Biochemical Engineering
Biochemical Engineering
Biochemical Engineering
References
1.0 Introduction
About 150 years ago, Louis Pasteur pointed out the important role of
living micro organisms in biochemical processes. In 1928, a major
breakthrough was achieved by Alexander Fleming in discovering
penicillin. The urgent need of penicillin throughout the 2nd world war led
to microbiologists, biochemists and chemical engineers in a “crash”
programme of developing and designing processes in areas which
were hitherto unfamiliar to them. Three American companies led the
way-Merck, Pfizer, and Squibb. Since then micro organisms have been
known to be used in the manufacture of a host of complex chemicals,
antibiotics, enzymes and vitamins.
1.3 MICRO-ORGANISMS
Types of micro-organisms
Bacteria
These are single cells, in the form of cocci, rods and spirals capable
of independent growth.
Fungi
Generally fungi are free living saprophytes but a few are parasitic
on animals and many are serious pathogens of plants.
Actinomycetes are intermediate between fungi and bacteria.
Protozoa
These are widely distributed in fresh and salty water, in soil and in
animals. They may be unicellular or multi cellular and exhibit a wide
range of morphological forms. Protozoa are either photosynthetic or
non-photosythetic while algae are capable of photosynthesis.
The best temperature for cultivation varies with species but organisms
occurring in the soil naturally grow best at temperatures between 25°c
and 30°c while those isolated from animals grow best at 37°c. Some
organisms are actually thermophilic e.g. those used in bio-digesters
(Lactobacillus), which grow best at 40-45°c.
Thus,
+ CO2 + H 2 O + Heat
The carbon substrate has dual role in the biosynthesis and energy
generation. The carbon requirement under aerobic conditions may be
estimated from the cellular yield coefficient(Y) which is defined as
WATER
Energy sources
Carbon sources
Nitrogen sources
Minerals
In many media magnesium, phosphorous, sulphur, calcium and
chlorine are considered as essential. These are added as a distinct
component. Others such as cobalt, copper, iron, manganese,
molybdenum and zinc are also essential but usually present as
impurities in major ingredients.
Vitamins sources
While many of the natural carbon and nitrogen sources contain all or
some of the required vitamin, any vitamin deficiency may be
eliminated by a careful blend of materials.
Nutrient recycle
In cases of large scale continuous culture fermenters, there is always
need for appropriate adjustment of nutrient levels. Phosphoric acid is
used as a reagent for flocculating bacteria.
Buffers
These are added to control the PH. The buffers include calcium
carbonate, phosphates, sodium hydroxide or sulphuric acid.
Precursors/inhibitors/inducers
Precursors help in the regulation of the product rather than support the
growth of micro organisms. Examples of precursors include corn steep
liquor which increases the yield of penicillin since it contains phenyl
ethylamine.
Oxygen
ANTI FOAMS
Before the process may be put into commercial operations, the toxicity
of the product and the organism must be checked and assessed. The
above account implies that cultures must be isolated from the natural
environments. However, the industrial microbiologists may also isolate
micro organisms from culture collections. It is probably cheaper to buy
a culture than isolate from nature.
Variations of micro-organisms
However, mutations have a beneficial side also, and to this end may
be produced artificially. A suspension of organisms is subjected to
treatment and desirable properties may be sought by examination of
individuals in the resultant population. The principal methods of
artificially induced mutations include:-
1. Exposure to ultra violet light
Ultra violet light is absorbed by the nucleoprotein material, which forms
the genetic material, resulting in the resonance and leads to
destructive changes. This method is not highly selective and is
accompanied by a good deal of damage to the rest of the cell.
2. Mustard gas
Mustard gas is a more drastic agent reacting with generic centres and
disrupts chromosomes. Once the generic centres are changes,
information on heredity is interrupted.
3. X-rays and Gamma rays
X-rays act almost the same way as the mustard gas, giving rise to free
radicals in the ambient medium. The free radicals are responsible for
further changes in genetic information. Gamma rays are the more
recent.
4. Use of radioactive substances
The use of radioactive materials is known to give higher mutants rates
with a lower killer rate. The classical isotope used is the sulphur-35 which
decays in 87 days to give chlorine-35. The major advantage of this
method is that mutations may occur without appreciable cell trauma.
It can also be simple to control. It also enjoys higher rates of survival,
almost about 100%. From the mutated cultures, conventional testing
techniques are used to obtain a higher yield of antibiotic or other
desired quality. For example, the yield of penicillin has been raised from
10units/ml in 1942 to over 2000units/ml in 1960. As an example of the
commercial use of mutant strain, Escherichia Coli has been mutated in
lysine fermentation.
Hybridization
A vessel fitted with coils or a jacket for heating and cooling. An agitator
may be fitted to aid heat exchange. The interconnecting piping
between the cooker and the main bioreactor must be sterilized at the
same time so that sterile medium may be transferred. This method
reduces the time in which the main reactor is unoccupied between
fermentations; against this is the higher cost of the extra equipment
involved, and the increased steam usage.
2) Continuous sterilization
This is a three section plate type heat exchange. This methods realizes
some important steam saving economy. It is necessary to avoid any
leakage or short-circuiting between the non-sterile medium entering
and the sterile medium leaving the system.
STERILIZING OF AIR
Since the time of Pasteur, air has been recognized as a source of
microbial contamination. At this time, it was discovered that a plug of
cotton wool would allow air to reach the culture but would prevent
other micro-organisms from contaminating it. Over the years, the
demand for pure air for industrial operation has been on the increase.
This calls for the sterilizing of air in terms of thousands of cubic feet of air
per hour. The most surely effective method is heating the air, but the
cost is prohibitive when large volumes of air have to be sterilized.
Therefore, other methods of sterilizing the air have been applied:-
1. Electrostatic precipitation
2. Exposure to UV light
3. Filtration through columns packed with glass wool, slug wool,
cotton wool, activated carbon or other filtering media coupled
or without counter-current scrubbing through phenols, caustic
soda, acids or other germicidal agents.
100( N 1 − N 2 )
effeciency % =
N2
Where N1 is the number of micro-organisms per unit volume of air
before filtration and N2 is the number of micro-organism per unit
volume of air after the filtration.
CHEMICAL STERILIZATION
1. Rideal_walker test
2.303 N
N t = N o e −κt or κ = log o
t Nt
where No, Nt are the concentration of organisms initially and at a
time t respectively, k is reaction velocity constant and t is time
usually in minutes
the hourly basis for air change has been calculated by
138 N
KA = log o
t Nt
III. the level of initial contamination- assuming the logarithmic
relationship, then it follows without doubt that absolute sterility
can only be approached asymptotically and arbitrary final level
must be quoted. The higher the initial contamination, the higher
the probability of including pathogenic or resistant micro-
organisms in th sterile environment.
IV. Concentration of the sterilizing agent- a general relationship
between the concentration of the sterilizing agent and the time
to reach a low count can be expressed
C1n t1 = C 2n t 2
Where n is considered as the concentration coefficient
V. Decay rates of the disinfectants-sterilising agent can
progressively loose their effectiveness as a result of chemical
change, adsorption and various other factors. For instance
hypochlorite (jik) is quite unstable or highly reactive compound
whose KA value approaches 20.
VI. Physiological effect of sterilising agents-unless there is clear
evidence to the contrary, it should be assumed that any
compound capable of disorganising a unicellular organism as to
cause its death, will also have a significant physiological effect
on larger creatures including man. These effects include allergy,
cumulative effect of ingestion in small amounts over a long
period of period. Caution needs to be exercised at all times!
Detergents
FERMENTATION PROCESSES
The seed is then transferred along the sterile connection into the
fermentor charged with mash. Aeration, agitation and temperature
control of the mash commences at least as soon as the seed is
received into a fermentor and continuous until the mash is harvested
between 3 and 5 days later.
The first step in harvesting consists of separating the mycelium from the
medium by the use of rotary vacuum filter in which the mycelium is
continuously stripped as felt.
YEASTS
Yeasts have uniquitous distribution throughout the plant and the animal
kingdom and also in the soils. From the industrial viewpoint, there re two
main genera of interest, the broad range of saccharomycetes and the
more limited candida or torulopsis yeasts. Industrial yeasts can be
classified broadly into six groups
a. Beer or ale yeasts-these are referred as top fermentation. They
rise to the surface of the wort as the head of foam, which can be
skimmed off for re-use
b. Lager yeasts- brewing lager is carried out at a much lower
temperature than beer and for longer periods. This yeast must
withstand the conditions and also settle out to the bottom of the
vessel towards the end of the fermentation
c. Distiller’s yeasts- they are selected strains of the beer/ale yeast
adapted for high sugar and alcohol tolerance and capable of
giving high conversion ratio.
d. Baker’s yeast-while its selected from beer/ale yeast, the baker’s
yeast is bland in flavour, lacking hop bitterness from beer, is more
rapid in action and has extended shelf life
e. Wine’s yeast
f. Food and fodder yeast
BREWING
Industrial Alcohol
Although a large proportion of industrial alcohol in the world is made
from petrochemicals, considerable amounts of fermentation spirits are
still made through the world the raw materials include grain, beet/cane
molasses etc.
After mashing the final wort is adjusted to conditions to suit the chosen
yeast. Temperatures are in the range of 23°c to 30°c and the initial PH is
adjusted to 4.5 to 6.0. The balance of nutrients and total concentration
are adjusted t provide only a limited growth but maximum conversion
of carbohydrates to ethanol.
Utilisation of by-products
Carbon dioxide-about ¾ is recovered for sale as compressed liquid or
dry ice. Since pure grade is required, the CO2 is scrubbed through
water, from which ethanol is recovered, then sulphuric acid and finally
deoderized an activated charcoal.
Fermentation residues- these are insoluble material filtered off and
dried to be used as animal feed.
CONTINUOUS FERMENTATION
The classical work of Monod, showed that the growth rate of many
organisms can be related to the concentration of the limiting
component by formula similar to Michaeli’s enzyme equation.
S
κ = km ( )
K+S
Where k= saturation constant, numerically equal to Km/2
⎛ S ⎞ ⎛ D ⎞
D = κm⎜ ⎟ where S = κ ⎜⎜ ⎟⎟
⎝K+S⎠ ⎝κm − D ⎠
MICROBIAL GROWTH KINETICS
ln X t = ln X o + µt
CONTINUOUS CULTURE
A media displaces an equal volume of culture from the vessel to
achieve continuous production. The flow of medium into the vessel is
related by the term dilution rate, D, defined as
F
D=
V
The net change in cell concentration over a time period may be
expressed as
dX
= growth − output
dt
dX
= µX − DX
dt
Under steady state conditions, the cell concentration remains
constant, thus dX/dt =0,
Then
µX + DX
and
µ=D
Thus under steady state condition, the specific growth is controlled by
the dilution rate.
The main objective of the first stage for the recovery of an extra-cellular
product is the removal of large solid particles and the microbial cells b
centrifugation or filtration. In the next stage the broth is fractionated or
extracted into major fractions using adsorptions or ion-exchange
chromatography liquid-liquid solvent extraction or precipitation, further
more precise chromatographic and crystallisation.
Due to the small size of microbial cells, filter aids are applied to improve
filtration rates while heat and flocculation are employed as techniques
for increasing sedimentation rates in centrifugation.
a. Foam separation- this is done by exploiting the differences in
surface activity of materials. The material is selectively adsorbed
or attached to the surface of gas bubbles, concentrated and
finally removed by skimming
b. Precipitation- It is possible to obtain some products from the
broth, either by adding a compound which leads to the
formation of insoluble complexes or salts or by adding a suitable
organic solvent.
c. Filtration- this is the commonest method of removing suspended
particles from a liquid or gas, using a porous medium which
retains the particles but allows the liquid or gas to pass. The
following factors influence the choice of the most suitable
filtration equipment to be used:-
Properties of the filtrate, viscosity and density
Nature of solid particles especially size, shape and size
distribution
Solids:liquid ratio
The need for recovery of the solid or liquid fraction or
both
The scale of operation
The need of batch or continuous operations
The need for aseptic conditions
The need for pressure/vacuum suction to ensure an
adequate flow rate of the liquid
d. Centrifugation-micro-organisms and other similar sized prticles
can be removed from broth by using centrifuge when filtration is
not satisfactory separation method. Although a centrifuge may
be expensive when compared with a filter it may be essential
when:-
a. Filtration is slow and difficult
b. The cells or other suspended matter must be obtained free
of filter aids
c. Continuous separation to a high standard of hygiene is
required
e. Liquid-liquid extraction- this is the separation of a component
from liquid mixture by treatment with a solvent in which the
desired component is preferentially soluble. The solvent is then
recovered by distillation.
f. Chromatography- this is used to isolate and purify relatively low
concentrations of metabolic products. This will involve the
passage and separation of different solutes as the liquid is
passed through a column. Depending on the mechanism by
which solutes may be differentially held in a column, the
technique can be grouped as
Adsorption chromatography-binding of the solute to
the solid phase primarily by weak van der waal forces
Ion-exchange-reversible exchange of ions between
liquid phase and solid phase which is not
accompanied by radical change in the solid structure
Gel-filtration-separates on the basis of size in which the
small particles diffuse through the gel rapidly
Affinity chromatography-depends on the interactions
between pairs of biological materials such as enzymes-
substrate
g. Ultra-filtration- the process in which solutes of high molecular
weights are retained when the solvent and the low molecular
weight solutes are forced under hydraulic pressure through a
membrane of very fine pore size.
h. Drying- this is often the last stage, which involves the removal of
water from the heat sensitive material ensuring that there is
minimal losses of viability, activity or nutritional value. Drying is
undertaken because:-
The cost of transport can be reduced
The material is easier to handle
The material can be more conveniently stored in dry
state
A spray drier is the most widely used for drying biological materials
when starting materials is in the form of a liquid or paste which can
be initially atomized into small droplets through a nozzle or by
contact with a rotating disc.
i. Crystallisation- This is best applied in the initial recovery of
organic acids and amino acids
FERMENTATION ECONOMICS
MARKET POTENTIAL