Microbes & Biotechnology

Role of microbes in the production of bulk chemicals, pharmaceuticals, and other

products:
Microbe(s)
Raw ingredient(s) Product
(Substrate)
Substrate:
- Starting materials or ‘ingredients’; includes the growth medium
- Should be available in large quantities
- Ideal if you can use waste from other processes as your substrate
- e.g. Whey – is a waste products of cheese production – can be used to produce
__________________________

Microbes:
- Must be able to make large quantities of the product
- Must _____________________________________________________
- Must be stable – can’t mutate (change) easily – want consistent results
(products)
- so cultures must be preserved
- e.g. _____________________________________________________
- The search is always on for new and improved strains of microbes which are
capable of producing greater quantities of the product or a superior product –
called __________________________

Example:
- The original Penicillium isolated in 1921 by Alexander Fleming was Penicillium
_______________ the problem with this organism was that it could not produce
large quantities of penicillin
- In 1943, a strain of Penicillium chrysogenum was isolated
 - apparently from a piece of fruit from a supermarket in Pretoria, Illinois
(USA)
- The strain produced much greater quantities of penicillin
- Existing strains of microbes can also be genetically manipulated to improve their
usefulness
- This can be achieved by treating the microbes with
__________________________________________________________
- The Penicillium chrysogenum strain originally isolated from fruit, for
example, was later treated in the laboratory with
__________________________________________________________
- These created mutations in the mold which resulted in a 20-fold increase
in penicillin production
Product:
- Need an efficient, economical way to recover and purify the desired end product
- At the end of the process, you may be left with:
o __________________________
o __________________________
o __________________________
o __________________________
o __________________________
- Have to be able to ‘recover’, therefore, the desired product from this mix
Note: These industrial processes frequently (but not always) involve fermentation
reactions.

‘True’ (scientific) definition of fermentation:

An energy-yielding process that takes place under __________________________
conditions – involves the catabolism of sugars and other organic molecules.

But, in industry, fermentation refers to:

…any large-scale cultivation of microbes in order to produce a substance; process
may be ____________________________________________________
Industrial Fermentation Process:
- Industrial fermentations are carried out in large vessels or tanks called
__________________________ or __________________________
- These are designed to allow control of growth/reaction conditions such as
____________________________________________________
- Many are continuously stirred – air is introduced at the bottom of the vessel; a
series of paddles and baffles keep the microbial suspension agitated so that it
doesn’t ‘sink’ to the bottom
- These tanks can be very large – hundreds of thousands of liters; as such, setting
up a large-scale fermentation involves several steps – can’t just add a loopful of
stock to a huge bioreactor and hope it works – must proceed in a series of steps
– referred to as a __________________________
- must make sure that growth product and production is first successful in
a small tank
- The product is usually harvested in either the log
phase(__________________________) or in the stationary phase
(__________________________)
- Primary metabolites are formed at the same time as new cells – they are related
to the synthesis and growth of microbial cells and include things like
________________________________________________________________
________________________________________________________________
- Secondary metabolites are not produced until growth has been completed; they
usually accumulate when the nutrients become limited and waste products
appear; they do not affect normal cell synthesis and growth; example –
__________________________

Batch fermentation:
- Grow cells all at once – in one big batch
- Harvest when maximum amounts of product have been produced
- Drain bioreactor – harvest product – clean up – start again with new seed culture
- e.g. __________________________
 Continuous culture:
- Is different from batch culture
- Is an ‘open’ rather than a ‘closed’ system
- Fresh growth medium (substrate) is constantly added
- Overflow of fluid occurs – spent media, waste products, and desired product are
removed continually
- Can keep the microbe in desired growth phase (_________________________)
for long periods; steady-state growth is maintained
- Can monitor cell density, pH, chemical composition, etc.
- Bioreactors used for continuous culture are also referred to as
______________________ or continuous reactors

Other systems:
- There are other ways to obtain mass culture of microbes
- E.g. __________________________ – the substrate is just kept moist to
maintain a thin surface film of water where microbes can grow
- __________________________ – microbes are associated with the surface of
support materials as biofilms; the media flows past

Bulk Chemicals
- certain chemicals produced with the help of microbes that are used for industrial
applications

1. Glycerol
- Has pharmaceutical and industrial uses – e.g used in the production of skin
lotions and creams (glycerine), suppositories, cough syrups (provides
‘body’)
Saccharomyces cerevisiae
Molasses (or other sugar source Glycerol
 2. Acetic acid
- Vinegar is 3-5% acetic acid
- Acetic acid is also used in food flavouring and preservation

Need 2 microbes and 2 reactions:

Saccharomyces (yeast)
Fruit juice or cider (sugar source) Ethanol

Acrobacter aceti (bacteria)

Ethanol Acetic acid

3. Citric acid
- Natural constituents of citrus fruits (oranges, lemons, etc.)
- Wide range of uses
o e.g. anti-coagulants, food flavouring, food preservation, pH
adjusters, anti-oxidants, emulsifier
- Found in hundreds of products including soft drinks, cosmetics and
pharmaceuticals
- Molds produce citric acid during their normal metabolism – it is produced as a
surplus intermediate compound of certain metabolic cycles
- More than 300,000 tons of citric acid is produced every year by Aspergillus
species
- The mold converts glucose (from a food substrate) to citric acid
- Can be synthesized chemically in the lab but not cheaply – the mold does a
better job!

Aspergillus niger or A. wentii

Molasses (or corn starch, beets, raw sugar) citric acid
 4. Acetone & Butanol

Clostridium acetobutylicum
Molasses acetone/butanol
(fermentation)

5. Biopolymers
- Microbially-produced polymers (usually polysaccharides) which are used
industrially to alter the viscosity of various liquids, act as gelling agents or
stabilizers, or to promote water retention in a product
- e.g. Erwinia (bacteria) produces certain polysaccharides that are used in paints

Fine Chemicals:
- Includes vitamins amino acids, antibiotics, enzymes, etc.
1. Vitamins:
- Can be made synthetically or by microbes (less expensive)
- Example:
o Vitamin B12 – made by Pseudomonas, Propionibacterium
o Riboflavin (B2) – produced by a mold (Ashbya gossypil)
o Vitamin C – produced by Acetobacter

2. Amino Acids:
- microbes may secrete large quantities of amino acids into the culture medium
- Can manipulate metabolic pathways to achieve this
- Example: glutamic acid
o Used in monosodium glutamate (MSG) - ‘flavour enhancer’ in food
o More than 600,000 tons produced annually
o Microbe involved? – Corynebacterium glutamicum
 o Same organism also produces lysine – used as a food supplement in
animal feeds and in cereals
o Industrial production is necessary because animals (including humans!)
cannot synthesize lysine (or methionine) and these amino acids are only
present in low levels in a normal diet
o Microbes are also used to produce the amino acids phenylalanine and
aspartic acid which help make up aspartame (NutraSweetTm) – artificial
sweetener in foods and beverages

3. Steroids
- Steroid hormones regulate metabolism
- Cortisone – relieves arthritis and other inflammatory reactions
- Can be chemically synthesized but…37 steps + expensive…$200/g to make!
- One step is very difficult and costly – therefore use a microbe to carry it out:
o Make a ‘pre-steroid’ in the lab and add it to a tank where Rhizopus
nigricans (mold) is growing
o The mold’s enzymes ‘modify’ the steroid structure and transform it to the
active form
o Filter out the mycelia (mold) and purify the steroid from the filtrate
o Cost is now $0.50/g
o A bacterium (Streptomyces) can also be used – adds a hydroxyl group to
the ‘pre-steroid’ to complete the synthesis
o These are examples of bioconversion where microbes are used to make
small (but significant) changes to an existing molecule
4. Human Proteins
- Genes coding for these are inserted into bacteria – are cloned
- Many copies are made because bacteria multiply so quickly
- Examples:
o Insulin (controls blood sugar levels in diabetics)
o Human growth hormone (stimulates growth in those with pituitary
disorders)
o Blood clotting factor VIII (used to treat hemophiliacs – individuals whose
blood can’t clot)

5. Antibiotics
- e.g. Penicillium chrysogenum – penicillin, Streptomyces – erythromycin,
streptomycin
- Making penicillin
o mold is grown in a large tank
o mold secretes penicillin into the growth media
o eventually, the mold mycelia is filtered out and the penicillin in the filtrate is
purified
- Problem with natural penicillin? – narrow spectrum of activity (effective only
against Gram positive organisms), destroyed by stomach acid (pH 2), susceptible
to penicillinases (enzymes produced by some microbes which inactivate
penicillin)
- Solution? – take the naturally produced penicillin to the lab and alter the side-
chain of the antibiotic chemically
- Result? – ampicillin, amoxicillin, methicillin, etc.
- These are referred to as semi-synthetic penicillins (microbe makes the base and
the rest is done in the lab)
- These are improved penicillins – have a broader spectrum of activity and are less
susceptible to stomach acid and penicillinases
6. Enzymes – numerous application

Examples of enzymes produced by microbes and their uses:

Enzyme Microbe(s) Use of Enzyme?
Amylase Aspergillus sp. (mold) Laundry detergents
Trichoderma viride
Cellulose Paper production
(yeast)
Aspergillus oryzae
Proteases Meat tenderizer
(mold)
Rennin Mucor (mold) Cheese production
Streptococcus
Streptokinase Lyses blood clots
(bacterium)