Ch 12: Microbial Biotechnology

Explain how bioreactors are used for biotechnology fermentation processes.

- allows large volumes of microbial culture to be grown under very specific conditions. - allows precise control over temperature, amount of aeration, and pH, to optimize the fermentation processes as required.

What problems may arise from running a chemostat or fed-batch fermentors?

- contamination, bacteriphages - fed-batch more common because smaller, short, period of time so mistake is easily recoverable

Discuss how transgenic plants are created via transformation by Agrobacterium and/or by biolistic approaches.

-Agrobacterium (nature's genetic engineer) causes crown gall on plants via a tumor-causing plasmid, scientists have taken out tumor genes and put genes of interest, so makes it a good "delivery system" for gene insertion into plants because bacterium loves to get inside plant cells and insert DNA; represents a cross-kingdom transfer of DNA (bacteria to plants) -other methods: removal of cell wall prior to DNA introduction (protoplast formation), metal fragments with DNA coating fired into plant cells (biolistics)

Explain how white biotechnology applications have had an impact on products that we use in our daily lives.

-Bioplastics: plastic use and buildup is tremendous and nonbiodegradable, but microbes can produce natural polyesters (PHB and PHA) -Enzymes (that come from microbes) for production of high fructose corn syrup, in laundry detergents, stain and/or odor removers -vitamins and amino acids: added to fortified food products, sold as over-the-counter supplements, used for nutritional supplementation (microbes produce a lot of vitamin B) and flavor enhancement products (MSG) -working on using microbes to convert grease to biodiesel and glycerol -ethanol and butanol through fermentation

Discuss the difficulties associated with the technology for producing recombinant human proteins in a bacterium.

-Introns have to be removed from eukaryal DNA -have to have all of the above on the plasmid in order for bacteria to express the gene

Define and discuss bioprospecting with respect to biotechnology application and ownership rights.

-bioprospecting: searching for useful new microbes to cultivate and add to collections; look for microbes with new characteristics such as antibiotic production -who owns these if industry finds them and keeps them to themselves? they spent the money, should they only profit from it or should the world?

Explain how white biotechnology applications have had an impact on fossil fuel utilization and controversies associated with implementing white biotechnology applications in this field.

-fungi ferment sugars to produce ethanol (can be used in internal combustion engines) -issues: commercial scaling and cost of feedstock use can be difficult to overcome, some lignin/cellulose feedstocks are more difficult to breakdown than others -what's a waste and what's not a waste, what can humans use, don't take away my corn my animals and kids can eat that and price of beef will go up -switchgrass and sugarbeat pulp being investigated to use as feedstock

Explain how green biotechnology applications have had an impact on herbicide and insecticide producing transgenic plant controversies associated with implementing green biotechnology applications in this field.

-genes can be inserted into plants to allow them to make their own herbicide/pesticide -these methods (transgenic plants) do have possible human health and environmental repercussions -public is very concerned with "Frankenfoods" like the FlavrSavr GMO tomato (commercially grown 1994, removed from market in 1997); tomato carried gene (that made antisense RNA) for enzyme that would make it much more difficult to go through rotting process- picked ripe instead of green so they'd be more flavorful and less open to damage

List and discuss the roles which microbes play in the white biotechnology sector.

-use microbial low-cost conversion of waste biomass to product with a higher value and industrial use (biofuels, bioplastics, industrial enzyme production, production of vitamins and amino acids); act on feedstock to produce useful materials in biorefineries

For the molecular genetic modification of microbes section, describe the different protocols which scientists employ (including their advantages/drawbacks) but you do not need to explain the processes.

1. Random mutagenesis: chemical/radiation exposure followed by screening for desired mutations; drawbacks= possibility of negative mutations, difficult, labor-intensive, and costly 2. Site-directed mutagenesis: specific mutations are made at specific DNA sites; at positions that might stabilize protein folding/bonds or open up binding sites in enzymes or substrates; much more cost-directed and time-saving but requires much more knowledge about molecular level of DNA and subsequent proteins (Ex. engineered protein form disulfide bonds that stabilize structure at high temperatures where it would normally denature) 3. Directed enzyme evolution mutagenesis: use gene cloning and progressive rounds of random mutation to "direct" and select for desired traits 4. Produce recombinant proteins: expression vectors transferred into bacteria to mass-produce (20% or more of total protein in cells) recombinant proteins (Ex. human insulin production)

Discuss the advantages, and disadvantages of industrial fermentation...

Advantage: predictable and desirable end-products & Controlled growth rate culture of microbes to produce desired substances (Bioreactors, Fed-batch reactors, Chemostats) Disadvantages:

For the production of recombinant proteins, explain why each of the specific needs on the expression vector is required: bacterial origin, bacterial promoter and operator, bacterial Shine Dalgarno, bacterial stop codon, selection marker.

Bacterial origin: Bacterial promoter: operator: bacterial Shine-Dalgarno: so bacterial ribosomes can recognize it and translate it, bacterial stop codon: selection marker: to make bacteria want to express this protein, grow in area where it needs that protein to survive so it doesn't kick plasmid out

Definition industrial fermentation.

Definition: controlled growth rate culture of microbes to produce desired substances

Compare and contrast different types of bioreactors used in fermentation: batch-fed vs. chemostat

Fed-batch: go through all phases (lag, stationary, etc.) and then harvest the whole batch (secondary metabolites) -provide a constant supply of specific nutrients to achieve high cell densities and minimize the production of non-desired side products. Chemostat: continuously add fresh medium and continuously drip out culture; keep things at logarithmic phase or stationary phase, up to you, which phase you choose depends on end products (primary metabolites) -allows the growth rate of a culture to be controlled by constantly feeding in a certain amount of medium while removing the equivalent amount of culture. This ensures that the physiological state of the culture can be maintained.

Biotech applications can be grouped into which 3 groups?

Green: Agrocultural Sector Red: Medical Applications White: Industrial Applications

Discuss the importance of microbial culture collections with regard to biotechnology applications.

Preserves specimens that other scientists can obtain Helps promote confirmation of findings through repeatable independent research

primary vs. secondary metabolites

Primary: product of metabolic processes REQUIRED for growth of the microbe (Ex. alcohol)- produced during log/exponential phase (when the organism is actively growing) Secondary: NOT required for microbial growth, often produced during stationary phase (Ex. antibiotic); produced to give microbes competitive advantage

Compare and contrast red vs. white vs. green biotechnology applications in a general sense.

Red: medical applications White: industrial applications Green: agricultural sector

Define Bioprospecting

Searching for useful new microbes to cultivate and add to collections, but who profits?

Identify the roles of natural secondary metabolites as well as recombinant human proteins produced in microbes in the red biotechnology sector.

Secondary metabolites: can be modified in the lab to produce semi-synthetic drugs (drugs that go through a series of reactions after being harvested from microbes ex. penicillin put though reactions); improvements have dramatically lowered production costs for drugs Ex. antibiotics and statins (natural products of fungi, inhibitors of cholesterol synthesis) Recombinant human proteins: cheaper but ethical issues: formerly derived from beef or porcine origins (pig insulin only had one AA different from humans) PETA was not a fan Ex. Type I interferons or human insulin or human growth hormone

Define Microbial Biotechnology

Use of biological processes and/or organisms for the production of goods or services