BTEC 22 Lecture Notes on Microbial Growth and Nutritional Requirements PDF

Btec 22 Lecture Module 2: Microbial Growth and Nutritional Requirements Chinelo M. Cardaño, MSc. In this module, we primarily discuss microbial growth, modes of microbial culture and the nutritional requirements of industrial microbial cultures.  Btec 22 Lecture Mo...

Btec 22 Lecture Module 2: Microbial Growth and Nutritional Requirements Chinelo M. Cardaño, MSc. In this module, we primarily discuss microbial growth, modes of microbial culture and the nutritional requirements of industrial microbial cultures.  Btec 22 Lecture Module 1: Lesson 1 Microbial Growth August 27- September 03, 2024 Chinelo M. Cardaño, MSc. Learning Outcomes ✓Understand the concept and phases of microbial growth ✓Differentiate the modes of microbial culture and understand how microbial growth occurs in batch and continuous cultures ✓Identify advantages and limitations of batch and continuous cultures and discern appropriate mode for the production of a specific metabolite ✓ right culture conditions Microbial catalyst-based industrial processes ✓ Right microbe Growth (1) ✓the ability of individual cells to multiply ✓to repetitively initiate and complete cell and organismal division ✓implies monitoring the increase in total number of discrete bacterial particles 1. microscopic enumeration 2. electronic enumeration (Coulter counter) 3. modern flow cytometry Can you think of an issue? Growth (1)…issue ✓Assessment of particle number would falsely include dead cells and detritus, which would tend to lower estimations of growth rate. ✓An increase in cell number is not exactly correlated with an increase in biomass or useful product. Growth (2) ✓An increase in colony-forming units (CFU) ✓Although the increase in the number of organisms capable of indefinite growth is the only important consideration for physiologist, not for biotechnology (i.e strain purity) ✓Fresh starter cultures for each new production Why do you think so? 1. avoid contamination and build-up of unwanted mutants 2. dead, dying cells and stationary cells may be the productive members of the culture in terms of product formation Growth (2) ✓An increase in colony-forming units (CFU) ✓Although the increase in the number of organisms capable of indefinite growth is the only important consideration for physiologist, not for biotechnology (i.e strain purity) ✓Fresh starter cultures for each new production Why do you think so? 1. avoid contamination and build-up of unwanted mutants 2. dead, dying cells and stationary cells may be the productive members of the culture in terms of product formation  Growth (3) ✓based on an increase in biomass (the practical definition) ✓macromolecular synthesis and increased capability for the synthesis of cell components ✓the ability of enzymatic systems to use available resources to form biomass usually limits growth ✓restriction of growth of the culture is usually the result of the depletion of resources or degradation of the local environment  Growth (4) ✓based on the organisms’ action in chemically changing their environments. ✓Could be a different consequence of the increase in biomass ✓allows the rate of the growth process and the biomass production to be estimated in indirect ways  Biomass ✓The result of growth ✓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 ✓In product formation, 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 Growth https://biologyreader.com/fed-batch-culture.html 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. Bioreactors An engineered system, deployed to facilitate the growth of biological mass through the transformation or degradation of material fed to the reactor -Singh, N.K. 2020 https://handling-solutions.eppendorf.com/fileadmin/_processed_/c/b/csm_228234_d0adf5dee2.jpg Bioreactors Are vessels that have been designed to provide an effective environment for enzymes or whole cells to transform biochemicals into products. -Erickson,L.E. 2019 Source: Tuklas Lunas Development Center, VSU Batch cultures Stirred-tank bioreactor (STBR) are the most widely employed for carrying out culturing of suspension cells and enzymatic processes, mainly in batch operations around 90% of the industrial bioprocesses, are carried out in this type of reactor. Borosilicate glass (lab- Stirred Tank Bioreactor (STB) scale, 10 liters) Agitator Stainless steel (larger scale, industrial scale) Baffles – obstructing vertical plates that Sparger (w/ ring) – converts rotational flow to breaks incoming air into axial mixing small bubbles Divided into: 1. Working volume - Consists of the total volume from the medium, microbes and gas bubbles (70-80% of total bioreactor volume) 2. Headspace volume - Remaining volume Batch cultures most batch cultures are agitated by mechanical mixing using impellers or bubbling gas (i.e. airlift reactor) Some cultures perform better with no agitation Batch cultures The typical growth curve of a batch culture Growth curve (Lag phase) Growth that usually begins moments after a microbial culture is inoculated into a fresh medium may be brief or extended, depending on the history of the inoculum and the growth conditions 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 Growth curve (Lag phase) Also occurs when a microbial population is transferred from a rich culture medium to a poorer one (e.g. complex medium to a defined medium) upon transfer to a medium where essential metabolites must be biosynthesized, time is needed for the production of the new enzymes that will carry out these reactions. Growth curve (Log phase) Exponential phase 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 are typically in their healthiest state and hence are most desirable for studies of their enzymes or other cell components Growth curve (Log phase) rates may vary Influence by environmental factors (i.e. temperature, culture medium composition) and its inherent genetic characteristics In general, prokaryotes grow faster than eukaryotic microorganisms and small eukaryotes grow faster than large ones. Growth curve (stationary phase) no net increase or decrease in cell number the growth rate of the population is zero 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 Cryptic growth- Some cells in the population grow, whereas others die, the two processes balancing each other out. Growth curve (stationary phase) It occurs when? ✓nutrient limitation (i.e.if an essential nutrient is severely depleted, population growth will slow; e.g. aerobic organisms) oxygen is not very soluble and may be depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth. The cells beneath the surface will not be able to grow unless the culture is shaken or aerated in another way. Growth curve (stationary phase) It occurs when? ✓Accumulation of toxic waste products seems to limit the growth of many anaerobic cultures Growth curve (death phase) Results from detrimental environmental changes, nutrient deprivation, build-up of toxic waste Decline in the number of viable cells Death rate is usually (not all the time) logarithmic (constant number of cells die every hour) Death is defined to be the irreversible loss of the ability to reproduce Growth curve (death phase) death phase curve may be complex death rate may decrease after the population has been drastically reduced (i.e. extended survival of particularly resistant cells) Batch cultures Deviations from this dynamic can occur – Diauxie phenomenon 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)] Growth dynamics of a batch culture Batch culture variation Diauxie- a phenomenon wherein cells display a different growth rate on a secondary nutrient after the primary nutrient is depleted Variations in batch culture  Do lab scale processes always work when scaled up? No, problems may be encountered during scale-up e.g. O2 requirement in cultures Nutrient depletion minimal cell density fewer product Fed-batch culture a concentrated solution of another nutrient, is slowly fed to the cells at a consistent but growth- limiting rate Allows for: efficient gas exchange, address problems with heat- exchange, overcome toxin build-up or undesirable product formation Fed-batch allows processes allow batch cultures to achieve much higher densities than batch cultures VI note: fed-batch methods also add a layer of complexity to a process that may reduce economic viability on the industrial scale Fed-batch culture (a) a hypothetical batch process that requires oxygen (b) the feedstock is fed slowly & consistently to the culture so that growth rate is limited Batch(a) vs fed-batch (b) What makes batch culture attractive? established and reliable Inexpensive to set- up simple to maintain Are there limitations? microbes are exposed to a constantly changing environment due to the consumption of nutrients and the buildup of waste products → small optimal window of ideal environmental conditions for biosynthesis of desired product batch cultures eventually reach an endpoint and must be restarted May require longer turn-around time to empty, clean, and refill the reactor for the next batch Continuous culture and the Chemostat determine the best culture conditions for the production of the desired end product and maintain the cells under these conditions in a steady state Continuous culture Continuous production of a desired product Utilizes a chemostat Continuous culture and the Chemostat (a) 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) How chemostat works Chemostat a single automated bioreactor in which spent medium is continuously replaced with a fresh medium where one nutrient is found in limiting quantities Chemostat variations Remember: in batch cultures, growing microbial biocatalyst to high density is important to maximize product yield But, in chemostat, cell density is constrained and/or controlled by the concentration of the limiting nutrient (i.e. often cannot be increased due to toxic effect) Retentostat - cell densities that exceed saturation can be achieved by recycling the cells that are removed (i.e. chemostat with cell recycle) - also allow for dilution rates that are higher than the growth rate without causing washout. - when do you think this is most useful? Limitations of continuous cultures cells do not grow evenly in suspension (e.g. filamentous fungi) susceptible to contamination from outside strains that infiltrate from the nutrient feed → thus, must be periodically stopped, sterilized and restarted In scale-up, vigorous mixing is required for homogeneity of the culture and the feed (i.e. industrial scale) Wall growth - when cells adhere to, and form biofilms on the inner surfaces of the bioreactor → adds a layer of complexity in achieving the right conditions Chemostats are more expensive to set- up and maintain despite its theoretical advantages  Btec 22 Lecture Module 2: Microbial Growth and Nutritional Requirements Chinelo M. Cardaño, MSc. In this module, we primarily discuss microbial growth, modes of microbial culture and the nutritional requirements of industrial microbial cultures.  Btec 22 Lecture Module 2: Lesson 2 Nutritional Requirements October 10, 2024 Chinelo M. Cardaño, MSc. Learning Outcomes ✓Understand the principal functions of each component of the substrate and other nutrient requirements in microbial cultures. ✓ Identify the different nutritional types of microorganisms. Media (singular: medium) nutrient solutions containing nutritional requirements for cell growth and production of metabolites Medium Essential requirement the choice of the correct medium can make or break a research program or an industrial process. Medium design take note of: cost availability of substrates reliability of substrate supply, handling, storage, ease of preparation storage transportation of components health and safety considerations primary consideration: to supply the microorganism or cell line with a source of necessary nutrients in a readily utilizable form Common & essential nutrient requirements Macronutrients or macro elements C,H,O,N,P,S - are components of carbohydrates, lipids, proteins, and nucleic acids. Potassium (K+) - required in a number of enzyme activities, including some of those involved in protein synthesis Calcium (Ca2+) - contributes to the heat resistance of bacterial endospores Magnesium (Mg2+) - serves as a cofactor for many enzymes, complexes with ATP, and stabilizes ribosomes and cell membranes Iron (Fe2+ and Fe3+) - a part of cytochromes and a cofactor for enzymes and electron-carrying proteins. Common & essential nutrient requirements Micronutrients or trace elements Manganese Zinc Cobalt Molybdenum Nickel Copper So little that often contaminants in water, glassware, and regular media components may be adequate difficult to demonstrate a micronutrient requirement are ubiquitous and do not usually limit growth Common & essential nutrient requirements Growth factors Amino Acids Vitamins Nucleic Acids Nutritional Types of microorganisms Microorganisms also require energy and electrons for growth Grouped into nutritional classes: Depending on preferred C source: ✓ heterotrops Based on these sources, ✓ Autotrophs microorganisms can be classified as a mix of their Depending on preferred energy source: various preferences – see Learning guide for details ✓ phototrophs and examples (e.g. ✓ Chemotrophs photoorganoheteroptrophs) Depending on preferred electron source: ✓ lithotrophs “rock-eaters” ✓ Organotrophs