Use of Ultrasound in Cell and Effects of Concentration, Power and Temperature in the Breaking Algae - Research Proposal Example

The use of ultrasound in cell and the effects of concentration, power and temperature in the breaking algae Aims and objectives Aim The aim of this study is ton establish the use of ultrasound in cell and the effects of concentration, power and temperature in the breaking algae. Specific objectives To determine the effect of concentration on the breaking algae To determine the effect of power on the breaking algae To determine the effect of temperature on the breaking algae Background information Several methods of cellular disruptions have been investigated in algae using various species including Euglena sp. The methods include autoclaving, freeze fracture, surfactant (TWEEN 80), cell grinding, microwaving and use of solvents such as octanol, butanol and isopropanol (Joyce, Wu, and Mason 863). Freeze thaw fracture method entails creation of ice crystals with cellular structure during freezing and rupturing of cells during thawing process which causes cellular expansion which catalyses rupture. Surfactant (TWEEN 80) is a surface active agent which is the main ingredient in soaps and works by increasing the solubility of the cellular membrane of the algae. Cell grinding is the preferred method for cellular disturbance in plant tissues (Manson 149). Microwaving is a unique method which provides the effect of heat on cellular disruption. However the method is limited due to it being a highly energy intensive hence is not economically viable on a large scale. Autoclave method entails exposing cells to high heat and high pressure for cellular disruption. This method also energy intensive disruption technique and hence cannot be economical on large scale (Clark and Macquarrie, 66). Solvents such as octanol, butanol and isopropanol work by disintegrating cellular membrane. The solvents are preferred in cell disruption of algae because it allows visible analysis of pigment extraction and microscopic observation of cellular disturbances (Bellinger and Sigee 104). Octanol is the most promising and most environmentally friendly of the three solvents mentioned above. Thus, in this study algae of the Euglena species will be suspended in octanol and exposed to ultrasound to to test for effects of concentration, power and temperature on the breaking algae. Literature review Ultrasound is cyclic sound pressure whose frequency is greater that the upper limit of human hearing. This frequency limit is usually greater than 20,000 hertz. Ultrasonication is often used in microbial studies to break cells in order to release intracellular products. High intensity ultrasound has been found to be effective in breaking microbial cells. However, low intensity ultrasounds have been found to improve productivity of some fermentation processes without cells being damaged. Studies have also indicated that ultrasound may also alter metabolic process of healthy cells. Production of ultrasound is usually carried out by the use of magneto-restrictive or piezoelectric transducers which are involved in conversion of the alternating current of an electronic oscillator to mechanical waves which are transmitted to the broth via cylindrical rod shaped probe or sonotrode (Lee, Nakano, and Matsumara 385). Ultrasonication usually generates alternating high pressure and low pressure waves in the exposed liquid. Vacuum bubbles are usually created by ultrasonic waves during the low pressure cycle. Once created, the vacuum bubbles collapse violently during high pressure cycle in a process called cavitation. The explosion of cavitation bubbles results in a strong hydrodynamic shear forces. These shear forces are capable of disintegrating fibrous and cellulosic materials into fine particles and breaking walls of the cell structure (Joyce, Wu, and Mason 865). As a result of the shear forces, much of intracellular material such as sugar or starch is released into the liquid. Furthermore, the cell walls materials are usually broken into small debris by these shear forces (Manson 146). Ultrasonic disintegration can be tested in any scale whether at lab scale for 1mL to about 5L using UP400S with 22mm sonotrode, bench top scale at about 0.1 to 20L/min using UIP1000hd with 34mm sonotrode and flowcell or production scale starting at 20L/min using UIP4000 or UIP16000. Ultrasonic process mechanism is influenced by energy supplied, the frequency of the ultrasonic and the nature of the influent (Mason and Tiehm, 103). Studies have shown that the disintegration of cells is proportional to supplied energy (Rouke et al. 147). High frequencies have been found to promote oxidation by radicals while low frequencies have been found to promote physical and mechanical phenomena like pressure waves. Complex influents have been found to decrease radical performance. Generation of ultrasonic waves in a liquid suspension can be done through either direct sonication where the ultrasound probe is immersed into the suspension or indirectly where the sample container with the suspension is introduced into a bath containing a liquid through ultrasonic waves are propagated (Heng, Jun, Wen-jie, and Guibai, 192). The recommended sonication is the direct sonication since the physical barriers to delivery of the power to the dispersion is reduced. Direct sonication yields a higher effective energy output into the suspension. Sonication can result in either agglomeration or cluster breakdown in addition other effects such as chemical reactions. Optimal sonication conditions must be established for any given system by assessing the effect of a variety of sonication parameters on the dispersion state of the suspension under a broad range of relevant conditions. Furthermore, the total effective acoustic energy utilized is influenced by various instrument and system specific parameters (Zhang, Zhang, and Fan 335). The extreme local heating cycles which occur at micro scale bubble interface during sonication as a result of cavitation leads to a bulk heating of the liquid over time. Substantial liquid evaporation due excessive bulk heating may result in changes of the liquid volume or even degradation of the material or medium components (Lee, Nakano, and Matsumara 387). The side effects of temperature may be minimized by immersing the suspension container in a cooling bath. This ought to be immersed to a level which is roughly equal to that of the internal suspension. Ice water bath is usually preferred for prevention of excessive evaporation. Working with containers which are good thermal conductors ensure rapid heat release from the suspension. Some recommended containers for reducing thermal conductivity include aluminum, stainless steel, glass and plastic. The chemical compatibility of the container and the suspension components should also be considered when choosing the containers to avoid explosions or corrosion. In this study since we intent to establish the effect of temperature on cell breakage, containers which reduce thermal conductivity will be used in all experiments. The total amount of energy (E) delivered to a suspension depends on both applied power (P) and the total amount of time (t) that the suspension is subjected to the ultrasonic treatment: E=P x t. thus two suspensions treated at the same power for different times can show significantly different dispersion states. Since this study is interested in establishing the effect of power on algae breakage, time to which algae suspension will be exposed to ultrasonic treatment will be kept constant while the power will be varied. Ultrasonic disruptors operate in either pulsed or continuous mode. Pulsed mode involves alternation of ultrasonic intervals with static intervals (Joyce, Wu, and Mason 866). The on and off interval durations can be regulated. Temperature increase is usually retarded by operation in pulsed mode hence reducing unwanted side effects and allow for better temperature control than when operating in continuous mode. However, since we are interested in the effect of temperature on algae breakage, continuous mode will be employed rather than pulsed mode. The sonication power and time describes the total amount of energy delivered to the suspension (Clark and Macquarrie 87). The response of suspensions of different volumes and particle concentrations to the same amount of delivered energy can vary significantly (Tatsuhisa et al. 417). Thus to measure the effect of concentration of suspension on algae breakage the volume of suspension and energy delivered to it will be held constant while the concentration will be varied. Studies have shown that at constant volume, higher particle concentrations results in an increased particle collision frequency (Sharma and Mudhoo, 87). Theoretically, increased collision frequency is expected to enhance particle breakage due to an increase in particle-particle impact events (Mason and Tiehm, 113). However such collisions can also result in agglomeration or aggregate formation as particles collide and coalesce. Thus the effect of concentration depends on both delivered energy and physiochemical properties of the suspension. Thus this study will establish whether increase in concentration of algae increase algae breakage or agglomeration. Consequential effects/Implications/Ethics Successful establishment of optimum conditions of cell disruptions of algae using ultrasound could increase the economic viability of bio-resource extraction from algae on large scale since existing methods have various disadvantages of either consuming great amounts of energy or being slow in cell disruption (Lee, Nakano, and Matsumara 388). The use of octanol as a solvent in which the sample is to be suspended is environmental friendly and hence its impact on the environment is not expected to harm it. Occupational exposure to ultrasound of excess of 120 dB may result in hearing loss. In addition, exposure in excess of 155 dB may result in production of heating effects that are harmful to the human body while exposures greater than 180 dB may result in death. The experiments will ensure that no one is exposed to ultrasound and that only the samples are the ones subjected to ultrasound. Other than exposure to ultrasound no other foreseeable ethical issue is expected to arise since the study will not involve human subjects. Project methodology and Justification Algae material Fresh Euglena sp. Algae will be acquired from commercial suppliers. Ultrasonic bath Ultrasonic bath with the operating frequency of 35kHz and with a rated output power of 170 W will be used. The bath to be used will have 240x137x100mm3 dimensions. The efficiency of the bath will be determined by calorimetric method as 28.8% that will indicate actual power dissipated in bulk solution to be 49 W. In order to maintain the temperature at desired value within ±0.5 K a cooling coil will be kept inside the reactor and water will be pumped at the required pressure. The ultrasonic bath will have an electrical heater in its back wall that will help to maintain the desired temperature. Mechanical stirrer will be used in the solution with constant revolution per minute (150 r.p.m) to enhance the effect of ultrasonic irradiation. a) The effect of concentration on the breaking algae In each experiment a certain volume of Euglena sp. Algae with desired concentration will be poured into ultrasonic bath and octanol will be added to the Euglena solution to prepare the reaction mixture. In order to analyze Euglena concentration, at certain reaction intervals 5 ml of sample will be withdrawn and absorbance will be determined by UV-vis spectrophotometer at 617 nm and a calibration plot based on Beer Lambert’s law to relate the absorbance to concentration. In order to analyze the effect of concentration on breaking algae, the withdrawn sample will be centrifuged at 14,000 r.p.m for 10 minutes and the upper layer which contains octane and dissolved carotene pigment of Euglena used to determine the pigment concentration by use of UV-vis spectrophotometer at 617 nm. b) The effect of power on the breaking algae In order to determine the effect of power on breakage of algae, calorimetric method will be used to alter the actual power dissipated in bulk solution at an interval of 5 W above and below the initial 49 W. at each power interval, the breakage will be determined via centrifuging the sample at 14,000 r.p.m for 10 minutes and the upper layer which contains octane and dissolved carotene pigment of Euglena used to determine the pigment concentration by use of UV-vis spectrophotometer at 617 nm. c) The effect of temperature on the breaking algae In order to determine the effect of temperature on breakage of algae, the electrical heater will be adjusted to maintain temperatures at different intervals. At each temperature interval the effect of temperature on breaking algae will be determined by centrifuging the sample at 14,000 r.p.m for 10 minutes and the upper layer which contains octane and dissolved carotene pigment of Euglena used to determine the pigment concentration by use of UV-vis spectrophotometer at 617 nm. Risk assessment The study is likely to incur limited risk because octane to be used is environmentally friendly and the ultrasound used will be below that which is known to cause adverse human side effects such as hearing impairment. However, care will be taken to ensure that the investigator is not exposed to any ultrasound waves unnecessarily. Resource planning Acquisition of algae Acquisition of ultrasound Printing and photocopies Spectrophotometer Bench fee Publication fee Time line Proposal writing May-June 2011 Proposal presentation to the tutor June 2011 Acquisition of algae June 2011 Determination of the effect of concentration on breaking algae June 2011 Determination of the effect of power on breaking algae June 2011 Determination of the effect of temperature on breaking algae June 2011 Data analysis July2011 Report writing and publication July-August 2011 Works Cited Bellinger, E. and Sigee, D. Freshwater algae: identification and use as bioindicators. New York: John Wiley and Sons, 2010 Clark James and Macquarrie Duncan. Handbook of green chemistry and technology. London: Wiley-Blackwell, 2002. Heng Liang, Jun Nan, Wen-jie He, and Guibai Li. Algae removal by ultrasonic irradiation–coagulation. Desalination, 239.1- 3 (2009): 191-197 Joyce Eadaoin, Wu Xiaoge, and Mason Thomas. Effect of ultrasonic frequency and power on algae suspensions. Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 45.7 (2010): 863-866 Lee, T., Nakano, K., and Matsumara, M. Ultrasonic Irradiation for Blue-Green Algae Bloom Control. Environmental Technology, 22.4 (2001): 383-390 Manson Timothy. Large scale sonochemical processing: aspiration and actuality. Ultrasonics Sonochemistry, 7.4 (2000): 145-149 Mason, T., and Tiehm Andreas. Advances in sonochemistry: Ultrasound in environmental protection. New York: Elsevier, 2001. Rouke Bosma, Wim A. van Spronsen, Johannes Tramper and René H. Wijffels. Ultrasound, a new separation technique to harvest microalgae. Journal of Applied Phycology, 15. 2-3, 143-153, Sharma Sanjay, and Mudhoo Ackmez. Green chemistry for environmental sustainability. New York: CRC Press, 2010. Tatsuhisa Hamada, Yasumasa Yamada, Shigekatsu Endo and Hajime Ogawa. Research on Inactivation of Blue-Green Algae Using Water Hammer Pressure. Advances in Water Resources and Hydraulic Engineering, 2, (2009): 417-422, Zhang Guangming, Zhang Panyue, and Fan Maohong. Ultrasound-enhanced coagulation for Microcystis aeruginosa removal. Ultrasonics Sonochemistry, 16.3 (2009): 334-338 Read More