BIOSEPARATION

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

Yeast extract /NaNO3

1g

Glucose

20g

Polysorbate 20(Tween 20)

4ml

Wheat Bran

975.8g

Distilled water
1L
Autoclave at 120C for 50min (pH 6.5)
Table 2.1: Composition of mycelium medium (MM) for 1 liter.
2. Fungal mycelia

Rule 1: Separate the most plentiful impurities first

Downstream processes for extracellular and intracellular product are different where, for the case
intracellular product formation, cell harvesting is the first step since the product are formed
inside the cell while the outside of the cells are the most plentiful impurities. But for case of

lipase enzyme produced extracellularly by fusarium sp., the lipase enzymes are produced outside
of the cell. So, this rule is neglected.
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 Gel-filtration
Chromatography to remove
tween 20

Final Purification

Phenyl-Sepharose Gel-filtration
Dehydration
Chromatography as final
Diagram: block diagram for downstream
process ofoflipase
enzyme production based on the
purification
enzyme

generalized block diagram of downstream processing from Bioseparation Sceince and

Dehydration or
Solvent removal by
drying

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

in household dishwashers, where their function is in the removal of fatty

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

cocoa butter substitutes.

Pulp and paper

Pitch control is an important aspect in pulp and paper manufacture, and the

industry

first example where microbial biotechnology provided successful solutions in

this industrial sector. Triglycerides cause deposits in softwood mechanical
pulping,

and

both

microbial

and

enzymatic

products

have

been

commercialized to be applied on wood and pulp, respectively. The former are

based on colorless strains of sapstain fungi. The latter are improved lipases,
including thermostable variants from directed evolution. These enzymes are
among the additives of choice in pulping of high-resin-content softwoods.
Organic Synthesis

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

archived using lipases

Enzymes in organic media without a free aqueous phase are known to display

aqueous media

useful unusual properties, and this has firmly established non-aqueous

enzyme systems for synthesis and bio-transformations. Lipases have been
widely investigated for various non-aqueous bio-transformations.

resolution of

Stereo-selectivity of lipases has been used to resolve various racemic organic

racemic acids and

acid mixtures in immiscible biphasic systems. Racemic alcohols can also be

alcohols

resolved into enantiomerically pure forms by lipase-catalyzed transesterification.

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

Use of lipases in oleochemical processing saves energy and minimizes

industry

thermal

degradation

during

alcoholysis,

acidolysis,

hydrolysis,

and

glycerolysis. Although lipases are designed by nature for the hydrolytic

cleavage of the ester bonds of triacylglycerol, lipases can catalyze the reverse
reaction (ester synthesis) in a lowwater environment. Hydrolysis and
esterification

can

occur

simultaneously

in

process

known

as

interesterification. Depending on the substrates, lipases can catalyze

acidolysis (where an acyl moiety is displaced between an acyl glycerol and a
carboxylic acid), alcoholysis (where an acyl moiety is displaced between an
acyl glycerol and an alcohol), and transesterification (where two acyl moieties
are exchanged between two acylglycerols).

c) Propose a RIPP scheme for desired products

The RIPP scheme

Stage Objectives Typical unit

Unit Operation used

Recovery

Remove cells debris /other particulate.

Reduce volume

Rotary Vacuum Filter

(separation of
insoluble)
Isolation of
product
Purification

Remove material have properties widely Precipitation

different from those desired in product.
Reduce volume
Remove remaining impurities which typically Ion Exchange Chromatography,
similar to desired product in chemical

Polishing

functionality & physical properties.

Remove liquid.

Drying,

Consideration that need to be taken during developing a bioseparation process:

1. The nature of starting material. The starting material is wheat bran. Its original state is
in the solid state. Wheat bran is rich in carbohydrate which are very suitable as substrate
for fusarium sp. additional nutrients are added for a better growth of fusarium sp.
2. The initial location of the target product: product are formed at extracellular
3. The volume or flow-rate of the starting material. The volume of starting material is
5000m3/l. The flow rate of starting material is let to flow until maximum allowable
volume for tray bioreactor is reached.
4. The relative abundance of the product in the starting material, lipase enzyme is
absence in the starting material. The relative abundance of the product will start to
increase in the fermentation trays when the medium is inoculated.
5. The susceptibility to degradation e.g. its pH stability, sensitivity to high shear rates
or exposure to organic solvents. Lipase degrades at 70oC, pH stability at pH8.8. Since
fungus suitable environment is solid state, stirring no stirrer or impeller are installed.
6. The desired physical form of the final product. Final product is desired to be in
powdered form.
7. The quality requirements. 99% pure lipase enzyme.
8. Process costing and economics. economic
A RIPP scheme is commonly used in bioseparation. This strategy involves use of low resolution
techniques first for recovery and isolation followed by high resolution techniques for purification

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

Concentration of lipase enzyme =

Purity of lipase enzyme

6.82 kg/ batch

6697.044 L /batch

6.82kg /batch
6632.285 kg /batch

= 1.02 x 10-3 kg/L

x 100% = 0.103%

Precipitation
Crude lipase mass flow rate
Total mass flow rate
Total volume flow rate

:
:
:

Concentration of crude lipase enzyme =

Purity of lipase enzyme

Q-sepharose Chromatography
Crude lipase mass flow rate
Total mass flow rate
Total volume flow rate
Concentration of lipase enzyme =

Purity of lipase enzyme

6.486 kg/batch
2260.443 kg/batch
1496.059L/batch
6.486 kg/batch
1496.059 L/batch

6.486 kg /batch
1496.059 kg /batch

:
:
:

4.151 kg/batch
1446.684 kg/batch

Concentration of lipase enzyme=

x 100% = 0.4335%

4.151kg/batch
1446.684kg/batch
967.781L/batch

4.151 kg/ batch

967.781 L/batch

Phenyl-Sepharose Chromatography
Pure lipase mass flow rate
:
Total mass flow rate
:
Total volume flow rate
:

= 4.335 x 10-3 kg/L

= 4.29 x 10-3 kg/L

x 100% = 0.3 %

0.830kg/batch
1443.363kg/batch
954.426L/batch

0.830 kg/batch
954.426 L /batch

= 8.696 x 10-4 kg/L

Purity of lipase enzyme

Drying
Pure lipase mass flow rate
Total mass flow rate
Total volume flow rate

:
:
:

x 100% = 0.0575 %

0.664kg/batch
0.664kg/batch
0.679L/batch

0.664 kg /batch
0.679 L/batch

Concentration of lipase enzyme=

Purity of lipase enzyme

0.830 kg /batch
1443.363 kg /batch

0.664 kg /batch
0.664 kg /batch

= 0.98kg/L

x 100% = 100%

Purity summary table

Rotary Filtration
Precipitation
Q-sepharose chromatography
Phenyl-sepharose chromatography
Drying

0.103%
0.4335%
0.3%
0.0575%
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.

Diagram 3.3: Valveless drum schematic

Filtrate pipe
No valvebody
No radial position control of vacuum
Special System Designs
There are 5 types of common system design for RVF
1. Cake Wash

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.

Filtrate Pump Capacity

Most precoat filter applications will have a process rate of filtration considerably lower than that
of the precoating mode.
Filteraid
Precoat Slurry Mix
Basic rule for design precoat slurry mix:
1) The mix tank must be large enough to hold the entire charge of filteraid for a full precoat
cake at an appropriate slurry concentration (a satisfactory design for small filters); or
2) The required amount of filteraid can be added so as to maintain the desired slurry
concentration during a 30 minute time span.
3) Note that the desired slurry concentration (wt:wt basis) is typically 5% - 8%. The %
concentration should be
i) Constant throughout the precoating mode or
ii) decrease uniformly to zero, as with recirculation systems.
Since the basis from the bioreactor is in greater amount, bigger filter is designed and the filteraid
is added to maintain the desired slurry during a 30 minute span. The amount of filteraid chose to
add is 5% from the slurry concentration and the concentration are set to be constant throughout
the precipitation precoating.
Make-up Water Recirculation
If a recirculation line is used, very little make-up water will be needed during precoating.
Filter Cloth/Septum Design
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.
Filteraids is a group of inert materials that can be used in filtration pretreatment. There are two
objectives related to the addition of filteraids. One is to form a layer of second medium which
protects the basic medium of the system. This is commonly referred to as precoat. The second
objective of filteraids is to improve the flow rate by decreasing cake compressibility and
increasing cake permeability. This type of usage is termed as admix or "body feed". Filtration
without filter aid, with precoat, and with precoat and body feed is shown in Fig. 1 (Eagle-Pitcher
Minerals, Inc., 1970).

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).

Diagram: Knife Cut analysis

Basic RVF Operation

Vat level and drum speed are the two basic operating parameters for any rotary vacuum drum
filter. These parameters are adjusted dependently to each other to optimize the filtration
performance.
Vat Level
Vat level determines the proportion of the filter cycle, such as one drum revolution, dedicated to
cake formation and cake drying. In the absence of any other contradicting factors, vat level
should be adjusted to maximum higher vat level is equal to greater filtration. The two basic
reasons for reducing the operating vat level are:
Hard to filter slurries which form thin, gelatinous or slimy cakes; or
Slurries with very high suspended solids content which form very thick cakes.
Summary of Operating Vat Level Cycles
High Vat Level
Maximum filtration time
Maximum solids formation per cycle
Maximum cake thickness
Maximum cake moisture content
Highest filter output
Low Vat Level
maximum cake drying/washing time
minimum solids formation per cycle
minimum cake thickness
minimum cake moisture content
lowest filter output

Diagram: Drum of Rotary Vacuum Drum Filter

As the drum rotate through the feed in the filter tank, vacuum is applied to dewater cake picked
on the media. Vacuum cutoff occurs just prior to the cake discharge point.

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.

To calculate the area of drum submerged

Rate of cell broth = 2000L/h
Vacuum Pressure= 70kPa
Cycle time =60s
Cake formation time = 15s
Viscosity of broth = 2.0cp
Cake solid (dry basis) per volume= 10g/Specific cake resistance 9 x 1010 cm/g
We can use the nitrated form of the filtration equation, with Rm =0
t/(V/A) =(o/2p)(V/A)
We solve forA2 to obtain
A2=ocV2/2pt
In applying this equation it is helpful to focus on the area of the drum that is submerged, which is
where the cake is being formed and where filtrate is being obtained. Thus, A is the area of that
part of the drum that is submerged. We can calculate the volume of filtrate that needs to be
collected during the cake formation time of 15s.
V15s=2000L/h x 15s x 1h/3600s =8.33L
We use this volume of filtrate with time t =15s in the equation for A2 to obtain
A2 =0.02g/cm.s x (9 x 1010cm/g) x 10g/L x (8.33L)2 x (cm.s2kPa/ 104 g) x 103cm3/L x(m/102cm)4
2 x 70kPa x 15s
=0.595m4
A = 0.771m3
Area A of the entire rotary vacuum filter, can be calculated from the cake formation time and the
total cycle time as
A = (60s/15s) x 0.771m2 = 3.08m2
This is the medium- sized rotary vacuum filter, with possible dimensions of 1m diameter by 1m
long.

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.

Polishing is process of removing liquid and

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.