160 presentation.ppt

ADVANCEMENTS IN INDUSTRIAL MICROBIOLOGY

FermentationMaintenance and Preservation of Cultures

Blue-White ScreeningBt Technology

I. FERMENTATION

• Beer undergoes fermentation to develop various flavors and aromas (most time consuming part of beer production)

• "The taste and quality of the beer depend to a large extent on the type of yeast used, and the applied fermentation process,“

---Konrad Müller-Auffermann of the Research Center Weihenstephan for Brewing and Food Quality.

I. FERMENTATION

• Breweries typically use large, cylindroconical stainless steel tanks (CCTs) for fermentation with only one inlet and outlet at the base.

• For ever fermentation cycle, the carbon dioxide, beer and yeast must be removed from the tank. Afterwards, the tanks is cleaned thoroughly before it can be filled by the next batch.

I. FERMENTATION• This made scientists come up with

interconnected tanks each with a central pipe with an open bottom to allow a cascade of reactions to occur and eliminate the downtime.

I. FERMENTATION• Bottom portion - used to discharge yeast cells

and other particles. • Central pipe - aids in transferring the beer to

the next fermentation tank. • The continuous transfer makes it easy to

extract the yeast as it sets.

Natural biopolymer for preservation of microorganisms during sampling and storageIryna Sorokulova, James Watt, Eric Olsen, Ludmila Globa, Timothy Moore, James Barbaree, Vitaly Vodyanoy. (2012)

• B. anthracis Sterne (substitute for pathogenic strain) and S. aureus 1, resistant to methicillin (MRSA)

• Different materials were used for storage of test-microorganisms:– cotton, wool, polyester, rayon, charcoal cloth (Zorflex),

and Whatman paper 41– Half pieces of each type of material were filled with sterile

water solution of acacia gum (150 μL added per piece), while untreated pieces were used as controls

• Samples of each material with acacia gum and samples of the same material without acacia gum were inoculated with MRSA or B. anthracis and were exposed for tested temperatures

Results

• Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5–45 °C for MRSA and 40– 90 °C for B. anthracis).

• Higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum

III. BLUE-WHITE SCREENING

• One of the most common molecular techniques that allow detecting the successful ligation of gene of interest in vector.

• Process:1. DNA of interest is ligated into a vector2. The vector incorporates gene into a competent cell

(bacteria)3. The competent cells are grown in the presence of X-

gal (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside)

III. BLUE-WHITE SCREENING4. Cells transformed with

vectors containing recombinant DNA will produce white colonies and cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies.

III. BLUE-WHITE SCREENING

• Molecular Mechanism– β-galactosidase hydrolyzes X-gal to form

colorless galactose and bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo

– Recombinant cells appear colorless while non-recombinant cells appear blue

– α–peptide disruption in recombinant cells; non-recombinant cells retain an intact α–peptide

– Detection of β-galactosidase activity

Blue-White Screening Limitations

• Use of a screening procedure (discrimination) rather than a procedure for selecting the clones.

• A few white colonies might not contain the desired recombinant but a small piece of DNA to be ligated into the vector's MCS might change the reading frame for LacZα.

• Some cases, blue colonies may contain the insert.• This complex procedure requires the use of the

substrate X-gal which is very expensive, unstable and is cumbersome to use.

Isolation and Characterization of native Bacillus thuringiensis from Syrian Soil

Ammouneh, H. et al. (2010)

• ConclusionThe use of the isolates in the production

of novel biopesticides or the engineering of pest-resistant plants could play a crucial role in controlling important insect pests and lead to the decrease in the usage of some chemical insecticides in Syria.

Beginnings of Bt

• 1995– The first genetically engineered plant, corn, was

registered with the EPA.

IV. Bt TECHNOLOGY

• http://www.mb.com.ph/in-defense-of-biotech-crops/• http://ati.da.gov.ph/rtc1/news/2011/pangasinan-farmers-await-release-bt-eggplant• http://blog.friendseat.com/france-bans-monsantos-genetically-modified-corn/• http://www.science.ph/full_story.php?type=News&key=11426:-in-defense-of-biotech-crops

http://www.mb.com.ph/in-defense-of-biotech-crops/

http://ati.da.gov.ph/rtc1/news/2011/pangasinan-farmers-await-release-bt-eggplant

http://blog.friendseat.com/france-bans-monsantos-genetically-modified-corn/

http://www.science.ph/full_story.php?type=News&key=11426:-in-defense-of-biotech-crops

Why do crops need protection?

• Many vegetables and fruits are wasted each year due to insect damage.

e.g. It is estimated that 40 million tons of corn never reach the market and thus wasted each year due to insect damage.

• If not controlled, corn borer and other crop insect larvae will turn into moths and the cycle continues.

Why do crops need protection?• Scientists have taken the Bt gene responsible

for the production of the insecticidal protein and incorporated it to plants.

Thus these plants have built-in mechanism of protection against pests.

http://www.chemistrylearning.com/benefits-of-genetic-engineering/



What is Bt?• Bt or Bacillus thuriengensis

– Crystalliferous spore-forming gram-positive soil dwelling bacterium found worldwide.

– First isolated in Thuringia, region of Germany.

– Unique characteristic includes the production of crystal like insecticidal proteins known as Delta-endotoxins.

– The protein is poisonous to insect stomachs resulting to paralysis of the larvae of some harmful insects (e.g. Colorado potato beetle, cotton bollworm, and Asian and European corn borers).

http://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htm

Bacillus thuringiensis viewed by phase contrast microscopy. The vegetative cells contain endospores (phase bright) and crystals of an insecticidal protein toxin (delta endotoxin). Most cells have lysed and released the spores and toxin crystals (the structures with a bipyramidal shape).

http://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htm

• Mode of Action

• AS OF TODAY...– With the advancement in molecular biology, it

soon became feasible to move the gene that encodes the toxic crystals into a plant.

– GM (genetically modified) crops including, potato and cotton are planted throughout the world.

Results of insect infestation on Bt (right) and non-Bt (left) cotton bolls. Source: USDAhttp://www.bt.ucsd.edu/gmo.html

http://www.genetic-id.de/en/products-and-services/gmo-testing/gmo-test-parameters.html#GM Potato

http://www.bt.ucsd.edu/gmo.html

http://www.genetic-id.de/en/products-and-services/gmo-testing/gmo-test-parameters.html#GM%20Potato

http://www.genetic-id.de/en/products-and-services/gmo-testing/gmo-test-parameters.html#GM%20Potato

Process1. DNA ISOLATION First, the strain

that can kill the targeted insect must be known first and then the DNA must be isolated from the bacterium.

http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553


Process2. GENE CLONING

The gene responsible for the production of the protein that causes the death of the insects must be separated from the extracted DNA.



Process2. GENE CLONING

A gene conferring resistance (antibiotic or herbicide) is also attached.



Process3. TRANSFORMATION

The BT gene with the resistance gene is inserted into plant cells using various techniques (gene gun, agrobacterium, microfibers, and electroporation).




Plant cells are grown in the presence of antibiotic or herbicide and then the cells that are unaffected by the antibiotic or herbicide are selected.



The plant cells are then grown into whole plants using tissue culture.


• The cells that do receive the new gene are called transgenic and are selected from those that are not transgenic.

• Many types of plant cells are totipotent meaning a single plant cell can develop into an entire plant.

• Therefore, each transgenic cell can then develop into an entire plant which has the transgene in every cell. The transgenic plants are grown to maturity in greenhouses and the seed they produce, which has inherited the transgene, is collected.

Benefits of Bt Technology

1. Improved pest management Bt crops provide season long

protection against several damaging insect pests and reduce or eliminate the need for insecticide sprays. Thus less money used for insecticide and more time other farm management duties.


2. Reduction in insecticide use US Department of Agriculture

reported that 8.2 million pounds of pesticide active ingredients were eliminated by farmers who planted Bt crops in 1998. Same in China where in pesticide reduction was reported to reduce from 60 to 70%.


3. Greater net return Lower input costs due to Bt crops

contribute to a higher net return compared to conventional crops. Better and additional quality yield ensure more profit.


4. Improved conditions for non-target organisms

Lower insecticide or pesticide use encourages the proliferation of beneficial organisms. These beneficial organisms can help control other secondary pests.


5. Less mycotoxin in corn Bt crops have lower incidences of

opportunistic microbial pathogen such as fungus, Fusarium. Fusarium produces mycotoxins that are deadly to livestock and also cause cancer in humans.

Other products containing Bt (commercially available)

• Powders containing a mixture of dried spores and toxin crystals. They are applied to leaves or other environments where the insect larvae feed.

• Represents about 1% of the total agrochemical market (fungicides, herbicides, insecticides) across the world.

Isolation and Characterization of native Bacillu Thuringiensis from Syrian Soil

Ammouneh, H. et al. (2010)

SAMPLING

ISOLATION

ASSAY

IDENTIFICATION

Issues on Bt crops

Issues on Bt crops

• At present...– Bt crops include corn, cotton, rice, and potato.

– Bt corn is already adopted in Argentina, Canada, France, Germany, Honduras, Portugal, Spain, South Africa, Uruguay, US, and Philippines.

Issues on Bt

• Bt in different countries– Bt corn in the US approved in 1996, consumed up

to present.– In South Africa, Bt variety of their staple food

white corn was made available in 1998 and is being consumed up to now.

– In the Philippines, Bt corn was approved in 2002 and was propagated in 2003.

Issues on Bt

• Bt corn was found to have significant economic benefits. – In the Philippines, a study by Gonzales in 2009

concluded an up to 75% income advantage per hectare of Bt corn.

– In the US, it was found out that Bt corn provided farmers with almost $7 billion cumulative benefit from 1996 to 2009.

Issues on Bt

1. Is it safe for the environment?

An international agreement among 135 countries called the Cartagena Protocol on Biosafety was established in 2000. It is a legal global protocol which ensures that products of modern biotechnology are safe for the environment, especially to biodiversity.

Issues on Bt

2. Is it safe for human consumption?

The Bt protein, in order to take effect should attach to specific receptors in the digestive system of target organisms. Non-target organisms like humans, do not have these receptors, so the Bt protein will not be able to work. The Bt protein also needs an alkaline condition and specific enzymes to be activated. Therefore, with the acidic digestive system of humans, Bt protein will be degraded and will thus not cause harm.

Issues on Bt


Before releasing biotechnology products in the market, scientists ensure that the product has passed many tests and biosafety examinations and assessments.

In the Philippines, products are evaluated in four stages:

1) contained research in laboratory and screen houses, 2) small confined trials, 3) multi-location field trials, and 4) commercial release. The Fruit and Shoot Borer

Resistant-Eggplant or FSBR-Eggplant went through these stages.

The National Committee on Biosafety of the Philippines or NCBP was responsible for confined and contained trials while the Bureau of Plant Industry under the Department of Agriculture was in charge of the assessment on large-field trials.

Issues on Bt


“GM foods currently available on the international market have passed risk assessments and are not likely to present risks for human health. In addition, no effects on human health have been shown as a result of the consumption of such foods by the general population in the countries where they have been approved.”--World Health Organization

Issues on Bt

3. How about for other animals?

In the US, the Extension Toxicology Network or Extoxnet, a project of several universities, which tried the Bt on humans, reported that Bt protein did not have any harmful effect on mammals such as dogs, rats, and guinea pigs, amphibians such as frogs and salamanders, and birds.

Issues on Bt

3. How about for other animals?

There was an issue that came out in 1999 regarding the threat posed by Bt corn to monarch butterfly population. Monarch caterpillars feed on milkweed leaves. It was found that they suffered when fed with milkweed leaves heavily dusted with Bt corn pollen. The Agricultural Research Service, US Department of Agriculture, conducted studies regarding this issue and they concluded that there is no significant risk to monarch butterflies and this was published in PNAS.

REFERENCEShttp://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htmhttp://www.bt.ucsd.edu/bt_history.html (University of California, San Diego, USA)http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?

P=NCR553http://passel.unl.edu/pages/informationmodule.php?

idinformationmodule=957879329&topicorder=2&maxto=9&minto=1http://phys.org/news/2013-05-sustainable-brew-beer-non-stop-fermentation.htmlIryna Sorokulova, James Watt, Eric Olsen, Ludmila Globa, Timothy Moore, James

Barbaree, Vitaly Vodyanoy, Natural biopolymer for preservation of microorganisms during sampling and storage, Journal of Microbiological Methods, Volume 88, Issue 1, January 2012, Pages 140-146, ISSN 0167-7012, http://dx.doi.org/10.1016/j.mimet.2011.11.002. (http://www.sciencedirect.com/science/article/pii/S0167701211003903)

Ammouneh, H. et al. (2010). Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against some insect pests. Turk J Agric For. 35 (2011). 421-431.

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