Biotechnology and Bioreactor PDF

Biotechnology: is a multidisciplinary field that involves the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, and molecular analogues for products and services. The concept of biotechnology encompasses a wide range of procedur...

Biotechnology: is a multidisciplinary field that involves the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, and molecular analogues for products and services. The concept of biotechnology encompasses a wide range of procedures for modifying living organisms for human purposes, going back to domestication of animals, cultivation of the plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization. Modern usage also includes genetic engineering, as well as cell and tissue culture technologies.  The American Chemical Society defines biotechnology as the application of biological organisms, systems, or processes by various industries to improvement of the value of materials and organisms, such as pharmaceuticals, crops, and livestock. Industrial biotechnology: is one of the most important fields in working for the implications of health and medicine. Industrial Biotechnology is the use of livingorganisms and enzymes to make bio-based products in various sectors such food, food ingredients, chemicals, paper, textiles, and many other industries. Another example is using naturally present bacteria by the mining industry in bioleaching, and also to produce biological weapons. Classification of microorganisms All organisms that are very small or microscopic in size, and cannot be seen with the naked eye are referred to as microorganisms. Microorganisms are visible under the microscope. Microorganisms include bacteria, archaea, algae, fungi, protozoa, etc. Although viruses are not considered living organisms, sometimes they are also included in the microorganisms category. Most organisms used by industry are harmless and many are used directly for the production of human and animal food. Only a small number of dangerous microorganisms have been used by industry in the manufacture of vaccines or diagnostic reagents, e.g. Bordetella pertussis (whooping cough), Mycobacterium tuberculosis (tuberculosis) and the virus that causes foot and mouth disease.  In the recent years recombinant DNA techniques have been the most successful ones for genetic alteration of microorganisms. Also, these techniques are the cause of much concern to the public. This natural worry has been ameliorated by several evidences: a. Risk assessment studies have failed to demonstrate that host cells can acquire novel hazardous properties from DNA donor cells. b. Considerable experimentation has shown no observable hazard.  However, care must be adopted when using recombinant DNA molecules  A classification of the degree of potential hazard of microorganisms has been drawn up by the European Federation of Biotechnology. Class Description Class A Microorganisms that have never been identified as causative agent of disease in human and that offer no threat to the environment. Class B Microorganisms that may cause human disease and might therefore offer a hazard to laboratory workers. They are unlikely to spread in the environment. Prophylactics are available, and treatment is effective. Class C Microorganisms that offer a sever threat to the health of laboratory workers but a comparatively small risk to the population at large. Prophylactics are available, and treatment is effective. Class D Microorganisms that cause severe illness in human beings and offer a serious hazard to laboratory workers and to people at large. In general, effective prophylactics are not available and no effective treatment is known. Class E This group contains microorganisms that offer a more severe threat to the environment than to people. They may be responsible for heavy economic losses. National and international lists and regulations concerning these microorganisms are already in existence in contexts other than biotechnology (e.g. for phytosanitary purposes). Media development The medium is the substrate used for fermentation to produce desired product. To obtain a good product from fermentation, the medium in which the microorganisms are grown must be supplied with enough energy sources and nutrients. Several factors must be kept in mind before designing or choosing the growth medium for fermentation. To obtain primary metabolites such as citric acid and ethanol, the media should be rich in components that support good growth. Similarly, for secondary metabolites such as alkaloids and antibiotics, the substrate requirement for product formation must be kept in mind.  While doing fermentation on a small scale, such as in laboratories, pure graded chemicals are used that are expensive. However, in large scale industrial fermentation, cheaper and unrefined chemicals are used. Therefore, the choice of media for fermentation is a crucial step that requires a lot of thought processes. Criteria for media selection The medium which used for fermentation should be: Suitable to produce maximum yield of product. Minimum yield of undesirable product. Cheap in price and available throughout the year. Cause minimal problems during medium sterilization. Aspects of production process such as aeration, agitation, downstream processing, and waste treatment. Types of media According to chemical composition the medium characterized to synthetic media, semi-synthetic media and crude or complex media.  In the laboratory research synthetic media was used but in case of industrial fermentation for economic reasons other media types were used. Synthetic media: is fully chemically defined, their components known and specific concentration of each of the components. Media are quite simple containing carbon source, nitrogen source, and a range of salts. Semi-synthetic media: largely chemically defined but one or more poorly specified components of variable but a controlled composition e. g; yeast extract, beef extract.  Plant, animal, fish and microbial extracts routinely been used in the past to supply vitamins and essential growth factors for specific organisms.  Crude or Complex media: largely composed of substances that are usually of plant or animal origin, some of the common ingredients include extracts of plant and animal tissues, e.g., fruits, vegetables, egg, milk, blood, body fluids, yeast, malt and manure extract etc.  The chemical composition and concentration of a complex medium is not well defined. Media Components  The fermentation media can either be liquid, known as broth, or it can be a solid-state fermentation. The media should satisfy all the nutritional requirements of the microorganism and should also obtain the target molecule.  A typical media requires a carbon source, a nitrogen source, salts, water and micronutrients. Let us look at them one by one. Carbon source Typically sugars and carbohydrates are used as carbon sources, but alcohols may also be used in making products such as vinegar. For laboratory uses, refined and pure carbon sources such as glucose, sucrose and glycerol are used that give a uniform product. However, in the case of industrial fermentation, inexpensive carbon sources such as whey, malt extract, molasses, corn steep liquor or sugar cane juice are used. Factors influencing the choice of carbon source 1) Main product of a fermentation process. 2) Rate at which it is metabolized and cellular yield coefficient. Nitrogen source  Nitrogen is an essential nutrient for growth and metabolic activity: it is the building block for ribonucleic acids, amino acids and proteins, and other vitamins and minerals used by microorganisms. It also is required for energy production during fermentation.  The nitrogen source for microorganisms may be used in the form of organic or inorganic compounds. Inorganic sources include ammonium salts or the free form of ammonia. Organic sources include urea, soybean, peanut, cottonseed meal, corn pulp and steep, meat extract, peptone, fish meal, yeast extract, and yeast peptone.  Inexpensive nitrogen sources are used for bulk production, such as tryptone, peptone, soy meal, corn steep liquor and yeast extract. Mineral o All microorganisms require certain mineral elements for growth and metabolism. In many media, macro element such as magnesium, phosphorous, potassium, sulphur, calcium and chlorine are essential components and must be added. o Others (micro elements or trace elements) such as cobalt, copper, iron, manganese, molybdenum and zinc are present in sufficient quantities in the water supplies and as impurities in other media ingredients but may need to be added when using pure sources. Vitamins Growth factors o Vitamins and other growth factors are important components in the fermentation media. o Many bacteria can synthesize all necessary vitamins from basic elements. For other bacteria, filamentous fungi and yeasts, they must be added as supplements to the fermentation medium. o Most natural carbon and nitrogen sources also contain at least some of the required vitamins as minor contaminants and yeast extract is consider a good source of vitamins. o Necessary growth factors, amino acids, nucleotides, fatty acids and sterols are added either in pure form or for economic reasons, as less expensive plant and animal extracts. Water o All fermentation processes, except solid state fermentation (SSF) require vast quantities of water. o Not only is water a major component of all media, but it is important for ancillary services like: heating, cooling, cleaning and rinsing. o A reliable source of large quantities of clean water, of consistent composition, is therefore essential. o The suitability of water is assessing through: 1. pH 2. Dissolved salts 3. Effluent contamination o Reuse of water is important, it reduces water cost by 50%. Oxygen Depending on the amount of oxygen required by the organism, it may be supplied in the form of air containing about 21% (v/v) oxygen or occasionally as pure oxygen when requirements are particularly high. The organism’s oxygen requirements may vary widely depending upon the carbon source. For most fermentations the air or oxygen supply is filter sterilized prior to being injected into the fermenter. Miscellaneous  Antifoaming agents: are necessary to reduce foam formation during fermentation. Foaming is largely due to denaturation of media proteins to form stable foam that is not easily disrupted. To solve the problem, antifoaming agents are also added to the fermentation medium. The antifoam should have the following properties: 1. Active at low concentrations 2. Long acting in preventing new foam 3. Should not be metabolized. 4. Should not be toxic. 5. Cheap, and not cause problem in fermentation.  Buffering agents: to stabilize pH of the media, mineral buffering salts such as phosphates and carbonates are also added.  Chelating agents: the addition of chelating agents may also be required when high concentrations of metals are present in the media. Some chelating (EDTA, citric acid and phosphates) agents form complexes with metal ions which are gradually utilized by microorganisms.  Instrumentation Instrumentation is concerned with the equipment and processes used in biotechnology and the biology-related and medical engineering-related field. The essential equipment and requirements which are essential for biotechnology, whether for research, development, or commercialization. Microbiological instrument: equipment for microbiological methods including microscope, incubator, laminar, shakers, autoclaves, cell & colony counter, colony pickers, ovens, water baths, etc.). Biochemical and molecular instrument: equipment for biochemical and molecular methods including spectrophotometer, PCR machines, and electrophoresis. Cultivating techniques: Bioreactors or fermenters for a wide spectrum of aerobic as well as anaerobic cultivations enabling online measurements and control of pH, oxygen content in gaseous phase, dissolved oxygen, redox potential, CO2, temperature, etc. Separation instrument: equipment for separation techniques including distillation, rectification, centrifuging, and filtration. Analytical instrument: equipment for gas chromatography (GC), high performance liquid chromatography (HPLC), mass spectrometers, NMR Spectroscopy and Flow Cytometers. The bioreactor Bioreactor is the most suitable containment for each biotechnological process must be designed to give the correct environment for optimizing growth and productivity, in other word bioreactors are machines that can be made to transform biological-based materials into desirable products. A bioreactor is a device that consists of various features such as an agitator system, an oxygen delivery system, a foam control system, and a variety of other systems such as temperature & pH control system, sampling ports, cleaning, and sterilization system, and lines for charging & emptying the reactor. Bioreactors principles Reactors are machines that can be made to transform biological-based materials into desirable products. The reactors can be engineered or manufactured based on the growth requirements of the organisms used. The bioreactor is provides an environment for microorganisms to obtain optimal growth and produce metabolites for the biotransformation and bioconversion of substrates into desirable products. They can be used for the production of various enzymes and other bio- catalysis processes. Bioreactor design The design and mode of operation of a bioreactor are based on the o Production of an organism o Optimum conditions required for desired product formation o Product value o Its scale of production. A good bioreactor design will help to improve productivity and provide higher quality products at lower prices. The essential guidelines in bioreactor design are: 1. The bioreactor should be designed to exclude entrance of contaminating microorganisms as well as containing the desired organisms. 2. The culture volume should remain constant (no leaking or evaporation). 3. The dissolved oxygen level must be maintained above critical levels of aeration and culture agitation for aerobic organisms. 4. The material used for the construction of a bioreactor must have the following important properties: o It should not be corrosive. o It should not add any toxic substances to the fermentation media. o It should tolerate the steam sterilization process. o It should be able to tolerate high pressure and resist pH changes. 5. Environmental parameters such as temperature, pH, etc., must be controlled; and the culture volume must be well mixed. 6. The sizes of the bioreactor vary widely depending on the application. Parts of the bioreactor and their function 1. Fermenter Vessel A fermenter is a large cylinder closed at the top and bottom connected with various pipes and valves. The vessel is designed in such a way that it allows to work under controlled conditions. Glass and stainless steels are two types of fermenter vessels used. The glass vessel is usually used in small-scale industries. It is non-toxic and corrosion-proof. Stainless steel vessel is used in large scale industries. It can resist pressure and corrosion.  2. Heating and cooling apparatus The fermentor vessel’s exterior is fitted with a cooling jacket that seals the vessel and provides cooling water. Thermostatically controlled baths or internal coils are generally used to provide heat while silicone jackets are used to remove excess heat. A cooling jacket is necessary removal of the heat generated during fermentation in the fermentor. 3. Aeration system An aeration system is one of the very important parts of a fermentor. It contains two separate aeration devices (impeller and sparger) to ensure proper aeration in a fermentor. Impellers are used to provide uniform suspension of microbial cells in different nutrient mediums. Impeller blades attached to a motor on the lid.  A sparger is a system used for introducing sterile air to a fermentation vessel. It helps in providing proper aeration to the vessel. 4. Feed ports They are used to add nutrients and acid/alkali to the fermentor. Feed ports are tubes made up of silicone. In-situ sterilization is performed before the removal or addition of the products. 5. Foam control The level of foam in the vessel must be minimized to avoid contamination, this is an important aspect of the fermentor. Foam is controlled by two units, foam sensing, and a control unit. A foam-controlling device is mounted on top of the fermentor, with an inlet into the fermentor. 6. Valves Valves are used in the fermentor to control the movement of liquid in the vessel. 7. Controlling devices for environmental factors A variety of devices are utilized to control environmental elements like temperature, oxygen concentration, pH, cell mass, essential nutrient levels, and product concentration. 8. Use of computer in fermenter For an efficient process, monitoring, and data collecting, fermentors are generally coupled with modern automated and semi-automated computers and database. Different shapes of bioreactors Structure of the bioreactor system has not changed for many years but recently, novel forms have been developed to suit the needs of specific bioprocesses. Stirred tank fermentor It consists of the motor driveshaft and a variable number of impellers (more than one). The impellers have a 1/3rd diameter of the vessel. The height and diameter ratio of this bioreactor is between 3:5. Air passes into the culture medium through a single orifice from the tube attached externally. It enables better distribution of the contents throughout the vessel.  Its basic functions include: 1. Homogenization 2. Suspension of solid material 3. Aeration to the medium 4. Heat exchange In stirred tank fermentor, rotating stirrer and baffle are found either at the top or the bottom. It mainly uses the batch process of fermentation. Airlift Fermentor It consists of a single container inside which a hollow tube is present. This hollow tube called ‟Draft tube”. There is a gas flow inlet present at the bottom of the fermentor, which allows the passage of oxygen. Gas flow inlet is attached with the perforated disc or tube that allows continuous distribution of air.  This type of bioreactor lacks the mechanical stirring arrangements for agitation. As from the name airlift, it is clear that the air lifts the medium upwards. There are internal liquid circulation channels, which enable continuous circulatory motion of the medium. Here, the fermentation occurs at a fixed rate of volume and circulation. Fluidized bed fermentor The top portion of this bioreactor is more expanded. This expansion reduces the velocity of the fluid. Its bottom part is slightly narrow.  It is designed in such a way where: The solid retain inside the vessel. Liquid flows out. An adequate amount of gas introduces into the medium to form a suitable gas-liquid-solid fluidised bed. By using this kind of biofermentor one should note that the suspended particles should not be too light or too heavy and secondly, there should be continuous recycling of the medium for good bioprocessing. Packed bed fermentor It consists of a cylindrical vessel and a packed bed with biocatalysts. The solid matrix that is used for the packed bed fermentor possess the following properties: 1. Porous or non-porous 2. Highly compressible 3. Rigid In this kind of biofermentor, a nutrient broth continuously flows over the immobilized biocatalyst. After that, a product releases into the fluid at the bottom of the culture vessel and finally, it can be removed. Here, the flow of fluid can be upward or downward. Photo fermentor This fermentor works under the principle of light energy that involves direct exposure to the sunlight or through some artificial illumination. It is extensively used for the production of p- Carotene, astaxanthin etc. This type of bioreactor is basically made of glass or plastic. Photo fermentor consists of: 1. A single container 2. Number of tubes or panels  Tubes act as “Solar light receivers or trappers”. In this, culture transfers through solar trappers with the help of a centrifugal pump. Inside photo fermentor, adequate penetration of sunlight is maintained and after that, it is cooled when there is a rise in temperature. Here, the microalgae and cyanobacteria are the common microorganisms that are used. These microorganisms grow in the presence of solar light and then product forms during the night. Bioreactor monitoring and control Bioreactor control measurements are made in either an on- line or an off- line manner. In on- line measurement the sensor is placed directly with the process stream. In an off- line measurement a sample is removed aseptically from the process stream and analyzed. Bioreactor processing is limited by a shortage of reliable instruments capable of on- line measurement of important variables such as DNA, RNA, enzymes and biomass. Off- line analysis is essential for these compounds, but the results cannot be obtained until several hours after sampling. Therefore, they cannot be used for immediate control purposes. However, on- line measurement is readily available for temperature, pH, dissolved oxygen and CO2 analysis.