BTE 404: Bioprocess

technology
CHAPTER 1
Subarno Hossain Turja,
Lecturer, Biotechnology Program
 Introduction
to
bioprocess technology,
industrial biotechnology
 Introductio
n
• A bioprocess is any process that uses complete living cells or their
components (enzymes) to obtain desired products.
• The technology that deals with the design and development of
equipment and processes for the manufacturing of products used in
agriculture, food, feed, pharmaceuticals, chemicals, polymers and paper via
biological means is known as ‘Bioprocess Technology’.
• Waste treatment and recycling is also facilitated by bioprocess technology.
• ‘Bioprocess Technology’ and ‘Industrial Biotechnology’ are very similar terms
and interchangeable in their meanings.
• Industrial biotechnology uses the application of biotechnology for industrial
purposes, such as industrial fermentation.
• The practice of using cells such as micro-organisms, or components
of cells like enzymes, to generate industrially useful products in
sectors such as chemicals, food and feed, detergents, paper and pulp,
textiles and biofuels is called ‘industrial biotechnology’ .
• For example, lipase enzyme is commercially used in leather and
textile industries. This enzyme is extracted from an abundant number
of microorganisms in an optimum grand scale.
• So, by definition, this is a ‘bioprocess technique’ as it uses biological
component (lipase producing microbes).
• This is also an example of ‘industrial biotechnology’ because the
production is done on a massive industrial level to meet up the huge
demands of lipase.
• Industrial biotechnology employs a combination of
mathematics, biology and knowledge of engineering.
• A combination of knowledge of different backgrounds are
incorporated into a successful biotechnology process.
• Choosing a viable microorganism strains, determining the optimum
condition to get maximum products- these types of works require
knowledge of biotechnology and microbiology.
• On the other hand, designing of bioreactors, and maintaining of air
pressure, temperature and oxygen saturation in the bioreactor
require knowledge of engineering and mathematics.
• In older times, the term ‘fermentation’ was equivalent to
‘bioprocess’.
Fermentation
• Conversion of carbohydrate (eg. sugar) into acid or alcohol by yeast
or bacteria- this is the basic definition of fermentation; but it has a
way broader understanding.
• Production of foods such as cheeses, yoghurts, fermented pickles
and sausages, soya sauce and other oriental products, and beverages
such as beers, wines and derived spirits- all these happen by
fermentation.
• In today’s consumer world, all of the mentioned products are
produced at massive level by using ‘industrial biotechnology’/
‘bioprocess technology’.
• When done at industrial level, increasing the size of the reaction
vessel (bioreactor, or fermenter) is required for the large-scale
growth of microorganisms.
 Bioreactor
and
upstream & downstream
processing
• A bioreactor refers to any system that supports a biologically active
environment.
• For example, a 1000 liter container in which a chemical process is carried out
involving organisms or biochemically active substances derived from such
organisms is a bioreactor, but a huge one.
• On the other hand, an ‘E. coli’ which has a plasmid containing a transgene can
also be called a bioreactor, but it would be a very small one.
• Generally, a fermentation/ bioprocess is divided into 2 parts: upstream and
downstream processing.
• Upstream Processing refers to the initial steps in which biomolecules are grown,
usually by bacterial or mammalian cell lines, in bioreactors.
• So, the steps that are involved in growth of microbial cells at high
density are all parts of upstream processing.
• Upstream processing involves preparation of liquid medium,
separation of inhibitory chemicals from the medium, sterilization, air
purification etc.
• Upstream processes include selection of a microbial strain
characterized by the ability to synthesize a specific product having the
desired commercial value.
• Downstream processing: The downstream part of a bioprocess refers
to the part where the cell mass from the upstream are processed to
meet purity and quality requirements.
• This involves separation of cells from the fermentation broth,
extraction and purification of desired product and waste disposal or
recycling.
Classification
of
fermentation
 Types of fermentation
• Microorganisms can be grown in batch, fed-batch, or continuous culture.
• Batch Fermentation: In this technique, the sterile growth medium is inoculated
with the appropriate microorganisms, and the fermentation proceeds without
the addition of fresh growth medium.
• During a batch fermentation, the composition of the culture medium, the
concentration of microorganisms, and the amount of target protein- all change
as a consequence of the state of cell growth, cellular metabolism, and
availability of nutrients.
• Under these conditions, six typical phases of growth are usually observed: lag
phase, acceleration phase, logarithmic (log) or exponential phase, deceleration
phase, stationary phase, and death phase.
• But generally 4 main phases (lag, log, stationary and death) are discussed
widely.
 A graphical
representation of
the 6 phases of
microbial life cycle
in batch
fermentation
 A graphical
representation of
the 6 phases of
microbial life cycle
in batch
fermentation
• Typically, there is no immediate increase in the numbers of microbial cells
after the inoculation into sterilized growth medium. This initial period is called
‘lag phase’.
• During the lag period, the microbial cells adapt to the new environmental
conditions (different pH or to a new level of available nutrients).
• During the log phase of growth, the cell mass undergoes several cell
doublings. With excess nutrient supply and no inhibition of growth, the
specific growth rate is independent of the substrate/nutrient concentration.
That means, the more amount of nutrient present in the medium, the more
the microbes will divide.
• After either the depletion of a critical growth substance, such as the carbon
source from the medium or the accumulation of metabolic end products that
inhibit growth, the increase in cell mass eventually ceases and the cells enter
the stationary phase.
• During stationary phase, although the amount of microbial cells remain
constant, but cellular metabolism often changes dramatically. That is because,
the condition is different than what it was during the log phase.
• In some instances, secondary metabolites that are of considerable commercial
interest are synthesized. For example, antibiotics are usually produced during
the stationary phase of the microbial growth cycle. The duration of the
stationary phase depends on the particular organism and the conditions of
growth.
• In the death phase, the energy reserves of the cell are exhausted, and
metabolic activity ceases.
• For most commercial processes, the fermentation reaction is halted and the
cells are harvested before the death phase begins.
 Fed-Batch Fermentation:
• In fed-batch fermentations, substrate is added in
increments at various times throughout the course of the
reaction.
• These additions prolong both the log and stationary phases,
thereby increasing the biomass and the amount of
synthesis of secondary metabolites of stationary phase,
such as antibiotics.
• However, microorganisms in stationary phase often produce
proteolytic enzymes (proteases), and these enzymes can
degrade proteins synthesized by a genetically engineered
microorganism.
• Therefore, when proteins are produced from a
recombinant microorganism, it is important that
the fermentation reaction not be allowed to
reach this part of the growth cycle.
•
Generally, fed-batch fermentations require
more monitoring and greater control than batch
fermentations and are therefore used to a lesser
extent.
 Continuous Fermentation:
• In the continuous fermentation process, fresh growth
medium is added continuously during fermentation, but
there is also concomitant removal of an equal volume of
spent medium containing suspended microorganisms.
• Continuous fermentations use smaller bioreactors than
batch fermentations to produce the same amount of
product.
• Continuous fermentation avoids the “down-time” between
batch runs, during which the bioreactor is prepared for
reuse.
• Continuous fermentations have less down time,
because a single reaction can be maintained for a
much longer period.
• The physiological state of the cells during continuous
fermentation is more uniform, so that yields of
product are more consistent.
• In batch fermentations, small differences in the
timing of cell harvest can lead to significant
physiological differences resulting in less
Diagram of 3 types of fermentation
 Types
of
bioreactor
 Types of bioreactors

• There are 3 main types of design of bioreactors:

• 1. Stirred-tank reactors (STRs): which have
internal mechanical agitation.
• 2. Bubble columns reactor: which rely on the introduction
of air or another gas (sparging) for agitation.
• 3. Airlift reactors: which have either an internal or an
external loop; the mixing and circulation of the culture fluid
in these reactors are the results of the motion of an
introduced gas.
 • In an STR, gas, usually air, is added to the culture medium
under
STR pressure.
• The gas is added through a device called a sparger,
• This sparger can be either a ring with many small holes or
a tube with a single orifice.
• With help of impeller/ agitators the gas which has been
introduced by sparger is dispersed within the STR.
• Due to introduction of gas into the reactor, bubbles are created
which is a problem for any bioprocess.
• Mechanical agitation created by the impellers breaks larger
bubbles into smaller ones and disperses the bubbles throughout
the medium.
• Because of the corrosive nature of many culture media and
sterilization procedures, STRs are usually constructed from
stainless steel or glass.
 • Often by the agitation and by the metabolism of large
STR number of microbial growing cells, heat is generated as a
consequence.
• Too much heat raises the temperature and alters the
physiological state of the cells, and decreases the product
yield.
• So, removal of such heat is necessary for maximum
production.
• Heat can be removed by using a external cooling jacket
around the reaction vessel or by internal coils.
• Although internal cooling coils are more effective than
jackets in keeping the fermentation reaction close to the
desired temperature, but they can become coated with
microorganisms, which prevents cooling, and they
sometimes interfere with the proper agitation of the
fermentation broth.
 Bubble • In bubble columns, the air is introduced
under high pressure near the bottom.
columns
• The smaller bubbles combine into larger
ones as they rise through the column.
• This large bubbles lead to uneven
gas distribution.
• In addition, the use of high-pressure
air tends to cause excessive foaming of
the medium.
• These disadvantages restrict the flexibility
or effective range of operating
conditions, as well as the potential size
of bubble columns.
 Airlift • In an airlift reactor, the gas is introduced
into the bottom of a vertical channel
reactor (riser).
• There are two main types of airlift
bioreactors: Internal-loop airlift reactors are
simple in design, but once they are
constructed, both the volume and the
circulation rate are fixed for all fermentation
processes.
• In contrast, the external-loop airlift reactors
can be easily changed or modified, e.g., by
altering its volume, to suit the requirements
of different fermentations.
Important activities of
downstream processing
 Separation of microbial cells
• The first step in the downstream processing is purification of desired
product.
• To do that, separation of the cells from the culture medium is needed.
• Recombinant and native microbial cells can both be harvested with the same
type of equipment.
• As a consequence of physiological changes, (alterations in cell size and the
production of extracellular polysaccharides), conditions that have been
established for non-transformed cells may not be optimal for recombinant
cells expressing a foreign protein.
• For large volumes, either high-speed centrifugation, which is the current
method of choice, or membrane microfiltration is used to separate cells from
 Disrupting Microbial
Cells
• After the product-producing transformed cells are separated from the media,
the next step is to disrupt the cells to extract the desired products.
• A large number of chemical, biological, and physical methods have been
developed for disrupting microbial cells.
• All of these procedures must be vigorous enough to break the microbial cell
walls yet gentle enough to ensure that the protein product is not denatured.
• In gram-positive bacteria, the cell wall consists of a thick peptidoglycan layer
of N-acetylglucosamine and N-acetylmuraminic acid residues cross-linked
by oligo-peptides.
• The cell wall of gram-negative bacteria has a thin peptidoglycan layer, and
a cytoplasmic membrane.
• The yeast cell wall is composed of a thick layer of partially phosphorylated
mannans and β-glucan.
• The chemical methods that disrupt microbial cell walls include treatment with
alkali, organic solvents, or detergents.
• Treatment with an organic solvent is a simple and inexpensive way to disrupt
cells and has been used for the isolation of enzymes from yeasts.
• Detergents increases bacterial cell permeability by solubilizing cell membranes
and membrane proteins.
• As a consequence of this activity, holes are formed, and proteins and other
molecules are released from the cells.
• Unfortunately, detergents are expensive, frequently denature the protein
product, and are often retained as contaminants throughout the purification
process.
• The major biological method for disrupting microbial cells is enzymatic lysis.
• The lysis technique depends on the strain of the microbe.
• For example, the cell walls of gram-positive bacteria are readily hydrolyzed by
the enzyme lysozyme.
• The cell walls of gram-negative bacteria are hydrolyzed by lysozyme and the
metal-chelating agent ethylene diamine tetraacetic acid (EDTA).
• Cell walls of yeasts are hydrolyzed by combinations of one or
more : glucanase, mannanase, and chitinase.
• Enzymatic treatments are highly specific, and the conditions for lysis are mild.
Currently, cost considerations limit the use of enzymes as cell lysis agents.
• Microbial cells can be physically disrupted either by non-mechanical methods,
which include osmotic shock and repeated cycles of freezing and thawing.
• Mechanical procedures, such as sonication, wet milling, high-pressure
homogenization, and impingement are also employed.
• Generally, after treatment by a non-mechanical method, many of the cells
remain intact.
• In contrast, mechanical disruption is highly efficient, which makes it the
preferred choice.
• A sonicator that generates high-pressure sound waves that cause cell
disruption by shear and cavitation (production of internal holes) is generally
useful for small volumes.
• Wet milling is quite commonly used for disrupting large quantities of cells.