Separation of bacterial spores from flowing water in macro-scale cavities by ultrasonic standing waves

Transcription

1 Separation o bacterial spores rom lowing water in macro-scale cavities by ultrasonic standing waves Abstract PSI-1564/SR-1414 B. Lipkens, J. Dionne Western New England College Department o Mechanical Engineering 115 Wilbraham Road, Box S-504 Springield, Massachusetts blipkins@wnec.edu M. Costolo, A. Stevens, E. Rietman Physical Sciences Inc. 0 New England Business Center Andover, Massachusetts costolo@psicorp.com, stevens@psicorp.com, erietman@gmail.com Prepared or upload to arxiv: June 010 The separation o micron-sized bacterial spores (Bacillus cereus) rom a steady low o water through the use o ultrasonic standing waves is demonstrated. An ultrasonic resonator with cross-section o m x m has been designed with a low inlet and outlet or a water stream that ensures laminar low conditions into and out o the resonator section o the low tube. A m diameter PZT-4, nominal -MHz transducer is used to generate ultrasonic standing waves in the resonator. The acoustic resonator is m rom transducer ace to the opposite relector wall with the acoustic ield in a direction orthogonal to the water low direction. At ixed requency excitation, spores are concentrated at the stable locations o the acoustic radiation orce and trapped in the resonator region. The eect o the transducer voltage and requency on the eiciency o spore capture in the resonator has been investigated. Successul separation o B. cereus spores rom water with typical volume low rates o ml/min has been achieved with 15% eiciency in a single pass at 40 ml/min. PACS numbers: 43.5 Qp; 43.35Zc Keywords: Acoustic radiation orce; laminar low; particle concentration; particle separation; ultrasonic standing waves 1

2 I. Introduction We previously reported 1 the use o ultrasonic standing waves or the separation o 6-micron diameter polystyrene beads rom a lowing water sample as the water passed through a 0.15-m long resonator cavity placed symmetrically across a m x m low channel. That system relied on a requency sweep method to translate the particles into the arm o the resonator cavity that is opposite the acoustic transducer. The collection eiciency o that resonator and low channel design was limited signiicantly by the acoustic streaming o the water as a result o the requency sweep. We report here an alternate system design in which the applied ultrasonic requency is kept ixed, the resonator cavity is much shorter (0.0356m rom transducer ace to the opposing relective wall), and the separation o micron-sized bacterial spores is achieved simply by their capture in the stable locations o the acoustic radiation ield. There is a large body o work on the use o the acoustic radiation orce or particle concentration and separation. A recent review paper by Marston and Thiessen presents an overview o the undamental principles and applications o the acoustic radiation orce. The theoretical ramework has been developed by King, 3 Yosioka and Kawasima, 4 Gor kov, 5 and Tolt and Feke. 6,7 They developed a separation process based on the acoustic radiation orce in a stationary ultrasonic standing wave ield. Tolt and Feke 6 used a requency sweeping method to translate the concentrated particles across the resonator. The requency sweep is over a range o 0, where 0 is the undamental resonance requency o a resonator with length L, i.e., 0 =c /L, where c is the speed o sound o the luid in the resonator. Hill et al. 8, 9 and Townsend et al. 10 developed a model or the calculation o particle paths or suspended particles in a luid. The particles are exposed to three orces, acoustic radiation orce, luid drag orce, and buoyancy orce. An electro-acoustic model is used to calculate the acoustic ield in the resonator. A separate model is used to calculate the low velocity vectors in the resonator. A third model considers the particle-acoustic ield interaction. A second example o using requency sweeps to translate particles across an ultrasonic resonator is presented in the work by Handl et al. 11 They used a our step requency sweep that alternated between the requencies o our consecutive acoustic resonance requencies o the cavity. They showed that under these conditions particles are translated across the resonator. The measured particle trajectories compared avorably with those predicted by a numerical model. II. Acoustic Radiation Force For design calculations a particle trajectory model has been developed. 1 In this model the acoustic ield generated by a piezoelectric transducer is predicted. The acoustic radiation orce is then calculated according to the ormulation o Gor kov. 5 The primary acoustic radiation orce F A is deined as a unction o a ield potential U where the ield potential U is deined as F A = ( U ), (1)

3 U p ( x, y, t) 3ρ u ( x, y, t) = V ρ c, () and 1 and are the monopole and dipole contributions deined by 1 1 = 1, Λσ ( Λ 1) = Λ + 1 (3) where p(x,y,t) is the acoustic pressure, u(x,y,t) is the luid particle velocity, Λ is the ratio o particle density (ρ p ) to luid density (ρ ), σ is the square o the ratio o particle sound speed (c p ) to luid sound speed (c ), and V o is the volume o the particle. For a one-dimensional harmonic acoustic wave, the expression or the acoustic radiation orce is: F Ax 3( ρ p ρ ) ρ = V0 U ρ p + ρ ρ c V 0 1 ρ pc p 1 ρ c M du dx P M M dp M, dx (4) where U M and P M are the magnitudes o acoustic velocity and pressure. The calculation o the acoustic radiation orce is implemented through a numerical calculation o the spatial derivative o velocity and pressure. The model is described in detail in Lipkens et al. 1 Understanding the numerical model is important or this work in that it illustrates the diiculty o capturing bacterial spores: they have extremely small volumes (V 0 about 3 x m 3 ), a density close to that o water (ρ p estimated at 1.07 g/cm 3 ) 13 which results in a small acoustic radiation orce on the particle. III. Design and Experimental Setup A. Acoustocollector design The acoustocollector, consisting o a low cell and resonator, is shown in Fig. (1). The low inlet and outlet channels are each m x m in cross-section and are attached to the resonator. The length o the inlet and outlet is designed to be at least the entrance length related to the Reynolds number associated with typical low rates o 150 ml/min, thereby ensuring laminar low conditions in the acoustoresonator; lengths o the inlet and outlet channels were anywhere rom 0.10 to 0.0 m. The inlet and outlet were manuactured in an Objet rapid prototype machine. 3

4 The ultrasonic resonator section is machined o aluminum; the ront ace is made o glass to allow visual observation. The resonator is a m long section inserted between the inlet and outlet sections, and has a square cross-section o m x m to match the inlet and outlet cross sections. The transducer is in direct contact with the water and is mounted in the top ace o the resonator, so that the acoustic ield direction is orthogonal to the low direction. A collection pocket, m x m and m deep is in the resonator ace that opposes the transducer; the resonator cavity thereore has a ace-to-ace distance o m. To enable gravitational settling o the spores into the collection pocket, the system is mounted so the low is horizontal, as shown in Fig. (1). Fig. 1. a) Isometric view and b) photograph o the acoustocollector, showing the inlet and outlet low channels, resonator cavity, transducer, and collection pocket. The low enters rom the let. The acoustic ield is in the vertical direction, with the transducer mounted on the top ace o the resonator. The action o the ultrasonic radiation orce is so as to trap the passing particles in the ield, as well as cause agglomeration. The spores can be harvested rom the collector pocket. The transducer was manuactured by UTX Inc., and is PZT-4 with a nominal resonance requency o MHz; its ace has a diameter o 0.75-in ( m). Similar -MHz transducers were also purchased rom The Ultran Group. The S11 curve (ratio o transmitted to relective power as a unction o the excitation requency) or each o the various transducers was determined using an Agilent E8356A vector network analyzer with the transducer ace immersed in water in the cavity o Fig. (1). A Tektronix unction generator was used to generate the drive signal which was ampliied by an Ampliier Research 100-W ampliier with a bandwidth rom 10 khz to 50 MHz. Typical voltages applied across the PZT-4 were in the range o 0 to 40 Vpp. A dual-head Cole-Palmer L/S Economy Variable-Speed Digital-Drive Peristaltic Pump is used to pump the water through the resonator. Flow rates used in the experiments varied rom 40 to 50 ml/min. 4

5 B. Diagnostic apparatus Two techniques were used to qualiy the acoustocollector. The irst is simply qualitative, and consists o a ProScope USB microscope with a 10x magniying lens that can be placed so as to image the captured spores in the resonator, and store the video images on a computer. Quantitative determination o the relative concentration o bacterial spores was achieved by using in-line optical scattering, shown in Fig. (). Fig.. Schematic o the optical-scattering apparatus used or in-line determination o relative spore concentrations. A green HeNe laser beam goes through a beamsplitter with the split beam picked up by a photodiode and used as a laser power reerence to actor in any luctuations in the laser intensity. The orward beam is chopped and then is incident on a cuvette which has the outlet water stream rom the acoustocollector suspension lowing through it. The light scattered at 90-degree scattering signal is picked up by a photodiode; the signal goes to a preampliier and then to a lock-in ampliier. The resulting light intensity data rom reerence and signal photodiodes are captured using an A/D board and custom LabVIEW sotware. C. Preparation o spore samples B. cereus spores were prepared by growing the bacteria (ATCC 14579) in nutrient medium at 30 C or three days, ollowed by collection o the spores by centriugation. The spores were thoroughly washed with sterile deionized water and stored at 4 C or several weeks in sterile DI water. Occasional centriugation o the sample and re-suspension in water during this time removed any residual cellular matter. Spores were stained by adding a small amount o Malachite Green to the sample in water, heating or about 10 min. to C, and then repeatedly washing the stained spores to remove excess dye. 5

6 A photomicrograph o a sample o the spores is shown in Fig. (3). The B. cereus spores are cylindrical, about 1.5 μm in length and 0.5 μm in diameter (volume V 0 =.9 x m 3 ); their density is estimated to be 1.07 g/cm 3 ; 13 their compressibility has not been measured. Fig. 3. Scanning electron micrograph o a preparation o B. cereus spores; the spores are about 1.5 μm in length and 0.5 μm in diameter. Dilute samples o spores in water were prepared using 0.05% Tween 0, a suractant that prevents the spores rom agglomerating in the stock solutions. Spore concentrations or these experiments were about 10 7 per ml. IV. Results A. Collection o B. cereus in the acoustocollector A 500-ml suspension o B. cereus spores in water (with 0.05% Tween) was pumped at a relatively slow low rate o 40 ml/min through the acoustocollector system shown in Figs. (1) and (). A column o captured spores orms in the resonator when power is applied to the transducer at a constant requency which is a cavity resonance; a photomicrograph o a column o captured spores is shown in Fig. (4). The column ormation occurs with an. MHz transducer requency and an applied voltage o 0Vpp across the transducer. As the system comes to thermal equilibrium, the applied requency must be adjusted (increased) to compensate or the change in the speed o sound o the luid with increasing temperature. In Fig. (4) the spores are stacked in planes parallel to the transducer ace; the planes correspond to the stable locations o the zeroes o the acoustic radiation orce. Additionally, there exists a radial component o the acoustic radiation orce that holds the spores in these planes against the luid low. A batch process can be implemented where spores are trapped and held until some volume o water has been processed, and the low o water and power to the transducer turned o so that the agglomerated spores all under the orce o gravity to the collector pocket. Alternatively, a slow requency sweep could be used to drive the particles into the collector pocket. 6

7 Fig. 4. Microphotograph o a column o captured B. cereus spores stacked in planes about 700 microns apart in the resonator. The spores have been dyed with Malachite Green to make them more clearly visible. The orientation o the acoustocollector is as shown in Fig. (1); the transducer is at the top, just out o the ield o view and acing downward, and the water low is moving to the right at 40 ml/min. Fig. (5) shows the optical scattering signal rom the outlet stream, normalized to the initial scattered intensity (proportional to the starting spore concentration); the x-axis units are in terms o bottle turnovers, since each bottle consists o a 500-ml sample volume; the outlet rom the collector is ed back into the sample bottle. Fig. (5) indicates that in the initial pass through the system, ~15% o the spores were trapped. Note, however, the reduction in slope at approximately 0.5 bottle turnovers; this decrease in capture eiciency was accompanied by a spontaneous increase in the voltage measured across the transducer rom 0V pp to 30V pp (constant driver voltage and ampliier gain, i.e., applied power), which occurred even with adjustment o the applied requency. 7

8 Fig. 5. a) The scattering intensity in the outlet stream as a unction o bottle turnovers, with the intensity at t=0 set equal to one or normalization. b) The concentration actor C as given by Eq. (5), assuming a 4-ml collection volume or the captured spores. Data were taken with the transducer requency set to. MHz (with slight re-tuning o the requency as the temperature o the sample increased), a 0 V PP initial applied voltage (which shited to about 30 V PP around 0.5 a bottle turnover), a low rate set to 40 ml/min, and an initial spore concentration o about 10 7 per ml. The concentration actor C is calculated rom the total raction o the spores captured (n), the total volume o processed luid V and the volume o luid v in the collection pocket. C = ( 1 n)( V / v) (5) Fig. (5)b shows the concentration actor C as a unction o bottle turnovers or a 500-ml sample volume (V) and a 4-ml collection volume (v), and shows that a concentration increase o about a actor o 50 was achieved ater 8 passes through the concentrator. Ater lowing the spore suspension through the acoustocollector 8 times, the low was stopped and then the power to the transducer turned o. The captured spores readily ell into the collection pocket at the bottom o the resonator cavity, where they were removed using a pipet. In order to investigate the spore capture as a unction o the applied transducer requency and the resonator modes, the transducer requency was scanned rom.0 to.3 MHz in 1 khz steps or a ixed unction generator output and ixed ampliier gain (i.e., constant applied power). During the scan the voltage across the transducer was measured; simultaneously the spore capture was monitored with the digital microscope videocamera so that conditions producing a column could be noted. Fig. (6) shows the results o the requency scan or a transducer that was characterized by a S11 minimum at.11 MHz (in water, external to the resonator). 8

9 Fig. 6. Measured transducer voltage vs. drive requency or a ixed ampliier gain (total system power) or the transducer/resonator in the acoustocollector. The S11 minimum or this particular transducer in water (with no resonant cavity) was measured in a separate experiment and ound to be at.11 MHz, indicated by the vertical line. Spore capture tends to be initiated at requencies corresponding to local minima in the aluminum transducer voltage as a unction o applied requency (i.e., requencies in resonance with the aluminum resonator and that thereore result in standing waves), and at requencies closest to the S11 minimum (i.e., where the most power is delivered rom the ampliier to the transducer itsel). The column o captured spores remains stable i the requency is then tuned to a region that produces higher voltages across the transducer, but it does not begin ormation as readily at the requencies corresponding to local maxima in the graph o voltage vs. requency. Column ormation starts a ew minima to the low requency side o the S11 requency (.11 MHz) and continues through the S11 minimum. The requency ranges that include a secondary minimum on the high-requency side tend to cause intense bubble ormation, presumably due to very high acoustic pressures that result rom the applied requency being near the transducer resonance. Once bubbles begin to orm in the column, spore collection stops. We attribute this to the presence o the air bubbles eectively damping the acoustic waves, due to the large compressibility dierence o air and water. Eective spore capture takes place when the highest acoustic pressure is obtained, but without creating bubbles. For example, or the transducer/resonator system o Fig. (6), this corresponds to a requency o ~.16 MHz, three minima to the lower requency side o the S11 resonance. The voltage vs. requency curve o Fig. (6) shits to higher requencies with increasing temperature, so the requency corresponding to the voltage minimum must be ound experimentally each time an experiment is run. 9

10 V. Conclusions Successul separation o bacterial spores rom a ml/min water stream using a ixed acoustic requency is demonstrated in an acoustocollector with a m long aluminum acoustic resonator section inserted between inlet and outlet sections. A m diameter PZT-4, nominal -MHz transducer is used to generate ultrasonic standing waves in the resonator and capture the spores in the resulting acoustic pressure ields; the radial ield holds the spores against the water low. B. cereus spores can be captured with 15% capture eiciency in a single pass through an acoustic resonator at 40ml/min low rate. The resulting agglomeration o spores can be collected by stopping the water low and the transducer, allowing gravitational settling o the spores, and removing the spore sample rom a collection pocket at the bottom o the resonator. The acoustocollector is ideally suited or large-volume sampling o water supplies or concentration o spores and transer o the resulting concentrated sample to apparatus e.g., Raman or InraRed spectrometers or urther identiication and analysis. Acknowledgements This work was unded by the U.S. Army under STTR contract number W911SR-06-C We thank Dr. James Jensen or technical discussions, I. Konen or assistance assembling the laser scattering apparatus, J. Towle or assistance measuring the transducer S11 curve, A. Ferrante, D. Vu, and L. Harrison or advice and assistance in spore preparation, and B. Szczur and A. Trask or testing assistance at Western New England College. Reerences 1. B. Lipkens, J. Dionne, A. Trask, B. Szczur, A. Stevens, and E. Rietman, Separation o micron-sized particles in macro-scale cavities by ultrasonic standing waves, International Congress on Ultrasonics, Santiago de Chile, January 009, in: Physics Procedia, 00, , P. L. Marston and D. B. Thiessen, Manipulation o luid objects with acoustic radiation pressure, Ann. N. Y. Acad. Sci. 107, (004). 3. L. V. King, On the acoustic radiation pressure on spheres, Proc. R. Soc. London, Ser. A 147, 1-40 (1934). 4. K. Yosioka and Y. Kawasima, Acoustic radiation pressure on a compressible sphere, Acustica 5, (1955). 5. L. P. Gor kov, On the orces acting on a small particle in an acoustical ield in an ideal luid, Sov. Phys. Dokl. 6, (196). 6. T. L. Tolt and D. L. Feke, Separation devices based on orced coincidence response o luidilled pipes, J. Acoust. Soc. Am. 91(6), (199). 7. T. L. Tolt and D. L. Feke, Separation o dispersed phases rom liquids in acoustically driven chambers, Chem. Eng. Sci. 48(0), p. 57 (1993). 8. M. Hill and R. J. K. Wood, Modelling in the design o a low-through ultrasonic separator, Ultrasonics 38, (000). 10

11 9. M. Hill, Y. Shen, and J. J. Hawkes, Modelling o layered resonators or ultrasonic separation, Ultrasonics 40, (00). 10. R. J. Townsend, M. Hill, N. R. Harris, and N. M. White, Modelling o particle paths passing through an ultrasonic standing wave, Ultrasonics 4, (004). 11. B. Handl, M. Groschl, F. Trampler, E. Benes, S. M. Woodside, and J. M. Piret, Particle trajectories in a driting resonance ield separation device, Proc. o the 16th Int. Congress on Acoustics and 135th Meeting o the Acoust. Soc. Am., ISBN , vol. III, pp (1998). 1. B. Lipkens, M. Costolo, and E. Rietman, The eect o requency sweeping and luid low on particle trajectories in ultrasonic standing waves, IEEE Sensors Journal 8(6), pp (008). 13. Y. Huang, X.-B. Wang, F. F. Becker, and P. R. C. Gascoyne, "Introducing Dielectrophoresis as a New Force Field or Field-Flow Fractionation," Biophys. J. 73(), pp (1997). 11