MICROBIAL BIOTECH: apps of scientific and engineering principles to the

processing of materials by microorganisms to create useful products or processes

Applications of microorganisms in 4 categories: agriculture, industry, health, and
molecular biology research
Microorganisms: minute living things that are unseen by naked eyes / diverse and
unique life forms / distribute ubiquitously in nature.
Including: bacterium, virus, mold, yeast, algae, protozoan, archaeon
Microorganisms are used in the production of fermented foods / enzymes, bioactive
compounds (medicals and pharmaceuticals) / bioremediation, waste treatment.
How to get microbial products: naturally isolated / lab selected mutants / microbes
that are genetically engineered using recombinant DNA methods.
Deals with the prevention of deterioration of goods / environmental protection, waste
disposal system | production of antibiotics, enzymes, organic acids by fermentation.
Food and Agriculture
Genetically engineered plants with internal resistance to drought, flood, pest and
infestation
Reduction of chemical fertilizers / alternatives to expensive fertilizers
Replacement of chemical pesticides with microbial pesticides
Herbicide-tolerant crops
Products with high yield / enhance nutritional value
New biomass products using bacteria, algae, yeast, fungi
Food improved and preserved by fermentation
Fermentation: process releasing alcohol beverage / acidic products | spoilage of food
| large-scale microbial process occurring with or without air | release energy from
sugars, inorganic molecules.

lactic fermentation
alcoholic fermentation
2
lactic acid
Pyruvate
ethanol + CO

Product during fermentation: enzymes, vitamins, antimicrobial compounds, texture-
forming agents, amino acids, glutamic acid, organic acids, flavor compounds.
Examples: GM = genetically modified
GM beer: fermentation by GM brewers yeast containing glucoamylase gene that
enables production of low-carbohydrate beer without the use of extra enzymes.
GM wine: fermentation by GM yeast containing malolactic enzyme gene (from
bacterium) + malate permease gene (from fission yeast) soften the feeling of wine
to mouth (by decreasing of acidity) + reduce buttery flavor (due to lactic a 2
nd

metabolism)
GM Vitamin B
2
: Pure riboflavin produced by GM strain of Bacillus subtilis, used in food
fortification (amplification), small amount for colorants
GM red yeast produce large quantities of pigment astaxanthin, which is used as a
coloring agent
Molecular diagnostics: outstanding tools for detection, identification, and
characterization of strains improvement in fermentation + detection of microflora
(microbes causing food spoilage).
Genetically based method is more specific, sensitive, rapid that the classic one
Classic diagnostic methods Molecular diagnostic method
- Culture based
- Microorganisms grown on agar and
detected thru biochemical identification
- Tedious, labor-intensive, slow
- Enhancing specificity, sensitivity, and
speed: PCR, DNA-sequencer, microarray,
gene chip, ELISA
Detection pathogens, toxins, pesticides
Facilitating the process, fermentation controlling, monitoring the safety of raw
materials and by products
Functional Genomics aims to determine patterns of gene expression, and
interaction in the genome (based on knowledge of genomic sequence of an organism)
know that which gene codes which protein
Agriculture production of proteins from microorganisms improving plant, animal
productions, food processing properties
Examples:
GM Squash contains coat proteins from watermelon mosaic virus + zucchini yellow
mosaic virus (created by Agrobacterium-mediate transformation). The crookneck
squash demonstrates remarkable field against two viruses.
GM Grape introduced with coat protein of grapevine fanleaf virus (GFLV) thru a
bacterium vector exhibits resistance to infection by GFLV.
Herbicide tolerant plants eliminate the environmental risks of using traditional chemical
herbicides. Using rDNA, the gene coding for phytotoxins can be identified, isolated, and
modified by mutagenesis, and then re-introduced into plants to create herbicide-
tolerance.
Chemical Industry Production of specialty chemicals (enzymes, vitamin,
sweeteners, food additives, polysaccharides, fragrances) + conversion of biomass into
specialty chemicals
Amino acids
Produced by isolation of natural materials, hydrolysis of plant proteins, chemical or
microbial enzymatic synthesis / GM strains of bacterium Corynebacterium efficiens
increase the yield of glutamic acid production.
Enzymes are biological catalysts that facilitates, speeds up metabolic reactions in living
organisms.
Industrial applications include the production of cheese (chymotrypsin), clarification of
apple juice and wine (pectinase and protease), laundry detergents (subtilisin), pulp and
paper production (cellulase and xylanase), sewage treatment (lipase and protease).
Enhanced the activity, specificity, stability at unusual optimum conditions are
achieved using rDNA technique, and protein engineering
Polysaccharides produced by yeast, fungi, and bacteria, and is used in food,
cosmetics, chemical, medical, and oil industry
Lubricants, viscosifiers, flocculating and gelling agents in food processing and
stabilizing liquid suspensions
Medicine and Pharmaceutical Industry
Insulin treatment of diabetes
Vaccines production uses only a part of specific antigens of the pathogenic organisms
that eliminate disease-causing ability.
Interferon: proteins with 2 biological effects: inhibition of cellular proliferation /
modulation of immune sys.
DNA probes: identify defects or mutation / diagnostics of bacteria casing gum diseases
and genetic diseases
Gene therapy: modification of microorganisms to increase yield / improve action
of antibiotics, biological agents treatment of hypertension, coronary hearts disease,
obesity, inflammation, and cancer
Industry
Biofuels

biomass conversion
organic matters biofuels
Renewable, less environmentally hazardous

anaerobic condition
2 4
sewage sludge H , CH , bio- diesels
Ex: Streptomyces:
cellulase
cellulose ethanol
Electricity
Ex: Geobacter sp. Clean toxic waste, generate electricity, sport minuscule wires
Mining
Extraction of minerals from ores using bacteria to leach metals from mine dumps
increase recovery rate + lower costs. Ex: Sulfolobus sp
Environment
Bioremediation: the use of microorganisms to degrade wastes less or non-toxic
materials healthier, safer, and cleaner environment / Ex: S. cerevisiae clean up
radioactive wastes (uranium), Rhodococcus sp. Bioremediation of PCB
Waste, Wastewater Treatment detoxify wastes in water, soils, sediments, sludge
Ex: Pseudomonas stutzeri degrades CCl
4
, Azoarcus tolulyticus breaks down toluene,
Clostridium bifermentans degrades TNT

FERMENTATION & ANTIBIOTIC PRODUCTION
Industrial biotech: the use of microorganisms in large scale to produce valuable
commercial products or carry out important chemical transformations. This process
is called fermentation.
Antibiotics are the most important manufactured commercial products. They are
produced by microorganisms to kill other ones treatment of infectious disease
Aerobic and Anaerobic Fermentation
Aerobic requires:
Adequate aeration / Bioreactor: supplied with sterile air; mechanisms of stirring +
mixing cells + mediums Vitamins, enzymes, antibiotics
Anaerobic: the provision of air is not usually required lactic acid, ethanol, wine
Fermentor an apparatus (bioreactor) | Fermenter an organism that uses
fermentation as a metabolic process
Industrial fermentor: 125 250 m
3
/ Conditions are monitored to regulate cell growth
/ before fermentation, fermentors + tubes must be sterilized by flushing the system
with superheated steam.
Frequent aerobic processes require the fermentor to be well aerated. Sufficient
aeration is to mix with many cultures. Thick and sticky cultures require additional
stirring using impeller (motor driven paddle).
Initially, warm the culture to start the process. Once it has started, cooling system is
vital (a water jacket or a cooling coil)
Fermentation could be batch mode or fed batch mode (continuous)
Batch fermentation
Most fermentations are batch processes.
Nutrients, inoculum (and maybe anti-foaming agents) are added in sterile fermentor
and left to get on with it! | Once the desired amount of product is present, contents
are drained off and product is extracted. The fermentor is emptied, then cleaned to
prepare for a new batch.
Some products are made by continuous culture system, in which sterile medium is
added balancing with withdrawal the broth for product extraction.
ANTIBIOTIC PRODUCTION
Penicillin production
- Fleming: the ability to kill staphylococcus of the mold Penecillium notatum.
- 1
st
production in large scale during WW2. The importance is remarkable because
penicillin could kill bacteria. Therefore, it was used to treat many wounded soldiers and
saved many lives during WW2.
- 1
st
important commercial production by aerobic, submerged batch fermentation / 1
st

to be manufactured in bulk / Input for some semi-synthetic antibiotics
WW2, Penicillium notatum 1mgdm
-3
. Today, P. chrysogenum 50gdm
-3
, and
constant search is still conducted for better yield
Mode of Act: Penicillin inhibits the synthesis of peptidoglycan (an essential
component of cell wall) without interfere with the synthesis of other intracellular
components. This antibiotic does not affect human cells due to the lack of cell wall.
Spectrum of Activity:
Penicillin is active against Gram(+) bacteria. Amoxcillin is also active against
Gram(-) bacteria but not Pseudomonas aeruginosa.
How to increase the yield of penicillin?
- Improvement of composition of culture medium.
- Isolation of better penicillin producing mold (P. chrysogenum)
- Development of culture technique cultivate the mold in large volume
Primary and secondary metabolites (products of metabolic processes)
1 metabolites are produced during active cell growth, while 2 ones are produced on
the onset of stationary phase. Antibiotics, and penicillin in particular, are 2 metabolites
harvested on stationary phase.
Industrial production of penicillin is classified into 2 processes, which are upstream
and downstream.
Upstream: technology synthesis of products (= exploration + development +
production)

Inoculum preparation Spores sources of inoculum.
Raw materials
Carbon sources: lactose 6% (very satisfactory compounds), glucose, sucrose
Nitrogen sources: corn steep liquor (CSL), (NH
4
)
2
SO
4
, CH
3
CO
2
NH
4

Mineral sources: P, K, Mg, Cu, Zn, S (from corn steep liquor)
Calcium form chalk to neutralize the acidity of CSL
PAA precursor (phenylacetic acid)
Fermentation process
The medium is inoculated with suspension of conidia of P. chrysogenum / constantly
aerated + vibrated (and molds grow as pellets). After 7 days, pH | >8, suppress the
production of penicillin.
Downstream = extraction + purification of the products.
Including following stages:
Cell removal: 1
st
step: separate cells and other insoluble components using filtration or
centrifugation
Isolation of benzyl penicillin:
+ H
2
SO
4
or H
3
PO
4
to lower pH to 2 2.5, at which partition coefficient favors organic
phase the solvent butyl acetate. This step should be carried out quickly as penicillin is
unstable in acidic condition. Then, the antibiotic is extract back in buffer 7.5 solution,
which favors the aqueous phase. Next, the aqueous solution is re-acidified and re-
extracted back with an organic solvent These shifts between organic and aqueous
phase help in purification of penicillin.
Treatment of penicillin extraction varies according to objectives, but involves the
formation of penicillin salt.
The solvent recovered in previous stage is carefully extracted back with aqueous
NaOH charcoal treatment to remove pyrogens and by sterilizations.
Pure penicillin salt is then sterilized with dry heat then the sln of penicillin is subjected to
crystallization.
Further processing:
Parental used packed in vial of powder suspension.
Oral used tableted + film coating
Searching tests are performed on number of random samples to make sure it satisfies
all strict government standards before being marketed.
1/ CSL + yeast extract + other substrates fermentor
2/ After 40hrs, penicillin is secreted by the fungus
3/ Cell matters are filtered from products
4/ Penicillin is extracted with butyl acetate an organic solvent.
5/ Add K
+
salt penicillin + being washed + dried
Production of Penicillin V
Phenoxy methyl penicillin / Precursor is phenoxyacetic acid instead of PAA (addition of
different acyl group to the medium).

GENETIC ENGINEERING makes change to DNA to change the way living things work
GM (genetically modified) C: crops / O: organisms / F: foods
Genetic engineering: create new crops/animals, make bacteria to make new medicines,
human body parts, prevent genetic diseases & change humans
Direct manipulation of DNA
1/ Isolation of plasmid DNA of bacteria + DNA containing gene of interest
2/ Gene inserted into plasmid (thanks to restriction enzymes and ligase)
recombinant DNA
3/ Transfer rDNA to bacterium recombinant bacterium
4/ Cells cloned with gene of interest
5/ Identification of desired clone
copies of genes / proteins
6/ Various applications
Agrobacterium: infect plants; insert some of their DNA to plants genome, forming a
gall.
Ti plasmid = tumor inducing plasmind
Cointegrate Ti-plasmid: intermediate molecule that donor DNA and target DNA can bind
to.
Transgenic plants have new / different characteristics: resistance to stress (frost, cold,
drought), herbicides, and bacteria (Bt toxins)
Pros & Cons
Pros Cons
Crops
- Better taste / quality (more nutrients,
food, stress tolerance; improve resistance
to herbicides, insects, disease)
- Less time to ripen
- New products, growing techniques.
Animals
- Increase resistance, productivity,
hardiness, feed efficiency
Safety
- On humans: Adverse side-effects:
allergens, unknown effects
- On environment: unintended transfer of
transgenes, loss of flora/fauna biodiversity
Access and Intellectual Property
- Domination of food production by few
companies
- Dependence of developing countries on
- Improve health, diagnostic methods
- Better yield of egg, meat, milk
Environment
- Friendly
- Conversion (soils, water, nrg)
- Better natural waste management
- More efficient processing
Society
More food for growing populations
developed one
Ethics
- Violate natural organisms
- Mix genes among species
- Stress for animal
- Objections to consuming plant genes in
animals and vice versa
Labeling
- Not mandatory
- Mix GM and non-GM organisms
Examples: Strawberries with antifreeze-gene isolated form the flounder do not smell or
taste like fish. The gene is transferred using a second bacterium to infect the
strawberries.
Estimate: 70% foods have at least one ingredient genetically modified. Ex: GM
tomatoes dont have to be picked up early, and have better taste. GM corns are reduced
being eaten by insects.
Bananas are starchy fruits that have unique reproduction method, and are susceptible
to viruses, pests, and fungi. Agroscience is trying to find a way to enhance bananas
ability to ward off parasite by copying genes of other fruits and inserting them to
bananas genome.
GM rice = golden rice containing |-carotene (vitamin A precursor) [expresses enzymes
of |-carotene pathway in rice endosperm). 3 new genes are implanted: 2 from daffodils
+ 1 from a bacterium. Advantage: Amelioration (= improvement) the hunger, lower
malnutrition and vitamin A deficiency in developing countries.
GM foods we eat daily have all been tested for safety, but people are still concerning
about the long-term effects.
Challenges ahead: Overpopulation Deterioration of soil quality, climate change,
less cultivated land, diminishing of water resources, malnutrition & undernourishment
The role of biotechnology is to increase the yield ceiling + provide sustainable
production system.
Agriculture: from resource based science base
GM crops are genetically improved by having one or more genes from the same or
different species artificially inserted to their genome tissue culture &
transformation give it flexibility.
Soybeans have the largest acreage of GM crops
Bt-cotton: 1
st
GM crop in 2002 and 2
nd
largest global production increase yield and
decrease the use of pesticides
GM Food Classification
- Food containing living organisms.
- Food derived from GMOs: ex: soy oil, corn flour
- Food containing ingredients produced by GMOs: vitamins, essential amino acids
- Food containing ingredients processed by enzymes produced by GMOs: high
fructose corn syrup produced using recombinant glucose isomerase
The Risks
Human Health
- Alter the nutrition profile of conventional food
- Introduce new toxins / existing or new allergens
Environmental
- Horizontal gene transfer (genes from GM foods can spread)
- Effects on non-targeted organisms
- Development resistance by pests
Social
- Unfamiliar with the technology / Lack of reliable info
- Negative media option / objections by activist groups
- Mistrust of the industry
Tradition
- Rarely, foods we eat today are assessed for safety.
- Some foods with toxins or allergens are still being used base on experience & history.
What is safety?
OECD (Organization for Economic Cooperation & Development) defined: The one
which, as far as we know, and with the exception of some individuals, who may be
sensitive or allergic, when consumed in moderation over a period of time does not
result in identifiable harm to the consumer. & Absolute safety is difficult to prove
What has been done so far?
- GM chickens without feathers / GM mice that glow and have no furs / GM salmons that
grow faster
- GM mouse: gene that coding for glowing protein in jellyfish sliced into viral
[virus] cell (with no harm) [as vector] transferred to fertilized egg adult mouse that
glow faintly
Whats next: red, blue, glowing fish
Cabbages containing gene coding for poison at the tail of the scorpions become
insecticides.
Insertion of human gene sheep produce o-1-antitrypsin in their milk
Genetic engineering works because there is one language of life
Gene therapy: modification of microorganisms to increase yield or improve the
reactivity of antibiotic and other biological agents
Ex: Treatment of patients with cystic fibrosis, who contain a gene that cannot work
properly. The same gene from a healthy person is spliced into a virus, which infect the
patients lung and deliver working gene to patients cells make right proteins and thus
can work normally.
Mouse with human ear made from cartilage
First chimera (hybrid creature of human and animal) is made by Chinese scientists by
fusing human cells + rabbit eggs
Cloning
CC-cat = gene from three-colored female cat (tortoiseshell or calico) + egg cell whose
nucleus is remove embryo, which is implanted into Allie, the surrogate mother.
Scientists at Massachusetts Advances Cell Technology (ACT) have succeeded in
cloning a gaur, which is threat of extinction in South East Asia = gaurs skin cell + cows
egg embryos, which were then implanted to cow womb. Next step by ACT is to clone
an extinct animal, the bucardo. Scientists have discovered the last dead animal and
currently preserved and froze tissue samples.
Judging by the successful growth of the combined human-cow clone embryo suggests
that cows mitochondria may well be compatible with human embryonic development

ENVIRONMENTAL BIOTECHNOLOGY, BIOREMEDIATION

2
O consumption
controlled release of energy (slow burning)
bacteria
2
Cell growth (division+increase in biomass) Organic pollutants

+CO evolved+energy +nutrients (C,P,S,N,O,Fe...)
Requirements for biodegradation:
Nutrients: C, N, P, S + essential chemicals + micronutrients/ traced metals /
vitamins + electron acceptors (usually O
2
or inorganic molecules)

convert/burn carbon substrates

CO
2
+ energy + increase in biomass (GROWTH)
Electron acceptors are crucial for biodegradation reactions in environment.
Rate of biodegradation reaction:
FAST GROWTH O
2
/H
2
O NO
3
-
/NO
2
-
, N
2
SO
4
2-
/H
2
S Fe
3+
/Fe
2+
SLOW GROWTH
Anaerobic Growth:

fermented
methanogenesis
2 2 4 2 2
acetic acid
Organic matters
+CO +H CH +H O+CO

Fixation of O
2
is the first and key step in biodegradation
e
-
NADH/NAD
+
reductase Ferredoxin
NAP

ISP
NAP
O
2
/ H
2
O
Environment and Biotechnology
Environment is the term to describe everything surrounding a particular organism.
This includes:
- Other organisms
- Soil, water, air
- Temperature, humidity, radiation
Environmental biotechnology is the use, development, and regulation of biological
systems for bioremediation of contaminated environments and for environment-
friendly processes.
Bioremediation is the use of microorganisms to remedy environmental problems (i.e.
to remove the contaminants from the environment).
Events that triggered the environmental biotechnology: pesticide use, oil spill
Advents of the Industrial Revolution: More products, wastes / People moved to the city /
Increase in population
Regulations were passed, including:
Resource Conservation and Recovery Act in 1976:
- Identify hazardous wastes; establish standards for managing them properly
- Require companies to have permits, stating how to manage the wastes
- Chain of Custody
EPA (Environmental Protection Agency) initiated Superfund Program (1980):
counteract against careless negligent practices
Environmental Genome Project: study, understand the impacts of environmental
chemicals on human diseases
Waste:
- Solid: landfills, combusting-including waste-to-ngr plants, recovery (slurries,
composting)
Liquid: septic: sewage treatment, deep-well injection
Gas: fossil fuels, chlorofluorocarbon
Hazardous: anything that can catch fires, explode, release toxic fumes, or even particles
causing corrosion
Recycled largely by microbes
Biogeochemical Cycles is the major part of the Recycling Process
Carbon cycle: primary: organic compounds CO
2

Nitrogen cycle: proteins NH
3
/NH
4
+
NO
2
-
NO
3
-
NO
2
-
N
2
O N
2
NH
3
/NH
4
+

Sulfur cycle: numerous oxidation states (Winogradsky column)
Phosphorus cycle: soluble / insoluble form
Scientist learnt from nature in 1980s:
The Gaia concept: The whole world is a living organisms and what nature makes,
natures can degrade (bio-infallibility), only humans make xenobiotic compounds