Lecture 10 Test Questions – Flashcards
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| Describe the differences between cellular models versus reactor models |
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| cellular model--> specify model complexitiy--> stoichiometry--> kinetics Reactor model --> mass balances |
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| provide 5 reasons to study microbial kinetics |
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| 1. process scale- up (pilot to production scale) 2. process scale-down (minature fermentations for screening) 3. medium optimization (substrates, growth, yield) 4. trouble shooting, manufacturing support 5. microbial death (sterilization, killing by antibiuotics, other agents 6. screening 7. define growth characteristics of a strain for process reproducibility |
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| draw a diagram indicating the relationship between structuring at the population level versus at the cell level as it relates to modeling microbial metabolism. indicate the " black box" kinetic model |
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| 4 circles from left to right unstructured non segregated structured non segregated unstructured segregated structgured segregated the black box is unstructured non segregated |
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| what are the benefits of using unstructured non segregated models, unstructured segregated models and structured models? |
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| Unstructured non segregated models- good for small changes Unstructured segregated models good for modeling plasmid loss structured models useful for modeling intracellular pathways, altered gene expression, responses to environment |
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| Provide the name of the reactions between substrates and precursor metabolites, precursor metabolites and building blocks, building blocks and macromolecules and macromolecules as a functioning cell |
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| Reaction between substrates and precursor metabolites- fueling reactions reactions between precursor metabolite and building block- biosynthetic reactions reactions between building blocks and macromolecules - polymerization reactions reactions between macromolecules and functioning cell- assembly reactions |
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| what are four methods for measuring cell growth |
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| 1. measurement of cell number 2. measurement of viable cell number 3. measurement of cell mass (physical methods, chemical methods) 4. measurement of cell composition (enzyme levels, total protein levels, nucleic acid levels, ATP levels, plasmid copy number, chlorophyll content) |
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| list 3 methods for measuring cell growth, in addition to the measuring principle, specify the detection limit (DL), advantages, and disadvantages of each measuring technique. what is the significance of these detection limits? |
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| Dry mass- mass of separated and dried solids- DL 50, advantage provides direct unconditional estimate- disadvantage - interference from dead cells and noncell solids wet mass- mass of separated material- DL 50 - advantage simple, quick- disadvantage- variation of wet biomass bulk density Plating, growing of the colonies on a petri dishg DL- 10^-5- low cost, high sensitivity , time consuming, low precision |
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| when can optical density measurements not be used to generate growth curves? |
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| Cannot use optical density when using insoluble complex media |
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| what are three common methods for measuring viable cells? what are the corresponding limitations? |
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| 1. Tube dilution Colony forming units (CFU) - time consuming, not online, assuming each colony comes from one cell, cells that are stressed can recover and grow and produce a colony- always lower than true cell viability (2 cells close to each other can look like 1) 2. Filtration and selective plating on agar (CFU) - filter out the cells onto a membrane filter and then place it on an agar- cells are not single cells on the filter- assumption is that cells dont make it though the pores in the filter (low) - filtration is really looking for contaminants 3. Most probable number method - serial dilution and use statistics to guess what the growth is in that tube |
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| cell composition varies during ___ |
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| batch growth |
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| What type of information can be obtained from batch culture kinetics |
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| 1. growth phases 2. exponential growht-what affects microbial growth rate 3. yield coefficients 4. substrate uptake kinetics 5. growth models 6. growth on multiple substrates 7. product formation models |
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| produce a graph illustrating the idealized normal growth cycle for a bacterial population in a batch culture system. be sure to label the axes and various growth phases. describe each growth phase |
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| lag, growht, maximum stationary phase, death phase log number of viablie cells versus time |
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| Why is lag phase elimination one of the most important factors in efficient growth of microorganisms? |
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| increasing productivity |
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| How is the length of lag phase effected by the inoculum age of an amino acid medium and an ammonium sulphate medium? |
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| the older the inoculum the longer the lag phase- ammonium sulphate- there is a sweet spot cant be too old or too young |
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| during exponential growth phase, what effects microbial growht? |
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| temperature (most organisms have an optimum) and pH |
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| which organism is more growth-rate dependent on dissolved oxygen (DO) Azotobacter vinelandi or E. coli and why? |
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| azotobacter vinelandii because its strictly an aerobic organism- no matter how much oxygen you put in it will reach maximum specific growth rate whereas e.coli can grow at 70% of its aerobic growth rate anaerobically |
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| specific growth rate ___ as osmolaltiy ___ and water activity ___ |
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| peaks then decreases as osmolality increases and water activity decreases |
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| cell yield (Yx/s) provides information regarding pathways used for ____ |
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| energy generation |
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| will an aerobic or anaerobic organism have a higher cell yield? |
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| aerobic |
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| why does the same organism possess different characteristic values for Yx/s for different classes of substrates? |
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| oxygen is a potential substrate so you can have a cell yield coefficient based on availability of oxygen |
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| what is substrate limited growth? |
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| control microbial processes by limiting one of the key substrates (carbon, nitrogen etc)- advantage is that you don't need any sophisticated machine to control fermentation |
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| draw a graph illustrating the relationship between substrate concentration and soecific growth rate as it relates to the limiting nutrient concept. label axes. |
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| What does Ks tell you? |
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| saturation constant represents concentration of substrate that you need to put into the media to get 1/2 of the maximum specific growth rate how sensitive is system to concentration of substrates |
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| What happens when S<< Ks and Ki as well as when S>> Ks and Ki? |
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| Saturation constant something small is going to be very small (natural monod) if its the other way around- a lot of substrate S^2/Ki - specific growth rate is going to get smaller and smaller mu=mumax*s/(kw+S+S^2/ki) |
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| In the unstructured logistic growht model what does the term x inifinity represent? when is the inflection point reached? |
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| X infinity is the carrying capacity or maximum growth possible due to accumulation of a toxin it is reached at 1/2x inifinity |
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| The following is a schematic illustrating nutrient and product concentration profiles in a mycelial pellet. which profile (A or B) will be of interest if the product is synthesized in the center of the pellet versus if the cells in the center are dead or nutrient starved? |
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| A- if the product is synthesized in the center of the pellet B- if the cells in the center are dead or nutrient starved |
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| Provide the description and application (example) of stochastic, continuous time and discrete time models. |
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| Stochastic- for small numbers of organisms (n>100) for cases where differences in organisms are important example: different microbial species exhibit varying resistance to a sterilization process continuous time- systems where events are evenly distributed in time- most microbial models example: logarithmic growth rate model for a batch culture in which some micro-organisms are always undergoing division discrete time- systems where events occur at a limited number of times example: a synchronous culture in which all microorgs divide simultaneously at discrete times |
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| Provide the description and application (example) of descriptive (black box), predictive (grey box) and strucutred models |
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| Descriptive- curve fitting, interpolation of data, useful within the region where the model was tested experimentally example: use of the logistic equation to describe batch microbial growth grey box- exprapolation of data, with care can be used outisde of the region tested experimentally example: michaelis- menten (1913) equation for describing enzyme-substrate reaction rates Structured- systems in a transient state useful for modelling changes in internal strucutre of cells or structure of mixed microbial populations example: williams (1967) two-compartment (synthetic component and genetic component) modelo for biomass |
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| provide the description and application (example of unstructured, non segregated, segregated, and deterministic models |
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| Unstructured- does not account for internal structure steady-state systems lumped parameter models example: steady-state continuous flow microbial reactor (chemostat) where mass is the only property of the biotic material non-segregated- considers that the biomass is homogeneously dispersed throughout the reactor fluid mass concentration is the parameter which describes biomass population balance models example: chemical oxidation of microbial cells by chlroine where biomass destruction or solubilization, not disinfection, is of greatest concern segregated-uses a discrete parameter, the number of cells as the parameter to describe biomass example: evaluating disinfection or sterilization processes where number of organisms alive and dead is most critical deterministic models: output variables have values completely determined by the structure of the system applicable in cases where large numbers of organisms are involved (n>10000) example: activated sludge process for a chemostat in which saturation kinetcs describes substrate removal and consequently microbial growth |
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| What is fed batch cultures often used for in biomanufacturing? |
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| slow-growing cell cultures slow-growing antibiotic manufacturing proceses |
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| as culture volume increases what happens to mu, S, and P in a repeated fed-batch culture? |
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| V increases, mu decreases, Substrate concentration is constant and product accumulates |
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| ______ is the most often used system in industrial biomanufacturing |
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| fed batch |
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| What is the impact of a continuous upstream process on downstream product purification? |
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| they might not be able to process continuously- might need holding tanks until downstream is ready to process |
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| what is the governing equation for specific growht rate using the "black box' model? how can you determine the saturation constant from the generated graph? |
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| Michaelis-Menton (MM) enzyme kinetics |
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| What does Ks tell you? |
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| that it will change based on the relative production level of carbon source can calculate saturation constants as bacteria many Ks values are <1 mg/l |
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| Not a good idea to put too much of a limiting substrate |
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| it can start to shut down its specific growth rate in order to avoid the toxicity by the excess of substrates can start to produce secondary products |
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| The following is a graph depicting the growth of E. coli in medium containing initially equally amounts of glucose and xylose in a batch culture. Describe what is occurring within the culture as it relates to phase of growth and substrate utiliziation. What type of growth is this known as |
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| it has two limiting substrates diauxic growth |
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| What are the advantages and disadvantages of batch bioreactor opterations |
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| Advantages- versatiles since it can be used for many different processes low risk of contamination complete conversion of substrate possible disadvantages- high labour cost much idle time, due to cleaning and sterilsation after each fermentation |
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| what are the advantages and disadvantages of continuous bioreactor operations? |
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| advantages- high efficiency of the reactor capacity high productivity can be maintained for long periods of time automation is simple constant product quality disadvantages- problems with infection, possibility of the appearance of low levels of mutant production during long operation inflexibility since it can rarely be used for different processes without substantial retrofitting downstream processing has to be adjusted to the flow through the bioreactor (or holding tanks are required) |
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| what are the advantages and disadvantages of fed-batch |
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| advantages- allows operating at well-controlled conditions by controlling the feed addition allows very high cell densities and therefore high titers disadvantages some of the same problems as for batch and continuous reactor but generally the disadvantages are less pronounced |
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| define a continuous culture |
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| fed and continuously harvesting |
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| what are 2 benefits to using a continuous culture |
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| 1. operating at steady state- can help in development 2. material balances (substrate, cells) 3. productivity (production of cell mass) 4. measuring the maintenance energy coefficient 5. single stage chemostat with cell recycle to enhance productivity |
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| traditional CSTR is recommended to keep substrate concentration low; however, this is not advisable in free enzymes? why? what reactor configuration would you suggest in this case? |
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| what does it mean to be at steady-state in a continuous system? |
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| RNA changes even though it is a steady state- genes are activated or triggered at different times- to make use of substrate or getting ready for secondary metabolism |
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| The schematics below (ABCD are types of continuous cultures=provide the name of each type |
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| a. chemostate b. auxostat c. multi stage continuous culture d. continuous cell recycle (prefusion) |
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| why use a chemostat provide 4 reasons |
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| 1. investigate growth rate dependent or substrate concentration-dependent cellular responses using steady state kinetics 2. to investigate transients (suddent environmental changes or stress) and cellular responses to a non-homogenous environment 4. for applying selective pressure to isolate mutants 4. continuous generate biomass |
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| Single stage kinetics provide a relationship between ___ and ____ |
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| cell mass and dilution rate |
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| chemostat are used to measure cell yield and ____ |
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| maintenance energy |
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| the following graphs illustrate typical profiles and responses for continuous cultures deviating from ideal chemostat behavior- for graphs A, B, C, and D what external factor is affecting the yield |
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| non-ideal behavior in chemostats can differ depending on the ____ |
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| limiting nutrient |
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| in terms of glucose as the substrate rank the following organisms based on cell yields from highest to lowest and corresponding maintenance energy coefficients aspergillus awamori aspergillus nidulans candida utilis e.coli penillium chysogenum saccharomyces cerevisae |
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| 1.e coli 2. pennilium chrysogenum 3. cndida utilis 4. a. awamori 5. S cerevisae 6. a nidulans |
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| Why are chemostat transient experiments important? |
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| you can learn what to expect when you change different parameters in your continuous system can quantity the changes |
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| When utilizing chemostats with cell recycle ____ does not equal ____ and biomass productivity can be dramatically ___ |
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| growth rate does not equal dilution rate |
