Lecture notes on biochemical engineering

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Fundament Fundame Fundamen ntals of Bioche tals of Biochem als of Biochem mical Enginee ical Enginee ical Engineering ring ring Subject code Subject code-PCCH 4402 Chemical Engineering Department Chemical Engineering Department Chemical Engineering Department th 7 Semester Mrs. Ipsita D. Behera Mrs. Ipsita D. Behera Asst. Professor Asst. Professor I.G.I.T Sarang I.G.I.T Sarang Email: - ipsitadbeheragmail.com eragmail.comDisclaimer: This document does not claim any originality and cannot be used as a substitute for prescribed textbooks. The information presented here is merely a collection by the committee faculty members for their respective teaching assignments as an additional tool for the teaching-learning process. Various sources as mentioned at the reference of the document as well as freely available material from internet were consulted for preparing this document. The ownership of the information lies with the respective authors or institutions. Further, this document is not intended to be used for commercial purpose and the committee faculty members are not accountable for any issues, legal or otherwise, arising out of use of this document. The committee faculty members make no representations or warranties with respect to the accuracy or completeness of the contents of this document and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose.SYLLABUS Module I Overview of microbiology, Aerobic & Anaerobic fermentation processes, fermenter design, sterilization of microbial medium, kinetics of microbial growth, enzymes and its kinetics, immobilization of enzymes, chemostats. Module II Transport phenomena in Biochemical Engineering, Heat and Mass transfer in Bioprocessing, oxygen transfer in fermenter, monitoring and control of fementation process. Module III Downstream processing: - Recovery and Purification of products, allied unit operation for product recovery, production of biogas and ethanol, Effluent treatment by biological method Text book 1. Bailey JB and oillis OR, Biochemical Engineering Fundamentals 2. Aiba S, Biochemical Engineering, Academic press 3. Rao D G, Introduction to Biochemical Engineering, Tata Mc Grow Hill 4.Michael L. Shuler/ Fikret Kargi, Bio Process Engineering , Pearson EducationLESSON PLAN Class No. Brief description of the Topic 1. Introduction of FBE 2. Overview of microbiology 3. Application of Microbiology 4. Aerobic & Anaerobic fermentation processes 5. SSF and SmF and applications 6. Fermentation Design 7. Microbial sterilization 8. kinetics of microbial growth 9. Kinetics of microbial growth 10. Problem discussion 11. Problem discussion 12. Enzyme and its characteristics 13. Enzyme kinetics 14. Problem discussion 15. Immobilization of enzymes & applications 16. Chemostats 17. Question Discussion 18. Heat transfer in bioprocessing 19. Mass transfer in bioprocessing 20. Oxygen transfer in fermenter 21. Monitoring and control of fementation process.22 Question discussion 23 Unit operation for product recovery 24 Effluent Treatment Physical methods 25 Effluent Treatment Chemical methods 26 Effluent Treatment Biological methods 27 Production of biogas 28 Production of ethanol 29 Question discussions 30 Semester question discussionFundamentals of Biochemical Engineering 1 Module-I INTRODUCTION Biotechnology is the art and science of converting reactants (substrate) into useful products by the action of microorganisms or enzymes. Microorganisms have been honestly serving the mankind. Thus any process in which microbes or living organisms play a vital role in getting transformation of the feed into useful products is termed as BIOPROCESSING. For example the way of converting milk into curds, or fruit juice into wines, or sugar into alcohol. BIOCHEMICAL ENGINEERING is of more recent origin, since the biological industries did not recognize the importance of engineering inputs until the experience of penicillin manufacture. Microbiology  (Micro-small, bios-life) is the study of microscopic organism, which are defined as any living organism i.e. either a single cell(unicellular), a cell cluster or has no cell at all(a cellular). This includes eukaryotes, such as fungi and protista and prokaryotes.  Microbiology is a broad term which includes virology, mycology, parasitology etc.  It is the study of microorganisms which are not only microscopic and exist as single cells, but also that ultramicroscopic organism which are not cellular and hence cannot exist independently. E.g. viruses  Microbiology deals with study and functioning of cells, their interaction with environment, other living organism and man.  It is studied with respect to two major aspects. a) As basic biological science b) As applied biological science Basic biological science: It provides a system to understand the nature of life processes, the principle behind it and the genetics which is involved in the heritance of traits to next generation.it has several sub streams such as i) Medical microbiology: Study of pathogenic microorganism, the causes of diseases and way to eliminate them. Fundamentals of Biochemical Engineering 2 ii) Agricultural microbiology: Study of plant diseases, understanding various beneficial interactions with plant system like soil fertility, crop-protection and increasing field. iii) Environmental microbiology: Study of relationship of microorganisms with its habitat, pollution effect and its impact on environment from the stand point of ecology balance and health. iv) Food and dairy microbiology: Study of microorganisms that produce various food and dairy products. Applied biological science: It deals with study of useful microorganisms as well as that of pathogenic organism. i) In dairy and food industry: Food microbiology not only includes the study of those microbes which provides food to their high protein content but also includes other those microbes which use our food supply as a source of nutrients for their growth and result in deterioration of the food by increasing their number, utilizing nutrients, contributing of flavours by means of breakdown of food. ii) Medical microbiology: Microbes causes infections resulting in diseases among human and animals. On other side they help in creating a “disease free world”, where people are from pain by this disease. Application of microbiology  Microbes in food and dairy industries  Microbes in production of industrial products  Microbes in genetic engineering and biotechnology  Microbes in environmental microbiology  Microbes in medical microbiology  Microbes in agriculture  Microbes in bioterrorism a. Food and dairy industry:  Provides food due to high protein content.  Food nutrient for their growth (deterioration of food), enzymatic changes, contributing flavour.  Certain moulds used for manufacturing of food and ingredients of food.  Some moulds used in production of oriental food, soya sauce etc. Fundamentals of Biochemical Engineering 3  Used for enzyme making like amylase.  Yeast: These are used in manufacturing of foods such as bread, beer, wines, vinegar, surface ripened, and cheese. Some yeasts are grown for enzymes and food.  Bacteria: Some pigmented bacteria cause changes in colour on the surfaces of liquid food. Acetobacteria oxidises ethyl alcohol to acetic acid. Some bacteria causes ropiness in milk and slimy growth cottage cheese. b. Microbes in production of industrial products:  Enzymes amino acid, vitamins, antibiotics, organic acid and alcohol are commercially produced by microorganism.  Primary microbial product: These products are used by microorganism for their growth. E.g. amino acids, enzymes, vitamins  Secondary microbial products: Not used by cell for their growth. E.g. alcohol, antibiotics, organics acids.  Microbes produce some important amino acids such as glomatic acid, lysine, and methionine. c. Microbes in Genetic Engineering and Biotechnology:  Microbes used for mammalian proteins such as insulin and human growth factor.  Making vaccine for microbial and viral genes and induce a new strain of microbes.  Vaccine and diagnostic kits also depend on the improved strains of microorganism.  Lactic acid as food preservative.  Acetic acid plays a major role in tanning and textile industry.  Interferons are produced in animal cell if included by viral infection. These are used in testing interleukins (which stimulate T-lymphocytes).  Production of viral, bacterial or protozoan, antigen for protecting against dysentery, typhoid, bacteria etc.  N fixing bacteria reduce nitrogen gas to ammonia required plant growth. 2 Fundamentals of Biochemical Engineering 4 d. Environmental Microbiology:  Plays an important role in recycling of biological elements such as oxygen, carbon, N sulphur, and phosphorous. 2,  Microbes in biochemical cycle: Photosynthesis and chemosynthesis microorganism convert CO into organic carbon. Methane is generated 2 anaerobically from CO and H by metheogenic archaea. The organic 2 2 forms of N are interconverted by metabolic activities of microorganism 2 which maintain the natural N balance. 2  Microbes in pollution microbiology: Biological sewage treatment and self-purification. Both results in mineralisation of organic, pollutants and in utilisation of dissolved oxygen. e. Medical Microbiology:  Vaccination: Small pox, diphtheria, whooping cough. f. Computer application:  Optimisation via computer: Computers are used on scale of, to store, evaluate effects of individual parameters on metabolic behaviour of culture.  Control via computer: Control fermentation process. g. Microbes in agriculture:  During compost formation by the crop residue like wheat straw, rice straw, sugar cane bagasse are very difficult to degrade due to presence of highly resistant lignified tissues. So, breakdown of these complex organic materials can be done by microbes by a short span.  Biogas production through anaerobic fermentation is must reverent to fulfil their energy demand in rural population.  The productivity of leguminous crop largely depend on efficient and suitable management of the ecosystem by specific rhizobial association.  Some bacterial helps in killing a wide range of insects like beetles, mosquitoes, flies, aunts, termites which is very useful for agricultural industries h. Microbes in Bioterrorism: It has been defined as deliberate release of disease causing germs, microorganism with the intent of killing large number of people. Accordingly microorganisms are used as weapons of mass destruction of people and causes small pox, plague, cholera and also anthrax. Fundamentals of Biochemical Engineering 5 Mode of transmission:  Air droplets and dust  Food fruits and vegetables  Drinking water Fundamentals of Biochemical Engineering 6 FERMENTATION Fermentation is the word derived from the Latin verb FERVERE (to boil), which describe the evolution of carbon dioxide bubble in anaerobic conditions by the action of yeast on fruit juice. General Requirements of Fermentation Process:- Since fermentation is a biochemical process brought about by the intervention of living organisms, it is essential that any fermentation process should have:  A microorganism for carrying out the bioconversion.  A substrate to get converted into useful products.  Maintenance of fermentation conditions.  Effluent treatment section.  A provision for recovery and purification of the products.  Facilities for packaging and marketing. Fermentation carried out in the presence of air / O is known as aerobic 2 fermentation, where as in absence of air is called anaerobic fermentation. Anaerobic Fermentation:-  Yeast fermentation process to produce alcohol requires a small amount of aeration for the cells to multiply. After word no air is required.  On the other hand air is detrimental for the process which will otherwise oxidise the substrate.  Most of the anaerobic fermentation produces carbon dioxide gas.  Many times gas covers the surface and acts as a blanket to prevent the effect of O 2.  The evolved CO will also help in better mixing conditions, which is more 2 evident in large industrial tanks because of longer pathways for the gas bubbles to go before they leave the fermenter. Aerobic Fermentation:- Sparaging air /O is very common phenomenon in fermentation process to 2 supply O for cells to meet their specific O demand .Such fermentation process 2 2 which are associated with the bubbling of O are termed as aerobic 2 fermentation. Fundamentals of Biochemical Engineering 7 Solid state and submerged fermentation and their Applications Solid state fermentation:- SSF is a method of growing microorganisms in an environment of limited moisture without having free flowing water. The microorganisms grow on a solid surface which is moistened and which has also got free access to air. It is also known as “ Koji” fermentation , for the production of soya products such as tempeh, soya sauce etc. certain metabolites. It has some advantages that lower manufacturing cost because of the use of crude solid agro wastes like wheat bran. The solid surface directly comes in contact with the air and hence the aeration costs are avoided. The other economic advantages are  Low capita investment and recurring expenditures.  Low water utilization and hence negligible outflow of water.  Low energy requirements for the fermentation process because of absence of agitation.  Absence of foam formation because of absence of excess water.  High reproducibility of the result.  Simple fermentation media.  Less fermentation space, and les complex plant and machinery.  Absence of rigorous control techniques.  Any level of scale of operation.  Absence of elaborate aeration requirements.  Ease in controlling bacterial contamination.  Facilities of using wet and dry fermented solids directly.  Ease in induction and suppression of spores.  Lower costs of downstream processing. Submerged Fermentation:- In case of submerged fermentation (SmF) the microorganisms and the substrate are present in the submerged state in the liquid medium, where a large quantity of solvent is present. This has many advantages over SSF.  Since the contents are in submerged state in the liquid medium, the transfer of heat and mass is more efficient, and is amenable for modelling the process.  The scaling-up the process is very easy. Fundamentals of Biochemical Engineering 8 Differences between SSF and SmF Characteristics feature SSF SmF Condition of microorganisms and static Agitated substrate Status of the substrate Crude Refined Nature of the microorganism Fungal system ______ Availability of water Limited High Supply of oxygen By diffusion By bubbling/ sparging Contact with oxygen Direct Dissolved O 2 Requirement of fermentation medium Small Huge Energy requirement Low High Study of kinetics Complex Easy Temp and concentration gradient Steep Smooth Controlling of reaction Difficult Easy Chances of bacterial contamination Negligible High Quantity of liquids to be disposed Low High Pollution problems Low high Applications:-  Citric acid can be produced by both SSF and SmF. Generally, the later technique is used in industrially. SSF method has not yet become commercial success because of its labour intensity.  Soya-based oriental foods like tempeh and soya sauce are produced by SSF alone.  Production of Roquefort cheese from sheep milk is a classical example of SSF.  Mushroom cultivation is another example of the growth of fungus on solid medium like paddy straw.  Fish and meat production are preserved in the form of sausages as fermented foods. Fundamentals of Biochemical Engineering 9 FERMENTER DESIGN A fermenter is a type of bioreactor for containing and controlling microorganisms during a fermentation process. BASIC FUNCTIONS OF FERMENTERS The main function of a fermenter is to provide a controlled environment for growth of a microorganism, or a defined mixture of microorganism, to obtain a desired product. (Bioreactors refer to production units of mammalian and plant cell culture) CRITERIA USED IN DESGINING AND CONSTRUCTING A FERMENTER-  Vessel should be capable of being operated aseptically and should be reliable for long term operation  Interplay of the transport parameters  Supply of adequate quantity of oxygen so that cells do not suffer from inadequacy of oxygen supply  Adequate aeration and agitation to meet the metabolic requirements of the microbes  Adequate amount of mixing should be ensured without causing damage to the cells  Vessel geometry should be such that it should facilitate scale-up  Flexibility in operation of the fermenter for various purposes, so that the vessel should be suitable for a range of processes  Low power consumption  Temperature and pH control  Low evaporation losses  Minimal use of labour in operation, harvesting, cleansing and maintenance  Proper sampling facility  Cheapest and best materials should be used  Adequate service provisions must be available for individual plants. Fundamentals of Biochemical Engineering 10 TYPES OF FERMENTER- Based on shape it can be classified as- (i) Tabular & (ii) Stirred tank (Cooling coils are provided to maintain constant temperature inside the bioreactor. It can be operated aseptically for many days and simple in construction. Disadvantages-  high power requirement  shearing on the organisms caused by vigorous agitation and inhibition exercised by the product) (i) Fluidized Bed Bioreactor: - It is more popular in chemical industry rather new to biochemical industry. These are mostly used in conjuction with immobilized cells or enzyme system and are operated continuously. (ii) Loop or Air Lift Bioreactor: - In the conventional bioreactor, oxygen is supplied by vigorous agitation of the bioreactor content. The heat is generated which is a problem in conventional type. In this cooling becomes simpler due to the position of inner or outer loop. (iii) Membrane Bioreactor: - These consist of a semipermeable membrane made up of cellulose acetate or other polymeric materials. The primary purpose of the membrane is to retain the cells within the bioreactor, thus increasing their density, while at same time allowing metabolic products to pass through the membrane. (iv) Pulsed Column Bioreactor: - It has a column bioreactor generator connected to the bottom of the column. It can be utilised as an aerobic bioreactor, enzyme bioreactor or as separation unit. (v) Bubble Column Bioreactor: - Multistage bubble column bioreactor are suitable in the equivalent batch process. In this it is possible to provide different environmental conditions in various stage. It is not suitable for fungal fermentation due to oxygen demanding system. (vi) Photo Bioreactor: - For the growth and production of photosynthetic organisms, a light source is required. In this, there is an important ‘reactant’, the photons which must be absorbed in order to react and produce products. The design of the light source is critical in the Fundamentals of Biochemical Engineering 11 performance of this type of bioreactor. Example- Annular Reactor. In this source of radiation is a cylinder with a annular section, which enclose the lamp completely. The nutrient passing from the product is removed from the top. It is used for Spirulina(SCP) and other algal protein production. (vii) Packed tower Bioreactor: - It consists of cylindrical column packed with an inert material like wood shavings, twigs, cake, polythene or sand. Initially, both medium and cells are fed into the top of the packed bed. Once the cells adhered to the support and were growing well as a thin film fresh medium is added at the top of the packed bed and the fermented medium removed from the bottom of the column. It is used for vinegar production, sewage effluent treatment and enzymes conversion of penicillin to 6-amino penicillanic acid. The design of fermenter involves the co-operation between experts in microbiology, biochemistry, mechanical engineering and economics. CONSTRUCTION OF FERMENTERS The criteria considered before selecting materials for construction of a fermenter are: (a) The material that have no effect on sterilisation (b) Its smooth internal finish-discouraging lodging of contamination. (c) Internal surface should be corrosion resistant. There are two types of such materials: (i) Stainless Steel, and (ii) Glass. The construction of the fermenter depends upon the following- (i) Control of Temperature. Since heat is produced by Microbial Activity and the mechanical agitation, thus it is sometimes necessary to remove it. On the other hand, in certain processes extra heat is produced by using thermostatically controlled water bath or by using internal heating coil or jacket meant for water circulation. Fundamentals of Biochemical Engineering 12 (ii) Aeration and Agitation. The main purpose is to provide oxygen require to the metabolism of microorganisms. The agitation should ensure a uniform suspension of microbial cells suspended in nutrient medium. There are following necessary requirements for this purpose: (a) The agitator (impeller) for mixing: The size and position of the impeller in the vessel depends upon the size of the fermenter. More than one impeller is needed if adequate aeration agitation is to be obtained. Ideally, the impeller should be 1/3 or 1/2 of the vessel diameter (D) above the base of the vessel. The number of impeller may vary from size to size to the vessel. (b) Stirrer glands and bearings meant for aseptic sealing: Four basic types of seals assembly have been used-  The packed gland seal  The simple bush seal  The mechanical seal and  The magnetic drive. (c) Baffles for checking the vortex resulting into foaming: The baffles are incorporated into the agitated into the agitated vessels to prevent a vortex ant to improve aeration efficiency. They are metal strips roughly one-tenth of the vessel diameter and attached radially to the walls. Fundamentals of Biochemical Engineering 13 (d) The sparger (aeration) meant for introducing air into liquid: A sparger is a device for introducing air into the liquid into a fermenter. It is important to know whether the sparger is to be used on its own or with mechanical agitation as it can influence equipment design to determine initial bubble size. Three basic types of sparger are: (i) Porous sparger (ii) Orifice sparger (iii) Nozzle sparger (e) Microbial sensors: It consists of a microorganism immobilized on a membrane and an electrode. The principle of working is the change in respiration or the amount of metabolites produced as a result of the assimilation of substrate by the microorganism. A wide range of thermophilic microbes have been used for the manufacturing of microbial sensors as mentioned in the table below. Immobilised yeast, Trichosporoncutaneumhas been used to develop an oxygen probe for BOD estimation in sewage and other water samples. The BOD sensor includes an oxygen electrode that consists of a platinum cathode and an aluminium anode bathing in salt KCl solution and a Teflon membrane. Immobilised yeast cells are crapped between the pores of a porous membrane and the Teflon sensor can measure BOD at 3-60/mg/litre. Methanotrohic bacteria is used in measuring methane as well as oxygen. Similarly, ammonia and nitrate biosensors consist of immobilised nitrifying bacteria. This is used to determine ammonia in waste water based on the conversion of nitrate to N O by an immobilised denitrifying Agrobacterium sp.The nitrate 2 biosensor has been used to measure nitrate profiles in biofilm. APPLICATIONS: Microbial biosensors have several uses in:  clinical analysis,  general health care monitoring,  veterinary and agricultural applications,  industrial product processing,  monitoring and control of environment pollution and  in military and defence for detection of chemical and biological species used in weapons. Fundamentals of Biochemical Engineering 14 DESIGN AND OPERATION The basic purpose of design of a fermenter or bioreactor is to visualise the size of the unit to deliver the product both qualitatively and quantitatively. After the size is designed, the next task is to achieve the transport properties i.e;  Fluid mechanics  Heat transfer  Mass transfer effects.  Fermenters are designed to provide support to best possible growth and biosynthesis for industrially important cultures ant to allow ease of manipulation for all operations associated with the use of fermenters.  These vessels must be strong enough to resist the pressure of large volume of agitating medium.  The product should not corrode the material nor contribute toxicity to the growth medium. This involves a meticulous design of every aspect of the vessel parts and other openings, accessories in contact, etc.  In fermentations, provisions should be made for the control of contaminating organisms, for rapid incorporation of sterile air into the medium in such a way that the oxygen of air is dissolved in the medium and therefore, readily available to the microorganisms and CO produced 2 from microbial metabolism is flushed from the medium. Fundamentals of Biochemical Engineering 15  Some stirring devices should be available for mixing the organisms through the medium so as to avail the nutrients and the oxygen.  The fermenter has a possibility for the intermittent addition of antifoam agent.  Some form of temperature control efficient heat transfer system is also there for maintaining a constant predetermined temperature in the fermenter during the growth of organism.  The pH should be detected.  Other accessories in the fermenter consist of additional inoculum tank or seed tank in which inoculum is produced and then added directly to the fermenter. Media Design Any fermentation process proceeds through the action of microorganisms which perform in the presence of a medium. Hence, proper design of the medium is an essential component in the design of a fermentation process. Thus, detailed investigations are needed to identify the most suitable medium for any fermentation process to proceed. Medium Requirements Since the medium is desired to support the functioning of microorganisms, the requirements of the medium are decided by those of the microorganisms. They are: • carbon • nitrogen • energy source • minerals • other nutrients like vitamins, etc. • oxygen/air for aerobic processes. • water. The medium used in a laboratory-scale process, or for that matter even in the pilot plant-scale level, can be reasonably composed of pure components; but such a luxury is not affordable in the case of commercial production, where the cost of production rules the economic viability, and hence the commercial viability of the process. Thus, for large-scale productions, we look for a medium, which has the following attributes: • It should be cheap, and easily available at a consistent cost and quality.

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