Ultrasonics 38 (2000) 711–716

www.elsevier.nl/locate/ultras

Improvement of biological activity by low energy

ultrasound assisted bioreactors
O. Schläfer *, M. Sievers, H. Klotzbücher, T.I. Onyeche
Clausthaler Umwelttechnik-Institut, Leibnizstraße 21+23, D-38678 Clausthal-Zellerfeld, Germany

Abstract

Bioreactors are broadly applied in biotechnology and wastewater treatments. The so-called advanced bioreactor systems should
be optimised in a more compact, more efficient and more effective form. One method of optimisation is the improvement of the
solid–liquid interface of the sludge flocs and the mass transfer rate of gas and nutrients in the liquid. This could be obtained by
the intake of ultrasound in the bioreactor at a frequency of 25 kHz and a power input of 0.3 W l−1. An increase in the biological
activity of the process investigated with alternating ultrasound as well as a decrease without ultrasound were measured. © 2000
Elsevier Science B.V. All rights reserved.

Keywords: Activated biomass; Improved wine fermentation; Process-water recycling; Ultrasound assisted bioreactor

1. Introduction and stability of operation are necessary. A suitable

Specific biochemical reactions and their economical
operations are the main advantages of bioreactors. They
are widely applied in the industrial sector such as in the
food and pharmaceutical industries, in biotechnology
and in wastewater purification processes.
In wastewater purification, biological treatment sys-
tems are usually the most cost efficient processes for
reducing dissolved biodegradable organic components.
A potential need for compact, cheaper and efficient
process-water purification systems such as bioreactors
was discovered from a market survey in the area of
processing agricultural products, especially the wine and
juice industry in southern Europe.
As regards the production processes, there are many
variations in the quantities of wastewater and their
concentrations of compounds. Not all wastewater con-
taminants are biodegradable with existing treatment
systems. Therefore, the conventional biological treat-
ment processes either have to be designed very large
with high operational costs and many control techniques
to guarantee a constant water quality or they are not
efficient enough to recycle the wastewater for re-use in
the production process.
To integrate a biological process-water treatment
system in industrial production, high safety measures
reaction of the purification system after interruption of
production is required. Moreover, the process must be
flexible with respect to varying load conditions with
less space.
The first step in developing a process-water purifica-
tion plant which fulfils these requirements was to activate
the biomass by improving the solid–liquid interface of
the sludge and the mass transfer rate of gas and nutrients
in the liquid.
Recent studies [1,2] have shown that low energy
ultrasound wave irradiation could be used to optimize
the efficiency in ethanol production by yeast and fermen-
tation of mixed office waste paper in bioreactors. For a
2 l laboratory reactor, an energy input of 2.9 W l−1 at
a frequency of 43 kHz was found to decrease the dis-
solved carbon dioxide (DCO ) concentration with simul-
2
taneous maximization of the cell density.
In this study, the effects of low energy ultrasound
irradiation on biomass in a model process-water treating
plant were investigated.
2. Materials and methods
2.1. Organisms

For fermentation and ultrasound exposition, the

* Corresponding author. strain Saccharomyces cerevisiae C-2324 was used.

0041-624X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
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712 O. Schläfer et al. / Ultrasonics 38 (2000) 711–716
2.2. Medium and preculture
A synthetic glucose medium was used to simulate the
wastewater from the wine and juice industry. It consisted
of 10 g of glucose, 4 g of peptone from casein and 2 g
of yeast extract in 1 l distilled water. After dissolving
the compounds, the medium was sterilized with steam
at 121°C for 25 min. S. cerevisiae was grown in this
medium at 27°C in 500 ml Erlenmeyer flasks in a shaking
incubator to a cell density of 2×107 ml−1. The experi-
ment was started with an inoculum of 1% preculture
and then grown up to an optical density of 1.0 at
wavelength of 550 nm (OD ) before ultrasound
550 nm
treatment was initiated.
2.3. Materials
The used chemicals were from the Merck Company
(Germany) with FermtechA quality for microbiology.
2.4. Bioreactor plant and fermentation
To fulfil all the requirements mentioned above, the
special bioreactor plant (shown in Fig. 1) was
constructed.
The laboratory-scale reactor plant comprised two
identical 5 l cylindrical containers made of laboratory
glass with a flanged bottom plate made of stainless steel
where different ultrasound transducers could be fixed
and a top flange with all pipes and measurement equip-
ment were inserted. These two identical reactors were
built to enable parallel test series with and without
ultrasound treatment. Additional tests were run under
sterile and temperature controlled conditions. The
Fig. 1. Functional diagram of the experimental plant.
treated volume of the medium was 4 l and the stirring
unit in the reactor was operated at 120 rpm.
In order to obtain reproducible results in some experi-
ments, it was important to keep the micro-organisms in
the pile for a longer time in a fixed state with almost
constant biomass concentration. Consequently, the bio-
reactors were combined with a continuous system. A
20 l container with nutrient solution supplied the reactor
with fresh solution. The inflow and outflow quantities
were controlled with a calibrated peristaltic pump. To
make the fermentation processes anaerobic, the reactor
and the media were gassed out with nitrogen. After
inoculation, it was necessary to regulate all parameters
to reach a steady state before starting the ultrasound
experiments.
2.5. Ultrasonic treatment
The ultrasound acoustic irradiation of the reactor
with micro-organisms in the culture medium was made
by a specially constructed flange with an attached inter-
changeable transducer at the bottom of the reactor as
shown in Fig. 2. The tranducer has a diameter of 5 cm
and was glued on a steel plate of 10 cm diameter. The
range of the frequency generator was adjustable between
15 and 100 kHz with a variable performance up to
0.6 W. A subsequent ultrasound amplifier increases the
power up to a maximum of 150 W. An oscilloscope was
installed on the secondary circuit to directly control the
power at the transducer. After testing several different
frequencies and power levels, the subsequent experi-
ments were carried out at 25 kHz and 0.3 W l−1 for low
energy ultrasound and at 12 W l−1 for high power
ultrasound. This was because the best output as regards
 O. Schläfer et al. / Ultrasonics 38 (2000) 711–716
Fig. 2. Flange construction for interchangeable ultrasound tranducers.
optimal energy transmission and narrow frequency band
had been achieved.
5. Measurements
The reactor was supplemented with measurement
instruments. The extinction was measured in a steady-
flow cuvette at OD with a Lambda II UV–VIS
550 nm
spectrometer from Perkin-Elmer ( USA) as a correlation
parameter to the biomass concentration. The ethanol
concentration was detected by a gas chromatograph
from Perkin-Elmer ( USA) with Headspace H101 and
GC 8700 FID. The glucose concentration was measured
on-line enzymatically with Olga 2b from IBA (Germany)
and intermittently with glucose test sticks. The measure-
ment of the ultrasonic field inside the bioreactor was
Fig. 3. Effect of low energy ultrasound on biological activity.
713
carried out with standard calibration ultrasound-needle-
hydrophone from UltraTech (Denmark). The temper-
ature and the pH of the broth were also monitored.
6. Results
6.1. Effect of ultrasound on biological activity and
decrease of glucose
Experiments to detect the effect of low power ultra-
sound on biological activity were carried out with S.
cerevisiae in the bioreactor plant with a continuous flow.
As shown in Fig. 3, the extinction of 0.6 (without
ultrasound ) rose up to 0.9 after 7 h of ultrasound
exposure.
This trend of increased biological activity went on
for several hours. Thereafter, a second exposition for
5 h of ultrasound brought the extinction to 1.2 indicating
an increase in biomass concentration from 0.12 to
0.4 g l−1 while the glucose concentration decreased from
0.8 to 0.5 g l−1. The best results were obtained at a
power input of 0.3 W l−1 and varying time intervals
with and without ultrasonic treatment. Consequently, a
steady increase in activity could be achieved. Steady
irradiation with ultrasound delivers no significant
enhancement of biological activity (Fig. 4). The positive
influence of ultrasound seemed basically to be due to
the effect of the alternating treatment stages.
In accordance with Ref. [2], in which an optimum for
treating biomass with low power ultrasound was also
found, the results were verified with high power ultrasound
experiments raising the power in steps up to 12 W l−1.
These experiments were carried out discontinuously and
in parallel in two identical reactors. One reactor was run
without ultrasound as a control. Glucose concentration
was measured every 10 min in each reactor. In Fig. 5, it
was remarkable that increasing the energy input does not
achieve increased biological activity. With an alternating
714 O. Schläfer et al. / Ultrasonics 38 (2000) 711–716

Fig. 4. Effect of continuous low energy ultrasound on biological activity.

Fig. 5. Effect of high power ultrasound on fermentation.

treatment at an ultrasound intensity of 12W l−1, there was
no recognizable difference in the biological activity with
and without ultrasound.
6.2. Increase of ethanol concentration by ultrasonic
irradiation
An increase in the ethanol production from the tested
biological process with low power ultrasound, as well
as a decrease without ultrasound, were recorded as
shown in Fig. 6. The ethanol production without ultra-
sonic treatment varied between 3 and 12 g l−1. At an
ultrasound frequency of 25 kHz and a power input of
0.3 W l−1, the ethanol concentration increased to values
between 18 and above 30 g l−1. In accordance with [1],
the highest ethanol concentrations were obtained with
a cycle regime of ultrasound exposure and a pause.
Lower ethanol concentrations were obtained at short
time intervals and higher frequencies (spikes), while with
continuous ultrasound irradiation no stimulation in the
ethanol fermentation process was recorded.
6.3. Ultrasonic field in the reactor
To acquire information on the grade of ultrasound
intensity as function of the distance of the micro-
 O. Schläfer et al. / Ultrasonics 38 (2000) 711–716 715

Fig. 6. Effect of low energy ultrasound on ethanol production.

organisms from the ultrasound transducer, we carried 7. Discussion

out a series of ultrasound measurements with the stan-
dard calibration needle-hydrophones. The Fig. 7
describes the shape of the ultrasound distribution in the
reactor. It was obvious that the ultrasonic transmission
was uniformly distributed in the reactor. In the case of
the tested reactor, there were apparently some reflections
of the ultrasound waves at the reactor cover and sides.
This could lead to a partial amplification and cancella-
tion of the ultrasonic transmission.
Because of the very low power ultrasound, no cavita-
tion occurred in the medium. The results of the ultra-
sound treatment seemed to be a combination of very
complex and dynamic effects which were influenced by
many factors including temperature, dissolved gases,
suspended particles, structures and metabolism of micro-
organisms, etc. Moreover, the ultrasound had several
effects on the different factors described above (i.e.

Fig. 7. Ultrasonic field at 25 kHz and 0.3 W inside of the reactor.

716 O. Schläfer et al. / Ultrasonics 38 (2000) 711–716
degassing of CO , dispersing particles and gases in the
2
medium, vibrating organisms, etc.). So the results
detected by measurement instruments may be influenced
by secondary effects.
Low cell concentrations of S. cerevisiae tend not to
build clusters in the medium, but some particles sank to
the bottom in the interval with no ultrasonic irradiation.
Hence the extinction measurement could be influenced
by dissociation of organisms. This effect should not be
confused with biological activity. The dissociation effect
appears a very short after switching on ultrasound and
also after short spikes with high and low power.
An increase in biological activity only appears several
hours after long-term irradiation and always coupled
with a decrease in glucose concentration. The exact
mechanism of biological activation is not yet clear. It is
a surprising phenomenon that ultrasound-activated cul-
tures continued with their higher activity for some hours
after irradiation stopped. Perhaps changes in the mem-
brane permeability or enzymatic differences of the organ-
isms were generated. Interestingly, discontinuous
ultrasonic treatments were more beneficial for activating
fermentation than the continuous exposure. It is sug-
gested that only a few steps in intracellular metabolisms
(e.g. enzymatic biosynthesis) are supported by ultra-
sound and others are not or are even inhibited.
Therefore, pauses are needed between ultrasound expo-
sures. In order to understand this phenomenon, further
experiments need to be carried out.
The results achieved in cell growth and ethanol
production suggest a tendency for acceleration with
ultrasound treatment, but remarkably the fermentation
process and the consequent changes after ultrasound
treatment are quite complex. The use of low power
ultrasound in bioreactors may present a significant
improvement in cost reduction in the biotechnology
industry especially in process-water treatment plants
for winerys.
8. Future actions
The next step in the project’s research activity will be
the construction of a bioreactor for a pilot plant as an
adaptation unit for practical operation in the food
processing industry. The volume of this bioreactor is
calculated to be 50 l. Simultaneously, the optimal effect
of ultrasound on biological activity will be specified with
further test results. Economic potentials should be exam-
ined as regards the costs (capital expenditures) and
power consumption (operating costs) of the ultrasonic
unit.
Acknowledgements
The authors are grateful to the European Commission
for supporting this research project on ‘Advanced
Wastewater Treatment by Enhanced Operation of
Separation and Improved Biological Activity
( WAMBIO)’ in the framework of FAIR program
( FAIR4.CT 973257). The special task of this EU project
is to develop a new liquid stream bioreactor based on a
combined biology/membrane system with the applica-
tion of ultrasound transduction. The authors also appre-
ciate the contributions of the project partners especially
TUD (Department of ultrasound) and RESON, for
their assistance in providing the ultrasound equipment.
References
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anol production during the simultaneous saccarification and fer-
mentation of mixed waste office paper, Biotechnol. Prog. 13
(1997) 232–237.
[2] K. Matsuura, M. Hirotsune, Y. Nunokawa, Acceleration of cell
growth and ester formation by ultrasonic wave irradiation, J. Fer-
mentation Bioeng. 77 (1) (1994) 36–40.