Fossil fuels have been the primary energy source for society since the Industrial Revolution. They provide the raw material for the manufacture of many everyday products that we take for granted, including pharmaceuticals, food and drink, materials, plastics and personal care. As the 21st century progresses we need solutions for the manufacture of chemicals that are smarter, more predictable and more sustainable. Industrial biotechnology is changing how we manufacture chemicals and materials, as well as providing us with a source of renewable energy. It is at the core of sustainable manufacturing processes and an attractive alternative to traditional manufacturing technologies to commercially advance and transform priority industrial sectors yielding more and more viable solutions for our environment in the form of new chemicals, new materials and bioenergy. This course will cover the key enabling technologies that underpin biotechnology research including enzyme discovery and engineering, systems and synthetic biology and biochemical and process engineering. Much of this material will be delivered through lectures to ensure that you have a solid foundation in these key areas. We will also consider the wider issues involved in sustainable manufacturing including responsible research innovation and bioethics. In the second part of the course we will look at how these technologies translate into real world applications which benefit society and impact our everyday lives. This will include input from our industry stakeholders and collaborators working in the pharmaceutical, chemicals and biofuels industries. By the end of this course you will be able to: 1. Understand enzymatic function and catalysis. 2. Explain the technologies and methodologies underpinning systems and synthetic biology. 3. Explain the diversity of synthetic biology application and discuss the different ethical and regulatory/governance challenges involved in this research. 4. Understand the principles and role of bioprocessing and biochemical engineering in industrial biotechnology. 5. Have an informed discussion of the key enabling technologies underpinning research in industrial biotechnology 6. Give examples of industrial biotechnology products and processes and their application in healthcare, agriculture, fine chemicals, energy and the environment.
Recovery and purification of small molecules
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來自 University of Manchester 的課程
Industrial Biotechnology
123 個評分
從本節課中
Biochemical and Bioprocess Engineering
Biochemical and bioprocess engineering is concerned with the design of processes which involve biological transformations to manufacture a range of bio-based chemicals, biopharmaceuticals and biofuels. Through applying knowledge of process constraints, which are usually described mathematically, biochemical engineers are able to design a series of integrated process steps or “unit operations” which together make up a bioprocess.
This module will give an appreciation of the key role biochemical engineering has in translating discoveries coming from life sciences and synthetic biology, such as improved microbial platforms for product expression, into economically viable full scale production processes. Key engineering concepts and the problem solving approach required for the design of bioprocesses will be taught by a group of biochemical engineers from The University of Manchester, University College London and Technical University of Denmark.
與講師見面
Prof. Nicholas TurnerDeputy Director
Manchester Institute of Biotechnology
Prof. Nigel ScruttonDirector
Manchester Institute of Biotechnology
Hello, my name is James Lawrence from the Department of Biochemical Engineering
at University College London and I'll be talking through this video on recovery and
purification of small molecules.
So all products derived from biological processes will need to be recovered and
purified before they can be sold or used in the manufacture of a different product.
The design of the process we use to do this recovery and
purification will depend on the type of product, the product's location, and
the nature of the contaminants present.
For the purpose of this course, we've broken down the type of product into two
categories, small molecules and large molecules.
By small molecule, we mean a molecule of with a low molecular weight.
So for example, ethanol or insulin.
And by large molecules, we mean molecules with a high molecular weight.
So for example, enzymes or antibodies.
In this video we will discuss the recovery and purification of small molecules, and
we'll move onto large molecules in the next video.
The location of the products will also impact the recovery and
purification process.
When we say location what we mean is whether it is retained within the cell
that has produced it, i.e., whether it's intracellular.
Or if it is secreted into liquid surrounding the cells, so
if it is extracellular.
All recovery and
purification processes will be made up of a series of separation of steps.
Each of which will exploit some difference between the desired products and
the other compounds that are present which we term the contaminants.
This difference can be any one of density, size, charge, hydrophobicity (which
is the degree to which the products or contaminate repels water), the affinity for
a particular molecule, or a combination of several of these differences.
It's worth noting that contaminants in a cell based or
biocatalyst based process will be different.
In a cell based process we will need to remove the cells and possibly cellular
debris as well as the culture fluid components all from our product.
We might also need to extract the products from the cells before we can start
the purification process.
If the process is biocatalyst based, we will need to remove the enzymes,
if they haven't been immobilized,
as well as any unreactive substrates or any cofactors required.
It's also worth bearing in mind that most of our processes will take place in water,
and our product concentration will typically be very low.
One of the first priorities of any recovery and purification process then
is going to be to remove as much of this water as possible, as soon as possible
to minimize the size and cost of operation of the latter stages of the process.
Recovery is the name given to the initial stages of the separation process in which
we will separate out and concentrate our product
and remove the bulk of the contaminants and water from it.
We typically use filtration or centrifugation for
this purpose, as they can handle large particulates like cells.
Filtration is driven by a difference in size of particles,
while centrifugation is driven by a difference in density.
So in filtration we pass the fluid through a membrane with small pores of
a defined size.
Only liquids or particles smaller than these pores will be allowed to pass
through the membrane and into the permeate.
And larger particles will be held back in the membrane in what we call
the retentate.
Filtration is quite a useful step for separating cells from culture fluids.
As the bulk of the culture fluid will be allowed to pass through the membrane,
most of the cells themselves will be retained within the retentate.
The filtration units can typically be operated in two modes,
by the normal flow or tangential flow.
In normal flow mode, the fluid will be driven directly into the surface of
the membrane, whilst in tangential flow mode,
the fluid is driven over the surface of the membrane, flowing in parallel to it.
In both cases, there is a pressure gradient from the retentate side to
the permeate side, which drives the fluid and small particles through the pores.
However, operating in tangential flow can be advantageous,
as the flow of fluid over the surface can prevent larger particles from
accumulating on the surface and blocking transmission through the membrane.
Centrifugation is the other main choice for the recovery of cells and
is very commonly used where large volumes of fluid needs to be processed.
As centrifuges are typically capable of much higher throughputs,
so they can process more fluid per hour.
There are two main types of centrifuge, the tubular bowl centrifuge and
the disc-stack centrifuge,
but both operate on the same principle.
this being the use of centrifugal force to displace particles of a certain density.
So the centrifuge has a rapidly rotating chamber into which our fluid flows.
The rotation of this chamber subjects the fluid and
the particles within it to force many times that of gravity.
This force causes particles to move away from the center of the chamber.
Most of the fluid itself, is allowed to flow through relatively unimpeded.
The denser the particles are,
the more rapidly they'll settle out of the fluid toward the walls of the chamber.
In the case of the tubular bowl centrifuge,
they will accumulate on the side of the bowl.
And in the case of the disc-stack centrifuge the particles will accumulate
towards the corners of the centrifuge.
We can adjust the rotational speed and the flow rate through the centrifuges to
obtain particles of a particular density, usually our cells,
whilst allowing the rest of the fluids and less denser particles to pass through.
If our product is intracellular, we will need to extract it from our cells by
breaking them open after the recovery stage.
This process is called lysis and it can be performed using chemicals, to dissolve
the cell membrane, or high temperatures or pressures to destabilize the membrane.
In industrial biotechnology, we typically use pressure to lyse the cells, as it
is the most efficient method and doesn't require the addition of further chemicals.
We use a device called a homogenizer which uses pressure to break the cells open
by forcing them through a partially closed valve and into the so called impact ring.
The constricted space of the valve accelerates the cells up to a high
velocity so
that when they collide with the impact ring the cell membranes will break open.
This releases our product into the surrounding fluids, but
it will also release cellular debris along with it.
So on further purification using a filter or
centrifuge is usually required to remove the bulk of this debris.
So the recovery stage of an industrial biotechnology process typically has a very
similar structure, regardless of the product that we are trying to produce.
But the purification stage will vary quite widely for different small molecular
products, as there are quite a lot of unit operations to choose from.
So rather than going through all of these steps in this video, we'll look at
a couple of case studies for the manufacturing of two different products.
The first being L-glutamate and the second being bioethanol.
So L-glutamate or glutamic acid is an extra-cellular product which is used as
a precursor for pharmaceutical compounds and also for flavourings like MSG.
It's produced by fermentation using a few different bacteria.
And after the fermentation and recovery stages are complete,
there are two main steps which are required for the purification stage.
These are crystallization and filtration.
So crystallization exploits the varying solubility
of compounds in different conditions to cause them to form crystals,
precipitating them out of solution as solids.
The concentration of the product that we are trying to crystallize,
as well as the pH of the solution, and the temperature of the solution,
can all be used to affect the product's solubility.
Since different compounds will have different solubilities under different
conditions, we can choose our parameters such that
only our L-glutamate molecules will crystallize.
The crystallization process consist of two main steps.
First is nucleation, where the crystal starts to form nuclei over few molecules.
And then we have crystal growth, where further molecules
join the structured nuclei and form larger and larger crystals.
Once the crystallization is complete, the suspension of our glutamate crystals can
be filtered to separate them out from the contaminants and the liquid.
The crystallisation is not always perfect and sometimes we will end up
with other contaminating molecules in our glutamate crystals.
But if this happens, we can simply redissolve the crystals and
repeat the crystallisation process until we reach our desired level of purity.
Bioethanol which is a somewhat lower value product than glutamate,
is also an extracellular product which is
typically produced by the fermentation of yeast or E.coli strains.
The main challenge of bioethanol production is that the ethanol is toxic to
the cells and will cause them to die if the concentration of the ethanol within
the solution is too high.
So to prevent this we need to remove the ethanol as it is generated.
And we can do this in one of two ways, either by continuous mode fermentation,
as we discussed in the previous video on cell culture, or by high temperature
fermentation, where the cells are grown at about 50 to 60 degrees C,
which causes the ethanol to evaporate out of solution as it is formed.
If we use continuous mode fermentation,
then the liquid leaving the fermenter will need to go through a recovery step
to extract the cells before it can be purified.
If instead we use the high temperature fermentation method,
then we can simply condense the ethanol vapor from the fermenter and
skip straight to the purification steps.
So the first step of our ethanol purification will be distillation,
which exploits the various boiling points of the different
compounds within the solution to effect separation.
So a gradient of different temperatures is applied over the length of the column,
with lower temperatures at the bottom, and higher temperatures at the top.
The fluid containing ethanol is put into the middle of the column and
the ethanol itself will evaporate and rise to the top of the column.
Whilst the water and other compounds which have a lower boiling point should condense
and drop to the bottom.
Additional vapor can be extracted from the column but
we can only ever reach about 95% purity with normal distillation.
Now this is because a solution of 95% ethanol and water actually forms a complex
that has a lowering boiling point then a solution of pure, 100%, ethanol.
So now matter how many times you put this complex of 95% ethanol
in water through our distillation pump we'll never be able
to separate out that remaining 5% of water.
So if you want to get to a higher purity of ethanol,
we need to use techniques called molecular sieving or reverse osmosis.
Both of these techniques utilise materials with nano-scopically porous structures
which are so small that only water molecules can pass into or through them.
So molecular sieves are particles made up of these nanoscopic structures which could
be added to the ethanol and water solution that comes out of our distillation column.
The water molecules will diffuse out of the solution and
into the pores of the sieve particles, and
the particles can then be removed leaving pure ethanol behind.
Reverse osmosis, on the other hand,
is essentially a filtration process which uses membranes with nanoscopic pores.
In this case, the fluid is flowed into the membrane, but
only water molecules are able to pass through.
So we'll be left with our solution of pure ethanol.
