ADVANCEMENTS IN INDUSTRIAL

MICROBIOLOGY
Fermentation
Maintenance and Preservation of Cultures
Blue-White Screening
Bt 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 storage
Iryna 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 vector
2. 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 SCREENING
4. 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)

• Conclusion
The 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
 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/be
nefits-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 http://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htm
Delta-endotoxins. Bacillus thuringiensis viewed by phase contrast
– The protein is poisonous to insect microscopy. The vegetative cells contain
endospores (phase bright) and crystals of an
stomachs resulting to paralysis of insecticidal protein toxin (delta endotoxin). Most
cells have lysed and released the spores and
the larvae of some harmful insects toxin crystals (the structures with a bipyramidal
(e.g. Colorado potato beetle, shape).

cotton bollworm, and Asian and

European corn borers).
• Mode of Action

Protein binds to
Delta-endotoxin Bt Protein activated
specific receptors
ingested by the in the insect gut's
on the insect mid-
larva alakaline condition
gut

Protein inserts into

Pores disrupt the the membrane and
Death of the insect
digestive process forms ion-specific
pores
• 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 http://www.genetic-id.de/en/products-and-

non-Bt (left) cotton bolls. Source: USDA services/gmo-testing/gmo-test-parameters.html#GM
Potato
http://www.bt.ucsd.edu/gmo.html
 Process
1. 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
 Process
2. 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.

http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553
 Process
2. GENE CLONING
A gene
conferring
resistance
(antibiotic or
herbicide) is also
attached.

http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553
 Process
3. TRANSFORMATION
The BT gene with
the resistance gene
is inserted into
plant cells using
various techniques
(gene gun,
agrobacterium,
microfibers, and
electroporation).
http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553
 Process
3. TRANSFORMATION
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.

http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553
 Process
3. TRANSFORMATION
The plant cells are
then grown into
whole plants using
tissue culture.

http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553
• 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.
 Benefits of Bt Technology
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%.
 Benefits of Bt Technology
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.
 Benefits of Bt Technology
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.
 Benefits of Bt Technology
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

IDENTIFICATION

ASSAY
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
2. Is it safe for human consumption?

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
2. Is it safe for human consumption?

“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.
REFERENCES
http://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htm
http://www.bt.ucsd.edu/bt_history.html (University of California, San Diego, USA)
http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=NCR553
http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=95787932
9&topicorder=2&maxto=9&minto=1
http://phys.org/news/2013-05-sustainable-brew-beer-non-stop-fermentation.html
Iryna 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.