Biotechnol. Prog. 2008, 24, 148−153
148
Effect of Moderate Electric Field Frequency on Growth Kinetics and Metabolic
Activity of Lactobacillus acidophilus
Laleh Loghavi,† Sudhir K. Sastry,*,† and Ahmed E. Yousef‡
Department of Food, Agricultural, and Biological Engineering, The Ohio State University, 590 Woody Hayes Drive,
Columbus, Ohio 43210-1058, and Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Road,
Parker Hall, Columbus, Ohio 43210
Moderate electric fields (MEF) have been previously shown to alter the metabolic activity of
microbial cells; thus, the effect of frequency and electric field would be of considerable interest.
We investigated herein the effects of MEF frequency on microbial growth kinetics and bacteriocin
(Lacidin A) production of Lactobacillus acidophilus OSU 133 during fermentation. The following
fermentation treatments were compared: conventional (for 40 h), MEF (1 V cm-1, for 40 h),
combination of MEF (1 V cm-1, for the first 5 h) and conventional (for 35 h) at various frequency
levels (45, 60, and 90 Hz) all at 30 °C, and control (conventional) fermentation at 37 °C. MEF
treatments with purely sinusoidal waveforms at all frequencies at 30 °C produced a shorter lag
phase than conventional fermentation. However, no lag phase reduction was found for a 60 Hz
waveform that contained high-frequency harmonics. There was, however, a significant increase
in the bacteriocin production under early MEF treatment at 60 Hz with high-frequency harmonics.
On the basis of these observations, the fermentation process is accelerated by applying pure
sinusoidal MEF at the early stage of growth while a significant increase in the bacteriocin
production occurs when sinusoidal field at 60 Hz with harmonics is applied at the early stage
of the growth.
Introduction
The applications of moderate electric fields (MEF) at 60 Hz
have shown some effect on metabolic activity of prokaryotes
(1, 2). MEF at different frequencies have shown interesting
influences on the permeability and diffusion across cell membranes in eukaryotes (3-9). This suggests that the effect of
electric field frequency on prokaryotes is worthy of investigation. Most studies on the effect of alternating electric fields on
microbial growth have been performed at a frequency of 60 Hz
(1, 2, 10).
Several studies have been conducted on the stimulation effects
of alternating electric current at different frequencies on
eukaryotes. Imai et al. (4) studied the effect of frequency (50
Hz-10 kHz) on heat generation in Japanese white radish. They
found 50 Hz as the most effective for rapid heating. They
suggested electroporation as the main reason for reduction in
impedance and therefore quick heating. Jemai and Vorobiev
(5) found that an increase in field intensity and pulse duration
enhanced the cell membrane permeabilization process, leading
to an increase in diffusion coefficient.
Kulshrestha and Sastry (7) studied the effect of frequency
[0, 10, 50, 250, or 5000 Hz] and voltage [from 0 to 23.9 V
cm-1] on diffusion during MEF treatment. They observed that
except for direct current (DC) diffusion of dye from beet cubes
was enhanced as field strength increases and frequency decreases. They also suggested electroporation as the mechanism
for enhanced diffusion. In their study, there appears to be a
* Address correspondence to this author. Phone: 1-614-292-3508. Fax:
1-614-292-9448. E-mail: [email protected].
† Department of Food, Agricultural, and Biological Engineering.
‡ Department of Food Science and Technology.
10.1021/bp070268v CCC: $40.75
threshold potential above which significant increases in permeabilization occur. The threshold potential level for permeabilization was found to increase as the frequency increased,
except for DC. They mentioned that this observation is
consistent with the theory that at low frequency there is sufficient
time for significant charge buildup on the cell membrane.
Several other studies have demonstrated the effect of frequency
and waveform on the electrical conductivity, drying rate, and
juice extraction of eukaryotes (8, 11).
These findings suggest that frequency and waveform may
play a significant role in MEF fermentation. Listed studies in
this area illustrate that the effect of different field frequencies
and waveforms on the microorganisms has not received much
attention. Accordingly, our objective was to understand the effect
of electric field frequency under a constant temperature on
growth kinetics and bacteriocin (lacidin A) production during
fermentation of Lactobacillus acidophilus.
Materials and Methods
Experimental Treatments and Procedure. We comparatively investigated the following treatments: (1) conventionals
no electric field was applied during the course of fermentation
at 30 and 37 °C respectively; (2) MEFselectric field strength
of 1 V cm-1 at 45, 60, and 90 Hz was applied during the course
of a 40 h fermentation at 30 °C; and (3) early MEFsMEF (1
V cm-1 at 45, 60, and 90 Hz) was applied for the first 5 h and
conventional fermentation for the remainder (35 h) of the
fermentation at 30 °C. Rationale: Previous works showed that
electric fields associated with ohmic heating may accelerate the
early stage of fermentation of Lactobacillus acidaphilus (1) and
may increase the bacteriocin production of Lactobacillus
acidophilus (2) but inhibit the late stage of the fermentation
(1) at temperatures lower than the optimum growth temperature.
© 2008 American Chemical Society and American Institute of Chemical Engineers
Published on Web 01/10/2008
Biotechnol. Prog., 2008, Vol. 24, No. 1
149
Cell Growth Analysis. Bacterial growth data were fitted to
the Gompertz model (1, 2, 13, 14):
y(t) ) A + C exp{-exp[-B(t - M)]}
(1)
where y(t) ) log count at time t, A is log number at t ) -∞, B
is the maximum specific growth rate, M is the time at which
the bacterial culture achieves its maximum growth rate, and C
is the final log increase in the population. To estimate the model
parameters, growth data were fitted to the Gompertz model of
the JMP statistical program (SAS Institute, (Version 6.0), Cary,
NC). The lag period (hours) equals M - (1/B), and the final
log count is A + C. Data on pH change were fitted to the
Logistic model (1, 2):
Yh ) A1 + C1/{1 + exp[- B1(X - M1)]}
Figure 1. Moderate electric field fermentor schematic.
Treatments were carried out in the fermentor described below,
without aeration. Expect for conventional treatment at 37 °C,
all treatments were at 30 °C, previously identified as most
associated with lag phase reduction (1) and bacteriocin production (2) in the presence of an electric field. The average room
temperature was 25 °C during these treatments. All treatments
were conducted in triplicate.
The experimental procedure used was the same as that used
by Loghavi et al. (2), except for using variable frequency levels.
MEF was achieved by passing alternating current at frequencies
of 45, 60, and 90 Hz at a field strength of 1 V/cm.
Moderate Electric Field Fermentor. The MEF fermentor
apparatus (Figure 1) was equipped with two platinized titanium
plate electrodes. Treatments at 45 and 90 Hz were conducted
using a variable frequency power supply (Elgar Corporation,
Model 1751, San Diego, CA) through a function generator
(Hewlett-Packard, model 3114 A, Santa Clara, CA). Electrodes
at 60 Hz were supplied with power from a power supply (0110 V, 60 Hz) through a transformer. For conventional
fermentation, a similar fermentor assembly was used, but the
power supply was inactive.
Bacteria Preparation. The preparation procedure for Lactobacillus acidophilus OSU 133 and Lactobacillus leichmannii
ATCC 4797 was as described by Loghavi et al. (2). Both
cultures were obtained from the microbial culture collection of
the department of Food Science and Technology at The Ohio
State University.
Bacteriocin Activity Measurements. Bacteriocin activity
was measured using the bioassay method described by Yousef
and Carlstrom (12). The concept was to indirectly measure the
amount of bacteriocin released during the fermentation by
determining the antimicrobial activity of the fermentation
medium against Lactobacillus leichmannii ATCC 4797, which
is sensitive to lacidin A. A series of twofold dilutions of filtered
fermentate were prepared. Small volumes (5 µL) of these
dilutions were spotted on a layer of soft MRS medium seeded
with Lactobacillus leichmannii ATCC 4797. After incubating
the agar plates for 24 h, clear areas appeared where solutions
with antimicrobial actives were spotted. The highest dilution
factor that generated a clear zone indicated the strength of
bacteriocin activity. This information was used to calculate
bacteriocin activity as given by Yousef and Carlstrom (12).
Biomass Monitoring. Viable cell numbers (expressed as
CFU/mL) were enumerated by the technique described by
Loghavi et al. (2). We also measured the OD600 of the samples
and used an OD600 vs CFU/mL calibration curve to estimate
bacterial counts (CFU/mL).
(2)
where Yh ) 14 - pH, X ) time (hours), and A1, B1, C1, and M1
are model parameters, with M1 being the time at the inflection
point of the pH change curve. To estimate model parameters,
the nonlinear modeling procedure of the JMP statistical program
was used. Data on growth curve and pH change parameters were
analyzed statistically using an Analysis of Variance approach
(ANOVA). When treatment factors were significant, Student’s
t-statistics were used for multiple comparisons of means.
Results
Effect of MEF Frequency on Growth Kinetics of Lactobacillus acidophilus. Frequency had significant effects on lag
time in the MEF fermentations. Means and standard deviations
of growth parameters and pH change for different treatments
are presented in Table 1. There was a significant difference in
lag phase between treatments at various frequency levels at
30 °C (growth curves for two of these treatments are presented
in Figure 2). A shorter lag phase was observed for treatments
at 45 and 90 Hz compared to conventional treatment at the same
temperature. However, no significant difference was observed
for treatments at 60 Hz compared to conventional treatment at
the same temperature. The growth curve at 37 °C (the optimum
growth temperature for this microorganism) had a shorter lag
phase than conventional fermentation at 30 °C. A higher lag
time was observed at 90 Hz compared to 45 Hz under MEF
and early MEF. Lag phase periods at both these frequencies at
30 °C were comparable (p > 0.05) to that at 37 °C conventional
fermentation and were significantly lower (p < 0.05) than the
lag time at 30 °C conventional. The greatest reduction in lag
phase at 30 °C was observed at 45 Hz followed by 90 Hz;
however, no significant reduction was observed in lag phase at
60 Hz. The bacterial population change, maximum specific
growth rates, and pH changes for fermentations at 30 °C were
lower than those for conventional fermentation at 37 °C. The
minimum generation time and the time for the inflection in the
pH decline curve at 30 °C were greater (p < 0.05) than
conventional fermentation at 37 °C. It is clear that the lag phase
at suboptimum growth temperature decreased especially at 45
Hz compared to all other treatments at the same temperature.
Effect of MEF on Bacteriocin Activity. The amount of
bacteriocin activity under each treatment, expressed in AU/mL,
over time is shown in Figure 3. There was a significant
difference in the bacteriocin production between 30 and 37 °C.
Less bacteriocin production was observed under conventional
fermentation at 37 °C compared to all other treatments. A slight
reduction in bacteriocin production was observed at 37 °C during
the stationary phase. Maximum bacteriocin activity was observed when MEF at 60 Hz was applied at the early stage of
Biotechnol. Prog., 2008, Vol. 24, No. 1
150
Table 1. Growth Parameters and pH Changes for Different Treatments
growth parametersa
lag
time (h)
treatment
MEF
early MEF
conventional
temp
(°C)
30
30
30
30
30
30
30
37
freq
(Hz)
45
60
90
45
60
90
0
0
SEb
0.046
0.053
0.05
0.047
0.065
0.066
0.078
0.044
mean
0.73°
1.46†‡
1.15‡*
0.65°
1.51†‡
1.17‡*
1.87†
0.79*°
max specific
growth rate
SDd
mean
0.195
0.177
0.129
0.055
0.344
0.323
0.362
0.115
0.160‡
0.157‡
0.16[‡
0.159‡
0.158‡
0.152‡
0.158‡
0.43†
pH changesa
min generation
time (h)
SDd
mean
0.009
0.006
0.000
0.021
0.017
0.005
0.01
0.008
2.013‡
2.043†‡
2.037†‡
1.963‡
1.95‡
2.14†‡
2.067†‡
0.647*
biomass
productionc
SDd
mean
0.129
0.09
0.055
0.235
0.185
0.078
0.038
0.031
2.55‡*
2.56‡*
2.51‡*
2.64‡
2.67‡
2.52‡*
2.59‡*
2.96†
total
decline
SDd
0.114
0.136
0.064
0.039
0.026
0.046
0.15
0.099
mean
2.213‡
2.013*°
2.107‡*°
2.173‡*
1.973°
2.13‡*°
1.977°
2.42†
inflection
time (h)
SDd
mean
SDd
0.046
0.071
0.093
0.095
0.105
0.036
0.155
0.157
16.69‡**
0.635
0.835
0.962
0.777
0.238
0.515
0.448
0.082
15.94°
17.06†‡*
16.62‡*°
16.19*°
17.32†‡
16.4‡*°
6.06°
a Means within each column not connected by the same superscripts (‡, *, °, †) are significantly (p < 0.05) different. b SE: standard error (standard
deviation of response mean) of the Gompertz model fitted to the experimental cell growth data. c Expressed as log change in OD. d SD: standard deviation
(().
increased when MEF at 60 Hz was applied at the early stage of
the fermentation at 30 °C, compared to all other fermentations
(Figure 3).
Discussion
Figure 2. Sample cell growth curves presented in OD600 change during
fermentation of L. acidophilus OSU 133 at 30 °C for conventional and
MEF treatment at 45 Hz.
the fermentation. The presence of an electric field at 45 and 90
Hz reduced the bacteriocin activity compared to conventional
fermentation at 30 °C. It is clear that bacteriocin activity
Surprisingly, lag time decreased at suboptimum growth
temperature when MEF was applied at 45 and 90 Hz but not at
60 Hz, despite all other growth kinetic parameters not being
significantly different at the various frequency levels (Table 1).
Several researchers (7, 11, 15) have observed that mass
transfer improves in eukaryotes as field strength increases and
frequency decreases. They have suggested that temporary,
reversible electroporation may be the main cause for enhanced
diffusion. Kulshrestha and Sastry (7) mentioned that this
observation is consistent with the theory that low frequency
allows more time for charges to build up on the cell membrane;
consequently higher electropore formation is expected at lower
frequencies. Therefore the slight increase in lag time at 90 Hz
compared to 45 Hz may be explained by this theory. Similarly,
lag phase is also expected to increase as the frequency increases
Figure 3. Production of bacteriocin, measured in arbitrary units (AU/mL), during fermentation of L. acidophilus OSU 133 for different experimental
treatments (T: temperature (°C)).
Biotechnol. Prog., 2008, Vol. 24, No. 1
151
Figure 4. Spectral analysis of 60 Hz standard US power source. The table on the right shows the top peak frequency harmonics.
from 60 to 90 Hz; however, our results indicate a longer lag
phase at 60 Hz compared to 90 Hz, which is not consistent
with the low-frequency theory.
We noted that while we had used a standard US electric power
source at 60 Hz, we had used a variable-frequency power supply
for the 45 and 90 Hz treatments. We hypothesized that the power
source difference may have caused some variation in waveform
(Lima and Sastry (11) showed both waveform and frequency
dependence for electrically assisted mass transfer). To verify if
current and voltage waves were purely sinusoidal and consistent
between all treatments, we examined the wave shapes at
different frequencies with an oscilloscope (Tektronix, TDS 5052,
Beaverton, OR). The results (Figure 4) indicate that highfrequency components (harmonics) exist in the 60 Hz standard
US power source. Further, waves at 45 and 90 Hz produced by
the variable-frequency supply were purely sinusoidal, while
waves at 60 Hz were not purely sinusoidal. This suggests that
cells may respond differently to different waveforms, and in
particular, to the presence of high-frequency harmonics. We
therefore hypothesized that the presence of high-frequency
components in harmonics sine waves may decrease the electroporation effect based on low-frequency theory leading to
lower diffusion rate and higher lag time.
We investigated this possibility further by conducting fermentations with a purely sinusoidal 60 Hz frequency field, using
the same power source as for the 45 and 90 Hz treatments
(Figure 5).
We found that the lag time for the 60 Hz pure sinusoidal
wave (Table 2) was decreased significantly compared to the
lag time at 90 Hz. This result is consistent with the lowfrequency theory. Lag periods under 45, 60, and 90 Hz pure
sinusoidal waves are comparable to those under optimum growth
temperature with no electric field. However, pure sinusoidal
waves at 45 and 60 Hz reduced the lag phase most significantly.
This suggests that there might be a low-frequency spectrum to
which cells react. Therefore exposure of a cell membrane to an
external electric field at low frequencies may cause temporary
pores, an increase in transmembrane conductivity and/or diffusive permeability of nutrients, and autoinducers (16) around
the cell membrane.
Figure 5. Current and voltage waves for 60 Hz harmonic (standard
US power source) (a) and pure sine wave (b).
Our results show a significant increase in the bacteriocin
production as temperature decreases from the optimum value.
High bacteriocin production is commonly associated with the
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152
Table 2. Growth Parameters and pH Change for MEF Treatment at 60 Hz Pure Sine Wave
growth parameters
lag time (h)
max specific
growth rate
pH change
max generation
time (h)
log change in
cell populationa
total
decline
inflection
time (h)
mean
SDb
mean
SDb
mean
SDb
mean
SDb
mean
SDb
mean
SDb
0.56
0.092
0.151
0.004
2.255
0.049
2.45
0.064
2.22
0.12
18.2
0.93
a
Expressed as log change in OD. b SD: standard deviation (().
growth stage of bacteria, optimum bacteriocin production pH,
and a supply of nutrients specific for a species/strain (2, 1719). This effect may be attributed to the slow pH decline at
suboptimum growth temperature, allowing cells to experience
their optimum bacteriocin production pH for a longer time (2).
The highest amount of bacteriocin production was observed
when MEF at 60 Hz with high-frequency harmonics was applied
at the early stage of the fermentation. The highest bacteriocin
activity at 60 Hz pure sine wave was comparable to all other
MEF treatments at 30 °C (3200 AU/mL). The difference
between bacteriocin activities at suboptimum temperature might
be related to the frequency, waveform, and the manner that the
electric field was applied. Since stress stimulates production of
defensive molecules such as bacteriocin (20), it is possible that
stress caused by electric field at certain frequencies and
waveforms induces an increase in production of bacteriocin.
On the basis of this information, it is possible that certain
frequencies corresponding to the bacteriocin dielectric relaxation
time enhance the rate of the bacteriocin production in the
cytoplasm.
To investigate if our frequency corresponded to the relaxation
time of the bacteriocin, we adapted a mathematical model (21)
based on the Debye formula to estimate the relaxation time of
the bacteriocin:
τ ) (8πηARB3)/2kT
(3)
where τ is the dielectric relaxation time (s), ηA is the viscosity
(Pa s) of the solvent (cytoplasm or periplasm), RB is the radius
of the bacteriocin molecule (m), k is the Boltzmann constant
(1.3807 × 10-23 J/K), and T is the absolute temperature (K).
We assumed the viscosity of the cytoplasm of L. acidophilus
to be about an average of 210 Pa s ((143 Pa s) (22, 23) and
the radius of the bacteriocin molecule to be around 2.5 nm based
on nisin dimensions stated by Jack et al. (24). The characteristic
frequency (f) was then estimated as 1/τ. We found f to range
from 60 to 320 Hz. A spectral analysis plot for our waveform,
presented in Figure 4, shows that the strongest harmonic occurs
at 300 Hz, which is within the range of our calculated values.
Since the pure 60 Hz sine wave did not result in such high
bacteriocin activities, this suggests that harmonics at specific
frequencies may alter the rate of bacteriocin production. We
recognize that the above analysis has some limitations: notably,
the physics at the nanoscale size of the bacteriocin molecule
may not follow classical physics laws; the molecular dimensions
and data may only be approximations; and stress caused by MEF
may affect other molecules than just the bacteriocin molecule.
It is nevertheless instructive to conduct such an analysis to
attempt to understand the reasons for differing results in such
a complex system.
Despite the growth kinetic parameters not being significantly
different between MEF and early MEF treatments at 60 Hz with
high-frequency harmonics, there were significant differences in
bacteriocin activity between these treatments. The reasons for
this variation are not clear. It is possible that the presence of
MEF during fermentation may interfere with regulation of
biosynthesis of bacteriocin in the manner detailed below.
Regulation of biosynthesis of lacidin A (a nonlantibiotic
bacteriocin) consists of two signal producing proteins: histidine
protein kinase (HPK) and response regulator (RR). In this
regulating system HPK autophosphorylates when it senses a
certain concentration of bacteriocin in the environment. The
phosphoryl group is transferred to the RR protein. This transfer
triggers the RR protein to activate the formation of prebacteriocin (25). Prebacteriocin gets activated and released to the
environment while being translocated by the ABC-transporter
proteins in the membrane. The presence of MEF at 60 Hz with
harmonics at the early stage of the fermentation may increase
the concentration of bacteriocin in the environment to the level
required for autophosphorylation of HPK, triggering prebacteriocin production. However, if MEF is applied during the entire
growth time, the transport of pre-bacteriocins may occur through
cell membranes due to temporary electroporation, thereby
bypassing the critical translocation process by ABC transporter
proteins that are necessary for activation of bacteriocin.
Therefore some of the bacteriocin molecules may be released
in the environment without getting activated. Further work is
needed to test this hypothesis.
Another explanation for the increase in bacteriocin activity
under early MEF treatment (60 Hz with harmonics) may be
that the relatively long lag time associated with this electric
field may have kept the cells from exposure to an electric field
at the early exponential phase when bacteriocin activity is
expected to greatly increase. Reduction in bacteriocin activity
under the continuous presence of MEF has been previously cited
by Cho et al. (1) and Loghavi et al. (2).
A small reduction in bacteriocin production at 37 °C may be
attributed to degradation of bacteriocin due to the release of
proteases in the media from an increasing population of aging
and dead cells (26). Cells grown at optimum temperature for
24 h would be well into their stationary phase, while cells grown
at suboptimum temperature for 40 h would have just completed
their exponential growth phase during which bacteriocin production would have occurred.
Conclusion
The application of MEF at different frequencies and waveforms during fermentation altered some of the growth kinetics
and the bacteriocin activity of the Lactobacillus acidophilus.
The results indicate that MEF fermentations can add economic
benefits by decreasing the fermentation operation time, and
under a modified set of conditions, potentially increasing
bacteriocin production.
These results provide evidence that pure sinusoidal waves at
45 and 60 Hz reduced the lag phase most significantly. This
suggests that there might be a low-frequency spectrum to which
cells react. Our results indicate a longer lag phase at 60 Hz
with harmonics than 60 Hz pure sine wave. However, a
significant increase in bacteriocin production at 60 Hz with
harmonics was observed when MEF was applied at the early
Biotechnol. Prog., 2008, Vol. 24, No. 1
stage of the fermentation. This may be due to slow pH decline
at suboptimum temperature, longer lag time, and the role of
the electric field in alternating the mechanism of production
and release of prebacteriocin. An analysis of bacteriocin
dielectric relaxation time shows that this time corresponds to
the top peak frequency harmonics in our experiment. Therefore,
it is possible that these high-frequency components influence
the mechanism of bacteriocin formation, while the lower
frequency component enhances diffusion of bacteriocin in the
cell cytoplasm and periplasm.
Acknowledgment
Salaries and research support provided in part by the Ohio
Agricultural Research and Development Center. References to
products and trade names are made with the understanding that
no endorsement or discrimination by The Ohio State University
is implied. Fellowship support provided by the USDA National
Needs Graduate Fellowship Grant No. 1998-5813 is gratefully
acknowledged.
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Received August 9, 2007. Accepted November 19, 2007.
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