LG_Module 2 Lesson 1 PDF - Microbial Growth & Nutritional Requirements

For instructional purposes only 1st Semester SY 2020-2021 19 Module 2: Microbial Growth and Nutritional Requirements Module Overview In this module, we primarily discuss microbial gro...

For instructional purposes only 1st Semester SY 2020-2021 19 Module 2: Microbial Growth and Nutritional Requirements Module Overview In this module, we primarily discuss microbial growth, modes of microbial culture and the nutritional requirements of industrial microbial cultures. Motivation Question How can efficiently grow microbes to overproduce metabolic products of economic value? Module Pretest Instruction(s): Answer each question briefly. Limit your discussions with less than 100 words per question. 1. What are the phases of microbial growth? Illustrate and briefly discuss each phase. 2. Differentiate a batch culture from a continuous culture. 3. List and discuss essential nutrient requirements of microbial cultures. Page 19 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  20 Btec22: Fundamentals of Bioprocessing Lesson 2.1: Microbial Growth Lesson Summary This lesson comprise a comprehensive review of microbial growth and the different approaches to the culture of microorganisms for industrial applications along with the benefits and problems associated with each approach. Learning Outcomes At the end of the lesson, students would be able to; 1. Understand the concept and phases of microbial growth. 2. Differentiate the modes of microbial culture and understand how microbial growth occurs in batch and continuous cultures. 3. Identify advantages and limitations of batch and continuous cultures and discern appropriate mode for the efficient production of a specific metabolite. Motivation Question How fast and efficient are microbes in growth and production of important metabolites in industrial bioprocesses? Discussion Introduction For many industrial processes that depend upon microbial biocatalysts, there are two central issues that must be addressed in order for the process to become economically viable. First, one must find the right culture conditions for efficient biocatalysis. Second, one must find the right microbe for the job — an organism that has the appropriate genotype to perform the particular biocatalytic task of interest. These two considerations — culture conditions and microbial genotype — are intimately related and, often, one must be altered to accommodate the other in order to get a biocatalyst to grow vigorously under the conditions that are required for process optimization. In this lesson, different approaches to the culture of microorganisms for industrial applications along with the benefits and problems associated with each approach are discussed. Page 20 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  For instructional purposes only 1st Semester SY 2020-2021 21 Definitions of Growth The most basic definition of growth is based on the ability of individual cells to multiply, that is, to repetitively initiate and complete cell and organismal division. This definition implies monitoring the increase in total number of discrete bacterial particles. There are three basic ways to do this: by microscopic enumeration of the particles, by electronic enumeration of the particles passing through an orifice (Coulter counter), and by modern flow cytometry. Assessment of particle number would falsely include dead cells and detritus, which would tend to lower estimations of growth rate. The rate would be artificially raised by the progressive dissolution of aggregates of bacteria and the fragmentation of non-growing filamentous organisms. An increase in cell number is not exactly correlated with an increase in biomass or useful product. Commonly, at the end of an exponential growth phase, cell division overshoots biomass production and the cells become smaller. A second definition of growth involves determining the increase in colony-forming units (CFU). Because some cells may be dead or dying, this definition of growth may be different from the one based on the detection of discrete particles as a function of time. Although in the long run, the increase in the number of organisms capable of indefinite growth is the only important consideration for the physiologist, this is not so for the biotechnologist, for several reasons. First, for strain purity, each new production run starts afresh from starter cultures; these cultures would have been specially treated much differently than the cells in the actual production run. This care is to avoid contamination and the buildup of unwanted mutants. Second, dead, dying cells and stationary cells may be the productive members of the culture in terms of product formation. This second definition is the reason that colony counting and most probable number (MPN) methods of measurement are so important. A third definition of growth (and the practical one) is based on an increase in biomass. Macromolecular synthesis and increased capability for the synthesis of cell components are the obvious basis for the measurement of growth by everyone interested in microbes and what they can do. From our point of view, the whole process of chromosome replication and cell division is an essential but minor process that seldom limits growth; what usually limits growth is the ability of enzymatic systems to use available resources to form biomass. Moreover, the restriction of growth of the culture is usually the result of the depletion of resources or degradation of the local environment. A fourth definition of growth is based on the organisms’ action in chemically changing their environments. This category can be simply considered as a different consequence of the increase in biomass. However, it allows the rate of the growth process and the biomass production to be estimated in indirect ways. Most important in industry, the measurements, and growth of cultures can be followed usefully and effectively. Biomass is the result of growth; it has various different definitions according to purpose. In most biotechnology, biomass is organic material that is cheap and can be converted into fuel, or heat, or Structural materials. It is necessary that such sources be self-generating, cheap, and available. Whether biomass is alive or dead is less relevant; the assumption is that it was, or at least once was, alive. If one has a mixture of coal and fresh plant Page 21 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  22 Btec22: Fundamentals of Bioprocessing material, the term biomass is fully ambiguous. For product formation, there is less ambiguity; microbial biomass requires the expenditure of resources, and useful products may be produced by the organisms that may be living or dead according to which of the definitions of growth is used. Modes of Microbial Culture Batch Cultures Batch cultures are closed systems that are essentially characterized by inoculating into a culture vessel, or bioreactor, containing a finite amount of nutrients and letting the culture grow to saturation over time. Although most batch cultures are agitated by means of mechanical mixing or by bubbling gas through the solution from the bottom (called airlift reactors), some cultures perform better with no agitation. A typical growth curve of a batch culture is shown in Figure 3. The growth curve describes an entire growth cycle, and includes the lag phase, exponential phase, stationary phase, and death phase. Figure 3. Typical growth curve for a bacterial population. [Adapted from Tortora et al. (2015)] Lag Phase When a microbial culture is inoculated into a fresh medium, growth usually begins only after a period of time called the lag phase. This interval may be brief or extended, depending on the history of the inoculum and the growth conditions. If an exponentially growing culture is transferred into the same medium under the same conditions of growth (temperature, aeration, and the like), there is no lag and exponential growth begins immediately. However, if the inoculum is taken from an old (stationary phase) culture and transferred into the same medium, there is usually a lag even if all the cells in the inoculum are alive. This is because the cells are depleted of various essential constituents and time is required for their biosynthesis. A lag also Page 22 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  For instructional purposes only 1st Semester SY 2020-2021 23 ensues when the inoculum consists of cells that have been damaged (but not killed) by significant temperature shifts, radiation, or toxic chemicals because of the time required for the cells to repair the damage. A lag is also observed when a microbial population is transferred from a rich culture medium to a poorer one; for example, from a complex medium to a defined medium. To grow in any culture medium the cells must have a complete complement of enzymes for synthesis of the essential metabolite not present in that medium. Hence, upon transfer to a medium where essential metabolites must be biosynthesized, time is needed for production of the new enzymes that will carry out these reactions. Exponential Phase During the exponential phase of growth each cell divides to form two cells, each of which also divides to form two more cells, and so on, for a brief or extended period, depending on the available resources and other factors. Cells in exponential growth are typically in their healthiest state and hence are most desirable for studies of their enzymes or other cell components. Rates of exponential growth vary greatly. The rate of exponential growth is influenced by environmental conditions (temperature, composition of the culture medium), as well as by genetic characteristics of the organism itself. In general, prokaryotes grow faster than eukaryotic microorganisms, and small eukaryotes grow faster than large ones. Stationary Phase In the stationary phase, there is no net increase or decrease in cell number and thus the growth rate of the population is zero. Although the population may not grow during the stationary phase, many cell functions can continue, including energy metabolism and biosynthetic processes. Some cells may even divide during the stationary phase but no net increase in cell number occurs. This is because some cells in the population grow, whereas others die, the two processes balancing each other out. This is a phenomenon called cryptic growth. Microbial populations enter the stationary phase for several reasons. One obvious factor is nutrient limitation; if an essential nutrient is severely depleted, population growth will slow. Aerobic organisms often are limited by O2 availability. Oxygen is not very soluble and may be depleted so quickly that only the surface of a culture will have an O2concentration adequate for growth. The cells beneath the surface will not be able to grow unless the culture is shaken or aerated in another way. Population growth also may cease due to the accumulation of toxic waste products. This factor seems to limit the growth of many anaerobic cultures (cultures growing in the absence of 02). Death Phase Detrimental environmental changes like nutrient deprivation and the build up of toxic wastes lead to the decline in the number of viable cells characteristic of the death phase. The death of a microbial population, like its growth during the exponential phase, is usually logarithmic (that is, a constant proportion of cells dies every hour). This pattern in viable cell count holds even when the total cell number remains constant because the cells Page 23 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  24 Btec22: Fundamentals of Bioprocessing simply fail to lyse after dying. Often the only way of deciding whether a bacterial cell is viable is by incubating it in fresh medium; if it does not grow and reproduce, it is assumed to be dead. That is, death is defined to be the irreversible loss of the ability to reproduce. Although most of a microbial population usually dies in a logarithmic fashion, the death rate may decrease after the population has been drastically reduced. This is due to the extended survival of particularly resistant cells. For this and other reasons, the death phase curve may be complex. Figure 4 shows the dynamics of a batch culture. It describes the relationship between population density, nutrients and end products as the culture progresses. Figure 4. Growth dynamics of a batch culture. The solid black line shows the increase in population as the cells consume feedstock and grow. The dotted red line shows the decrease in nutrients in the culture medium as the culture progresses. The dotted/dashed green line indicates the amount of end product produced by the cells in culture. Letters refer to distinct phases of the growth curve. A, lag phase; B, logarithmic phase; C, stationary phase; and D, death phase [Adapted from Moo-Young (2011)]. Although many cultures display classic logarithmic growth curve, deviations from this dynamic occur for a variety of reasons. A common cause of this is a phenomenon known as diauxie, in which cells display a different growth rate on a secondary nutrient after the primary nutrient is depleted [Figure 5(b)]. A classic example of this is the diauxic shift during ethanol fermentation by yeast. Although glucose is abundant, the cells show rapid growth and the production of ethanol. However, as glucose is depleted, the cells begin to consume the ethanol as a secondary carbon source, which sustains a slower growth rate. The result is two distinct stage of logarithmic growth. Since ethanol is often desired end product of yeast fermentations, its consumption represents a real complication for biotechnological applications. Batch Culture Variation Often, a biocatalytic process must take full advantage of different growth phases to enhance product yield and utilize variations of batch culture method to achieve the best results. For example, some end products are only Page 24 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  For instructional purposes only 1st Semester SY 2020-2021 25 produced en masse until the cells enter stationary phase. In such cases, it is often important to limit one essential nutrient, such as nitrogen, to induce the transition into stationary phase and add a carbon source from which the product will be made in excess [Figure 5(a)]. However, as the amount of product is ultimately proportional to the number of cells, a productive logarithmic phase is required to produce the maximal number of cells. The process can be thought of as having two stages. In the first stage, the goal is to achieve the maximum possible cell density and, in the second stage, the goal is to produce the maximum amount of product. Such multistage process can also take advantage of diauxic growth to circumvent problems associated with catabolite repression, which occurs when the preferred feedstock (most often glucose) represses genes involved in the biosynthesis of the desired end product. Under these conditions, glucose can be the preferred carbon source to produce the maximum number of cells and a second carbon source can be used to produce the end product. Figure 5. Variations on batch culture. (a) End products produced during stationary phase after cells have stopped growing due to nitrogen depletion. Depletion of carbon sources occurs in two stages. In the first, carbon is used to produce microbial biomass. In the second, it is diverted into product production. (b) Overcoming catabolite repression by using a multistage culture with a primary, but repressing, carbon source to produce microbial biomass and a secondary carbon source to produce the desired end product. [Adapted from Moo-Young (2011)] Sometimes, a particular biocatalytic process works well on the laboratory scale but encounters problems upon scale-up where the dynamics of gas and heat exchange are markedly different. For example, if a hypothetical process requires O2 and there is poor gas exchange in the bioreactor, then microbial growth and oxygen consumption may exceed the replenishment of oxygen from the atmosphere or from air bubbles that are sparged through the solution. The result is the depletion of an essential nutrient, in this case O2, before the culture is able to achieve maximal cell density. Fewer cells also means less product [Figure 6(a)]. The solution to this problem is a variation on the batch process known as fed-batch, in which a concentrated solution of another nutrient, most often the carbon source such as glucose, is slowly fed to the cells at a consistent but growth-limiting rate. By slowing down microbial growth, one allows gas exchange to catch up with consumption [Figure 6(b)]. Variations on the fed-batch methodology can also be used to alleviate problems with heat exchange in large bioreactors, to Page 25 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  26 Btec22: Fundamentals of Bioprocessing overcome toxicity of contaminants or to prevent the formation of undesirable side products. Fed-batch processes allow batch cultures to achieve much higher densities than batch cultures. The reason is that many nutrients (e.g., iron) are essential and are also toxic at high levels. As a result, the amount of said nutrient in the initial batch limits the maximum achievable cell density — yet, more nutrient cannot be added in batch culture due to inherent toxicity. By slowly feeding such nutrients, much greater biomass yield can be attained. As more biomass means greater product yields, it is not surprising that fed-batch is often preferred over batch cultures. However, it is important to note that fed-batch methods add a layer of complexity to a process that may reduce economic viability on the industrial scale. Conversely, batch culture is established and reliable — inexpensive to set up and simple to maintain — making it an attractive option despite its limitations. Figure 6 Batch vs. fed-batch cultures. (a) A hypothetical batch process that requires oxygen, in which oxygen consumption is faster than exchange with air. The result is oxygen limitation and poor biomass and product yields. (b) The same process in which the feedstock is fed slowly and consistently to the culture so that growth rate is limited. Oxygen consumption no longer exceeds exchange with air and the culture can grow to much higher biomass yields and, consequently, make more product. [Adapted from Moo-Young (2011)] Limitations of Batch Culture Batch cultures certainly have their advantages; however, they also have important limitations. First, in batch, microbes are exposed to a constantly changing environment due to the consumption of nutrients and the buildup of waste products. Therefore, there is usually only a small optimal window in which the environmental conditions are ideal for the biosynthesis of the desired end product. Second, batch cultures eventually reach an endpoint and must be restarted. For large bioreactors, there is a significant turnaround time required to empty, clean, and refill the reactor for the next batch. Page 26 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  For instructional purposes only 1st Semester SY 2020-2021 27 Continuous Cultures and the Chemostat In theory, the ideal situation for biotechnology would be to determine the best culture conditions for the production of the desired end product and maintain the cells under these conditions in a steady state, so that the product can be made continuously — a process known as continuous culture. A chemostat is a single automated bioreactor in which spent medium is continuously replaced with fresh medium where one nutrient is found in limiting quantities (Figure 7). If the rate of medium replacement — the dilution rate — is lower than the growth rate of the microorganism inside, then the cell density will increase. If the dilution rate is higher than the growth rate, then the cell density will decrease and, eventually the cells will wash out. If the rate equals the growth rate, then a steady state is achieved. In the standard chemostat, the dilution rate is calculated based on the known growth rate of the microbe within and is fixed at the beginning of the experiment. Chemostat Variations In batch cultures, it is often important to grow microbial biocatalysis to high density in order to maximize product yield. In the chemostat, cell density is constrained by the concentrations of the limiting nutrient, which often cannot be increased beyond a certain concentration due to toxic effects. Cell densities that exceed saturation can be achieved in chemostats by recycling the cells that are removed (chemostat, with cell recycle, or retentostat). In addition to allowing higher cell densities, and subsequently greater product yields, such bioreactors also allow for dilution rates that are higher than the growth rate without causing washout. This is particularly useful when the product is toxic and needs to be rapidly removed. Another useful feature of this method is the fact that the growth rate decreases to near zero due to severe nutrient depletion. In essence, they maintain cells in stationary phase for long periods of time. This is useful if a particular product is synthesized in stationary phase. One of the central limitations of the chemostat is that the culture is essentially on its own once the experiment has begun — the user has no opportunity to alter parameters once inoculated. This led to the development of chemostat variations that allow for the modification of dilution rate depending on real-time monitoring of changes in the culture conditions. For example, the turbidostat continuously measures cell density using turbidimeter, which directly controls the dilution rate. However, turbidostats operate under the assumption that light diffraction correlates linearly with cell density, yet this is only true for transmission values that lie in the linear range of the particular turbidimeter. Significant changes in cell size also increase light diffraction and consequently, turbidimeters may not accurately report the status of a particular culture. Turbidostats are also not particularly useful for measuring cell density with microbes that do not grow evenly in suspension, such as filamentous fungi, or for cultures in which the substrate is particulate, such as biomass. Finally, microbes have the unfortunate habit of adhering to reactor surfaces — including the optics of the turbidimeter — often tricking the turbidimeter into thinking cell density is higher than it really is. In these cases, it is often only possible to track microbial growth using a secondary reporter that is closely tied to growth, such as changes in pH or the consumption of essential nutrients such as oxygen or glucose. Devices that Page 27 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  28 Btec22: Fundamentals of Bioprocessing take this approach are called auxostats and their detectors directly control dilution rate. Figure 7. Continuous culture via chemostats and related devices. (a) The concept behind continuous culture is similar to fed- batch, In which fresh medium is continuously added to the bioreactor. The difference lies in the fact that saturated medium is removed from the bioreactor at a rate that is equivalent to that of medium addition. The dilution rate is ideally matched to the growth rate of the microorganisms to maintain constant cell density. (b) In the chemostat, fresh medium is added to the bio- reactor at a rate that is equivalent to the removal of saturated medium. The medium is thoroughly mixed and the culture needs to be maintained at a constant temperature. Gas exchange is generally achieved by bubbling the appropriate gas mixture through the solution. Some physical barrier is needed to prevent chemotactic backgrowth of the cells into the reservoir containing fresh medium. In variations of the chemostat, dilution rate is not calculated based on growth rate of the cells, but rather on continuously modified based physical measurements of the culture, such as pH, dissolved oxygen, glucose levels, or cell density [Adapted from Moo-Young (2011)] As with batch cultures, it is often useful to set up chemostats or related devices with multiple stages when the production of biomass must be separated from the biosynthesis of the desired end product or to alleviate problems associated with catabolite repression. This can be achieved by altering the nutrient feed after the culture has reached equilibrium so that one nutrient mix is used to produce biomass and another is used to synthesize product. Alternatively, several chemostats could be set up in series, where the effluent from one, which is saturated with cells, serves as the inoculum for a downstream chemostat. One of the benefits of this approach is that increased residence time in the downstream bioreactors gives the biocatalysts another shot at complete consumption of excess substrate that would normally be lost in the effluent or at secondary substrates that can only be utilized once a primary substrate is consumed. Multistage continuous culture is also helpful when the feed or environmental conditions, such as temperature or gas composition, for the downstream chemostat are significantly different from the upstream chemostat. Page 28 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02  For instructional purposes only 1st Semester SY 2020-2021 29 Limitations of Continuous cultures Despite its advantages, continuous culture has several limitations that may restrict or prevent its use for industrial applications. One problem is encountered with cells that do not grow evenly in suspension such as filamentous fungi. Such cells grow as hyphal masses that are difficult to homogenize and remove as part of the effluent. The same problem is encountered if the microbial feedstock is particulate, as would be the case for biomass particles. In addition, although continuous cultures can theoretically be maintained indefinitely, they are susceptible to contamination from outside strains that infiltrate from the nutrient feed. Thus, they must be periodically stopped, sterilized, and restarted. Other limitations of continuous culture are only revealed upon scale- up. As it takes up to five volumes of the reactor to achieve maximal density, the nutrient feed reservoir needs to be large.Vigorous mixing is also required to ensure homogenization of the culture and the feed. On the lab scale, these are not serious problems; on this industrial scale they may be considerable limitations. The most crucial limitation of continuous culture is the phenomenon called wall growth in which cells adhere to, and form biofilms on the inner surfaces of the bioreactor. For microbial cells that require a solid surface to mediate growth, chemostats cannot be effectively used because maximal biomass yield is limited by the inner surface area of the bioreactor and not by the limiting nutrient feed. Even when growing microbes that prefer to grow as planktonic populations, variants will rapidly appear that stick to the bioreactor walls and avoid being washed out during dilution, essentially turning the continuous culture into a selection scenario for adherent populations. Indeed, wall growth can occur within hours of establishing the culture and chemostats will quickly produce a heterogeneous mixture of planktonic and adherent populations that experience vastly different environmental conditions. This can complicate attempts to maintain the right conditions for end product biosynthesis. The end result is that chemostats cannot be maintained for long periods of time and chemostats must be periodically emptied and cleaned to remove wall growth. Not surprisingly, chemostats are expensive to set up and maintain and despite their theoretical advantages, their practical limitations make them less desirable from an industrial perspective. Because batch cultures are simple, they are easy to establish on the commercial scale and still represent the most economical way of facilitating biocatalysis. Page 29 of 52 Vision: A globally competitive university for science, technology, and environmental conservation. Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-02 V0 07-15-2020 and innovative technologies for sustainable communities and environment. DBt-IM-02