Ultrasonic cell disruption

The treatment of microbial cells in suspension with inaudible ultrasound (greater than about 18 kHz) results in their inactivation and disruption. Ultrasonication utilises the rapid sinusoidal movement of a probe within the liquid. It is characterised by high frequency (18 kHz - 1 MHz), small displacements (less than about 50 mm), moderate velocities (a few m s⁻¹), steep transverse velocity gradients (up to 4,000 s⁻¹) and very high acceleration (up to about 80,000 g). Ultrasonication produces cavitation phenomena when acoustic power inputs are sufficiently high to allow the multiple production of microbubbles at nucleation sites in the fluid. The bubbles grow during the rarefying phase of the sound wave, then are collapsed during the compression phase. On collapse, a violent shock wave passes through the medium. The whole process of gas bubble nucleation, growth and collapse due to the action of intense sound waves is called cavitation. The collapse of the bubbles converts sonic energy into mechanical energy in the form of shock waves equivalent to several thousand atmospheres (300 MPa) pressure. This energy imparts motions to parts of cells which disintegrate when their kinetic energy content exceeds the wall strength. An additional factor which increases cell breakage is the microstreaming (very high velocity gradients causing shear stress) which occur near radially vibrating bubbles of gas caused by the ultrasound.

Much of the energy absorbed by cell suspensions is converted to heat so effective cooling is essential. The amount of protein released by sonication has been shown to follow Equation 2.9. The constant (k) is independent of cell concentrations up to high levels and approximately proportional to the input acoustic power above the threshold power necessary for cavitation. Disintegration is independent of the sonication frequency except insofar as the cavitation threshold frequency depends on the frequency.

Equipment for the large-scale continuous use of ultrasonics has been available for many years and is widely used by the chemical industry but has not yet found extensive use in enzyme production. Reasons for this may be the conformational lability of some (perhaps most) enzymes to sonication and the damage that they may realise though oxidation by the free radicals, singlet oxygen and hydrogen peroxide that may be concomitantly produced. Use of radical scavengers (e.g., N₂O) have been shown to reduce this inactivation. As with most cell breakage methods, very fine cell debris particles may be produced which can hinder further processing. Sonication remains, however, a popular, useful and simple small-scale method for cell disruption.