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1 Ultrasonically-Assisted Fermentation for Bioethanol Production by Kathrin Hielscher, Hielscher Ultrasonics, Germany 1. Fermentation Fermentation can be an aerobic (= oxidative fermentation) or anaerobic process, which is used for biotechnological applications to convert organic material by bacterial, fungal or other biological cell cultures or by enzymes. By fermentation, energy is extracted from the oxidation of organic compounds, e.g. carbohydrates. Sugar is the most common substrate of fermentation, resulting after fermentation in products such as lactic acid, lactose, ethanol and hydrogen. Ethanol especially for use as fuel, but also for alcoholic beverages - is produced by fermentation. When certain yeast strains, such as Saccharomyces cerevisiae metabolize sugar, the yeast cells convert the starting material into ethanol and carbon dioxide. The chemical equations below summarize the conversion: C 6 H 12 O 6 2 CH 3 CH 2 OH + 2 CO 2 C 12 H 22 O 11 + H 2 O 4 CH 3 CH 2 OH + 4 CO 2 If the starting material is starch, e.g. from corn, firstly the starch must be converted into sugar. For bioethanol used as fuel, hydrolysis for the starch conversion is required. Typically, the hydrolysis is speeded up by acidic or enzymatic treatment or by combination of both. Normally, fermentation is carried out at around C. Overview over various fermentation processes: Biogas/ ethanol: - improvement of biogas/ bioethanol production from biomass - biogas production from sewage sludge Food: Drugs: - production & preservation - dairy (lactic acid fermentation), e.g. yogurt, buttermilk, kefir - lactic fermented vegetables, e.g. kimchi, miso, natto, tsukemono, sauerkraut - development of aromatics, e.g. soy sauce - decomposition of tanning agents, e.g. tea, cocoa, coffee, tobacco - alcoholic beverages, e.g. beer, wine, whiskey - production of medical compounds, e.g. insulin, hyaluronic acid Various research papers and tests in bench-top and pilot size have shown that ultrasound improves the fermentation process by making more biomass available for the enzymatic fermentation. In the following section, the effects of ultrasound in a liquid will be elaborated. 2. Effects of Ultrasonic Liquid Processing By high-power/ low-frequency ultrasound high amplitudes can be generated. Thereby, high-power/ low-frequency ultrasound can be used for the processing of liquids such as mixing, emulsifying, dispersing and deagglomeration, or milling. When sonicating liquids at high intensities, the sound waves that propagate into the

2 liquid media result in alternating high-pressure (compression) and low-pressure (rarefaction) cycles, with rates depending on the frequency. During the lowpressure cycle, high-intensity ultrasonic waves create small vacuum bubbles or voids in the liquid. When the bubbles attain a volume at which they can no longer absorb energy, they collapse violently during a high pressure cycle. This phenomenon is termed cavitation. Cavitation, that is "the formation, growth, and implosive collapse of bubbles in a liquid. Cavitational collapse produces intense local heating (~5000 K), high pressures (~1000 atm), and enormous heating and cooling rates (>109 K/sec)" and liquid jet streams (~400 km/h). (Suslick 1998) There are different means to create cavitation, such as by high-pressure nozzles, rotor-stator mixers, or ultrasonic processors. In all those systems the input energy is transformed into friction, turbulences, waves and cavitation. The fraction of the input energy that is transformed into cavitation depends on several factors describing the movement of the cavitation generating equipment in the liquid. The intensity of acceleration is one of the most important factors influencing the efficient transformation of energy into cavitation. Higher acceleration creates higher pressure differences. This in turn increases the probability of the creation of vacuum bubbles instead of the creation of waves propagating through the liquid. Thus, the higher the acceleration the higher is the fraction of the energy that is transformed into cavitation. In case of an ultrasonic transducer, the amplitude of oscillation describes the intensity of acceleration. Higher amplitudes result in a more effective creation of cavitation. In addition to the intensity, the liquid should be accelerated in a way to create minimal losses in terms of turbulences, friction and wave generation. For this, the optimal way is a unilateral direction of movement. Changing the intensity and parameters of the sonication process, ultrasound can be very hard or very soft. This makes ultrasound a very versatile tool for various applications. Soft applications, applying mild sonication under mild conditions, include degassing, emulsifying, and enzyme activation. Hard applications with high intensity/ high power ultrasound (mostly under elevated pressure) are wet-milling, deagglomeration & particle size reduction, and dispersing. For many applications such as extraction, disintegration or sonochemistry, the ultrasonic intensity requested depends on the specific material to be sonicated. By the variety of parameters, which can be adapted to the individual process, ultrasound allows finding the sweet spot for each individual process. Besides an outstanding power conversion, ultrasonication offers the great advantage of full control over the most important parameters: Amplitude, Pressure, Temperature, Viscosity, and Concentration. This offers the possibility to adjust all these parameters with the objective to find the ideal processing parameters for each specific material. This results in higher effectiveness as well as in optimized efficiency. 3. Ultrasound to Improve Fermentation Processes, explained exemplarily with the bioethanol production Bioethanol is a product of the decomposition of exclusively biomass or

3 biodegradable matter of waste by anaerobic or aerobic bacteria. The produced ethanol is mainly used as biofuel. This makes bioethanol a renewable and environmentally friendly alternative for fossil fuels, such as natural gas. To produce ethanol from biomass, sugar, starch, and lignocellulosic material can be used as feedstock. For industrial production size, sugar and starch are currently predominant as they are economically favorable. How ultrasound improves a customer-individual process with specific feedstock under given conditions can be tried out very simple by feasibility tests. At first step, the sonication of a small amount of the raw material slurry with an ultrasonic laboratory device will show, if ultrasound does affect the feedstock Feasibility Testing In the first testing phase, it is suitable to introduce a relatively high amount of ultrasonic energy into a small volume of liquid as thereby the chance increases to see if any results can be obtained. A small sample volume also shortens the time using a lab device and cuts down the costs for the first tests. The ultrasound waves are transmitted by the sonotrode s surface into the liquid. Beneth the sonotrode surface, the ultrasound intensity is most intense. Thereby, short distances between sonotrode and sonicated material are preferred. When a small liquid volume is exposed, the distance from the sonotrode can be kept short. The table below shows typical energy/volume levels for sonication processes after optimization. Since the first trials will not be run at optimum configuration, sonication intensity and time by 10 to 50 times of the typical value will show if there is any effect to the sonicated material or not. Process Energy/ Volume Sample Volume Power Time Simple <100Ws/mL 10mL 50W < 20 sec Medium 100Ws/mL 10mL 50W 20 to 100 sec Hard > 500Ws/mL 10mL 50W >100 sec Table 1 - Typical sonication values after process optimization The actual power input of the test runs can be recorded via PC-interface or by powermeter. In combination with the recorded data of amplitude setting and temperature, the results of each trial can be evaluated and a bottom line for the energy/volume can be established. If during the tests an optimal configuration has been chosen, this configuration performance could be verified during an optimization step and could be finally scaled up to commercial level. To facilitate the optimization, it is highly recommended to examine the limits of sonication, e.g. temperature, amplitude or energy/volume for specific formulations, too. As ultrasound could generate negative effects to cells, chemicals or particles, the critical levels for each parameter need to be examined in order to limit the following optimization to the parameter range where the negative

4 Picture 1 - ultrasonic lab device (100 watts) for feasibility tests effects are not observed. For the feasibility study small lab or bench-top units are recommended to limit the expenses for equipment and samples in such trials. Generally 100 to 1,000 Watts units serve the purposes of the feasibility study very well. (cf. Hielscher 2005) 3.2. Optimization The results achieved during the feasibility studies may show a quite high energy consumption regarding the small volume treated. But the purpose of the feasibility test is primarily to show the effects of ultrasound to the material. If in feasibility testing positive effects occurred, further efforts must be made to optimize the energy/volume ratio. This means to explore the ideal configuration of ultrasound parameters to achieve the highest yield using the less energy possible to make the process economically most reasonable and efficient. Chart 1 Flow chart for the optimization of an Ultrasonic Process

5 To find the optimal parameter configuration - regarding minimal energy input to obtain the intended benefits the correlation between the most important parameters amplitude, pressure, temperature and liquid composition have to be investigated. In this second step the change from batch sonication to a continuous sonication setup with flow cell reactor is recommended as the important parameter of pressure cannot be influenced for batch sonication. During sonication in a batch, the pressure is limited to ambient pressure. If the sonication process passes a pressurizable flow cell chamber, the pressure can be elevated (or reduced) which in general affects the ultrasonic cavitation drastically. By using a flow cell, the correlation between pressure and process efficiency can be determined. Ultrasonic processors between 500 watts and 2000 watts of power are most suitable to optimize a process Scale-Up to Commercial Production If the optimal configuration has been found, the further scale-up is simple as ultrasonic processes are fully reproducible on a linear scale. This means, when ultrasound is applied to an identical liquid formulation under identical processing parameter configuration, the same energy per volume is required to obtain an identical result independent of the scale of processing. (Hielscher 2005). That makes it possible to implement the optimal parameter configuration of ultrasound to the full scale production size. Virtually, the volume which can be processed ultrasonically is unlimited. Commercial ultrasonic systems with up to 16,000 watts per unit are Picture 2 Industrial ultrasonic processor with 16,000 watts power

6 available and can be installed in clusters. Such clusters of ultrasonic processors can be installed parallel or in series. By the cluster-wise installation of high power ultrasonic processors, the total power is almost unlimited so that high volume streams can be processed without problem. Also if an adaption of the ultrasonic system is required, e.g. to adjust the parameters to a modified liquid formulation, this can be mostly done by changing sonotrode, booster or flow cell. The linear reproducibility and the adaptability of ultrasound makes this innovative technology to an efficient and cost-effective. 4. Parameters of Ultrasonic Processing Ultrasonic liquid processing is described by a number of parameters. Most important are amplitude, pressure, temperature, viscosity, and concentration. The process result, such as particle size, for a given parameter configuration is a function of the energy per processed volume. The function changes with alterations in individual parameters. Furthermore, the actual power output per surface area of the sonotrode of an ultrasonic unit depends on the parameters. The power output per surface area of the sonotrode is the surface intensity (I). The surface intensity depends on the amplitude (A), pressure (p), the reactor volume (VR), the temperature (T), viscosity (η) and others. I[W / mm²]= ( A[µm], p[bar], VR [ml], T[ C], η[cp],...) The impact of the generated cavitation depends on the surface intensity. In the same way, the process result correlates. The total power output of an ultrasonic unit is the product of surface intensity (I) and surface area (S): 4.1. Amplitude P [W] I [W / mm²]* S[mm²] The amplitude of oscillation describes the way (e.g. 50 µm) the sonotrode surface travels in a given time (e.g. 1/20,000s at 20kHz). The larger the amplitude, the higher is the rate at which the pressure lowers and increases at each stroke. In addition to that, the volume displacement of each stroke increases resulting in a larger cavitation volume (bubble size and/or number). When applied to dispersions, higher amplitudes show a higher destructiveness to solid particles. Table 1 shows general values for some ultrasonic processes. Process Amplitude Dispersing/Deagglomeration Emulsifying Primary Particle Reduction 10 to 30 micron 20 to 60 micron 40 to 120 micron Table 2 - General Recommendations for Amplitudes

7 4.2. Pressure The boiling point of a liquid depends on the pressure. The higher the pressure the higher is the boiling point, and reverse. Elevated pressure allows cavitation at temperatures close to or above the boiling point. It also increases the intensity of the implosion, which is related to the difference between the static pressure and the vapor pressure inside the bubble (cf. Vercet et al. 1999). Since the ultrasonic power and intensity changes quickly with changes in pressure, a constant-pressure pump is preferable. When supplying liquid to a flow-cell the pump should be capable of handling the specific liquid flow at suitable pressures. Diaphragm or membrane pumps; flexible-tube, hose or squeeze pumps; peristaltic pumps; or piston or plunger pump will create alternating pressure fluctuations. Centrifugal pumps, gear pumps, spiral pumps, and progressive cavity pumps that supply the liquid to be sonicated at a continuously stable pressure are preferred. (Hielscher 2005) 4.3. Temperature By sonicating a liquid, power is transmitted into the medium. As ultrasonically generated oscillation causes turbulences and friction, the sonicated liquid in accordance with the law of thermodynamics - will heat up. Elevated temperatures of the processed medium can be destructive to the material and decrease the effectiveness of ultrasonic cavitation. Innovative ultrasonic flow cells are equipped with a cooling jacket (see picture). By that, the exact control over material s temperature during ultrasonic processing is given. For the beaker sonication of smaller volumes an ice bath for heat dissipation is recommended. Picture 3 Ultrasonic transducer UIP1000hd (1000 watts) with flow cell equipped with cooling jacket typical equipment for optimization steps or small scale production

8 4.4. Viscosity and Concentration Ultrasonic milling and dispersing are liquid processes. The particles have to be in a suspension, e.g. in water, oil, solvents or resins. By the use of ultrasonic flowthrough systems, it becomes possible to sonicate very viscous, pasty material. High-power ultrasonic processor can be run at fairly high solids concentrations. A high concentration provides the effectiveness of ultrasonic processing, as ultrasonic milling effect is caused by inter-particle collision. Investigations have shown that the breakage rate of silica is independent of the solid concentration up to 50% by weight. The processing of master batches with highly concentrated material s ratio is a common production procedure using ultrasonication Power and Intensity vs. Energy Surface intensity and total power do only describe the intensity of processing. The sonicated sample volume and the time of exposure at certain intensity have to be considered to describe a sonication process in order to make it scalable and reproducible. For a given parameter configuration the process result, e.g. particle size or chemical conversion, will depend on the energy per volume (E/V). Result = f (E /V ) Where the energy (E) is the product of the power output (P) and the time of exposure (t). E[Ws] = P[W]*t[s] Changes in the parameter configuration will change the result function. This in turn will vary the amount of energy (E) required for a given sample value (V) to obtain a specific result value. For this reason it is not enough to deploy a certain power of ultrasound to a process to get a result. A more sophisticated approach is required to identify the power required and the parameter configuration at which the power should be put into the process material. (Hielscher 2005) 5. Ultrasonically Assisted Production of Bioethanol It is already know that ultrasound improves the bioethanol production. It is recommendable to thicken the liquid with biomass to a highly viscous slurry that is still pumpable. Ultrasonic reactors can handle fairly high solid concentrations so that the sonication process can be run most efficient. The more material is contained in the slurry, the less carrier liquid, which will not profit from the sonication process, will be treated. As the input of energy into a liquid causes a heating of the liquid by law of thermodynamics, this means that the ultrasonic energy is applied to the target material, as far as possible. By such an efficient process design, a wasteful heating of the excess carrier liquid is avoided. Ultrasound assists the extraction of the intracellular material and makes it thereby available for the enzymatic fermentation. Mild ultrasound treatment can enhance enzymatic activity, but for biomass extraction more intense ultrasound will be required. Hence, the enzymes should be added to the biomass slurry after the sonication as intense ultrasound inactivates enzymes, which is a not desired effect.

9 Current results achieved by scientific research: The studies of Yoswathana et al. (2010) concerning with the bioethanol production from rice straw have shown that the combination of acid pre-treatment and ultrasonic before enzymatic treatment lead to an increased sugar yield of up to 44% (on rice straw basis). This shows the effectiveness of the combination of physical and chemical pretreatment before the enzymatic hydrolysis of lignocelluloses material to sugar. Chart 2 Ultrasonic enhancement of ethanol yield during fermentation (Yoswathana et al. 2010) The chart above illustrates the positive effects of ultrasonic irradiation during the bioethanol production from rice straw graphically. (Charcoal has been used to detoxify the pretreated samples from acid/ enzyme pretreatment and ultrasonic pretreatment.) In another recent study, the influence of ultrasonication on the extracellular and the intracellular levels of β-galactosidase enzyme has been examined. Sulaiman et al. (2011) could improve the productivity of bioethanol production substantially, using ultrasound at a controlled temperature stimulating the yeast growth of Kluyveromyces marxianus (ATCC 46537). The authors of the paper resumes that intermittent sonication with power ultrasound (20 khz) at duty cycles of 20% stimulated biomass production, lactose metabolism and ethanol production in K. marxianus at a relatively high sonication intensity of 11.8Wcm 2. Under the best conditions, sonication enhanced the final ethanol concentration by nearly 3.5-fold relative to control. This corresponded to a 3.5-fold enhancement in ethanol productivity, but required 952W of additional power input per cubic meter of broth through sonication. This additional requirement for energy was certainly within acceptable operational norms for bioreactors and, for high value products, could be easily compensated by the increased productivity.

10 6. Conclusion: Benefits from Ultrasonically-Assisted Fermentation Ultrasonic treatment has been shown as an efficient and innovative technique to enhance the bioethanol yield. Primarily, ultrasound is used to extract intracellular material from biomass, such as corn, soybeans, straw, lingo-cellulosic material or vegetable waste materials. Increase in bioethanol yield Disintegration/ Cell destruction and release of intra-cellular material Improved anaerobic decomposition Activation of enzymes by mild sonication Improvement of process efficiency by high concentration slurries The simple testing, reproducible scale-up and easy installation (also in already existing production streams) makes ultrasonics a profitable and efficient technology. Reliable industrial ultrasonic processors for commercial processing are available and make it possible to sonicate virtually unlimited liquid volumes. Picture 4 Setup with 1000W ultrasonic processor, flow cell, tank and pump

11 References: Hielscher, T. (2005): Ultrasonic Production of Nano-Size Emulsions and Dispersions. in: Proceedings of European Nanosystems Conference ENS'05. Jomdecha, C.; Prateepasen, A. (2006): The Research of Low-Ultrasonic Energy Affects to Yeast Growth in Fermentation Process. At: 12 th Asia-Pacific Conference on NDT, , Auckland, New Zealand. Kuldiloke, J. (2002): Effect of Ultrasound, Temperature and Pressure Treatments on Enzyme Activity an Quality Indicators of Fruit and Vegetable Juices; Ph.D. Thesis at Technische Universität. Berlin, Mokkila, M., Mustranta, A., Buchert, J., Poutanen, K. (2004): Combining power ultrasound with enzymes in berry juice processing. At: 2nd Int. Conf. Biocatalysis of Food and Drinks, , Stuttgart, Germany. Müller, M. R. A.; Ehrmann, M. A.; Vogel, R. F. (2000): Multiplex PCR for the Detection of Lactobacillus pontis and Two Related Species in a Sourdough Fermentation. Applied & Environmental Microbiology. 66/ pp Nikolic, S.; Mojovic, L.; Rakin, M.; Pejin, D.; Pejin, J. (2010): Ultrasound-assisted production of bioethanol by simoultaneous saccharification and fermentation of corn meal. In: Food Chemistry 122/2010. pp Sulaiman, A. Z.; Ajit, A.; Yunus, R. M.; Cisti, Y. (2011): Ultrasound-assisted fermentation enhances bioethanol productivity. Biochemical Engineering Journal 54/2011. pp Suslick, K. S. (1998): Kirk-Othmer Encyclopedia of Chemical Technology. 4 th ed. Wiley & Sons: New York, pp Yoswathana, N.; Phuriphipat, P.; Treyawutthiawat, P.; Eshtiaghi, M. N. (2010): Bioethanol Production from Rice Straw. In: Energy Research Journal 1/ pp