CHEM-E Bioprocess technology II Elisa Alasuvanto Risto Hertzberg Saija Pajari Cell Disruption Methods

Transcription

1 CHEM-E Bioprocess technology II Elisa Alasuvanto Risto Hertzberg Saija Pajari Cell Disruption Methods

2 Contents 1 Abstract Background Methods Mechanical Methods High-pressure homogenization Freezing Sonication Bead milling Non-mechanical methods Enzymatic cell disruption Chemical cell disruption Physical cell disruption Case studies Aspartase production Lipid extraction from microalgae Lactase Production Conclusions... 7 References... 8

3 1 Abstract This review introduces different methods for cell disruption. The review focuses mainly on high-pressure homogenization, freezing, sonication, bead milling and enzymatic disruption of cells, which are commonly used cell disruption methods. First, the more common, mechanical methods are introduced, which is followed by introduction to non-mechanical methods, focusing on the enzymatic method for cell disruption. Some additional methods are also briefly discussed. Three case studies are also discussed. Mechanical methods provide effective ways to release intracellular products. They can be used in various different downstream processes and are also suitable for industrial applications. However, nonmechanical methods are more specific and more gentle. On the other hand, non-mechanical methods require more time and unique procedures for each product and process. Cell disruption can cover major part of the overall process costs, which creates a demand for economically feasible methods. The more detailed analysis contains information about the pros and cons, notes and highlights about the methods and some evaluations of how they affect the economics of the processes. 2 Background All cells have a cytoplasmic membrane that separates the intracellular organelles of the cell from the environment it is growing in. In addition to the cytoplasmic membrane, some cells contain a cell wall. When producing substances in living cells, many products are not secreted through the cytoplasmic membrane to the growth media, but they stay inside the cell. This makes the extraction and purification of the products problematic. In cell disruption, the cytoplasmic membrane and the cell wall are broken down so the intracellular products can be extracted. There are various cell disruption methods (Figure 1). The method of choice depends on the cells that are used as well as the products. To find the most suitable disruption method, one must know the structure of the cell wall and cytoplasmic membrane of the organism used in the process. Disruption methods include non-mechanical and mechanical methods. Non-mechanical methods are further divided into physical, chemical and enzymatic disruption methods. In mechanical cell disruption, the cell is physically broken down with for example high-pressure homogenization or freezing and grinding the cells. Mechanical methods have higher efficiency and broader applicability than the other methods. However, non-mechanical methods are very specific and more gentle. For pharmaceutical, agrochemical and food industries, cell disruption is often the first step in the downstream processing of intracellular products. Development of the cell disruption methods targets high product recovery and economical feasibility. (Chin et al. 2008), (Geciova et al. 2002), (Singh, 2013). The cell disruption methods are fairly new techniques, that still need improvements. The energy usage for different mechanical methods can be fairly high, around MJ/kg of dry biomass. The recent studies have shown that the force of the mechanical methods is usually unnecessarily high and therefore it wastes energy. For the chemical methods, the contamination prevention and the need for purifying the product still needs improvement and specification. Usually, the cell disruption strategies are a mix of different mechanical and 1

4 chemical cell disruption methods. The scope of these combined strategies is to optimize the energy consumption to minimum, maximize the yield and have pure products. (Lee, 2012) Figure 1. Different types of cell disruption. (Lee, 2012) 3 Methods 3.1 Mechanical Methods The most common different mechanical methods are sonication, high pressure homogenization, high-speed homogenization, microwave technology, freeze drying and hydrodynamic cavitation. (Lee, 2012) Mechanical methods are nonspecific, but they usually have higher efficiency than non-mechanical cell disruption methods. They also have more applications than non-mechanical methods, mostly based on cost and effectiveness. (Geciova, 2002) In this chapter, high-pressure homogenization, freezing, sonication and bead milling are introduced in detail High-pressure homogenization High-pressure homogenization (HPH) is one of the most widely used mechanical cell disruption methods. High pressure forces the cells through a narrow hole or a channel, which creates a shear force to the cells. Upon discharge from the channel, the cells expand instantly. This instant expansion after the shear force disrupts the cells. A picture of a typical high-pressure homogenizer can be seen in figure 2. The cells can be run through the homogenizer multiple times to ensure total lysis. (Chin, 2008). HPH-method is fast, continuous and easy, but it can have disruptive effects on heat sensitive products, such as enzymes and proteins, because of the temperature increase. The temperature increase per pressure is suggested to be around 2 o C per 10 MPa. (Lee, 2012) High-pressure homogenization is routinely used in pharmaceutical applications for disrupting bacteria and 2

5 baker s yeast (S. cerevisiae) for releasing for example human insulin. (Geciova, 2002) (Soavi, 2011) Figure 2. A typical high-pressure homogenizer. (Seok-Cheol, 2012) Freezing Freezing and thawing are linked mechanical disruption methods in which the cell disruption is continuous. The formation and melting of the ice crystals are the driving forces. If the freezing is carried out slowly and gradually, the ice crystals become larger and the cell damage is also higher and might cause severe damage to the organism e.g. by rupturing the cell walls (Figure 3). If the product is intracellular, grinding can also be introduced in combination with freezing to further improve cell breakage. Freezing method is not too largely applied because of the low yields and high energy requirements. (Mohammadi, 2015) It s also suggested that in at least animal cells the freezing-thawing will greatly increase the number of apoptotic cells up to 24% versus the 3% to untreated cells. Osmotic shock is also greatly related to thawing, because of the sudden change in solute concentration within and around the cell. The osmosis will also cause shear stress to disrupt the cell walls. The osmotic shock can be avoided by reduced pressure to have the ice sublimed to gas. (Odintsova, 2017) The process is very costly, slow and usually only used to obtain valuable heat sensitive proteins or enzymes. (Lee, 2012) 3

6 Figure 3. Dead cells, disrupted by freezing (left) and as comparison, cell that went through cryo induced-necrosis (right). n, nucleus, l, lipofuscin granules (Odintsova, 2017) Sonication In sonication method, pressure waves are generated by ultrasound. It is suitable especially for small, 1 ml to 2 L, reactors. The mechanism is based on around 25 khz sonic waves that generate stable and transient cavitation within the reactor. Transient cavitations will begin to oscillate and then collapsing, generating implosions. The implosions cause a localized shock wave and temperature increase. The increased temperature ionizes its surroundings and the wave causes enough force shear and disrupt the micro-organisms outer membranes. Stable cavitations are continuous oscillates that cause micro-scale eddies that can disrupt microorganisms. (Lee, 2012), (Zhang et al, 2007) Sonication is a relatively simple method, and its advantages include also low operating costs. (Singh, 2013). Sonication doesn't lead to the protein denaturation because cell disruption can be performed at relatively low temperatures. Chemicals or beads, which have to be removed afterwards, are not needed in the method. Ultrasonicators can also be scaled-up. However, when ultrasonication is used for a long time radicals may be produced, which can affect quality of the product. Therefore sonication conditions should be optimized. (Gerde et al, 2012) Bead milling Bead mill is composed of grinding chamber filled with cell suspension. Agitation shaft transfers kinetic energy to the grinding elements, such zirconia beads. Shear forces and collisions of the beads disrupt the cells. Smaller beads are used to release enzymes in cytoplasm whereas larger beads release enzymes in cytoplasmic membrane. Increased bead loading improves efficiency of cell disruption. However, also heating and power 4

7 consumption are increased. Optimal bead loading is usually %. Also impeller tip speed and milling time have an effect on the level of cell disruption. Higher disruption is achieved with longer milling time. (Geciova et al. 2002) Advantages of bead milling include high disruption efficiency, good temperature control and high biomass loading. There are also equipment available for laboratory scale as well as for commercial use. (Jahanshahi & Najafpour, 2007) Wide range of applicability makes bead mill well suited for the dairy industry (Geciova et al. 2002). A picture of a bead mill can be seen in Figure 4. Figure 4. Grinding chamber of the bead mill. (from Non-mechanical methods Enzymatic cell disruption In enzymatic cell disruption, enzymes are used to break down the cell walls and membranes. The enzymes used can be produced by the cell itself (autolysis) or foreign (lytic). Autolysis can be achieved with for example antibiotics, but foreign lytic enzymes are more extensively studied. (Geciova, 2002) Enzymatic cell disruption is gentle, specific and easy to scale up but often time-consuming. The disruption process has to be individualized for each product. The process conditions have to be suitable for the enzymes, which means that for example temperature, ph and pressure have to be adjusted carefully. Specific activity is a major factor in choosing enzymes for cell disruption. The enzymes should target the cell wall and membrane but they should not affect the product. For this, the structure of the cell wall and the membrane must be known. In addition to intracellular product release, lytic enzymes are used in many antimicrobial applications, for example in antibiotics. (Chin, 2008), (Salazar, 2007) (Zheng, 2011) The enzyme of choice depends on the product and microbes used in the production. For example lysozyme enzymes are often used for bacterial cell disruption. Lysozymes are one of the most studied and used lytic enzymes, but they usually can not affect Gram-negative bacteria, because of the outer cell membrane of the bacteria. The cells can be pretreated 5

8 with a detergent, which dissolves the outer membrane, or the lysozyme itself can also be modified to be effective in lysing Gram-negative bacteria. (Salazar, 2007) Attaching fatty acids of polysaccharides covalently to the enzyme has been proven to enhance the enzymes lytic properties towards Gram-negative bacteria. For example, labiase and achromopeptidase are commercially available enzymes which are able to disrupt some lysozyme-resistant bacteria. (Aminlari, 2005) Lysozymes are often used with algal cells, too. Other enzymes used for algal cells include cellulase and snailase. (Zheng, 2011) The yeast cell wall is composed mainly of mannoproteins, glucans and chitin, so several enzymes, including glucanases, mannanases and chitinases, are needed for disruption of yeast cells. These enzymes are often derived from organisms, that are naturally predatory towards yeast. (Salazar, 2007) Chemical cell disruption In chemical cell disruption, chemical agents, such as urea, butanol or isopentanol, are used to disrupt membrane structures of the cells. The chemicals interact with molecules in the cell wall or cell membrane resulting in bond breakage in the structures. Chemical treatment is often followed by a mechanical homogenization method and separating the product with for example chromatography or gel electrophoresis. These necessary steps are time consuming and therefore make the disruption method less efficient. There is also the danger of toxic residues left in the product. (Seetharam, 1991) Physical cell disruption Physical cell disruption methods are more gentle than mechanical methods. The difference to mechanical methods is, that the cell wall is not entirely torn apart when using physical methods. For example in decompression method, the gases inside the cell expand rapidly because of decompression, which results in point puncture of the cell wall and release of an intracellular product. Other physical methods include microwaving, freeze-drying and osmotic shocks. (Lee, 2012) (Middelberg, 1995) 4 Case studies Numerous potential microbial products are intracellular, which makes cell disruption methods important. (Geciova et al. 2002) Two examples of a production where cell disruption plays a major role are aspartase production in E. coli and lipid production in algae. 6

9 4.1 Aspartase production Aspartase is a highly specific enzyme, which is industrially used for the production of L- aspartic acid (precursor of aspartame). It is an intracellular enzyme. Singh (2013) studied release of aspartase from E. coli using beadmill, sonication and french mill methods. Bead mill and ultrasonication resulted in over 90 % release of aspartase, while french mill was the most effective method (released 98 % of aspartase). (Singh, 2013) 4.2 Lipid extraction from microalgae Microalgae can be used as a biodiesel source because of fast biomass production and high oil content of the cells. Biodiesel production from microalgae includes cultivation, harvest, extraction of the lipid product and transesterification. Cell disruption is needed to extract the lipid effectively, and cell disruption step represents a major proportion of the overall process costs. Prabakaran & Ravindran compared different methods for lipid extraction from microalgae. They found sonication the most suitable method for lipid extraction in the large scale. (Prabakaran & Ravindran, 2011). The estimated cost of microalgae production is around 5.8 $/kg and the contrasted canola oil is around 1.3 $/kg. With the process yield being around 80%, it still needs improvement to be commercially competitive. For example, the theoretical minimum of energy required to the microalgae disruption is estimated to be around 10-5 of the experimental energy input. (Lee, 2012) 4.3 Lactase Production Lactase is used in hydrolysing lactose from dairy products or from the consumed food products. Lactose intolerance is growing phenomena in the world with over half of the population suffering from it. Most of the lactase production is coming from bacterial origin. The producing microorganisms can be, for example, Lactobacillus delbrueckii, which is thermophilic, gram-positive bacteria. These microorganisms produce lactase intracellularly, so cell disruption methods are significant part of the downstream process. (Vasiljevic, 2001) After the fermentation, the end-use determines, how the downstream process is handled. The Lactobacillus delbrueckii is deemed to be GRAS or generally recognized as safe (to eat, for example). So, they can be used as such if they re going to dairy products. But when going to lactase medication, then the lactase must be isolated and purified. For example, ethanol is used in disruption the cells outer membrane and the products are then processed with high pressure ultrafiltration (HPUF) and then separated further. (Vasiljevic, 2001) 5 Conclusions Mechanical methods, such bead mill and sonication are effective ways to release intracellular products. They are nonspecific methods, that have a broader range of applications than non-mechanical methods, which need to be adjusted carefully for every 7

10 process. Bead mill is also well suitable method for industrial use because of accessible scale-up. The mechanical separation techniques can usually be adjusted quite easily and quickly, so they can be optimized better than the chemical and enzymatic methods. Non-mechanical methods are more specific and gentle, as they do not cause large shear forces to the cells. The methods often require additional separation steps after the disruption. Enzymatic cell disruption can be a gentle and highly specific method to disrupt cells. The method is also easy to scale up, which is an important aspect from a commercial point of view. The method can be complicated and time-consuming, though. Certain cells require certain enzymes, so the method has to be optimized for every process. Many cells require several enzyme systems for efficient cell disruption, which makes the disruption step more complicated. Enzymes also need stable conditions to work properly, so more emphasis on environment controlling is needed. Lastly, the downstream processing costs may increase, when the product needs to be separated from enzymes and other substances in the broth. Cell disruption methods are still a new kind of tool for bioprocess engineers to harvest intracellular products. With further improvements, these methods can have significant reduction on cost of synthetic chemicals, since the cell disruption step is responsible for a major proportion of the overall production costs. Finding a suitable cell disruption method is extremely important, because the disruption can affect the product quantity as well as quality. Product stability, yield and cost of the unit operation are important aspects to be considered when designing a commercial and industrial process involving cell disruption. References Aminlari, M., Ramezani, R. & Jadidi, F Effect of Maillard-based conjugation with dextran on the functional properties of lysozyme and casein. Journal of the Science of Food and Agriculture. Vol. 85, pp Chin, W., Wen, S., Wei, B., Tau. C., & Beng, T Comparative Evaluation of Different Cell Disruption Methods for the Release of Recombinant Hepatitis B Core Antigen from Escherichia coli. Biotechnology and Bioprocess Engineering. Vol. 13. pp Geciova, J, Bury, D & Jelen, P International Dairy Journal. Vol 12:6. pp Gerde, A., Montalbo-Lomboy, M., Yao, L., Grewell, D., Wang, T Evaluation of microalgae cell disruption by ultrasonic treatment. Bioresource technology. Vol pp Jahanshahi, M. & Najafpour, G Advanced downstream processing in biotechnology. Biochemical Engineering and Biotechnology, pp

11 Lee, A., Lewis, D. & Ashman, P Disruption of microalgal cells for the extraction of lipids for biofuels: Processes and specific energy requirements. Biomass and Bioenergy. Vol 46. pp Middelberg, A Process-scale Disruption of Migroorganisms. Biotechnology Advances. Vol. 13(3), pp Mohammadi, M. & Ghaffari-Moghaddam, M Recovery and Extraction of Polyhydroxyalkanoates (PHAs). Polyhydroxyalkanoate (PHA) based Blends, Composites and Nanocomposites. RSC Green Chemistry Vol. 30. pp ISBN Odintsova, N., Boroda, A., Maiorova, M, Yakovlev, K The death pathways in mussel larval cells after a freeze-thaw cycle. Cryobiology. Vol. 77. pp Prabakaran, P. & Ravindran, A A comparative study on effective cell disruption methods for lipid extraction from microalgae. Applied microbiology. Vol. 53:2. pp Salazar, O. & Asenjo, J Enzymatic lysis of microbial cells. Biotechnology letters. Vol. 29, pp Seetharam, R. & Sharma, S Purification and Analysis of Recombinant Proteins. Marcel Dekker, New York, USA. Seok-Cheol, C., Woon, Y,, Sung-Ho, O. & Hyeon-Yong, L Enhancement of Lipid Extraction from Marine Microalga, Scenedesmus Associated with High-Pressure Homogenization Process. Journal of Biomedicine and Biotechnology. Singh, RS A comparative study on cell disruption methods for release of aspartase from E. coli K-12. Indian journal of experimental biology. Vol. 51:11. pp Soavi, N Cells Disruption By Means Of High Pressure Homogenization. Pharmaceutical Online. Vasiljevic T., Jelen, P Production of -galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria. Innovative Food Science & Emerging Technologies. Vol. 2. pp Zhang, P., Zhang, G. & Wang, W Ultrasonic treatment of biological sludge: Floc disintegration, cell lysis and inactivation. Bioresource technology. Vol. 98:1. pp Zheng, H., Yin, J., Gao, Z., Huang, H., Xiaojun, J. & Chang, D Disruption of Chlorella vulgaris Cells for the Release of Biodiesel-Producing Lipids: A Comparison of Grinding, Ultrasonication, Bead Milling, Enzymatic Lysis, and Microwaves. Appl Biochem Biotechnology. Vol. 164, pp