Culture & Metabolic Engineering Flashcards
Describe 4 ways to measure microbial growth
- Cell dry weight, pellet, wash, dry and weigh. Provides mass/unit volume but doesn’t indicate cell viability
- Cell number. Either as a total count of dead and live cells or as a viable count: Perform a serial dilution, usually upto 10^6, plate and count the number of colonies/volume used
- Optical Density (OD), 595/600/610nm wavelength usually used, requires a standard curve to relate OD to cell count, photosynthetic bacteria can skew the results
- Specific cell components, measuring the mass of chloroplasts, mitochondria, or specific proteins, this doesn’t account for biological variation.
Tell me about batch culture
It is a culture of a fixed volume in a flask or culture vessel. Unrestrained growth isn’t possible due to the depletion of nutrients and accumulation of autoinhibitory waste products.
Describe the phases of a batch culture growth curve
- Lag phase, cells prepare machinery for growth, the length depends on the change in environment.
- Log phase, exponential growth
- Stationary phase, cells stop growing, growth machinery shuts down and stress responses turn on
- Death phase, cells die with a half life.
What are the limitations of batch culture?
It is an artefact of lab growth, it doesn’t represent a natural growth curve.
It is in a closed system, there are massive variations in the physiochemical environment of pH, number of cells, [O2], [CO2] and medium composition.
There is significant variation between 2 points in log phase.
What’s the main difference between chemostats and turbidostats?
Chemostats involve spent medium being removed and fresh medium being added continuously whereas this happens periodically in turbidostats
What are the benefits of continuous culture?
Substrates and nutrients are added for maintained growth.
Autoinhibitory waste products are diluted.
Bacterial populations can be maintained at a constant OD in log phase.
Growth rate and cell density can be controlled independently.
Chemostats allow the reproducible cultivation of microbes at submaximal growth rates at different growth limitations.
Tell me about common chemostat/Turbidostat properties and limitations
Common properties:
Fresh media is added with the culture volume remaining constant
It is well-mixed, there is aeration and agitation to ensure the even distribution of cells, nutrients and oxygen tension.
pH is kept within a predetermined level alongside a constant temperature.
Limitations:
Wall growth & foaming, usually remedied with anti-foam chemicals.
Mutations can affect growth rate.
Describe steady-state establishment in chemostats
- Initially, the growth rate > dilution rate. The cell number increases
- As the concentration of the growth-limiting nutrient decreases, the growth rate < dilution rate.
- Eventually, growth rate = dilution rate and the steady state has been achieved.
What is the chemostat steady state?
Specific growth rate (µ), cell density and growth-limiting nutrient concentrations are constant. Varying the dilution rate can vary the growth rate while maintaining a constant OD, cell density.
When does the chemostat steady state break down?
At low and high dilution rates:
a) At low dilution rates, an increasing proportion of the growth-limiting nutrient is used in basic house-keeping processes reduces the amount available for growth taking the culture out of log phase
b) At high dilution rates, more cells are siphoned away than divide leading to a washout
Tell me about turbidostat culture
Turbidity is maintained; there is a preset OD level, monitored by a spectrophotometer that feedbacks into the system to add and release medium to return to the preset turbidity/OD when it reaches a threshold. There is no growth-limiting nutrient.
What is industrial microbiology and microbial biotechnology?
Industrial microbiology is the large-scale production of commercial products by microorganisms.
Microbial biotechnology is the engineering of microbes to produce non-native compounds.
Give some examples of microbes that produce antibiotics
Penicillin by Penicillium chrysogenum.
Tetracycline by Streptomyces
Give some examples of microbes that produce enzymes
Lipase by Candida cylindracae
Amylases by Bacillus subtilis
Lactase by Kluyveromyces lactis
Give some examples of microbes that produce food additives
Vitamin riboflavin by Ashbya gossypii and Bacillus subtilis
Amino acids by Corynebacterium glutamicum
Give some examples of microbes that produce chemicals
Citric acid by Aspergillus niger
Bioethanol by Saccharomyces cerevisiae
Butanediols by Escherichia coli
Give some examples of microbes that produce terpenes
Artemisinin by Saccharomyces cerevisiae
Carotenoids by Chlorella
List some useful properties of industrial microbes
a) Produces substance of interest in a high yield
b) Can grow rapidly in a reproducible manner, produces product in a short period of time
c) Can grow and produce product in large scale culture or under bioreactor conditions
d) Metabolically flexible and adaptable
e) Doesn’t produce toxins or toxic by-products, not pathogenic to humans or animals
f) Amenable to genetic engineering & is genetically stable
g) Can be stocked or stored
h) Secretes the product into the media or is easy to handle or break
Tell me about fermentation, fermenters and fermentors
Fermentation refers to the growth of large quantities of fermenters (microbes) in a vessel called a fermentor or a bioreactor for the production of commodity chemicals, biofuels, pharmaceuticals, enzymes etc.
Most industrial fermentations are aerobic, the process relies on scalability with fermentors often being 10,000 to 500,000 Litre capacity where the fermenters can be subject to harsh conditions.
What’s the difference between batch, continuous and fed-batch fermentation?
a) Batch fermentation is where you add all the nutrients at the start, once they have been consumed, growth ceases and the fermentation has ended.
b) Continuous fermentation is essentially a large chemostat; culture is constantly added and removed
c) Fed-batch fermentation is where you provide nutrients in a batch culture medium, once consumed, a feed is initiated to provide the culture with additional nutrients allowing for further growth.
Describe the process of fed-batch fermentation with Pencillium chrysogenum
- Initial growth phase in a small fermentor inoculated with freeze-dried spores
- Scaled-up via 2 successively larger fermentors to provide a large enough inoculum for the production phase.
- Fermentation production phase is now a fed-batch process. High O2 levels and C/N levels are maintained.
- Monitored to keep Penicillium in production phase for 120-200 hours
- Penicillin is excreted into the medium & recovered at the end of the process.
What is bioprospecting?
The search for organisms, enzymes and natural compounds with potential for commercial application. Typically occurs in extreme environments as extremophiles are more likely to be tolerant of harsh culturing environments.
What is metagenomics?
The study of genes/genetic samples from environmental samples. Involves recovering nucleic acids, cloning them into BAC libraries, introducing BACs into E. coli and screening for phenotypes. Positive clones are then sequenced and analysed.
What is gene-mining?
The process of identifying and isolating genes from environmental samples without having to culture the organism.
