In most cases, one can't use wild type microorganisms in industrial processes - they either don't grow well enough, make enough product, support enough plasmids or bacteriophages, or they may be environmentaly or medically unsafe. Therefore, new strains incorporating the best features possible must be developed.
(biochemical markers)
1. Plate out (or other technique) and pick those organisms with the desired characteristics; as observed by tests and/or descriminatory media
2. Select for desired characteristics (those without them will not grow or will die under selection conditions) using selective media and/or the presence or absence of specific chemicals
This allows for new forms to be developed, which might be better
than the wild type organisms
1. Ultraviolet radiation
2. X-rays
3. Gama radiation, etc.
4. Chemical mutagens
1. Drying (on a filter?)
2. Lyophilization
3. Storage under liquid nitrogen
4. Use of low salt media and/or water agar
1. Classical method: protein --> mRNA --> cDNA --> hybridization to the chromosome at the site of the desired gene after digestion with restriction enzymes
(if this is a deleterious gene) - after identifying and obtaining a "restriction map" of the gene and its neighborhood, cut out the gene out with restriction enzymes and religate the chromosome
1. Identify useful gene
2. Cut out useful gene
3. amplify DNA - so that there will be enough to work with
a. Classical method - clone into bacteriophage
b. PCR (polymerase chain reaction)
4. Insert gene into host microorganism
a. Plasmid - utilizing restriction enzymes, ligation, etc.
i. E. coli (best known)
ii. B. subtilis (leaky)
iii. Yeast or other eukaryote
b. Genome (gene therapy?)
5. Production of gene product
Ideal is to maintain the optimal growth and production conditions for a large culture over an indefinate period so that the most product can be made. The growth process is called fermentation; this term is not related to the biochemical processes undergone by the organism when it is used in this manner. First, the process is developed in small, laboratory conditions, then 'scaled up' to large scale, production conditions. There may be unexpected physical and/or biological problems encountered during scaleup
1. Stirred
2. Lift tube (filtered air - or other gas mixture - provides stirring)
3. Solid state
4. Fixed bed reactor - microbes attached to particles in a fixed bed
5. Fluidized bed reactor - microbes attached to particles which become suspended in the medium
6. Dialysis culture unit - diffuse away wastes, diffuse in nutrients (substrates)
7. Continuous culture unit - (like chemostat) medium drips in, medium with cells drips out
Use as much low cost, redily availible constituents as possible. However, this can make
it difficult to keep a consistant formula for the growth medium as these constituents can vary in
composition from batch to batch, and they must be checked before use.
1. Formulia optimized for growth
2. Formulia may become limiting for a specific nutrient which might stimulate production and/or cause specific genes to become active.
a. Some factors (carbon source, etc.) must still be added constantly 'continuous feed' or in batches over time so that unwanted metabolites don't build up (as they can with excess substrate)
1. Temperature
2. pH - buffering woth phosphates (P source), chalks. carbonates
3. Oxygenation
4. Other gases (CO2 etc.)
1. Contamination
2. Maintainance of conditions and/or reliable cycling of conditions
3. Clumping and formation of filamentous mats by the micrroorganisms (especially fungi) and/or their products
a. Non-Neutonian broth - viscous, resists stirrring and/or aeration
b. Blocks pipes etc.
pharmaceuticals (antibiotics etc.); bio/medical (hormones,
steroids,etc.); solvents, organic acids (lactate, pyruvate, etc.); amino acids; enzymes; animal (and
human?) feeds
Made during growth phase and related to cell growth and cell division products and/or products engineered to be activated during growth phase (trophophase) - such as amino acids, nucleotides, fermentation products (ethanol, acids), some enzymes, and varieties of bioengineered products
Accumulate during stationary phase (idiophase), not related to cell growth, can be triggered by medium conditions - such as antibiotics, mycotoxind and some bioengineered compounds which have not been grafted to growth - related operator/promoter regions
In most cases, tricks involving limiting or adding substances to the medium, using cell mutants and/or bioengineering, are used to get the microorganisms to overproduce the desired product(s), as most cells will only make enough of any product as is necessary for maximal growth and no more
1. Penicillin (Penicillium chrysogenum) - Primary metabolite under less than maximal growth; low glucose and/or lactose with limited nitrogen to slow growth plus specific precursors --> removal of molds --> isolation of antibiotics --> chemical modifications (if any)
2. Streptomycin (Streptomyces griseus) - Secondary metabolite --> growth --> idiophase - -> rise of antibiotic concentration with nitrogen limitation, increasing pH --> removal of supernatent --> isolation of antibiotic
3. Bioconversion - Microbial transformations or biotransformations - sort of an inside - out fermentor. Since enzymes and living systems are used, the energy input is minimized
a. Biocatalysts
i. purposeful utilization of microorganisms to perform specific tasks
Bacteria + 4 Fe+2(aq) + O2(g) + 4 H+ --> 4 Fe3+(aq) + 2 H2O.
e.g. Thiobacillus thiooxidans,Thiobacillus ferrooxidans (Hardanger?) cause ferric iron reduction (in iron pyrites) when they oxidize sulfur, and are useful alone or, at best, together in purifying copper in mine tailings (which are usually acid). They work at acid pH (pH 1.6) and at 20 t0 35oC, and may attach themselvs to the pyrite crystals.
, sometimes used together in culture to get maximum yields.
Leptospirillum ferrooxidans
attaches directly to pyrite crystals and causes similar reactions. It is used in purifying Gold, Copper and Zinc from mine tailings.
b. Biodeterioration
i. problem causing, can be regulated using microbial inhibitors
c. Insecticides
d. Biosensors - used with microelectronics and microorganisms working together e.g. to monitor level of contaminants etc.