Week 2 Lecture Flashcards
Upstream Processing includes?
Microbial cells, substrates, fermentation systems, and process monitoring/control
Downstream Processing includes?
Separations and purifications, product produced and effluent waste
Do internal or external products require more steps to be extracted?
Internal products do
When was Pure strain isolation or pure culture first done?
In 1883
When was penicillin first made?
1940s during World War II
Fermentation products include:
Food, Beverages, health care products, microbial enzymes, industrial chemicals and fuels, environmental applications
Cells as a reactor
Depend on: growth and reproduction, utilization of energy from environment to produce highly ordered structures, variation within species based on natural selection (optimization), and chemistry (base of life)
Cells as a reactor basis for life
Acquisition of food (energy), conversion of food into the structure of the living system, the retention of information, and introduction of information
In unit operation is the conversion of reactants to desired products straightforward?
No
Microorganisms require?
Specific temperature, pH etc.
What are the environmental precursors?
CO2, H2O, and N2
Carbohydrates?
-Are a major source of metabolic energy, both for plants and for animals that depend on plants for food
-A structural material (cellulose)
-A component of the energy transport compound ATP
-Recognition sites on cell surfaces
-One of three essential components of DNA and RNA
What is more expensive to use cellulose or sucrose?
Cellulose is expensive to use compared to sucrose, have to be able to separate cellulose from other compounds
What are the 3 main areas that Upstream Processing can be classified into?
Upstream processing involves all the factors leading to and including the fermentation. It can be classified into 3 main areas
- The obtaining, maintaining, improving and inoculation of a producer microorganism
- The selection of a cost effective carbon & energy source along with any other required nutrients
- The actual fermentation process itself
Industrial Microorganisms
There are a multitude of microorganisms used to generate a number of significant food & beverage, health, environmental and industrial products. These can include: Yeasts, Bacteria, and sometimes fungi and algae
Yeasts
Growth form of eukaryotic microorganisms classified in the kingdom Fungi, with about 1500 species currently described they dominate fungal diversity in the oceans
Bacteria
A large group of unicellular microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals.
How are industrial microorganisms isolated from the natural environment and improved?
They are isolated from the natural environment and then improved through mutagenesis and breeding
What are GRAS microbes?
GRAS microbes means generally regarded as safe microbes. These are microorganisms that the food & beverage industries rely the most upon. Don’t want to use toxic microbes, want to use safe non-toxic microbes. Can be bacteria, yeasts and fungi
Fungi?
Fungi are eukaryotic microorganisms. They can grow in 2 ways: as filamentous hyphae or as unicellular yeasts
What are the two methods for microorganism isolation?
Shotgun and objective
Shotgun method
The sampling of free living microorganisms from animal/plant material, soil, sludge
Can be screened for desirable traits. Shotgun is a random selection
A series of microorganisms are collected and the target microorganism must be further isolated
Objective method
Sampling from specific sites, where more likely a given organism is to be found. This is a specific selection
A series of microorganisms are collected and the target microorganism must be further isolated
What are the benefits of enrichment cultures?
They can kill or suppress the growth of common organisms and encourage the growth of the target microorganism. This would cause larger populations of the target microorganisms to grow and make for easier isolation and screening. Enrichment cultures can be batch or continuous.
What are the fundamentals of isolating a pure culture?
Isolating a pure culture on a solid growth media involves choosing the appropriate media and growth conditions
-pure cultures must then be screened for the proprietary inhibitory compound, a specific enzyme etc. and also determine that it is stable and if necessary non-toxic
-If you want a specific strain you need optimum conditions; specific sugars, pH, is it aerobic or anaerobic?
What is the typical method used in enrichment cultures?
The isolating method is used for microbes which are in small numbers in the sample and whose growth is slow than other species present. Some bacteria have very specific requirements for their growth.
Why is it important to control the nutrient and culture conditions for enrichment cultures?
Controlling the temperature, air supply, light, pH etc. in such a way that it suits only to the given species, important for a target microorganism
-Strategies in enrichment cultures can be physical (temperature, O2 or lack of O2), chemical (pH), or biological (host for specific viruses)
What is selective media?
Media designed to inhibit the growth of undesired organisms so that only the desired type can grow
Is Isolating and screening easier for single or multiple microorganisms?
Isolation and screening is possible for single microorganisms but becomes much more difficult when trying to isolate many beneficial microorganisms
What are the prime functions of a culture collection?
-Maintain existing collections
-Continue to collect new strains
-To provide pure, authenticated culture samples of each microorganism
-To have access to microorganisms without going through the isolation process
The use of cryopreservation and freeze drying has aided in maintaining these collections
What are the methods in culture collection?
Cryopreservation, Lyophilization-Freeze-drying, Agar
Cryopreservation
Is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures (typically) 77 K or -196 *C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped.
Lyophilization-Freeze-drying
Is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport.
Freeze-drying works by freezing the material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas
Agar
-A gelatinous substance derived from seaweed; used as a solid substrate to contain culture medium for microbiological work. The gelling agent is an unbranched polysaccharide obtained from the cell walls of some species of algae
What traits should industrial microorganisms ideally have?
-Genetic stability
-Efficient production of the target product
-Limited or no need for vitamins and additional growth factors
-Utilization of a wide range of low cost and readily available carbon sources
-Amenability to genetic manipulation
-Safety, non-pathogenicity, no toxic byproducts
-Ready harvesting from the fermentation
-Ready breakage if the target product is intracellular
-Production of limited byproducts
Strain improvement
Strain improvement is ongoing to try to continue to reduce production costs and improve product quality.
What are the traits of strain improvement?
Specifically, these global targets can be obtained by finding a microorganism that has or can:
-Rapid growth
-Genetic stability
-Non toxic
-Large cell size for easy removal
-Use of cheaper substrate
-Eliminate undesired byproducts
-Improve cell permeability to improve product export rates
Additional strain improvement
-Don’t screw up batches in bioreactors, be 100% sure microbes won’t cause issue when running process
-You want to tweak microbes genetically so biomass can grow fast
-Making cells large enough so you can skim the particles easier out of liquid
-Modification of microbial cells allows you to use cheaper substrate
-Eliminating undesired byproducts and using less steps with chemical reactions is ideal
Strain improvement is done through?
Genetic recombination (natural method), mutagenesis (natural method), and genetic engineering (not natural method)
-Easier to get permission using natural methods
Natural Genetic Recombination
Brings together genetic elements from two different genomes into one unit to from new genotype
Bacterial DNA
Typically is a single chromosome and potentially 1000 plasmids (self replicating, pieces of DNA); Each plasmid has up to a few 100 additional genes carrying genetic information not found in the bacteria’s chromosome. This genetic information can be passed on to other bacteria through the processes of:
* transformation.
* transduction
* conjugation,
* (Bacteria do not have sexual reproduction cycle)
Transformation
-Is a naked piece of DNA that is taken up by the cell from the surrounding medium and becomes
Transduction
-A bacterial virus acts as a vector in transferring genes between bacteria
Conjugation
-A donor bacteria passes copies of all or part of its DNA through a protein structure called a sex pilus by cell to cell contact
Natural genetic recombination sexual cycle (in eukaryotes)
Eukaryotes – (natural) genetic recombination is through the sexual reproduction.
Crossing over and recombination during meiosis
Mutagenesis
-Is considered as a natural method
-Mutations result from a physical change in the DNA of a cell such as: deletion, insertion, duplication, inversion, translocation of a piece of DNA
-Subjection of microorganisms to repeated rounds of mutagenesis, followed by subsequent selection and screening of the survivors is very effective in improving industrial microorganisms.
Genetic Engineering
Gene technology allows specific gene sequences to be transferred from one organism to another such as sequences that allow
-the production of plant and animal proteins
-new properties can be added
-Examples: human growth hormone, insulin, interferon
Why is Genetic Engineering important?
– Increased product yield by removing metabolic
bottlenecks in pathway
– Increased product yield by amplifying and modifying specific metabolic rates
– Microorganisms to synthesize and excrete enhanced ranges of enzymes
– to facilitate the production of new products
– to use cheaper complex substrates
Strain stability
Both natural recombined or genetically engineered cells / cultures need to be preserved to maintain the selected attribute; often done through storage in liquid nitrogen or lyophilization
