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a) Identify the types of desired product (Intra, Extra or Whole Cell). Apply the rules of
thumb for the process of bio-separation
What is our desire product?
At least 99.7% of pure Lipase enzyme
Where we can get it?
It is produced extracellularly by Fusarium sp. Extracellular is not as complicated as intracellular
product where the product are in mix with medium and the microorganism species.
What are our impurities?
1. Media
Constituent
Dipotassium phosphate
Magnesium Sulfate
Amount
1g
0.5g
Iron Sulfate
0.1g
Asparagine
1.5g
1g
Glucose
20g
4ml
Wheat Bran
975.8g
Distilled water
1L
Autoclave at 120C for 50min (pH 6.5)
Table 1.1: Composition of mycelium medium (MM) for 1 liter.
2. Fungal mycelia
Rule 2: For extracellular product formation, rule of thumb starts from second generic
heuristic. Remove the easiest to removed first
Fusarium sp. is the easiest to remove first. Leaving the fungal to later step and removing them
will be waste of treating unwanted mass which can lead to increase in operating cost. Removing
mycelia of Fusarium sp. is more difficult than growing Fusarium sp. since its a fungus that
develops in its vegetative form, generating hyphae. In agreement with the second generic
heuristic, remove the easiest to remove impurities first, fusarium sp. mycelia removal is the
first step of downstream processing of extracellular products. This step can be accomplished by
using rotary vacuum filtration since its the most suitable for mycelia separation.
Rule 4: Choose those processes that will exploit the differences in the physicochemical
properties of the product and impurities in the most efficient manner
In laboratory studies, Solid State Fermentation are generally carried out in Erlenmeyer flasks,
beakers, petri dishes, roux bottles, jars and glass tubes. For this laboratory scale fermentation, the
enzyme and solid fermented matter can be easily separated based on the different in physical
state. The fermented matter is in solid state and the enzyme is in moderately viscous liquid state
on the surface on the fermented matter. Hence the separation is just as simple as scooping out the
enzyme by spoon.
In industrial separation process, scooping each tray are inefficient. Hence other basic of
separation are need to be considered for the separation. Based on the chemical properties, lipase
enzyme has greatly different solubility in Ammonium Sulfate compared to the fermented matter
thus solubility would be a good basic of separation to separate lipase enzyme from solid
fermented matter. The chosen separation process that can separate lipase from fermented broth
based on solubility is precipitation.
Precipitation, which is the process of coming out of solution as a solid, is an important method in
the isolation of enzymes and protein that usually comes early in the purification process. The
primary advantages of precipitation are that it is relatively inexpensive, can be carried out with
simple equipment, can be done continuously, and leads to a form of the enzyme or protein that is
often stable in long-term storage. The goal of precipitation is often concentration to reduce
volume, although significant purification can sometimes be achieved.
Salt (Ammonium sulfate) precipitation is used to recover lipase enzyme from the fermented
matter. Salt concentration plays a role in the rate of reaction. At low concentrations, the presence
of salt stabilizes the various charged groups on a protein molecule, thus attracting lipase enzyme
into the solution and enhancing the solubility of lipase enzyme. The solubility of lipase enzyme
depends on salt concentration in the solution. However, as the salt concentration is increased, a
point of maximum lipase enzyme solubility is usually reached. Further increase in the salt
concentration implies that there is less and less water to solubilize lipase enzyme. Finally, lipase
enzyme starts to precipitate when there are not sufficient water molecules to interact with lipase
enzyme molecules. This phenomenon of lipase enzyme precipitation in the presence of excess
salt is known as salting-out. The precipitate then is dissolved in trs-HCl buffer.
Rule 3: Make the most difficult and expensive separations last
Chromatography is typically done later in a process in agreement with the third generic heuristic
make the most difficult and expensive separations last. With the previous separation steps, a
large fraction of contaminants are removed, which reduces the volume of material that needs to
be treated further. In fact, a 50-100 fold volumetric reduction is quite common for high value
biological products, resulting in a protein content of 1-5% w/v in the feed stream to
chromatographic units.
One type of molecule called tween 20 might not be completely removed in precipitation step.
Hence unnecessary proteins and Tween 20 were removed by Q-sepharose ion exchange
chromatography. The reason why Tween 20 must be removed first before final purification was
removed was that Tween 20 affected the next Phenyl-sepharose ion exchange chromatography
because it decreased the hydrophobic nature of proteins. As final purification, the lipase was
further purified by Phenyl-sepharose ion exchange chromatography.
Rule 5: Select and sequence processes that use different separation driving forces.
The downstream process was sequenced in block diagram below was based on the generalized
block diagram of downstream processing from Bioseparation Sceince and Engineering Book.
Rotary vacuum
filtration
Precipitation using
Organic solvent
(salt)
Precipitate
Tris-HCl
Fungal mycelia
removal
Product
Extraction
Solution
Q-Sepharose IonChromatography to remove
tween 20
Final Purification
Phenyl-Sepharose IonDehydration
Chromatography as final
Diagram: block diagram for downstream
process ofoflipase
enzyme production based on the
purification
enzyme
Engineering Book.
b)Industrial Application
Applications of lipases
Lipases are widely used in the processing of fats and oils, detergents and degreasing formulation,
food processing, the synthesis of fine chemicals and pharmaceuticals, paper manufacture, and
production of cosmetics, and pharmaceuticals. Lipase can be used to accelerate the degradation
of fatty waste and polyurethane. Most of the industrial microbial lipases are derived from fungi
and bacteria.
Industry that
uses lipase
Detergent
Description
industry
Lipases are added to detergents such as household and industrial laundry and
residues and cleaning clogged drains. The cleaning power of lipase detergents
increases markedly.
Enzymes can reduce the environmental load of detergent products as the
chemicals used in conventional detergents are reduced; they are
biodegradable, non-toxic and leave no harmful residues. Besides lipases,
other enzymes are widely used in household cleaning products and in
laundering.
Decompose fatty material. Lipase is capable of removing fatty stains such as
Food industry
fats, butter, salad oil, sauces and the tough stains on collars and cuffs.
Fats and oils are important constituents of foods. The nutritional and sensory
value and the physical properties of a triglyceride are greatly influenced by
factors such as the position of the fatty acid in the glycerol backbone, the
chain length of the fatty acid, and its degree of unsaturation. Lipases allow us
to modify the properties of lipids by altering the location of fatty acid chains
in the glyceride and replacing one or more of the fatty acids with new ones.
This way, a relatively inexpensive and less desirable lipid can be modified to
a higher value fat. Cocoa butter, a high-value fat, contains palmitic and stearic
acids and has a melting point of approximately 37 C. Melting of cocoa
butter in the mouth produces a desirable cooling sensation in products such as
chocolate. Lipase-based technology involving mixed hydrolysis and synthesis
reactions is used commercially to upgrade some of the less desirable fats to
Pitch control is an important aspect in pulp and paper manufacture, and the
industry
and
both
microbial
and
enzymatic
products
have
been
The use of enzymes for organic synthesis has become an interesting area for
organic and bio-organic chemists. Since many enzymes have been
demonstrated to possess activity against non-natural substrates in organic
media they have become widely used to carry out synthetic transformations.
Hydrolases are the most frequently used enzymes due to their broad substrate
spectrum and considerable stability. Additionally, many of them are
commercially available and they work under mild reaction conditions and
without the necessity for cofactors. Among the hydrolases, lipases are
considered the most popular and useful enzymes for asymmetric synthesis.
Applications for lipases include kinetic resolution of racemic alcohols, acids,
ester or amines as well as the desymmetrization of prochiral compounds.
They are alsosuccesfully employed in regioselective esterification or
transesterification of polyfunctional compounds, for instance in the
chemoenzymatic synthesis of nucleoside derivatives. Recently, nonconventional processes, such as aldol reactions or Micheal addition have been
Bioconversion in
aqueous media
resolution of
alcohols
Ester synthesis
Lipases have been successfully used as catalyst for synthesis of esters. The
esters produced from short-chain fatty acids have applications as flavoring
agents in food industry. Methyl and ethyl esters of long-chain acids have been
used to enrich diesel fuels.
Oleo chemical
industry
thermal
degradation
during
alcoholysis,
acidolysis,
hydrolysis,
and
can
occur
simultaneously
in
process
known
as
Recovery
(separation of
insoluble)
Isolation of
product
Purification
Polishing
Drying,
and polishing. The high-throughput, low-resolution techniques are first used to significantly
reduce the volume and overall concentration of the material being processed. The partially
purified products are then further processed by high-resolution low-throughput techniques to
obtain pure and polished finished products.
Tray bioreactor
Recover
y
High
throughput, low
resolution
Fungal mycelia
removal by Rotary
vacuum filtration
Precipitation by
Organic solvent
(salt)
Q-Sepharose ionChromatography to remove
Purificatio
tween 20
Phenyl-Sepharose
ionn
Chromatography as final
purification of enzyme
Dehydration or
Polishing
Low
Solvent removal by
throughput,
drying
high
Lipase enzyme in powder form are packed
anddiagram
labelled
Diagram 3.2: Process Flow
based on RIPP
Isolation
Rotary vacuum filtration which removes the fungal mycelia as the first step in downstream is the
recovery process. Precipitation using organic solvent (ammonium sulfate) is product isolation
process. These recovery and isolation process are high throughput, low resolution techniques.
Purification by chromatography and polishing by drying are low throughput, high resolution
technique.
c) Calculate the recovery (eg. concentrations, purity and / or efficiency) for each of the
unit operation in bio-separation process.
Rotary Filtration Vacuum
Crude lipase mass flow rate
Total mass flow rate
Total volume flow rate
:
:
:
6.82kg/batch
6632.285 kg/batch
6697.044L/batch
6.82 kg/ batch
-3
6697.044 L /batch = 1.02 x 10 kg/L
6.82kg /batch
6632.285 kg /batch
Precipitation
Crude lipase mass flow rate
Total mass flow rate
Total volume flow rate
:
:
:
Q-sepharose Chromatography
Crude lipase mass flow rate
Total mass flow rate
Total volume flow rate
Concentration of lipase enzyme =
Purity of lipase enzyme
x 100% = 0.103%
6.486 kg/batch
2260.443 kg/batch
1496.059L/batch
6.486 kg/batch
-3
1496.059 L/batch = 4.335 x 10 kg/L
6.486 kg /batch
1496.059 kg /batch
x 100% = 0.4335%
:
4.151kg/batch
:
1446.684kg/batch
:
967.781L/batch
4.151 kg/ batch
-3
967.781 L/batch = 4.29 x 10 kg/L
4.151 kg/batch
1446.684 kg/batch
x 100% = 0.3 %
Phenyl-Sepharose Chromatography
Pure lipase mass flow rate
:
0.830kg/batch
Total mass flow rate
:
1443.363kg/batch
Total volume flow rate
:
954.426L/batch
0.830 kg/batch
Concentration of lipase enzyme= 954.426 L /batch = 8.696 x 10-4 kg/L
Purity of lipase enzyme
0.830 kg /batch
1443.363 kg /batch
x 100% = 0.0575 %
Drying
Pure lipase mass flow rate
Total mass flow rate
Total volume flow rate
:
0.664kg/batch
:
0.664kg/batch
:
0.679L/batch
0.664 kg /batch
0.679 L/batch = 0.98kg/L
0.664 kg /batch
0.664 kg /batch
0.103%
0.4335%
0.3%
0.0575%
100%
x 100% = 100%
d) Design one unit operation only in the downstream process (eg. ultrafiltration,
sedimentation, centrifugation, chromatography, etc).
Rotary Vacuum Filtration (RVF)
1.
2.
3.
4.
5.
Discharge Design
Drum Design
Special System Design
Filter Cloth/ Septum Design
Basic RVF Operation
Discharge Design
The five basic discharge types are:
1. Scraper
2. Endless Belt
3. String
4. Roll
5. Precoat
Each is designed to be able to discharge specific types of formed cake solids. In essence, these
five mechanisms enable the rotary vacuum filter to efficiently handle mechanism such as filter
solid-liquid slurry and discharge the formed solids as a complete spectrum of process slurries.
Since media formulation for fermentation are designed as 80% moisture for a good growth rate
of fusarium sp., the fermentation broth are low solid concentration slurry. The problem faced is
during rotary vacuum filtration. According to Haug, G. (1999), precoat discharge is used if slurry
with very low solid concentration slurry is used that resulted in difficult cake formation or if the
slurry is difficult to filter to produce cake formation. Hence, filter with precoat discharge are
applied since it is the most suitable this case.
Drum Design
Any RVF utilizing a scraper, endless belt, string or roll discharge must have a drum with (1)
filtrate pipes and a (2) valvebody with bridge blocks. A filter with a precoat discharge can use (1)
a drum with filtrate pipes, (2) a drum with a valvebody, (3) a valveless drum or (4) a drum
without filtrate pipes. For this reason, precoat discharge filters have a wide array of designs,
specialty features and varying requirements for successful operation.
For all the discharge design, valvebody is a requirement, accept for precoat discharge. Valvebody
is a device which controls the radial position of application for form and dry zone vacuum
blowback pressure if required, and venting to the various surfaces of the drum as the drum
rotates through its cycle. The valvebody is the connection between the filter which is at the drum
and the vacuum system typically the vacuum receiver. Since the drum dont have vent and bridge
blocks, valvebody are not installed for a greater performance. Precoat specific drum designs
cannot control the vacuum level at various radial positions on the drum; the entire drum is at the
same vacuum level throughout the entire drum cycle [drum revolution] All liquid and air are
contained within the filtrate pipes.
2. Knock-out-receiver
3. Tilting Valt
4. Hydraulic Agitator
Precoat discharge applications are typically high foam generators. Since foam does not easily
separate out from the air or liquid stream coming out of the filter drum, there is a high tendency
for the foam to be swept through the vacuum receiver, into the vacuum pump and out of the
filtration system with the vacuum pump seal water. This can cause environmental problems,
vacuum pump operation problems and a loss of filtered product. By adding a second receiver
with a diameter sufficient to reduce the air flow velocity to 1 ft/sec (or less), most foam can be
dropped out of the air stream. Foam carry-over can also be eliminated by reducing the filter
operating vacuum level. However, this will reduce the filter throughput and increase operating
costs, especially with a precoat discharge filter. Hence among the other systems, Knok-OutReceiver is the most suitable for higher efficiency.
Makeup
Filter Aid
Rotary
Vacuum
Filter
Precoat
Slurry
mix
Primary or
main vacuum
receiver
Knock-out or
secondary
vacuum
receiver
To
Diagram
Vacuum Receiver
Check
Proceed
The purpose of thevalve
vacuum
receiver is to (1) separate the two phase mixture coming out of the
Illustrated:
3.4:Knock-out
Precoat
Filtrate
filter, the air and liquid
(filtrate).
If
foam
is
present,
the
receiver
must
also
be
capable
of
discharge
receiver design
pump
preventing carry over of foam to the vacuum pump. The vessel diameter is the critical dimension
for effecting the separation of the two phases; vessel height is to accommodate surges in flow.
Vacuum Pump Capacity
For the precoating mode, the pump must deliver at least 2.5 Vacuum
to 3.5 CFM per square foot of filter
pump
area. During the process mode, 2.0 to 3.0 CFM per square foot is satisfactory. It is usually
satisfactory to employ a single vacuum pump for these small filters. Pumps should be capable of
achieving 28 Hg vacuum and sized for the required CFM capacity at 20 Hg operating level.
a)Filtration without filteraid b)Filtration with filteraid c) Filtration with filteraid and admix
Diagram 3.5: Filteraid
The common filter aids are diatomaceous earth (DE), perlite, cellulose and others. DE is the
skeleton of ancient diatoms. They are mined from ancient seabed, processed, and classified to
make different grade of filter aids. There are different grades of commercial DE. A finer grade
may be employed to increase the clarity of filtrate. The smaller the filter aid particle size, the
smaller the process particles can be removed. However, the filtration rate is lower. There is
always a balance between initial filtrate clarity and filtration rate.
Diatomaceous earth
Since the filteraid is the actual filtering medium, careful attention must be paid to the single most
important selection criterion that is process solids penetration. For effective performance, any
filteraid must limit the degree of solids penetration into the precoat cake to 0.002 0.005.
Greater penetration requires too high of a knife cut to remove the spent filteraid resulting in
high filteraid and disposal costs. Conversely, if the filter aid is too tight, for example, too fine,
solids penetration will be minimized, but flow rate will also be forfeited. Using too tight of a
filteraid grade not only forfeits available flow (filtration) rates and reduces filteraid efficiency, it
may not yield any improved filtrate clarity compared to an optimum grade (in this case, more
open). In like manner, there may not be a degradation of filtrate clarity if the filter aid grade is
too open, but excessive quantities of filteraid would be required for the same output (flow rate)
compared to a tighter (optimum) grade.
Knife cut analysis must always be based on knife advance rate per drum revolution. Most precoat
discharge filters have knife advance drives which are independent of the drum drive. This system
design makes it necessary to adjust the knife advance rate whenever the drum speed is changed
(assuming that the original knife cut was an optimum one). If the drum speed is reduced, the
optimum cut will change to an excessive cut. If the drum speed is increased, the optimum cut
will change to an insufficient cut (without a knife advance rate change, the knife will advance at
a constant rate per time period, not per drum revolution).
From the article of Enhancing the Performance of Rotary Vacuum Drum Filter (T. Sivakumar,
2011), with the overflow weir set to a maximum the "apparent submergence" is normally 3335% so the slurry levels between 04.00 and 08.00 hrs. Once a sector enters submergence vacuum
is applied and a cake starts to form up to a point where the sector emerges from the slurry. The
portion of the cycle available for formation is the "effective submergence" and its duration
depends on the number of sectors, the slurry level in the tank.
Since for higher performance, highest of possible vat level that is 35% is designed.
Drum speed
With a precoat discharge filter, higher drum speeds also means lower filteraid efficiencies
(hence, higher production costs). Drum speed and vat level are usually adjusted dependently in
order to optimize filter performance. The drum speed is let to be 1 minute/ revolution of drum.
e) Discussion
Bioseperation in this project is to recover, isolate, purify and polish the lipase enzyme in the
downstream process to obtain highest percentage of purity. First of all the content from the
fermentation broth is identified first. Before designing the downstream process, the 5 thumb
rules of bioseparation is well understood.
5 rule of thumbs:
Rule 1: Separate the most plentiful impurities first
Rule 2: For extracellular product formation, rule of thumb starts from second generic heuristic.
Remove the easiest to removed first
Rule 3: Make the most difficult and expensive separations last
Rule 4: Choose those processes that will exploit the differences in the physicochemical
properties of the product and impurities in the most efficient manner
Rule 5: Select and sequence processes that use different separation driving forces.
By following all the thumb rules above, appropriate equipment are selected to design a high
performance downstream process with lowest cost as possible. The entire important unit
operations chose is in correct sequenced by referring to generalize block diagram in the rule of
thumbs.
Then, the by utilizing RIPP (Recovery, Isolation, Purification, Polishing) scheme, the sequence is
then arranged in more according order. Recovery is the process is removing cells; the Isolation is
the process of removing material has properties widely different from those desired in product.
Purification is removing remaining impurities which typically similar to desired product in
chemical functionality & physical properties.
convert product to crystallized form. For recovery, rotary vacuum drum filter, for isolation,
precipitation, for purification, ion exchange chromatography and for polishing, is drying.
For the design a unit operation, rotary vacuum filter(RVF) is chose. Discharge design, drum
design, special system design, filter cloth/ septum design basic RVF operation is considered in
the design. The chosen discharge design is precoat discharge since it is the most suitable for high
moisture (80%) solid state fermentation. The chosen drum design is valveless drum schematic
which have filtrate pipe no valve body and no radial position control of vacuum. Valveless drum
schematic is the more suitable for precoat discharge type. Chosen special system design is
knock-out receiver. This system design is designed especially for the drum uses precoat. Precoat
discharge applications are typically high foam generators. Since foam does not easily separate
out from the air or liquid stream coming out of the filter drum, there is a high tendency for the
foam to be swept through the vacuum receiver, into the vacuum pump and out of the filtration
system with the vacuum pump seal water. Precoat discharge filter has different requirements than
other discharge type because the septum is not the filter medium; the precoat cake is the filter
medium. The chosen filter aid is diatomaceous earth. In thhe basic RVF operation, the important
parameters are vat level and drum speed. The greater the vat level the greater the filtration.
Hence the highest possible vat level is choosed that is 35%. For drum speed, increase in drum
speed lower the filteraid efficiency. Hence the drum speed is let to 1 min per revolution of drum.
The area of drum submerges calculated for 1m Diameter and 1 m long is 3.08m2.