Review
Low-frequency ultrasound in
biotechnology: state of the art
Ekaterina V. Rokhina1, Piet Lens2 and Jurate Virkutyte1
1
Kuopio University, Department of Environmental Sciences, Yliopistonranta 1E, 70211 Kuopio, Finland
2
UNESCO-IHE, Department of Environmental Resources, PO Box 3015, 2601 DA Delft, The Netherlands
The use of low-frequency (10–60 kHz) ultrasound for
enhancement of various biotechnological processes
has received increased attention over the last decade
as a rapid and reagentless method. Recent break-
throughs in sonochemistry have made the ultrasound
irradiation procedure more feasible for a broader range
of applications. By varying the sonication parameters,
various physical, chemical and biological effects can be
achieved that can enhance the target processes in
accordance with the applied conditions. However, the
conditions that have provided beneficial effects of ultra-
sound on bioprocesses are case-specific and are there-
fore not widely available in the literature. This review
summarizes the current state of the art in areas where
sonochemistry could be successfully combined with
biotechnology with the aim of enhancing the efficiency
of bioprocesses, including biofuel production, biopro-
cess monitoring, enzyme biocatalysts, biosensors and
biosludge treatment.
Ultrasound in biotechnology
The rapidly developing field of biotechnology constantly
attracts new methods and solutions for further improving
bioprocess performance. Moreover, based on the principles
of green engineering and science, biotechnology has not
only profoundly transformed various areas of science –
such as medicine, agriculture, material science and chem-
istry – but has also provided entirely novel opportunities
for sustainable production of existing and new products
and services.
Ultrasound is a sound, pitched above human hearing,
that is used for a growing variety of purposes in diverse
areas. It is considered a ‘green’ technology owing to its high
efficiency, low instrumental requirements, significantly
reduced process time compared with other conventional
techniques and its economically viable performance [1,2].
Ultrasound can be classified according to the frequency
level [3] into: (i) high frequency and low power (2–10 MHz
range) ultrasound, also termed extended range or diag-
nostic ultrasound, which is used in medical imaging and
chemical analyses; and (ii) low frequency and high power
(20–100 kHz) ultrasound, the conventional type of ultra-
sound, which is used for cleaning and welding and also for
sonochemistry.
According to Suslick [1], chemical effects of ultrasound
occur not from a direct interaction with molecular species
Corresponding author: Virkutyte, J. (jurate.virkutyte@uku.fi).
298 0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.02.001 Available online 25 March 2009
but from the cavitation phenomenon: the formation, growth
and implosive collapse of cavities in liquids that release
large amounts of highly localized energy. Cavitation can be
classified into four types based on its mode of generation:
acoustic, hydrodynamic, optic and particle [4]. Of these, only
acoustic and hydrodynamic cavitation is able to generate the
intensities required to induce chemical or physical changes
in a system [5]. Ultrasound generates acoustic cavitation
with extremely high local temperatures (in excess of
5000 K), high pressures (500 atm) and heating/cooling rates
of a solution that are greater than 109 K s 1 [6]. During
acoustic cavitation, free radicals are formed owing to the
dissociation of vapors trapped in cavities; these radicals
might be utilized for acceleration of chemical reactions at
ambient temperatures that otherwise would require more
drastic conditions [7]. Furthermore, local turbulences and
liquid micro-circulations (acoustic streaming) are formed
that might be utilized for the enhancement of transport
processes and the elimination of mass transfer resistance in
heterogeneous systems [5,8]. Therefore, ultrasound irradia-
tion can actually be considered a combination of chemical
reactions utilizing the formed radicals and of physical effects
that are associated with an increase in temperature (pyrol-
ysis and combustion) and shearing [9].
Several ultrasound parameters, such as its frequency,
intensity, duty cycle and duration of irradiation, deter-
mine any potentially damaging effects on biological mol-
ecules, which might arise form the extent and severity of
possible cell disintegration due to bulk heating and chemi-
cal action of the free radicals, which are formed during
sonolysis and which are able to attack cellular material
[10]. These molecular effects of ultrasound are summar-
ized and illustrated in Box 1. The main reaction
parameters of various ultrasound-assisted bioprocesses
are presented in Table 1.
For years, ultrasound has been used in several biotech-
nology applications, for example with the purpose of cell
disruption to release intracellular enzymes and organelles
that are applied in industry and medicine [11,12]. Other
applications in the field of bioprocessing include facilitated
transfer from the cell into the solvent, for example intensi-
fication of mass transfer of cellulose and pectinase enzymes
during bio-processing of cotton textiles [13] and during
cholesterol oxidation to cholestenone by resting cells of
Rhodococcus erythropolis [14]. Ultrasound has also been
shown to influence reaction kinetics and reduce reaction
time of esterification, for example during isopropyl ester
synthesis from palm fatty acid distillate and medium chain
Review Trends in Biotechnology Vol.27 No.5

Box 1. Molecular effects of ultrasound

Physical changes Chemical changes

Thermal: pyrolysis and combustion Cavitation-induced generation of radicals

 Thermal effects typically don’t cause significant damage; however,  Release of compounds such as nitric acid, nitrous acid and
the extent of any cell damage will depend on absorbed energy, the hydrogen peroxide due to interaction of formed radicals with the
maximum temperature achieved and exposure duration. Observed cell (Figure Ivi).
cell damage can include partial and full lysis (Figure Ii).  Decreased cellular stability (Figure Ivii).

Non-thermal: cavitation and shearing Stress-induced changes

 Changes to ultra-structures within cells (Figure Iii).
 Altered enzyme stability (Figure Iiii).
 Cellular effects caused by altered growth properties, which could
lead to cell lysis (Figure Ii).
 Nucleus rupture and release of DNA (Figure Iiv).
 Breakage of extracellular polymer substances (Figure Iv).
Acoustic streaming
 Enhanced mass transport inside and outside the cell due to altered
membrane permeability (Figure Iviii).
 Alteration of cell surface charge (Figure Iix).
 Rupture of cell membranes (Figure Ivi).

Figure I. Molecular effects of ultrasound.

triglyceride synthesis from fatty acids and glycerol [15,16]. ultrasound has been successfully used to reduce enzymatic
Ultrasound has also been exploited to aid the transester- hydrolysis times of trypsin, pepsin and a-amylase [18] from
ification of glucose and divinyl dicarboxylates in organic typically 6 h to 30 min and to increase conversion rates of
solvents during glucose ester synthesis [17]. Moreover, waste paper in the presence of cellulase by 1.5-fold [19].

Table 1. Representative ultrasound-assisted bioprocesses and parameters used

Process Sonication parameters Sonication Refs
time
Transesterification of triglicerydes and free fatty acids for biofuel Frequency 20–48 kHz and power 120–1200 W 10, 60, [25,27,28,
production 140 min 31–35]
Emulsification of vegetable oil and methanol for biofuel production Frequency 40 kHz and power 700–1200 W 120 min [31,32]
Laccase-catalyzed decolorization of textile effluents Frequency 150–850 kHz and power 42–120 W 60–540 min [42–44]
Horse radish peroxidase-catalyzed degradation of phenol Frequency 423 kHz and power 5.5 W 20–60 min [48,49]
Protease-catalyzed oxidation of untanned leather waste Frequency 40 kHz 120 min [50]
Cellulase-catalyzed degradation of distillery wastewater Frequency 22.5 kHz and power 120 W 30–120 min [51]
Bacillus subtilis var. niger detection with immunosensor in USSW Acoustic pressure amplitude 460 kPa and 3 min [55,56]
system frequency 2–4 MHz
Tyrosinase-laccase immobilization on sonogel–carbon transducer Frequency 40 kHz and power 600 W NA [57–59,63]
Increase in dehydrogenate activity of waste activated sludge Frequency 35 kHz and power 80 W 10 min [64]
Anaerobic digestion of waste activated sludge Frequency 10–20 kHz, energy density 20–60 kJ l 1 NA [65,66]
Dewatering of waste activated sludge Power 100–600 W and energy density >2000 kJ l 1 NA [70,72]

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Ultrasound could also be used to improve productivity of
some fermentation processes [8], to alter filamentous fungi
growth morphology and metabolite production [20] and to
induce emulsification and crosslinking of proteins into
enzyme microspheres in aqueous media, which were
shown to sustain enzymatic activity of lysozyme [21],
amylase and a-chymotrypsin [22]. Moreover, ultrasound
has been used successfully to reshape sheet-like dipeptide
particles into elongated molecular assemblies, which are at
the underlying basis of solvent gelation via sonocrystalli-
zation [2,23]. This review will focus on the recent advances
in ultrasound-assisted bioprocesses and reveal the contri-
bution of ultrasound to enzyme characteristics, reactions
rates and duration, as well as mass transfer in various
industrially relevant applications, such as biofuel pro-
duction, biocatalysis for ultrasound-assisted bioremedia-
tion, enzyme biosensors and biosludge processing.
Biofuel production and process monitoring
Environmental concerns and the impending depletion of
oil reserves have resulted in governmental actions and
incentives to establish greater energy independence by
promoting research on more environmentally friendly
and sustainable biofuels. In the recent years, biofuel,
which is comprised of mono-alkyl esters of long chain
fatty acids (FAME) derived from vegetable oils or
greases, has been advocated as a promising fuel for the
future [24]. Its advantages over petroleum-based fuels are
that it is renewable, biodegradable and non-toxic and its
contribution to greenhouse gases is minimal because any
emitted CO2 is equal to the CO2 absorbed by the biomass
[25]. However, if vegetable oils are directly used as a fuel,
they can cause engine problems such as injector fouling
due to incomplete combustion and incorrect vaporization
characteristics and particle agglomeration due to their

Figure 1. Schematic representation of the beneficial effects of ultrasound for biofuel production. (a) Shown is the effect of ultrasound irradiation on emulsification of
vegetable oil (dark yellow) and methanol (white), which is essential before biofuel can be produced. As shown in (i), ultrasound can significantly facilitate emulsification
compared to mechanical stirring (ii), which also requires longer mixing times [31,32]. (b) Schematic illustration of the effect of ultrasound on catalytic transesterification of
vegetable oils to biofuel. Upon ultrasound irradiation, biofuel yield (shown in brown) as well as the reaction time is increased (i) compared with conventional
transesterification in the absence of ultrasound (ii), which requires higher amounts of catalysts (area above methanol) and of methanol [25,27,32–35]. The occurrence of
saponification (as indicated by a white cloud in the bottom panel) can be entirely avoided by use of ultrasound irradiation [25,34].

300
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high viscosity, which is 10–20 times higher than that of
petroleum fuel [26].
A traditional way to produce biofuels is by transester-
ification of natural oils, which is commonly carried out by
treating the pre-extracted oil with an appropriate alcohol
in the presence of an acidic or alkaline catalyst over one or
two hours in a batch reactor [27,28]. Because oils and
methanol are not completely miscible, the mixing efficiency
is one of the most important factors that determines biofuel
yield [29]. It has been widely demonstrated that low-fre-
quency ultrasound irradiation is a highly useful tool for
emulsifying immiscible liquids (Figure 1a). The induced
collapse of the cavitation bubbles disrupts the phase
boundary and causes emulsification through ultrasonic
jets that impinge one liquid to another [1]. Because of
these ultrasonic jets and localized extreme temperatures,
additional mixing and heating steps are not required to
produce biofuel in the presence of ultrasound [30]. Indeed,
it was recently reported that ultrasound irradiation
allowed for effective emulsification and mass transfer,
which resulted in an tenfold increase in the rate of ester
formation compared with ester formation in a conventional
stirred process [31,32].
Ultrasound can also be efficiently utilized to optimize
the conversion of triglycerides to biofuel. For instance, the
influence of low-frequency ultrasound (28 and 40 kHz) on
biofuel production from triglycerides [25,27,28,33,34], free
fatty acids [35] and fatty acid odor cut (C8–C10) [31] was
tested using either methanol or ethanol in the presence of
different catalysts, such as sodium hydroxide (NaOH)
[28,30], potassium hydroxide (KOH) [27,32] and sulfuric
acid (H2SO4) [31] and compared to a conventional trans-
esterification process (Figure 1b). These studies demon-
strated that the use of ultrasound significantly reduced
the amount of required catalyst [27,32–34], eliminated
saponification [25,34] and dramatically shortened the
reaction time from 2 h to 30 min [25,28,30,31,33]. In
addition, an up to threefold lower molar ratio of methanol
to fatty acids could be used, which resulted in high biofuel
yields of 95–97%, regardless of the initial material
[25,27,28,31–33,35].
In addition to accelerating biofuel production, ultra-
sound can be used to monitor the biofuel production pro-
cess. Currently, the quality of biofuels is typically assessed
by gas chromatography (GC), high performance liquid
chromatography (HPLC) and 1H nuclear magnetic reson-
ance (NMR) spectroscopy. However, these methods require
significant monetary investments, continuous sampling
procedures and time-consuming analysis [36]. Ultrasound
could be an efficient alternative for monitoring and con-
trolling various bioprocesses by providing non-invasive,
robust and rapid measurements that could be fully auto-
mized at a relatively low cost [37]. For example, to deter-
mine completion of the transesterification reaction, the
settling rates of by-products, such as glycerol, could be
monitored with ultrasonic systems [38]. Recently, Koc [36]
established that a low-intensity ultrasonic measurement
system could also be efficiently exploited to determine the
effects of different parameters that are important for the
overall biofuel quality, such as catalyst type and amount,
reaction temperature, mixing time and speed and alcohol
Trends in Biotechnology Vol.27 No.5
type and amount, on reaction rates and yields of transes-
terification of various vegetable oils.
Effect of ultrasound on enzymes
Enzymes are increasingly becoming the industrial work-
horse for a range of processes, such as biocatalysis and
bioremediation, with the aim of reducing energy and raw
material consumption and amounts of waste and toxic side
products [39]. An ultrasound wave, which is a periodic
pressure fluctuation, can control the enzyme character-
istics by altering its structure in response to a dynamic
perturbation [40]. Ultrasound irradiation of ten to several
hundred kHz would be able to perturb loop and domain
regions of an enzyme and thus affect its activity. This
property of ultrasound would allow control over desired
enzyme function. The tolerance of enzymes towards ultra-
sound might depend on the physiological location of the
enzyme in the cell and on its molecular weight. However,
very little is known about the actual effect of ultrasound on
enzymes because contradictory results of inactivation and
activation of enzymes upon ultrasound treatment have
been reported. Unlike traditional heat denaturation, the
sonication process does not destroy the active site of
an enzyme, and this has been clearly demonstrated for
a-amylase, horse radish peroxidise, laccase and alkaline
phosphatase [22,41–44]. However, ultrasound-mediated
deactivation of some enzymes, including alkaline protease,
glucose-6-phosphate dehydrogenase and trypsin, was
observed when high ultrasound power of 500 W was used
(the typical power used is 25–100 W) [43]. This was most
likely due to the reaction of hydroxyl or hydrogen radicals
formed with the protein backbone [43]. This in turn could
lead to enzyme aggregation, thereby obstructing the active
sites while at the same time also decreasing protein
stability [45]. Furthermore, shear forces that occur in
the fluid during ultrasound irradiation also have a signifi-
cant role in enzyme inactivation [41].
The overall effect of ultrasound on enzymes will there-
fore mainly depend on parameters such as energy input
and irradiation duration [17,43]. The presence of redox
mediators, such as polyvinyl alcohol, violuric acid and 2,2-
azinobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS),
that are able to increase the stability of the irradiated
enzyme [44,45] will also determine the outcome of ultra-
sound treatment, as well as the type of enzyme [41]. The
optimization of these parameters will allow the determi-
nation of the threshold conditions for ultrasound irradia-
tion of enzymes so that their activity is not adversely
affected.
Enzymes as biocatalysts in ultrasound-assisted
bioremediation
Bioremediation refers to processes that use microorgan-
isms or their enzymes for the clean up of contaminated
soils or waters. These enzyme-catalyzed reactions have
several advantages compared with conventional waste
treatment methods in that they are applicable to a variety
of xenobiotic compounds, such as polycyclic aromatic
hydrocarbons, polynitrated aromatic compounds, pesti-
cides [39], bleach-plant effluents, synthetic dyes [46], poly-
mers and wood preservatives, for example creosote and
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pentachlorophenol [47]. In addition, they can be operated
at high or low concentrations of contaminants and over a
wide range of conditions with regard to pH, temperature
and salinity. The absence of shock loading effects, which
are often associated with the acclimatization of biomass, is
another significant advantage, as are the reduced volume
of waste sludge (no biomass is actually generated) and the
ease and simplicity of the process control.
Over the last few years, ultrasound has been used with
the aim of enhancing enzyme-catalyzed waste treatment.
For instance, laboratory-scale application of sonobiodegra-
dation (ultrasound irradiation at 1.12 W cm 2 and 423 kHz
for 20–60 min) resulted in 80–100% degradation of phenol
and its halogenated substitutes in the presence of horse
radish peroxidase and hydrogen peroxide in a synthetic
wastewater [48,49]. However, despite successful labora-
tory-scale experiments, the scale-up of such systems is
complicated by the relatively short catalytic lifetime of
the enzyme, which is due to the attack of phenoxy radicals
that are generated during the process and that destroy the
catalytic ability of the enzyme [48]. The short catalytic
lifetime together with the high production costs of enzymes
will lead to unacceptably high treatment costs and thus
ways to overcome these drawbacks must be sought [49].
The ultrasound-mediated decolorization of various tex-
tile dyes with laccase from Trametes villosa was also
successfully attempted in laboratory- and pilot-scale exper-
iments [42–44]. For instance, Rehorek et al. [42] and
Tauber et al. [44] demonstrated that in the presence of
60–120 W ultrasound irradiation at 850 kHz, a complete
mineralization of azo dyes could be attained within 1–9 h.
However, an increase in ultrasound power from 90 to
120 W resulted in the partial deactivation of the laccase
enzyme, leading to a considerably shorter half-life time of
5 h (compared with a half-life time of 20 h without ultra-
sound treatment). Basto et al. [43] achieved 65 to 77%
decolorization of indigo carmine when they applied ultra-
sound of 47–72 W with an optimum frequency of 150 kHz
for 60 min. To avoid deactivation of enzyme and to increase
its stability, they used polyvinyl alcohol to create a pro-
tective hydrophobic layer around the enzyme in the reac-
tion solution.
Sonobiodegradation of solid untanned leather waste
could also be effectively performed at laboratory scale with
an alkaline protease from Bacillus licheniformis [50]. In
this study, the catalytic activity of the enzyme was unal-
tered by ultrasound irradiation of 0.64 W cm 2 min at
40 kHz frequency for 2 h [50]. It was also found that this
ultrasound irradiation accelerated the enzymatic hydroly-
sis and significantly improved the final conversion ratio to
84.1% by increasing the transport of the protease through
the pores of the skin, thereby impairing the integrity of
collagen, the main constituent of the untanned leather.
Furthermore, ultrasonic pretreatment of molasses
spent wash (MSW) from distillery wastewater was also
carried out on a laboratory scale with the help of cellulase
[51]. The pretreatment of MSW by ultrasound in the pre-
sence of cellulase resulted in an almost fourfold increase in
MSW oxidation rates over the initial 36 h compared with
aerobic oxidation without pretreatment over a similar
length of time [51].
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Trends in Biotechnology Vol.27 No.5
All the studies above provide evidence that a concurrent
application of enzymes and ultrasound was considerably
more effective in terms of reduced reaction time and treat-
ment efficiency than either ultrasound-assisted or enzyme-
catalyzed reactions alone, regardless of the enzyme or
process applied. However, to efficiently utilize sonobiode-
gradation on an industrial scale, the catalytic activity of
the enzyme needs to be protected from radical attack and
the stability of the enzyme needs to be increased so that it
can persist intact over industrial time scales.
Biosensors
Biosensors offer simplified, sensitive, rapid and reagent-
less detection methods for a wide range of biomedical and
industrial applications [52]. In biosensors, a biological unit
(e.g. an enzyme or an antibody), which is typically immobil-
ized on the surface of the transducer (electrode), interacts
with the analyte (which contains, for example, a target
bacterium or xenobiotic) and causes a change in a measur-
able property within the local environment near the trans-
ducer surface, thus converting a (bio)chemical process into
a measurable electronic signal [53]. The main factors that
influence the biosensor response are the mass-transport
kinetics of analytes and products, as well as loading of the
sensing molecule.
To improve the sensitivity and efficiency of biosensors,
ultrasound irradiation can be used to aggregate [54] and
drive [55,56] microorganisms towards antibody-coated
sensors using ultrasound standing waves (USSWs). Alter-
natively, ultrasound could be used to facilitate enzyme
immobilization on sonogel–carbon transducer electrodes,
which are frequently used to detect, for example, xeno-
biotics and organophosphorus pesticides in waste streams
[57–60].
Kuznetsova and co-workers [54] discovered that by
applying a 1.5 MHz USSW field, it was possible to attain
suspended particle aggregation in the solution within a few
minutes and, importantly, to control the size of these
aggregates, thus ensuring shorter analysis time and also
preventing sedimentation (Figure 2a). It is vital to prevent
sedimentation because particle aggregates might settle on
the surface of the ultrasonic transducer and alter the
radiation characteristics of the system [2]. USSWs might
also be used to drive and deposit bacterial spores onto
various sensors, such as bacteria-detecting immunosen-
sors and optical leaky waveguide sensors. For example, in
a study that employed a pressure amplitude of 460 kPa at a
frequency of 2.82 MHz and an initial spore concentration of
2  104 particles ml 1, it was shown that the use of USSWs
enhanced the deposition of Bacillus subtilis spores onto
immunosensors by 70-fold [61]. and a different study
reported a more than 200-fold increase in deposition at
frequencies from 2 to 4 MHz and a spore concentration of
6.6  106 particles ml 1 [55]. Zourob and co-workers used
USSW with an optical leaky waveguide sensor and were
able to improve the detection limit of Bacillus subtilis var.
niger to only 103 spores ml 1 and to shorten analysis time
from 20 to 3 min [56].
Compared with other biosensors, biosensors that utilize
immobilized enzymes offer several advantages, such as
their high selectivity, which is determined by the used
 Review Trends in Biotechnology Vol.27 No.5

Figure 2. Application of ultrasound for preparation of biosensors. (a) Effect of ultrasound on biosensor performance. Ultrasound can be used to induce aggregation of
bacterial spores (shown in red) and to drive these towards an antibody-coated biosensor surface (i), resulting in a significantly faster sensor response and improved
detection limit compared with biosensor response in the absence of ultrasound (ii) [54–56]. (b) Use of ultrasound irradiation for preparation of sonogel–carbon transducers
from graphite (shown as gray octagon) and sonogel (shown as yellow octagon). Transducers are subsequently coated with enzyme (represented in pink) to generate the
biosensor (i) [57]. Compared with conventional sol–gel transducers (ii), which are made by simply mixing components, ultrasound irradiation leads to faster preparation of
transducers and to enzyme-coated surfaces that exhibit significantly higher wear resistance, activity and longevity, as indicated by the lack of degradation of surface-bound
sensing enzyme [58–60,63].

enzyme and which can be tailored to a specific application,
and their faster response, which is due to their short
diffusion path as cell walls are absent [62]. However, these
biosensors also have some inherent disadvantages that are
associated with the requirement to stabilize the enzyme
and the potential need for co-factors to detect the desired
substances.
As a consequence, the most crucial part in manufactur-
ing of these types of biosensors is the immobilization of the
sensing enzyme onto an appropriate transducer (elec-
trode), because inadequate immobilization might lead to
a lower efficiency of the sensor caused by partial enzyme
destabilization that might occur in the process (Figure 2b).
Sonogel–carbon transducers, which are prepared by apply-
ing high-energy ultrasound directly to a mixture of sonogel
and spectroscopic grade graphite, might be able to effi-
ciently stabilize enzymes because they have been shown to
show a high degree of biocompatibility with enzymes [57].
For instance, ultrasound-assisted immobilization of some
enzymes, including laccase, tyrosinase and acetylcholin-
esterase, onto sonogel–carbon transducers was shown to be
remarkably successful in avoiding material shrinkage and
in preserving the integrity and shape of the enzyme, as well
as in achieving the desired response towards the analyte
[58]. For example, a biosensor containing immobilized
laccase and tyrosinase maintained 80% of its initial
activity for a minimum of three weeks [57]. Another sensor
with immobilized tyrosinase exhibited nearly 80% of its
initial activity after 100 measurements of phenol with a
detection limit of 3  10 7 M [63]. In another example, a
sensor for organophosphorous compounds based on
immobilized acetylcholinesterase retained 50% of its
initial response for 50 days (equalling 100 measurements
of 10 min incubation time each) with a detection limit of
2.5  10 10 M [59].
Biosludge processing
Activated sludge processes are widely used for the treat-
ment of industrial and domestic wastewaters. However,
these processes produce large quantities of biomass
(sludge), which require additional procedures to either
stabilize the sludge or to enable its environmentally
safe utilization or disposal [3]. Anaerobic digestion is the
standard technique for treatment of waste-activated
sludge and leads to a reduced content of organic matter
and biogas production. However, anaerobic digestion has

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some disadvantages that are mainly associated with the
requirement of post-treatment and the inability of the
process to treat sewage to levels suitable for direct dis-
charge [64]. Therefore, methods for enhancing the bio-
logical activity of the anaerobic sludge and improving
the efficiency of anaerobic digestion are urgently required.
To enhance anaerobic digestion, various pretreatment
methods might be applied, including heating, enzyme
addition for cell wall disruption, ozonation, alkaline
addition and mechanical disintegration; however, none
of these methods have so far been able to prevent excess
sludge production [65]. However, ultrasound was success-
fully used to increase the biological activity of the
anaerobic sludge, to disintegrate sludge to increase the
production of biogas, to prevent an excess sludge pro-
duction and to aid dewatering to some extent [9,64,66–68].
The removal of water from sludge, which constitutes 25–
40% of the operating costs of a wastewater treatment
plant, is an unavoidable and compulsory step to reduce
the volume of sludge and, consequently, to reduce the costs
of transporting the sludge to its ultimate disposal site [67].
Conventional mechanical techniques, such as centrifu-
gation, vacuum and belt pressure filtering and thermal
and chemical conditioning methods (e.g. addition of poly-
eletrolytes), are usually used to remove water from sludge;
however, the final water content in sludge still ranges from
45 to 87% [69,70]. In theory, the effects induced by ultra-
sound can assist the dewatering mechanism [3]. First,
ultrasonic stresses that arise in the sludge can facilitate
the migration of moisture through either naturally present
channels or channels that have been created by the wave
propagation. Second, air bubbles that are trapped in micro-
pores can grow by rectified diffusion and lead to a displace-
ment of the liquid out of these micropores. Third, cavitation
can help to separate the moisture that is colloidal or
chemically attached from the solid phase. However, contra-
dictory results have been reported for the actual applica-
bility of ultrasound to dewatering anaerobic sludge.
Despite the fact that ultrasound is able to destroy extra-
cellular polymeric substances (EPSs), such as proteins and
polysaccharides, and to decrease the dry solid (DS) content
of sludge, the overall dewaterability has been shown to be
reduced when the irradiation time or intensity is
increased, which is a consequence of the water-retaining
effect of EPSs [67]. Moreover, the external energy supplied
to the sludge caused a reduction of the average floc size,
which results in an increased surface area and in higher
water adsorption, as well as in sludge cake clogging,
thereby decreasing the filterability of the sludge and its
subsequent dewaterability [66]. One way to increase the
sludge dewaterability in the presence of ultrasound might
be to subject only a portion of sludge (at constant volatile-
solids content) to ultrasound and re-blend it at a later stage
with un-sonicated material [71].
Conversely, some research attempts demonstrated
beneficial effects of ultrasound on the dewaterability of
waste activated sludge. For example, Feng and co-workers
[70] determined the optimal specific ultrasound energy
that would give maximal dewaterability characteristics
to be 800 kJ kg 1 total solids (TS), which generated sludge
with optimal EPS concentration of 400–500 mg l 1 and
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Trends in Biotechnology Vol.27 No.5
particle size distribution with diameters in the range of 80–
90 mm. In addition, Na and co-workers [72] observed that
ultrasound (100–600 W and energy densities higher than
2000 kJ l 1) induced the dewatering of waste activated
sludge by fivefold, thus decreasing the capillary suction
time (CST), which is a good indicator of dewaterability.
However, any reduction in the DS fraction (e.g. 30%) would
result in considerable savings on further discharge costs,
which are currently s100 per tonne of DS [66].
Anaerobic digestion is typically achieved through differ-
ent enzymatic steps, such as hydrolysis, acidogenesis and
methanogenesis, with hydrolysis being the rate-limiting
step for sludge treatment [9]. Consequently, ultrasound
irradiation might be applied to improve the hydrolysis step
and thus the overall efficiency of anaerobic digestion.
Laboratory- and pilot-scale studies showed that the appli-
cation of ultrasound of low frequency (10–20 kHz) and
density (1000–3000 kJ kg 1 TS equal to 20–60 kJ l 1 of
sludge) led to a 20% increase in biogas production, which
was accompanied by a 4.5% reduction in organic DS and a
25–30% decrease in total phosphorus content [9,65,66].
Moreover, low-frequency ultrasound irradiation could also
significantly promote the biological activity of the
anaerobic sludge. For instance, the treatment of sludge
with ultrasound of 35 kHz frequency at 0.2 W cm 2 for
10 min increased the dehydrogenate activity almost two-
fold and reduced the effluent COD by more than 30%
compared with un-sonicated conditions [64].
The use of ultrasound for sewage-sludge treatment has
already been established in some full- scale wastewater
treatment plants. From these, it has been reported that
low-intensity ultrasound application is particularly useful
for improving sludge settling and for treating any foam
problems that occur, whereas high-intensity ultrasound
could cause the release of intracellular material and thus
increase the sludge digestion time by 20–50% with
increases in biogas yield of up to 25–50% [71].
Conclusions and future perspectives
Despite extensive scientific efforts, there is still room for
novel methods that might result in significant improve-
ments in bioprocesses efficiency. The application of low-
frequency ultrasound has proven to be an environmentally
friendly, time saving and economically viable approach for
several important applications, such as the enhancement
of emulsification and transesterification of fatty acids for
biofuel production and the improvement and acceleration
of bioprocess monitoring. Further important uses include
the improvement of biosensors through enhanced immo-
bilization of biological entities and the improved disinteg-
ration and dewatering of waste activated sludge with
concomitant biogas production. The economic viability of
ultrasound and subsequent industrial application is influ-
enced by the legislation, associated operating costs, incen-
tives for renewable energy, costs of enzymes, sludge
recycling routes and transport costs, dewatering costs
and the type of available wastewater treatment. However,
the fulfillment of the true potential of ultrasound has so far
been hampered by the lack of knowledge regarding the
exact molecular effects of ultrasound on enzymes and
living cells.
Review
In enzyme-based applications, ultrasound has been
shown to increase enzyme stability and catalytic activity,
as well as the longevity of enzyme biocatalysts. However, a
better understanding of the ultrasound effect on enzyme
properties is necessary to develop efficient biocatalysts with
further enhanced activity and long-term stability. Also, the
molecular changes that occur when ultrasound interacts
with various cellular parts need to be further studied to
allow the range of future applications to be widened.
The areas of biotechnology that have already profited
from ultrasound continue to expand, and further improve-
ments might be achieved by determining the optimum
reaction conditions and parameters for ultrasound-
assisted applications. If harnessed effectively, ultrasound
power can contribute to future advances in the areas of (i)
renewable energy production, (ii) biocatalysts in terms of
significantly reduced reaction time and (iii) analytical
chemistry in terms of lowered detection limits. Overcoming
the present limitations and difficulties of ultrasound-
assisted bioprocesses will probably boost their indus-
trial-scale applications.
Acknowledgements
The Academy of Finland (decision number 212649) is greatly
acknowledged for financial support.
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