for lyophilised yeast by the basic and
auxiliary methods, respectively, are

47

46

45

44

43

20

40
60
80
100
120
Ultrasound output power, kW m2

140

Fig. 5. Time period required for the formation of cavitation for

lyophilised yeast

800
Time period for formation of cavitation, s

302

790
780
770
760
750
740
730
20

40

60
80
100
120
Ultrasound outputpower, kW m2

140

Fig. 6. Time period required for the formation of cavitation for

dolomite

cavitation seconds for lyophilised yeast and dolomite as

a function of the applied ultrasound output, respectively.
The standard deviation from the average length of the
time period needed for the formation of cavitation
measured for lyophilised yeast, as shown in Fig. 5, was
in the range of 4
58
5%. From the data shown in Fig. 5,
the functional relationship [Eqn (9)] between the length
of the time period needed for the formation of cavitation
TC, in s and the ultrasound output power for lyophilised

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ULTRASONIC CELLULAR DISRUPTION OF YEAST

yeast is
T C 0
0267p 47

(9)

Table 1
Suspension concentrations applied in the biological examinations,
in 107 cell ml1 and cavitation threshold concentration Ca

with a value for R of 0
5333.

The standard deviation from the average length of the
time period needed for the formation of cavitation
measured for dolomite, as shown in Fig. 6, was in the
range of 0
933
15%. From the data shown in Fig. 6, the
functional relationship [Eqn (10)] between the length of
the time period needed for the formation of cavitation
and the ultrasound output power) for dolomite is
T C 0
53p 725

Multiple of Ca
1
1
5
1
7
2
2
3

Suspension concentration 107 cell ml1

1
79
2
68
3
04
3
94
5
37

(10)

with a value for R of 0
7363.

In order to determine the effect of the suspension
density in g l1 on the acoustic phenomena formed in the
ultrasound eld, the cavitation threshold concentration
was examined at different output powers. The results of
measuring the length of time periods required for the
formation of cavitation were obtained at particle
concentrations in the ultrasound eld that were 1
5
times higher than the respective cavitation threshold
concentrations for the different materials. Cavitation
threshold concentrations measured at different output
powers differ from each other signicantly. When
increasing the output power, the cavitation threshold
concentration was also increased proportionally. As a
consequence of this, the densities of suspensions used for
determining the time period for the formation of
cavitation were also very varied. In the studies to
determine the time period for the formation of cavitation, the added quantities of yeast, equal to 1
5 times the
cavitation threshold concentration, were 3 and 6
3 g l1
at ultrasound output powers of 30 and 120 kW m2,
respectively. The signicant similarity in the measured
time period for formation of cavitation for different
materials was caused by the fact that the particle
concentration in the ultrasound eld was increased by
the same factor relative to the cavitation threshold
concentration for every material and at every output
level. However, the results of measurement obtained for
pressed yeast were missing from the gures, as such
powerful bands were formed in the cluster planes of the
standing wave space, that no sedimentation took place
and no cavitation occurred at either output level even
after treating the suspension for 30 min.
4.3. Cell biological examinations
The measured cavitation threshold concentrations Cb
and Ca were 3
45 and 3
2 g l1 from the basic and
auxiliary methods, respectively. Multiples of the results
of the auxiliary measurement method were used in the
survival cell number determination experiments because

it turned out from the examination results of the

acoustic phenomena that if a suspension density higher
than the value for Ca is applied, different acoustic
phenomena (acoustic streaming, standing wave, cavitation) are formed in the ultrasound eld. On this basis, it
is possible to examine the biological effects of the
acoustic phenomena. Cell densities applied in the
examinations were 3
2 g l1 (for Ca); 4
8 g l1 (for
1
5Ca); 5
44 g l1 (for 1
7Ca); 7
04 g l1 (for 2
2Ca); and
9
6 g l1 (for 3Ca). Initial numbers of total cells at the
applied cell densities are shown in Table 1.

4.4. Relative change in surviving cell number

When the experiments were carried out at higher
suspension concentrations, three different acoustic
phenomena were observed in the ultrasound eld. They
always followed each other in the following sequence:
acoustic streaming, standing wave, cavitation. Figure 7
shows the changes in the relative numbers of living cells
measured in suspensions having different initial cell
concentrations during the ultrasound treatment. Trend
lines were drawn to relative cell numbers that belonged
to the points of time when the standing wave and
cavitation phenomena occurred. From the trend lines,
the absolute number of living cells was calculated in the
following way: at a given point of time t in s, the actual
absolute number of living cells nt in ml1 in the given
acoustic zone can be calculated as the product of the
relative number of living cells in % measured under the
conditions of standing wave and cavitation and of the
initial number of living cells n0 in ml1. Based on this,
percentages of the relative numbers of living cells NS
and NC measured at the borders of standing wave and
cavitation zones, respectively, can be determined from
the trend lines [Eqns (11) and (12)] shown in Fig. 7:
N S 0
775t 77
708

(11)

N C 77
006e0
0088

(12)

with values for R2 of 0
9906 and 0
9975, respectively.

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80
Relative cell survival, %

70
60
50
40
30
20
II

10

SW

III

CAV

0
0

60

120
180
240
Exposure period, s

300

360

Fig. 7. Zones of acoustic phenomena bordered by trend lines

connecting relative cell number points of time when the
formation of the standing wave (SW) and the cavitation
(CAV) occurred (I, acoustic streaming; II, standing wave;
III, cavitation zone): }, 3
2 g l1; &, 4
8 g l1; W, 5
44 g l1; 3,
7
04 g l1; , 9
6 g l1

Figure 7 is divided into three different acoustic zones

by the two trend lines; each of these zones shows an
acoustic phenomenon (I, acoustic streaming; II, standing wave; and III, cavitation). In the case of the
untreated control samples, no signicant change occurred in the number of cells even after hours of
preparing the suspension.
Zone I starts from the initial 80% living cell ratio and
ends at the moment when the standing wave is formed.
The dominant factor in this zone is the acoustic
streaming. Under the conditions of higher suspension
concentrations, the time interval of the existence of the
acoustic streaming phenomenon is longer. In case of a
given sample, the acoustic streaming remains in
existence until the moment when the radiation forces
trap the particles in pressure nodal planes and the
standing wave (zone II) is formed.
Zone II starts at the moment when the standing wave
is formed and ends when cavitation is formed (Fig. 7). In
a standing wave, the cells are concentrated in the sound
eld in planes in a clearly visible way. The distance of
these planes is equal to half of the wavelength. As a
result of the particle agglomeration effect of the
standing wave, sedimentation occurred, and the concentrated aggregates precipitated on the bottom of
treating vessel and as a result of this process, the number
of the adsorption and scattering centres located in the
sound eld decreased. This resulted in increasing the
amplitude of the acoustic pressure; thus, detectable
cavitation (zone III) occurred.
Zone III starts from the moment when the cavitation
occurs and lasts until innity. Cavitation occurs later as

the cell concentration increases, because the decrease of

the particle concentration in the sound eld forms larger
and larger acoustic pressure amplitudes and this acts
against the sedimentation.
In the experiment, where an initial concentration of
3
2 g l1 was applied, no standing wave and acoustic
streaming occurred. Acoustic cavitation was formed at
the beginning of the experiment and it lasted until the
end of the experiment. In the case of concentrations
higher than 3
2 g l1, the standing wave and the
cavitation occurred later as the concentration increased.
Reproducibility of the repeated experiments was good;
the calculated standard deviation values were low.
Sequential occurrence of the acoustic phenomena in
the sound eld was named phenomenoneffect chain
reaction.

4.5. Survival dynamics

The number of cells N in the suspension was 5
6 106
cells ml1 at the yeast concentration level of 1 g l1.
Based on this total cell count and the relative number
of living cells measured in the experiments (Fig. 7),
the survival dynamics were determined by all three
ranges of acoustic phenomena (I, acoustic streaming; II,
standing wave; and III, cavitation zone). In Fig. 8, the
absolute numbers of living cells belonging to the
individual biological sampling times, as determined in
each experiments, are shown. Based on the results of
Deak (1997) and Thacker (1973), separate exponential
trend lines were drawn through the cell number results
that belong to the different acoustic phenomena
occurring in the sequence of acoustic streaming, standing wave and cavitation. These trend lines become linear
in a log-normal diagram (Fig. 8). Therefore, the linear
trend lines are proportional to the disruption rate
of the Saccharomyces cerevisiae under the conditions of the different acoustic phenomena, as observed
in the experiments and display the survival curves of the
yeast. Sections with greater slope (larger values of D)
mean more rapid cell disruption than the sections with
smaller slope; therefore, the resistance of the cells to the
given phenomenon is less in the ranges with greater
slopes.
When delineating the average numbers of surviving
cells measured at initial cell concentrations exceeding
3
2 g l1 in Fig. 8, the sequence of the appearance of the
individual acoustic phenomena was always acoustic
streaming, standing wave and cavitation. This was
indicated by the slopes of the characteristic curves that
were greater, smaller and greater in the ranges of
acoustic streaming I, standing wave II and cavitation
III, respectively.

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305

ULTRASONIC CELLULAR DISRUPTION OF YEAST

It can be seen from Fig. 8, that the cell disruption rate

was the greatest in the cavitation phase in each case; the
length of the decimation period is the shortest in this
phase. The decimation period is slightly longer in the
range of the acoustic streaming and it is much longer in
the standing wave phase. Figure 8 is related to Fig. 7 in
the following manner: from the relative number of living
cells prevailing at a given moment, in the knowledge of
8
I

the total number of cells of 5
6 106 ml1, the absolute

number of living cells was calculated. If the numbers of
living cells n0 and nt measured at the point in time when
an acoustic phenomenon occurs at t0 and ends at tt,
respectively, are substituted into Eqns (1) and (2), then
the numeric values of the decimation period D and
disruption rate k can be obtained. These values were
determined in each experiment separately for each
acoustic phenomenon (Table 2). The basic form of the
survival curves from Fig. 8 shown in Eqn (13) is used to
calculate the actually living cell counts given in decimal
logarithm.
log10 nt log10 n0 eat

II

Log N

III

50

100
150
200
Exposure period, s

250

300

Fig. 8. Calculated survival curves belonging to the individual

initial cell concentrations, in multiples of the cavitation threshold
concentration by the different acoustic phenomena (I, acoustic
streaming; II, standing wave; III, cavitation): }, 3
2 g l1; &,
4
8 g l1; W, 5
44 g l1; 3, 7
04 g l1; , 9
6 g l1; N, living cell
count, cell ml1

(13)

where: nt is the actually living cell count; n0 constant is

the initial living cell count between the given examination and acoustical phenomenon; e is the number of
natural logarithm; a is the exponent; and t is the
exposure period in s. For the different ranges of the
specic acoustic phenomena, values of the n0 constant,
the a exponent and the correlation of the trend functions
are shown in columns 5, 6, and 7 of Table 2,
respectively.
Equations and correlation coefcients of the trend
lines shown in Table 2 and in Fig. 8 are also included. In
Fig. 8, it is clearly seen that denite survival dynamics
are prevailing in the different phases of the different
acoustic phenomena. Under the conditions of low cell
concentrations, the quickest cell destruction is caused by
cavitation; the decimation time is the shortest in this
phase. In the range of the acoustic streaming and the

Table 2
Survival dynamic characteristics (decimation time D in s and destruction rate k in s1) in different acoustic zones (I, acoustic streaming;
II, standing wave; and III, cavitation), and values of the n0 constant and the a exponent of the trend line equations tted to the survival
curves and their coefcients of determination R2
Initial cell
densities g l1

Acoustic
zone

Decimation
time (D), s

Constant
log10n0

Exponent a

R2

0
057

7
192

0
0039

0
97

Destruction
rate coefficient
(k), s1

3
2

III

40
3

4
8

I
II
III

155
6
1404
2
50

0
0148
0
00164
0
046

7
332
7
160
8
248

0
0009
0
0001
0
003

0
99
1
0
97

5
44

I
II
III

165
5
1112
57

0
0139
0
002
0
04

7
383
7
147
8
977

0
0008
0
0001
0
0031

0
99
0
95
0
97

7
04

I
II
III

145
75
919
3
94
77

0
0158
0
0025
0
0243

7
477
7
156
8
468

0
001
0
0002
0
0016

0
94
0
97
0
99

9
6

I
II
III

134
91
895
7
150
5

0
0171
0
0025
0
0153

7
560
7
157
8
332

0
001
0
0002
0
001

0
91
0
97
0
91

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standing wave, the decimation time was slightly longer

and much longer, respectively.
When the cell concentration is high, the aforementioned facts in Fig. 8 are modied to some extent. In
order to understand these changes, the results obtained
shall be interpreted in a wider framework. Sonolysis of
cells, which can be detected easily and clearly if the cell
concentration is low, takes place in a sluggish way if the
cell concentration is high.
When considering the survival changes in the ranges
of different acoustic phenomena, it can be observed that
in the range of acoustic streaming the survival dynamics
produces very similar values of D, almost regardless of
the initial cell concentrations; the calculations show that
the values of D may even be slightly lower at higher
initial cell concentrations (Fig. 9).
The functional relationship [Eqn (14)] between the
decimation intervals D and the initial cell concentrations
C in g l1 under the conditions of acoustic streaming
that is shown in Fig. 9 is
D 5
7043C 188
36

1500
1400
Decimation time D, s

306

1300
1200
1100
1000
900
800
6

8
Initial cell concentration C, g l1

10

Fig. 10. Decimation times (D) measured under the conditions of

standing wave at different initial cell concentrations (C)

(14)

160

170

140
120
Decimation time D, s

with a value for R of 0
808.

While examining the effect of the standing waves on
the survival of the cells, it can be established that there is
little difference in the survival features of the suspensions having different initial cell concentration. When
the initial cell concentrations are higher, the observed
values of D are slightly lower. This may be caused by the
fact that in this situation the cells spend longer time in
the sound eld (Fig. 10). The functional relationship
[Eqn (15)] between the decimation intervals and the
initial cell concentrations under the conditions of

100
80
60
40
20

165
0
Decimation time D, s

160
155

5
7
6
8
Initial cell concentration C, g l1

10

Fig. 11. Decimation times (D) measured under the conditions of

cavitation at different initial cell concentrations (C)

150
145

standing wave that is shown in Fig. 10, is

140

D 870
78 62758
65eC

135

with a value for R2 of 0
9896.

Based on these results, it can be established that the
effect of the cavitation on the decimation time of the
surviving cells signicantly differs from the effect of the
acoustic streaming and the standing wave on the same
variable if the initial cell concentrations are different. In
the case of samples having higher cell concentrations,

130

10
1

Initial cell concentration C, g l

Fig. 9. Decimation times (D) measured under the conditions of

acoustic streaming at different initial cell concentrations (C)

(15)

ARTICLE IN PRESS
ULTRASONIC CELLULAR DISRUPTION OF YEAST

the decimation time is longer, while if the initial cell

concentration is lower, the decimation time is shorter
(Fig. 11). The functional relationship [Eqn (16)] between
the decimation intervals and the initial cell concentrations under the conditions of cavitation that is shown in
Fig. 11 is
D 18
84e0
2173C

(16)

with a value for R2 of 0
9809.

In the experiments the equilibrium phase is characterised by the fact that the cavitation becomes stable
after the standing wave phase; thus, the effects of this
phase prevail until the number of the living cells
decreases to zero. In the individual experiments, if the
sections of lines are lengthened in Fig. 8, it indicates
that the living cell number decreases under conditions of the acoustic phenomena of standing wave until
the ordinate, this line intersects the axis at the starting
point of the experiment carried out at a cell concentration of 3
2 g l1, that is, the cavitation threshold
concentration, which means a cell number of log 7
14.
Survival dynamics in the standing wave range as a
continuous virtual line separates the survival dynamics
prevailing in the acoustic streaming and cavitation
ranges that are located above and below this line,
respectively.

5. Conclusions
(1) The method for determining the cavitation threshold
concentration and the length of the time period
required for the formation of cavitation provide an
opportunity for obtaining new research information
for the basic and applied material sciences.
(2) The difference in the measured time period for the
formation of cavitation for lyophilised yeast and
dolomite is caused by the lower density of the yeast
particles which move by acoustic streaming and
which are trapped more easily by the acoustic force
eld in the pressure node planes than the dolomite
particles whose density and moment of inertia are
higher.
(3) In the case where the initial cell suspension
concentrations were higher, the lengths of the
standing wave phase were longer and this made it
possible for longer manipulation of the cells in the
sound eld.
(4) There is an interaction between the suspension
concentration in the ultrasound eld and the
formation of the acoustic phenomena and, through
this, between the survival dynamics of the cell
suspension and the suspension concentration. This

307

acoustic phenomenon was called the biological effect

chain reaction.
(5) Acoustical phenomenon formed in the ultrasonic
eld clearly affect the survival dynamics of the cells
present in the sound eld. The formation of these
phenomena can be affected deliberately and this
makes it possible to affect the survival dynamics of
the cells.

Acknowledgement
We express our thanks for the help of Prof. Dr. Pal
Greguss from the Technical and Economic University of
Budapest.
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