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Review

Review
Review Low-frequency ultrasound in biotechnology: state of the art Ekaterina V. Rokhina 1 , Piet Lens

Low-frequency ultrasound in biotechnology: state of the art

Ekaterina V. Rokhina 1 , Piet Lens 2 and Jurate Virkutyte 1

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 (20100 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 ).

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 10 9 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 dura tion 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

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Box 1. Molecular effects of ultrasound

Physical changes

Thermal: pyrolysis and combustion Thermal effects typically don’t cause significant damage; however, the extent of any cell damage will depend on absorbed energy, the maximum temperature achieved and exposure duration. Observed cell damage can include partial and full lysis (Figure Ii).

Non-thermal: cavitation and shearing 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).

Chemical changes

Cavitation-induced generation of radicals Release of compounds such as nitric acid, nitrous acid and hydrogen peroxide due to interaction of formed radicals with the cell (Figure Ivi). Decreased cellular stability (Figure Ivii).

Stress-induced changes

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.
Figure I. Molecular effects of ultrasound.

triglyceride synthesis from fatty acids and glycerol [15,16]. Ultrasound has also been exploited to aid the transester- ification of glucose and divinyl dicarboxylates in organic solvents during glucose ester synthesis [17] . Moreover,

ultrasound has been successfully used to reduce enzymatic hydrolysis times of trypsin, pepsin and a -amylase [18] from typically 6 h to 30 min and to increase conversion rates of 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 production Emulsification of vegetable oil and methanol for biofuel production Laccase-catalyzed decolorization of textile efuents

Frequency 20 48 kHz and power 1201200 W

10, 60,

[25,27,28,

140 min

31

35]

Frequency 40 kHz and power 700 1200 W 120 min

[31,32]

Frequency 150 850 kHz and power 42 120 W

60540 min

[42 44]

Horse radish peroxidase-catalyzed degradation of phenol Frequency 423 kHz and power 5.5 W 2060 min [48,49]

Protease-catalyzed oxidation of untanned leather waste Cellulase-catalyzed degradation of distillery wastewater Bacillus subtilis var. niger detection with immunosensor in USSW system

Frequency

40 kHz 22.5 kHz and power 120 W

120 min 30120 min 3 min

[50]

Frequency

[51]

Acoustic pressure amplitude 460 kPa and frequency 2 4 MHz

[55,56]

Tyrosinase-laccase immobilization on sonogelcarbon 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 Anaerobic digestion of waste activated sludge Frequency 10 20 kHz, energy density 20 60 kJ l 1 NA Dewatering of waste activated sludge Power 100 600 W and energy density > 2000 kJ l 1 NA

[64]

[65,66]

[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 CO 2 is equal to the CO 2 absorbed by the biomass [25] . However, if vegetable oils are directly used as a fuel, they can cause engine proble ms such as injector fouling due to incomplete combustion and incorrect vaporization characteristics and particle agglomeration due to their

characteristics and particle agglomeration due to their Figure 1 . Schematic representation of the beneficial

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 [25,27,3235]. 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].

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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 (C 8 C 10 ) [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 (H 2 SO 4 ) [31] and compared to a conventional trans- esterification process ( Figure 1 b). 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 1 H 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

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 [22,4144]. 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 25100 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 2060 min) resulted in 80100% 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 [4244]. For instance, Rehorek et al. [42] and Tauber et al. [44] demonstrated that in the presence of 60120 W ultrasound irradiation at 850 kHz, a complete mineralization of azo dyes could be attained within 19 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 4772 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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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 sonogelcarbon transducer electrodes, which are frequently used to detect, for example, xeno- biotics and organophosphorus pesticides in waste streams

[5760].

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 10 4 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 10 6 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 10 3 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

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Review Trends in Biotechnology Vol.27 No.5 Figure 2 . Application of ultrasound for preparation of biosensors.

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) [5456]. (b) Use of ultrasound irradiation for preparation of sonogelcarbon 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 solgel 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 [5860,63].

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). Sonogelcarbon 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 2540% 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 400500 mg l 1 and

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particle size distribution with diameters in the range of 80 90 mm. In addition, Na and co-workers [72] observed that ultrasound (100600 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 s 100 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 (10003000 kJ kg 1 TS equal to 2060 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 2530% 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 2050% 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.

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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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