continuously agitating suspension. Due to the vacuum, liquid is sucked in and is filtered on the surface of the drum. A scraper installed at the other end of the drum removes the separated solids from the drum. Filter media can be
precoated with a filter aid like diatomaceous earth which can be continuously replenished. Examples of these kinds of filters are filters used in the separation of fungal mycelia in antibiotic fermentation.
Both these types of filters are unsafe from the angle of biosafety, as both are potential generators of aerosols and
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allergens and thus are unsuitable for processing toxic products, pathogens or certain recombinant DNA microorganisms and their products.
• What decides the choice of filter to be used? There are several factors. The properties of the filtrate, particularly its density and viscosity, the nature of the solid particles, particularly their size and shape, distribution and packing character, the solid: liquid ratio, the need to recover liquid fraction as well, the scale of operation, the need for batch or continuous fermentation, the need for asepsis, the need for pressure or vacuum suction ….. the list seems to be endless!
• What is membrane filtration?
The science of filtration has made remarkable progress over last few years. It is possible no to separate not only the finest of suspended solids but also the dissolved salts out of the solution. Modern methods of filtration involve absolute filters rather than depth filters. These consist of supported membranes with specified pore sizes that can be divided into three main categories. They are, in decreasing order of pore size, microfiltration, ultrafiltration and reverse osmosis membranes. The suspension to be filtered is pumped across the membrane (cross-/tangential-flow) rather than at a right angle to it, as occurs with conventional filtration methods. This retards fouling of the membrane by particulate materials. Particles whose size is below the membrane ‘cut- off ’ will pass through the membrane to become the ultrafiltrate or permeate, whereas the remainder is retained as the retentate. As filtration progresses, the flux across the membrane can slow due to membrane fouling. This may be caused by the accumulation of a layer of solute molecules on the surface of the membrane, referred to as concentration polarization. The presence of silicon antifoams may have a similar negative effect.
• Can you tell me more about these techniques? Sure.
Microfiltration is used to separate particles of 10-2 um to 10 um, including removal of microbial cells from the
fermentation medium. This method is relatively expensive due to the high cost of membranes, but it has several advantages compared with centrifugation. They include quiet operation, lower energy requirements, the product can be easily washed, good temperature control is possible, containment is readily achieved and no bioaerosols are produced. Consequently, it is suitable for handling pathogens and recombinant microorganisms.
Ultrafiltration is similar to microfiltration except that the membranes have smaller pore sizes, and are used to fractionate solutions according to molecular wt, normally within the range 2000-500 000 Da. The membranes have anisotropic structure, composed of a thin membrane with pores of specified diameter providing selectivity, lying on top of a thick, highly porous, support structure. A membrane manufactured with an exclusion size of 100 000 Da, for example, should produce a retentate of proteins and other molecules over 10000 Da and an ultrafiltrate of all molecules
below 10000 Da. However, non-spherical proteins may exhibit different exclusion reactions to the membrane. Flat membranes are available, but for larger-scale operations hollow-fiber systems are usually preferred .Several of these ultrafiltration units can be linked together to produce a sophisticated purification system These methods are extensively employed for the purification of proteins, and for separating and concentrating materials. Ultrafiltration is also effective in removing pyrogens (bacterial cell wall lipopolysaccharides), cell debris and viruses from media, and for whey processing.
Another variation on the ultrafiltration system is
diafiltration, where water or other liquid is filtered to remove unwanted low molecular weight contaminants. This can be used as an alternative to gel filtration or dialysis for removing ammonium sulphate from a protein preparation precipitated by this salt (desalting) for changing a buffer or in water purification.
Reverse osmosis is used for dewatering or concentration steps and has been employed to desalinate seawater for drinking. In osmosis water will cross a semipermeable membrane if the concentration of osmotically active solutes, such as salt, is higher on the opposite side of the membrane. However, if pressure is applied on the salt side, then reverse osmosis will occur, and water will be driven across the membrane from the salt side. This reversal of osmosis requires a high pressure, e.g. a pressure of 30-40 bar is needed to dewater a 0.6 mmol/L salt solution (note: 1 bar=100kPa=0.987 atm). Consequently, a strong metal casing is required to house this equipment. As the membranes have pore sizes of only 10-2 to 10-4 um diameter, solute molecules can deposit on the surface, causing a large resistance to solvent flow.
However, this fouling can be overcome by increasing the turbulence at the surface of the membrane. Various chemicals are also used to prevent and control of fouling of these membranes.
· Exercise: find out more about the antifoulant chemicals used in RO systems. Write briefly on their mode of activity, advantages and disadvantages. Use additional sheets if required. If possible make a visit to an industrial RO unit and study its operation. Write a report.
• What is the next technique used? Well, it is cell disruption.
Some target products are intracellular, including many enzymes and recombinant proteins, several of which form inclusion bodies, which are concentrated proteins with incomplete tertiary structure. Therefore, methods are required to disrupt the microorganisms and release these products. The breaking of the cell wall/envelope and cytoplasmic membrane can pose problems, particularly where cells possess strong cell walls. For example, a pressure of 650 bar is needed to disrupt yeast cells, although this may vary somewhat at different times during the growth cycle and depending upon the specific growth conditions.
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General problems associated with cell disruption include the liberation of DNA, which can increase the viscosity of the suspension. This may also affect further processing, such as pumping the suspension on to the next unit process and flow through chromatography columns. A nucleic acid precipitation step or the addition of DNase can help to prevent this problem. If mechanical disruption is used then heat is invariably generated, which denatures proteins unless appropriate cooling measures are implemented. Products released from eukaryotic cells are often subject to degradation by hydrolytic enzymes (proteases, lipases, etc.) liberated from disrupted lysosomes. This damage can be reduced by the addition of enzyme inhibitors, cooling the cell extract and rapid processing. Alternatively, attempts may be made to produce mutant strains of the producer microorganism lacking the damaging enzymes.
• How do we achieve this goal of breaking open the cells, then?
Cell disruption can be achieved by both mechanical and non- mechanical methods. The disruption process is often quantified by monitoring changes in absorbance, particle size, total protein concentration or the activity of a specific intracellular enzyme released into the disrupted suspension. First, let’s study about the mechanical cell disruption methods.
Several mechanical methods are available for the disruption of cells. Those based on solid shear involve extrusion of frozen cell preparations through a narrow orifice at high pressure. This approach has been used at the laboratory scale, but not for large-scale operations. Methods utilizing liquid shear are generally more effective. The French press (pressure cell) is often used in the laboratory and the high-pressure homogenizers, such as the Manton and Gaulin homogenizer (APV-type mill), are employed for pilot- and production- scale cell disruption. They may be used for bacterial and yeast cells, and fungal mycelium. In these devices the cell
suspension is drawn through a check valve into a pump cylinder. At this point, it is forced under pressure (up to 1500 bar) through a very narrow annulus or discharge valve, over which the pressure drops to atmospheric. Cell disruption is primarily achieved by high liquid shear in the orifice and the sudden pressure drop upon discharge causes explosion of the cells.
• What are the factors that decide the efficiency of cell disruption?
The efficiency of disruption is independent of the cell concentration, but is depends on the pressure exerted, the number of cycles through the homogenizer and the temperature. Disruption of yeast cell preparations, for example, typically requires three passes through the pressure cell at 650 bar, whereas wild-type Escherichia coli generally needs 1100-1500 bar. During processing the temperature rises by about 2.2-2.4°C per 100 bar, i.e. by approximately 20 0C over one pass at 800 bar. Consequently, precooling of the cell preparation is usually essential. The energy input necessary is approximately 0.35kW per 100bar and the throughput is up to 6000 L/h. A problem with this method
of cell disruption is that all intracellular materials are released. As a result, the product of interest must be separated from a complex mixture of proteins, nucleic acids and cell wall fragments. Some fragments of cell debris are not readily separated, making the solution difficult to clarify. In
addition, proteins may be denatured if the equipment is not sufficiently cooled and filamentous microorganisms may block the discharge valve. When used for certain categories of microorganisms, the homogenizers have to be contained to prevent the escape of aerosols.
• How can we carry out cell disruption on a small scale, say, in a laboratory?
On a small scale, manual grinding of cells with abrasives, usually alumina, glass beads, kieselguhr or silica, can be an effective means of disruption, but results may not be reproducible. In industry, high-speed bead mills, equipped with cooling jackets, are often used to agitate a cell
suspension with small beads (0.5-0.9um diameter) of glass, zirconium oxide or titanium carbide. Cell breakage results from shear forces, grinding between beads and collisions with beads. The efficiency of cell breakage is a function of agitation speed, concentration of beads, bead density and diameter, broth density, flow rate and temperature. Cell concentration is also a major factor (optimum 30-60% dry weight), which is an important difference from the liquid shear homogenizers described above. Maximum throughput in these systems is about 2000 Llh.
Ultrasonic disruption of cells involves cavitation, microscopic bubbles or cavities generated by pressure waves. It is
performed by ultrasonic vibrators that produce a high- frequency sound with a wave density of approximately 20kilohertz/s. A transducer converts the waves into mechanical oscillations via a titanium probe immersed in the concentrated cell suspension. However, this technique also generates heat, which can denature thermolabile proteins. Rod-shaped bacteria are often easier to break than cocci, and Gram-negative organisms are more easily disrupted than Gram-positive cells. Sonication is effective on a small scale, but is not routinely used in large-scale operations, due to problems with the transmission of power and heat dissipation.
Some newer disruption systems are being developed to give improved large-scale disruption, often with integral containment. They include a newly designed ball mill, the CoBall Mill; the Constant Systems high-pressure disrupter, which operates at up to 2700 bar; and two systems with no moving parts, the Microfluidics impingement jet system and the Glass-col nebulizer. The Parr Instruments cell disruption bomb is designed for disrupting mammalian cells. This is a relatively gentle method that works on the principle of nitrogen decompression and does not generate heat. Nitrogen is dissolved in cells under high pressure, and sudden pressure release then causes the cells to burst. Exercise
“Mechanical means of cell disruption with high energy inputs are effective, but pose a great threat to the safety of workers and
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environment, especially in the absence of proper containment processes”. Justify.
Now let’s see about the non-mechanical cell disruption methods.
Autolysis, osmotic shock, rupture with ice crystals (freezing/ thawing) or heat shock are some of the ‘non violent’ methods used for cell disintegration. . Autolysis, for example, has been used for the production of yeast extract and other yeast products. It has the advantages of lower cost and uses the microbe’s own enzymes, so that no foreign substances are introduced into the product. Osmotic shock is often useful for releasing products from the periplasmic space. This may be achieved by equilibrating the cells in 20% (w/v) buffered sucrose, then rapidly harvesting and resuspending in water at 4°C.
Wide ranges of other techniques have been developed for small-scale microbial disruption using various chemicals and enzymes. However, some of these can lead to problems with subsequent purification steps. Organic solvents, usually acetone, butanol, chloroform or methanol, have been used to liberate enzymes and other substances from microorganisms by creating channels through the cell membrane. Simple treatment with alkali or detergents, such as sodium lauryl sulphate or Triton X- 100, can also be effective.
Several cell wall degrading enzymes have been successfully employed in cell disruption. For example, lysozyme, which hydrolyses B 1,4 glycosidic linkages within the peptidoglycan of bacterial cell walls, is useful for lysing Gram-positive organisms. Addition of ethylene diamine tetraacetic acid (EDTA) to chelate metal ions also improves the effectiveness of lysozyme and other treatments on Gram-negative bacteria. This is because EDTA has the ability to sequester the divalent cations that stabilize the structure of their outer membranes. Enzymic destruction of yeast cell walls can be achieved with snail gut enzymes that contain a mixture of B-glucanases. These enzyme preparations are also useful for producing living yeast
spheroplasts or protoplasts.
The antibiotics penicillin and cycloserine may be used to lyse actively growing bacterial cells, often in combination.
Additionally, basic proteins like protamines etc. can also be used for cellular lysis.
After the products have been brought into an extractable form, our next job is to recover them in pure form. In the next lesson , we will see how to do that.
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Learning Objectives
In this lecture, you will learn• Applications of computers in fermentations
• Adaptive control
• Data logging, data analysis & process control
Why Computers?
This question is really irrelevant. While we can still imagine a world without computers, it would be a very underdeveloped one. It is therefore not surprising that computers are finding application in the field of fermentation technology as well. There are a number of advantages to be gained by coupling process instruments to digital computers. First, the computer can enhance data acquisition functions in several respects. Improved reliability and accuracy can be obtained by using statistical methods and digital filtering. Readings from several parallel sensors can be compared and analyzed to provide on- line recalibration and to identify sensor failure. With a computer, the number and sophistication of analysis systems can be increased. For example, a computer-controlled system may take samples automatically, conduct a chromatographic analysis, and interpret the results, using internally stored calibrations or algorithms to give output directly in convenient units. Although simple signal conditioning and correcting operations such as linearization can be done with particular electronic circuits, these functions are readily accomplished using a computer without the need for additional specific hardware. Another advantage of computers with respect to data acquisition is the ability to store large quantities of measured results in digital form which may be accessed conveniently, analyzed, and displayed later.
Using computers, data analysis and interpretation can be enhanced greatly. Results of several measurements may be combined to calculate instantaneously quantities such as oxygen utilization rate and respiratory quotient. Advanced Slate and parameter estimation methods may also be applied on-line to provide additional useful information on process status from the limited measurements available. More specifics and some examples of computer applications for data analysis are presented in the next section.
Computers expand opportunities tremendously for improved process control and optimization. One computer can replace many conventional analog controllers and control many individual valuables such as pH and temperature using standard feedback algorithms. Furthermore, more sophisticated multi variable control methods may be implemented easily with a computer. Controlled variables may include derived quantities such as RQ when a computer is applied. Computer methods may be used to evaluate and improve process mathematical models which may then be employed for determining optimum operating conditions and strategies. Then, the computer provides the memory and computation capability to implement the optimization method, such as variation of nutrient feeding rate or pH during a batch fermentation. Operation of a batch process requires a carefully controlled and coordinated sequence of valve openings and closings and pump starts and stops. While all of these functions have been done by