Industrial Biotechnology Lecture Notes PDF

**Industrial biotechnology** Industrial biotechnology is a branch of biotechnology that applies microbial sciences to create industrial products in mass quantities. There are multiple ways to manipulate a microorganism in order to increase maximum product yields. Introduction of mutations into an o...

**Industrial biotechnology** Industrial biotechnology is a branch of biotechnology that applies microbial sciences to create industrial products in mass quantities. There are multiple ways to manipulate a microorganism in order to increase maximum product yields. Introduction of mutations into an organism may be accomplished by introducing them to mutagens. Another way to increase production is by gene amplification, this is done by the use of plasmids, and vectors. - The manipulation of organisms in order to yield a specific product has many applications to the real world like the production of some antibiotics, vitamins, enzymes, amino acids, solvents, alcohol and dairy products. - They can also be used in an agricultural application and use them as a biopesticide instead of using dangerous chemicals or as inoculants and help plant proliferation. - Industrial biotechnology came into existence, primarily, based on a naturally occurring microbiological process called fermentation. Despite the necessity for team work, the microbiologist has a central and key role in his organization. Some of his functions include: a\) the selection of the organism to be used in the processes; b\) the choice of the medium of growth of the organism; c\) the determination of the environmental conditions for the organism's optimum productivity i.e., pH, temperature, aeration, etc. d\) during the actual production, the microbiologist must monitor the process for the absence of contaminants, and participate in quality control to ensure uniformity of quality in the products; e\) the proper custody of the organisms usually in a culture collection, so that their desirable properties are retained; f\) the improvement of the performance of the microorganisms by genetic manipulation or by medium reconstitution. Industrial fermentations comprise both **upstream processing** (USP) and **downstream processing** (DSP) stages, Figure 1. USP involves all factors and processes leading to the fermentation and consists of three main areas. 1\. Producer microorganism- suitable microorganism, strain improvement. 2\. Fermentation media- cost-effective carbon and energy sources, essential nutrients and optimization. 3\. Fermentation- growth of the organism or the production of a target microbial product under rigorously controlled conditions. **DSP** encompasses all processes following the fermentation. It has the primary aim of efficiently, reproducibly and safely recovering the target product to the required specifications (biological activity, purity, etc.), while maximizing recovery yield and minimizing costs. · It is the collective term for the processes that follows fermentation i.e. a\. cell harvesting b\. cell disruption c\. product purification from cell extracts or the growth medium Figure 1: Outline of Upstream and Downstream processing operations **Fermentation process** The definition of fermentation changes with reference to the context i.e. · In case of metabolism, fermentation refers to energy generating processes where organic compound acts as both electron donor and acceptor. · In context to industrial biotechnology, fermentation is defined as the process by which large quantities of cells are grown under aerobic or anaerobic conditions. The scaling of the aerobic processes also is more complicated than anaerobic processes. · The industrial microorganisms are grown under controlled conditions with an aim of optimizing the growth of the organism or production of a target microbial product. · Fermentation is carried out in vessels known as Fermenters. The types of fermenter ranges from simple tank to complex integrated system of automated control. The advantages in producing materials by fermentation are as follows: 1\. Complex molecules such as antibiotics, enzymes and vitamins are impossible to produce chemically. 2\. Optically active compounds such as amino acids and organic acids are difficult to prepare chemically. 3\. Though some of the products that can be economically derived by chemical processes, but for food purpose they are better produced by fermentation such as beverages, ethanol and vinegar (acetic acid). 4\. Fermentation usually uses renewable feed stocks instead of petrochemicals. 5\. Reaction conditions are mild, in aqueous media and most reaction steps occur in one vessel. 6\. Byproducts of fermentation are usually chemicals. The cell mass and other major by products are highly nutritious and can be used in animal feeds. However, it is beset with some drawbacks, which are as follows: 1\. The products are made in complex solutions in low concentrations as compared to chemically derived compounds. 2\. It is difficult and expensive to purify the product. 3\. Microbial processes are much slower than chemical processes, increasing the fixed cost of the process. 4\. Microbial processes, are subjected to contamination by competing microorganisms, requires the sterilization of the raw materials and the containment of the process to avoid contamination. 5\. Most microorganisms do not tolerate wide variation in temperature, pH and are also sensitive to upsets in the oxygen and nutrient levels. Such upsets not only slow the process, but fatal to microorganism. Thus careful control of pH, nutrients, air and agitation require close monitoring and control. 6\. Although nontoxic, waste products have high BOD and requires extensive sewage treatment. Some of the products such as ethanol, lactic acid and cell mass products are generally growth associated, while secondary metabolites, energy storage compounds, and polymers are non-growth associated. Primary metabolites are the essential compounds that are directly involved in the normal growth, development, and basic life-sustaining functions of an organism. They are produced during the primary metabolism, which is the set of chemical reactions that are necessary for the survival and reproduction of the organism. *Primary metabolism* refers to the set of essential biochemical pathways and processes that produce the fundamental, life-sustaining compounds necessary for the normal growth, development, and basic functions of an organism. *Secondary metabolism* refers to the set of biochemical pathways and processes that produce compounds known as secondary metabolites, which are not directly involved in the normal growth, development, or reproduction of an organism. Secondary metabolites have valuable applications, such as in the production of drugs, pesticides, and other industrial compounds. Note that the fermentation process is divided into a number of sections: · in-bound logistics which involves the delivery and storage of raw materials, · upstream processing which involves the processing of raw materials for the fermentation, · the fermentation, where the major conversion occurs, · downstream processing, which involves the purification and concentrating of the raw materials, · outbound logistics which involves the final packaging, storage and delivery of the purified product. In developing a product, a fermentation technologist must consider all stages of the manufacturing process. **Types of Fermentation Process** Fermentation in liquid media is of two types depending upon the mode of operation: A. Batch Fermentation B. Continuous Fermentation **Batch reactors** are simplest type of mode of reactor operation. In this mode, the reactor is filled with medium and the fermentation is allowed to proceed. When the fermentation has finished the contents are emptied for downstream processing. The reactor is then cleaned, re-filled, re-inoculated and the fermentation process starts again. Batch processes are dynamic processes that are never in a steady state. Often in batch processes the critical parameter is gas exchange or balance between respiration rate and oxygen transfer. In batch operation the sterilized media components are supplied at the beginning of the fermentation with no additional feed after inoculation. When cells are grown in a batch reactor, they go through a series of stages known as (Figure 2) · Lag phase · Exponential phase · Stationary phase · Death phase  Figure 1: Growth of a microorganism in a batch culture *Lag Phase* · In lag phase the microbial population remains constant as there is no growth. However, it is the period of intense metabolic activity. Factors Influencing the Lag Phase · Chemical composition of the fermentation media influences the length of the lag phase. Longer lag phase is observed if the inoculum is transferred into a fresh medium of different carbon source as that of the medium in which the cells are grown. This is because the cells need to induce the enzymes required for the metabolism of the new substrate. · Age of the inoculum. If the inoculum is in exponential growth phase, it will exhibit shorter lag in the fresh medium. · Concentration of the inoculum · Viability and morphology of the inoculum *Exponential Phase* · Cell divides with increasing frequency till it reaches the maximum growth rate (*μ~max~*) · At this point logarithmic growth begins and cell numbers or cell biomass increase at a constant rate. *Stationary Phase* · The specific growth rate of the microorganism continues decelerating until the substrate is completely depleted. · Overall growth rate has declined to zero and there is no net change in cell numbers/ biomass i.e. rate of cell division equals rate of cell death. · Microorganisms are still metabolically active, metabolizing intracellular storage compounds, utilizing nutrients released from lysed cells and in certain cases produce secondary metabolites. *Death Phase* · Cells die at constant rate and often undergo lysis. **Continuous reactors**: Fresh media is continuously added and bioreactor fluid is continuously removed. As a result, cells continuously receive fresh medium and products and waste products and cells are continuously removed for processing. The reactor can thus be operated for long periods of time without having to be shut down. Continuous reactors can be many times more productive than batch reactors. This is partly due to the fact that the reactor does not have to be shut down as regularly and also due to the fact that the growth rate of the bacteria in the reactor can be more easily controlled and optimized. In addition, cells can also be immobilized in continuous reactors, to prevent their removal and thus further increase the productivity of these reactors. Continuous reactors are as *yet not widely used in industry* but *do find major application in wastewater treatment*. **Fed batch reactor** is the most common type of reactor used in industry. In this reactor, fresh media is continuous or sometimes periodically added to the bioreactor but unlike a continuous reactor, there is no continuous removal. The fermenter is emptied or partially emptied when reactor is full or fermentation is finished. As with the continuous reactor, it is possible to achieve high productivities due to the fact that the growth rate of the cells can be optimized by controlling the flow rate of the feed entering the reactor. **Anaerobic Fermentation:** Where a fermentation process carried out in the absence of oxygen. There are two types of anaerobic microorganisms viz, obligate anaerobic microorganisms and facultative anaerobic microorganisms. Anaerobic conditions in the fermenter are created either by withdrawing the oxygen present in the head space by an exhaust pump and pumping some inert gases like nitrogen, argon etc. or by flushing it out, by the emergence of certain gases like carbon dioxide or hydrogen. **Aerobic Fermentation:** Where a fermentation process carried out in the presence of oxygen. In most of the commercial processes and majority of the products of human utility are produced by this type of fermentation. **Surface Fermentation:** where the microorganisms involved in the fermentation are grown on the surface of a solid or semi-solid medium rather than being submerged in a liquid medium. This method of fermentation is commonly used in the production of various food and industrial products, such as cheeses, vinegar, and certain antibiotics. **Submerged Fermentation:** Where the microorganisms involved in the fermentation are grown in a liquid medium, rather than on a solid or semi-solid surface. The culture conditions are made uniform with the help of sparger and impeller blades. **Solid Substrate/State Fermentation (**SSF)**:** It is generally defined as the growth of the microorganism on moist solid materials in the absence or near the absence of free water. For large-scale SSF bioprocess, three types of fermenters are in operation: \(a) Drum Fermenter, Figure 3. \(b) Tray Fermenter, Figure 4. \(c) Column Fermenter, Figure 5.  Figure 3: Drum Fermenter Figure 4: Tray fermenter  Figure 5: Column fermenter. **Mathematical Expression of Growth** It can be based on cell mass (x) or cell number (N). **Rate of Cell Growth based on Cell Biomass** Rate of change of biomass at a given time is.............(1).............(2) Where: *x* = concentration of biomass (g/l, or kg/m^3^) *μ* = specific growth rate (h^-1^) *t* = time (h) Integrating equation Eq. (1) leads to *x~t~ = x~o~ e ^μ\ t^*............(3) or ln *x~t~* = ln *x~o~* + *μt*..........(4) or *μ* =.............(5) where: *x~t~* = biomass concentration after time *t* *x~o~* = biomass concentration at the start of exponential growth For cells in exponential phase, a plot (ln *x*) against time, should yield a straight line with the slope (gradient) equal to *μ*, and the intercept = ln *x~o~*. **Growth Rate Constant based on Cell Number** If the cell population at the time of starting of exponential growth is 1, i.e. N~o~ =1, then undergoing binary fission the number of cells after time *t* becomes *N~t~*. The value of *N~t~* is given by the equation: *N~t~ =* 2^n^ *N~o~*.............(6) *N*~o~ = initial population size *N* = Number of generation or divisions Taking natural logarithms gives ln *N~t~ =* ln *N~o~* + *n* ln 2............(7) Number of divisions (*n*) is given by *n =*............(8) Growth rate or division rate constant is the average number of generations per hour. *K = n/t =*...........(9) **Calculation of Doubling Time on the Basis of Cell Biomass** If we consider the initial cell biomass to be *x~o~*, and after time *t*, the microbial biomass doubles i.e. *x~t~* = 2*x~o~* when *t* is the doubling time *t~d~*, Substituting these parameters into equation (*x~t~ = x~o~ e ^μ\ t^*) → 2*x~o~ = x~o~ e ^μ\ td^*.............(10) And *t~d~* =............(11) In the above growth calculation, we consider the growth to be indefinite, however in batch fermentations the nutrient provided is finite which will eventually be depleted. The implication on growth is that growth decreases and eventually stops. Monod showed that growth rate is an approximate hyperbolic function of the concentration of the growth limiting nutrient(s). In industrial processes we need to know the generation time or doubling time rather than division rate constant. Generation time or doubling time is the average time for population to double or time taken for one division. *t~d~* = *t/n =* 1*/K* = **Growth Parameters for Process Optimization** During the development of a batch process, key growth parameters can be determined that enable the production of a given microbial product to be optimized, whether it is the biomass itself or a specific metabolite. *Yield Coefficient* Yield coefficient (Y), is determined on the basis of the quantity of rate- limiting nutrient, normally the substrate converted into the microbial product. · In case of biomass production, the yield coefficient relates to the quantity of biomass produced per gram of substrate utilized and is depicted by the equation *x* = *Y~x~*~/s~ (*S -- S~r~*).............(12) where *x* = biomass concentration (g/L), *Y~x/S~* = yield coefficient (g biomass/g substrate utilized), *S* = initial substrate concentration (g/L), and *S*~r~ = residual substrate concentration (g/L) Therefore, the higher is the yield coefficient, the greater the percentage of the original substrate converted into microbial biomass. · In case of microbial metabolic products (*p*) the yield coefficient is related to the quantity of metabolite produced in relation to the quantity of substrate used (*Y~p/S~*). Determination of yield coefficient is important as it will decide how productive and how cost viable is the medium used. **Specific Growth Rate (*μ*) and Maximum Specific Growth Rate (*μ~max~*)** Determination of *μ* and *μ~max~* is important where product formation is related to growth i.e. it is a primary metabolite. To optimize the overall productivity of the system, the microorganism must usually be grown at its maximum potential. The operating substrate concentration has a major effect on the growth rate of a microorganism. By performing a series of batch fermentations, each with a different initial concentration of the limiting substrate, the specific growth rate (*μ*) for each experiment can be determined. These data can then be used to estimate both *μ~max~* and saturation constant (*K~s~*) by simply taking the reciprocal values in Monod equation............(13) Reciprocal of this Eq.:............(14) A plot of *μ~max~* against l/s should produce a straight line with the intercept on the *y*-axis at 1*μ~max~*.  *Ks* = saturation constant, i.e. concentration (g/L) of limiting nutrient enabling growth at half the maximum specific growth rate, i.e. *μ* = 1/2 *μ~max~* and is a measure of the affinity of the cells for this nutrient. **Continuous Fermentation** The concept of control in fermentation has its roots in the operation of chemostat, in which a continuous fermentation process is designed so that one nutrient will be limiting or controlling. Initially, continuous fermentation starts as batch cultures. When the batch culture reaches the exponential growth phase, it can be extended indefinitely by continuous addition of fresh fermentation medium. A **chemostat** is a type of bioreactor or continuous culture system used in microbiology, biochemistry, and biotechnology to maintain a steady-state culture of microorganisms or cells under controlled environmental conditions, Figure 5.  As with batch fermentations, the specific rate at, which the microorganism grows in continuous culture is controlled by the availability of the rate-limiting nutrient. Therefore, the rate of addition of fresh medium controls the rate at which the microorganisms grow. However, the actual rate of growth depends not only on the volumetric flow rate (*F*) of the medium into the reactor, but also on the dilution rate (*D*). This equals the number of reactor volumes (*V*) passing through the reactor per unit time and is expressed in units of reciprocal time, per hour..............(15) The term *D* is the reciprocal of the mean residence time or hydraulic retention time (h^-1^). Addition of fresh medium into the reactor can be controlled at a fixed value, therefore the rate of addition of the rate-limiting nutrient is constant. Within certain limits, the growth rate and the rate of loss of cells from the fermenter will be determined by the rate of medium input. Therefore, under steady state conditions the net biomass balance can be described as.............(16) Under steady-state conditions, and *μ = D*...........(17) For any given dilution rate under\_steady-state condition, the residual substrate concentration (*s~r~*) in the reactor can be predicted by substituting *D* for *μ* in the Monod equation ()...........(18) →.........(19) Consequently, the residual substrate concentration in the reactor is controlled by the dilution rate. Any alteration to this dilution rate results in a change in the growth rate of the cells that will be dependent on substrate availability at the new dilution rate. A **turbidostat**, where nutrients in the medium are not limiting. The specific growth rate of a turbidostat culture is at or very close to *µ~max~* and is controlled by the rates of **internal** cellular reactions as they are expressed in the optical density of the culture biomass (that is, the turbidity of the culture). In this case, turbidity or absorbance of the culture is monitored and maintained at a constant value by regulating the dilution rate, Figure 6.