Ultrasonic intensification as a tool for enhanced microbial biofuel yields

Ultrasound causes both physical and chemical changes through the process of cavitation. Chemically, highly reactive radicals can be generated from the dissociation of the entrapped vapour molecules in a cavitation bubble [27]. Physically, cavitation can cause intense convection in a bulk medium leading to microturbulence (an intense oscillating motion of liquid with low to moderate velocities) and shock waves (high pressurised waves emitted by the bubble, with amplitudes as high as 30–50 bar). During lipid extraction from biomass, the physical effects of ultrasonication can significantly enhance the lipid yield. Microturbulence can lead to a more efficient mixing of the biomass and solvent (without induction of shear stress), while shock waves can cause rupture of the cell wall [28]. Ultrasound can also generate intense local turbulence in the medium, pushing the extracted lipids away from the surface of the microbial cells, and thus, maintaining a constant concentration gradient for continuous diffusion of lipids from the cells [27].

The physical effects of ultrasonication can also enhance the transesterification process during biodiesel production [29–32]. Ultrasonication generates an enormous interfacial area between the oil and alcohol due to microturbulence leading to the formation of fine emulsions [33]. Kalva et al. [29] investigated the mechanism of the enhancement of transesterification by discriminating the physical and chemical effects of ultrasound. This was analysed by transesterifying soybean oil using methanol and sodium hydroxide as the base catalyst with ultrasound frequency set at 20 kHz and power output of 100 W. It was reported that the enhancement of transesterification was due to the physical effect rather than a chemical effect of ultrasound, i.e., production of radical species and acceleration of the reaction by these species [34–36]. In addition it was found that the transesterification yield increased when the alcohol to oil molar ratio was increased. This was due to the effect of low intensity microturbulence generated by the cavitation bubbles in the oil, which restricted the dispersion of oil in methanol at high alcohol to oil molar ratios [29].

Ultrasound may also enhance enzymatic reactions within the cell. Khanna et al. [30] reported that the nature of convection generated by ultrasound at raised static or ambient pressure in the medium provided oscillatory motion of liquid, accompanied by medium amplitude movement by the acoustic or shock waves emitted by the bubbles. This was concluded by investigating the mechanism of enhancement of glycerol conversion into 1,3-propanediol and ethanol using Clostridium pasteurianum MTCC 116 with the aid of ultrasonication. Mild shock waves of ultrasonication were shown to cause rapid movement of microbial cells in the fermentation broth. Inter-collisions and/or collisions with the walls of the test tube lead to the acceleration of enzymatic reactions inside the cells [30]. In addition, it was reported that the high velocity micro-streaming caused desorption of the CO₂ produced in the metabolic pathway and thus favoured a faster diffusion of glycerol into the microbial cells. As a result, the overall enzymatic reaction system is expected to be substrate saturated with enzyme being the limiting reactant. During ethanol formation in this system the Michaelis constant (K_m) was found to decrease with ultrasonication indicating a rise in the formation of enzyme–substrate complex. Hence, ethanol yields were found to increase from 0.024 to 0.04 mol mol⁻¹ (i.e., 83 % increase) and a decrease in substrate inhibition (K_I) contributed to the enhancement of 1,3-propanediol yields from 0.017 to 0.021 mol mol⁻¹ (30.89 % increase) [30, 36].

Ultrasonication has also been shown to enhance ethanol fermentation processes by increasing transport through the cell membrane and increasing enzymatic hydrolysis with strong micro-convection [37–40]. Recent studies have found that it could enhance the utilisation of substrate for cell growth, reduce the inhibition by substrate and decrease the specific death rate of cells leading to an increase of ethanol yield from 0.149 to 0.166 g g⁻¹ of raw biomass [41].

Traditionally, solvent based processes are utilised for extraction of many bioactive compounds [42, 43]. However, standard extraction mechanisms such as cell maceration and soxhlet extraction have limitations such as high solvent consumption, large operating cost and extended operation times which frequently result in lower yields [44]. The application of ultrasonic intensification can allow higher yields to be generated in a shorter time, with lower energy input [45] and without adding additional reagents to the extraction. In addition, the effects of increasing temperature on the extraction components can be avoided. Such modified extraction techniques have been developed recently for the extraction of macromolecules such as polysaccharides, proteins, terpenoids, flavonoids, carotenoids and phenolic compounds [46–49]. The ultrasonic intensification process can be effectively used to improve the extraction rate by increasing the mass transfer due to the formation of microcavities leading to higher growth and product yields. Ying et al. [49] reported that ultrasonic-based extraction is associated with two main physical phenomena, acoustic cavitation and diffusion through the cell wall. The conditions associated with cavitation, an increase in temperature and pressures up to 100 MPa, produce very high shear energy waves and turbulence in the cavitation zone. The combination of these factors (pressure, heat and turbulence) is used to accelerate mass transfer in the extraction process. Ultrasonic intensification also exerts a mechanical effect which leads to enhanced diffusion of solvents into the cell wall. In pure liquids, the microbubbles retain their spherical shape during the collapse, as their surroundings are uniform [50]. However, when the microbubbles collapse near a solid surface it occurs asymmetrically and produces shock waves toward the cell wall. These waves have a strong impact on the cell surface; therefore, enhance the solvent penetration into the cell. Another effect caused by the ultrasound wave is that it can facilitate the swelling and hydration of biomass and so cause an enlargement of pores in the cell wall which can improve diffusion processes and therefore enhance mass transfer which can enhance extraction yield [50]. Hence ultrasonic intensification can provide high extraction efficiency in a short time with less solvent consumption over other extraction techniques [51]. As an example, Rocco et al. [52] reported a 73 % increase in recovery of polychlorinated biphenyls from biomass generated through treatment of household wastewater with 30 min of ultrasonic intensification during the extraction process.

Algal cell walls are typically tri-layered rigid structures with high tensile strength [66], hence the release of intra lipids can be blocked. Homogenization can affect the outer cell walls with shearing force but not the interior of the cell. Extraction of lipids from cells may occur by either diffusion of lipids across the cell wall, if the algal biomass is suspended in the solvent with higher selectivity and solubility (or large partition coefficient) for lipids or disruption of the cell wall with release of cell contents in the solvent. The diffusive mechanism is less efficient due to slow diffusion of lipid across the cell wall while disruptive mechanism results in faster extraction with high yield as it causes direct release of lipid due to the rupture of cell wall [27]. Sonication can interfere with the cell interior via shock waves produced by imploding cavitation bubbles [67]. Thus, ultrasonic intensification can be an advantageous addition for lipid extraction processes involving algae. Park et al. [68] investigated the effect of homogenisation and ultrasonication in combination on lipid extraction from Chlorella vulgaris. The initial fatty acid content of C. vulgaris was 360 mg g⁻¹ cell. Lipid recovery was found to increase when both techniques were combined compared with the use of either alone. The results of the combined processes showed 100.5, 123.9, and 152.0 mg lipid g⁻¹ cell recovered for the 20, 40, and 60 min reaction times used, respectively. The yields were 5.3-fold, 6.6-fold, and 8.1-fold higher, respectively, than that of the control (single treatment by sonication or single treatment via homogenization). In this system [68], microalgal suspensions were allowed to circulate continuously between the homogenizer and ultrasonicator and it was concluded that cell walls damaged slightly by homogenization would be effectively disrupted by the subsequent ultrasonication-induced cavitation bubbles resulting in a superior extraction process [68]. When the cell concentration was increased to 40 g.L⁻¹, the lipid recovery yield for 1 h of sonication-assisted homogenization was increased to a very high 281.3 mg lipid g⁻¹ cell using the chloroform–methanol solvent. This system’s treatment of high cell concentrations makes it possible to enhance lipid recovery capacity based on unit time [68].

The length of ultrasonic treatment has a significant effect on product yields. In a study using c with both continuous and intermittent ultrasonication during fermentation it was found that the ethanol concentration was increased up to 30 g L⁻¹ with intermittent ultrasonic intensification, whereas with continuous ultrasound intensification, no increase was observed [89]. Similarly intermittent ultrasonic intensification (in fermentation broth was found to enhance the production of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672 by 1.82 fold compared to non-sonicated broth [90]. The intensity of ultrasonication can also affect yields obtained. In a study with molasses fermentation by Saccharomyces cerevisiae M30, the effect of ultrasonic intensification on ethanol production was examined. The ultrasonic frequencies were varied as 20, 25 and 30 kHz with a maximum specific ethanol production rate of 1.55 g g⁻¹ h⁻¹ being achieved at 25 kHz [91].

While low to high intensity pulsing can enhance productivities, increased microbial exposure to ultrasonic power causes cells to become fragile and reduces microbial viability due to mechanical stress on the microbial cell [92]. The mechanical shear caused by the ultrasonic waves can disrupt the cell wall at higher duty cycles and ultimately has a lethal effect [93]. Thus, numerous studies have shown that intermittent ultrasonic intensification is more advantageous, increasing product yield while keeping microbial viability high. In addition, the life span of the ultrasonic transducers is increased and temperature effects due to the transducer usage are minimised [94]. Thus, examination of the effects and the rates of ultrasound intensification utilised appear to be important for individual process strategies.

The effect of intensification on a bioprocess depends on the choice of ultrasonic parameters utilised. These include the ultrasonic mode, which can be either continuous or pulse, the frequency, the intensity, the processing temperature, the nature of the solvent utilised, the aeration and the design of reactor which determines the level and distribution of energy within the system [19]. The main parameters in ultrasonic reactor design are the type of reactor, its geometry; the design of the transducer set up and the volumetric scale of the feed stock [19]. In addition, the location of the ultrasonic probe in the reacting vessel has been found to influence the distribution of cavitational behaviour within the system [95]. The probe location is usually dependent on the reactor size and the working volume of the liquid reactants [96]. The vertical location of probes in the reactor has been shown to result in poor distribution of cavitation microbubbles with only 10 % distribution reported in a 2.5 L reactor [97]. In contrast, the horizontal location of a probe in a reactor with 82 % immersion length was shown to result in uniform cavitational behaviour over the entire reactor [98]. Another important factor is the shape and diameter of the probe tip. Ultrasonic probes with large diameter tips provide low ultrasonic energy density owing to a wide emitter surface [99]. Usage of larger tips allows higher reaction rates to be achieved. In a study utilising B. amyloliquefaciens producing α-amylase, rapid deactivation of α-amylase was observed to occur, due to the reaction between hydroxyl radicals and the enzyme but this was shown not to take place inside the bubbles. High density cavitational bubbles from a 1 mm sonotrode tip resulted in serious acoustic attenuation and high energy in each cavitational bubble being released when the bubbles collapsed, even though the delivered energy density was low [99]. The radiating face of the ultrasonic probe was shown to greatly affect the efficiency of enzyme inactivation in this case. It is of interest that using large sonotrodes may reduce the intensity of bubble collapse and hence are thus not recommended for intensification [99].

The amount of dissolved gas present in media undergoing sonication is a parameter which also influences the acoustic cavitation. Lowering the quantity of dissolved gas reduces the number of nuclei and thereby increases the cavitation threshold and results in a change in the type of cavitation, producing more vaporous cavities [95]. Such microbubbles collapse less violently and hence the rate of sonochemical reaction will be decreased. The selection of the correct ultrasonic frequency for the applications envisaged is also important. Higher frequency or combinations of lower frequencies is recommended for applications controlled by chemical effects whereas lower frequency is recommended for applications with physical effects [73]. The use of multiple transducers can lead to the formation of uniform cavitational behaviour and also minimise the required energy consumption in applied systems. This occurs as it would not be possible to dissipate the same power through a single probe in a large scale bioprocess operation [95]. In addition, the selection of liquids with low vapour pressure, low viscosity and high surface tension was found to enhance cavitational behaviour [95]. Therefore, the rational design of scaled-up ultrasonic reactors requires the quantitative prediction of acoustic streaming, power dissipation, mass transfer and cavitational activity in the reactor by theoretical simulations [19]. An effective scale-up of the ultrasound reactor can be achieved if the energy dissipation pattern in the reactor is known. One of the major disadvantages of ultrasound baths is the directional sensitivity of the ultrasound waves in the bath, which creates a non-homogeneous energy dissipation pattern. This can be overcome by using tubular reactors with ultrasound intensity concentrated at the core of the reactor, but any industrial scale operation has not yet been reported [100]. In an ultrasonic reactor, the input electrical energy undergoes many transformations while being converted into cavitation energy, which is dissipated in the medium to carry out the physical and chemical change. The cavitation intensity varies with the gas content of the medium [101]. The principles described above have influenced the design of several reactor types for ultrasonication-based processes. Coincidence of the compression half cycles of the two acoustic waves while using dual frequency sonochemical reactor has a favourable effect on the production of radicals, as it intensifies the transient collapse of the cavitation bubbles [102]. The dual frequency ultrasonic processor will not be able to contribute sonochemical effects, which is usually attributed to radical formation during transient cavitation. However, the moderate cavitation intensities in dual frequency reactors would favour the physical processes such as extraction and leaching [103]. Cintas et al. [104] used a reverberative flow reactor, in which two irradiation arrays made up of multiple ultrasonic probes were mounted on the upper and lower portion of a metal cuboid reactor, while liquid reactant was allowed to flow through the cuboid space [104, 105].The reflection of ultrasonic waves at the inner wall of the reactor chamber and its reverberation in the chamber caused the acoustic intensity to be multiplied, thereby giving a more non-uniform cavitation field than single probe systems. Similar technology has also been adopted in sonochemical polygonal reactors [106].

A liquid whistle ultrasonic reactor (frequency 5–30 kHz and intensity, 1.5–2.5 W cm⁻²) which causes hydrodynamic cavitation has been widely used for industrial wastewater treatment [107], oil emulsification [108], liquid–solid mixture homogenization [19] and fat hydrolysis [109]. This type of reactor can be operated at low cost and is suitable for continuous flow reactions, with scale-up also possible [19, 104]. With effective reactor design as describe, it is possible to maximise the beneficial effects of ultrasonic intensification in a specific process while minimising cost.