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D2 – Part C – Microbiology Cultivation Techniques- A

D2 – Part C – Microbiology



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A cultivation technique is when microbial culture is allowed
to multiply, in controlled lab conditions. Culture media is required in order
to cultivate microbes in a lab. Culture media is a mixture of various nutritive
substances, that is needed to cultivate microorganisms. The culture media helps
the microorganism grow almost acting as soil.

Isolation and Cultivation of Microorganisms

Large scale
industrial growth –

Antibiotics are produced by large fermenters that is used by pharmacy
companies. The large fermenters can work in aerobic or anaerobic conditions,
with nutrients added to it, under sampling and under aseptic conditions.
Real-time information is provided for fermentation by biosensors and infrared
monitoring. They detect specific substrates, metabolic intermediates and final


application for growth conditions-

To grow bacteria there are four phases. The lag phase is when
there is not much bacterial growth. The log phase is when the bacteria cells
have doubled at an exponential rate. And the stationary phase is when the
bacteria levels off and the death of the cells rate equals the cell division
rate. At death phase at an exponential rate cells die. This is because all
their energy reserves and exhausts the cell. Inoculum sources are also known as
spores. They are suspended in water or in a nontoxic lauryl sulphonate from
stock culture. They ate then added to a flask that has wheat bran and nutrient
soln. 4-day old shake flask culture is then inoculated into a seed tank for
about 3 days. The lag phase is when the organisms start to adjust, the growth
phase is when there is an exponential growth , the stationary phase is when the
nutrients decrease and organisms die at the same rates as the new one that is
being produced, the death phase also known as the decline phase is when the
nutrients exhaust and increase their levels of toxic waste products.

Compare and
contrast small scale labs and large-scale production-

The volume of a small-scale lab is 200dm3 to see if the
growth requirements grow the same. Large scale fermenting has affected our lives.
Industrial scale fermentation produces antibiotics and other therapeutic
proteins. However, for small molecule pharmaceuticals it is not really that common
and are usually used to ferment products used for flavours and fragrances. The
term fermentation originally was used to describe the anaerobic process to
convert starch grains into alcohol. This process is still used for first
generation biofuels.


of penicillin-

Penicillin is a penicillium specie that grows once the growth
of fungus has stopped growing due to stress. Alexander Fleming discovered
penicillin, making antibiotics production possible. There has to be an oxygen
supply also known as aeration which is limited in a bioreactor for the biosynthesis
of penicillin. Bioreactors have a sooth interior surface, an inlet to allow
filtration, a ph control , a temp control , antifoaming agents , nutrients and
a steam inlet that allow it to sterilise  The aeration speed is in-between 3.0 to 1.5. The
production of penicillin should be at 28 degrees Celsius for temperature.
Penicillin requires a high biomass concentration. This is done by increasing
the rate of the agitation and the power. The PH of the medium of the production
of penicillin is maintained at a neutral PH by calcium and magnesium carbonate
in the phosphate buffer. The PH of the medium is also controlled by adding
sodium hydroxide or sulphuric acid

Batch or
continuous fermentation-

There are 2 common methods for industrial fermentation for
scale production of microbial biomass or metabolites. Primary metabolites are
produced when microorganisms grow, for example alcohol by yeast. A secondary
metabolite is produces at the end of their growth phase for example penicillin.
One being batch fermentation the other being continuous fermentation. The
concentration of the microorganisms, the cells, the cellular components,
enzymes , temperature , PH and oxygen or no oxygen affects the rate of
fermentation. Batch fermentation is when there is fermentation carried out in
batches, fermentation media is then added to the fermentation, then the fermentation
media is sterilised and inoculated with an inoculum , carried out using their
optimum conditions, then it is shaped and the product that is desired will be
obtained. Batch fermentation is a closed bioreactor, nothing is added or
removed if there is contamination during fermentation it only affects one
batch. Continuous fermentation is a open bioreactor, where nutrients ae
continuously added and the PH, temperature and oxygen is controlled. With
continuous fermentation there is always more wastage and foaming compared to
batch fermentation and it also more cost effective than batch production.


Why it
might be used for penicillin-

Sugar is used to regulate the PH of penicillin. In the
production of penicillin, batch fermentation is widely used in laboratory scale
as well as industrial scale. For 30 minutes the fermentation media for the
production, is sterilised at 121 degrees. Changing the sterilisation time to
see how much fermentation media was converted from complex to simple.




 Growth requirements for penicillin-

There is a simple nutrient requirement for the growth of
penicillin. For a fast growth rate, 25% of carbon is needed. Penicillin is also
required to grow in a large vessel called a fermenter. Industrial fermenters
have an air supply that provides oxygen for the microorganism to respire, to
keep the microorganism in suspension the fermenter has a stirrer this also
maintains the temperature. Penicillin is an antibiotic that is produced by
growing in the penicillium mould in a fermenter. The medium has sugar and other
nutrients contained in it. When all the nutrients are used up for growth from
the penicillium then the penicillin is produced.


used for penicillin:

Substrates that are used for penicillin is Glucose, lactose
and carbon. The growth can differentiate depending of penicillin substrates
that can enhance or extend it. There are 4 distinct regions on a filamentous
hyphae which divides up the activities. There is an actively growing region, a
non growing region , a vacuole region and a degenerated or metabolically
inactive region. As the main carbon source, the fermentation medium contained
glucose and lactose monohydrates. Excess whey powder which is a source of
lactose, is used from the inoculum medium. Throughout the fermentation glucose
is provided continuously. When the residual glucose concentration of the medium
drops down to a low level, glucose and galactose is formed when lactose is
hydrolysed .

How is
penicillin harvested and purified?

Harvested- first the media is sterilised, then cooled at room
temperature before it has been inoculated, then the production media is moved
to a clean air laminar flow cabinet, then seed culture is added to it and then
shaken and then maintained in a shaking incubator and then harvested.

Purified – The media broth is filtered, then the insoluble is
removed , treated with carbon , then liquid-liquid extraction is used , the
penicillin is then extracted from the aqueous phase that uses butyl acetate solvent
and is then evaporated leaving a purified penicillin. They choose butyl acetate
as an aqueous solution for penicillin because it is not flammable and has a
negligible vapor pressure.

A harvested culture broth contains penicillin G and other
metabolites. To sperate the mycelium from the broth a vacuum filter is used on
a rotary conversion at a low PH and then phosphoric acid is added or sulphuric
acid. To remove the pigment of the penicillin or other solvent impurities that
is found it is treated with active charcoal. To form its sail potassium or
sodium hydroxide is added and is the extracted with water. This then produces
sodium or potassium penicillin once it has been crystalized.
















D3 – Part C – Microbiology



Growth Rate Calculation:

Is expressed as generation time or doubling time of the
bacterial population. To measure the growth rate of a bacteria is to measure
the optical density regularly and then plot it on a graph with an x axis and an
y axis. The x axis is usual the time and the y axis is usually the 10 log
(optical density). After the data has been plotted the growth rate looks like a
line with a positive slope. To work out the growth rate the equation is.


This applies to the biotechnological context as it is
measures (h-1, d-1) of the rate of growth or
multiplication of an organism or culture by the increase of mass divided by the
cell number per time unit referred to the unit of mass.

How is it applied to industrial context?

It is applied to industrial context by understanding the
relationships between cell behaviour and the surround environment.

Biotechnological creates useful products from microorganism
by using genetic modification. To produce useful agriculture, biotechnology is
used. It is a technique that uses genetic engineering and other techniques that
involve modification of genes. Biomedical engineering is applying engineering
principles to medicine for health care purpose. This combines engineering and
medicine to solve and treat health care issues by diagnosing. It has evolved
over the tears and is commonly used.



Narrow or broad spectrum of antibiotics:

They are active against a certain bacterial group. They are
used for the specific infection that is known. They are only effective against
a narrow range of bacteria. Penicillin for example is very affective to killing
gram positive bacteria but it is not affective for killing gram negative
bacteria. This is due to the ability of the antibiotic penetrating in the
bacterium. Gram positive bacteria has a looser outer wall do that antibiotics
can diffuse through them but a gram-negative bacteria has a complex outer layer
that stops the passage of larger molecules from getting through.

Fermentation is the production of ethanol. Yeast brakes
sugars down as well as other sugar substrates when making an alcoholic
beverage. By using yeast to ferment sugar solutions, aqueous solutions of
ethanol can be produced. Fruit juices like grape juice has a source of glucose
sugar which formula is C6H12O6. In the absence
of oxygen, the added yeast feeds on the sugar to form wine and C02. Fermentation
is a chemical reaction that happens to break down glucose into ethanol by the
enzymes found in yeast. 


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