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The POME being a waste from palm oil production should be very cheap or even free
of cost and it is abundantly available. Hence, the POME can be treated as a rospective
alternative raw material for the production of cellulase enzyme leaving an attractive
opportunity for potential investment.
Figure 1: Flow diagram of upstream palm oil mill with circle in shape showing the production
of palm oil mill effluent (Man Kee Lam, 2011)
Cellulolytic microbes are primarily carbohydrates degraders and are generally unable
to use proteins or lipids as energy sources for growth. Table 2 shows that different types of
cellulolytic microbes can utilize a variety of other carbohydrates in addition to cellulose.
the expression of genes which are involved in the degradation of less favourable and complex
carbon sources such as cellulose.
An ultrafiltration membrane retains molecules that are larger than the pores of the membrane
while smaller molecules such as salts, solvents and water, which are 100% permeable, freely
pass through the membrane. In continuous diafiltration, the buffer solution is washed out by
adding water at the same rate as filtrate was being generated.
The dialyzed solution is then separated from water by using an ion-exchange
chromatography column, I-100. Ion exchange chromatography is used to purify proteins and
other charged molecules. In cation exchange chromatography positively charged molecules
are attracted to a negatively charged solid support. Conversely, in anion exchange
chromatography, negatively charged molecules are attracted to a positively charged solid
support. The concentrated enzyme solution obtained from chromatography was stored in TK100. The concentrated enzyme solution was filled into 1L bottles and packaged into boxes.
Each box comprises of 12 bottles containing cellulase enzyme solutions.
POME
Sterilisation Tank
Nutrient
Tricoderma
Reesei
Water
Fermentor Tank
CO2
Storage Tank
Product
Air
Ammonium
Sulphide
Filtration
Water
Purification
Mixing Tank
Water
Filtrate
Centrifuge
Buffer
Mixing Tank
Figure 3: Block Flow Diagram of Production of Cellulase from POME by using Trichoderma
Reesei species.
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Cellulases have been successfully used for the biostoning of jeans and biopolishing of cotton
and other cellulosic fabrics. During the biostoning process, cellulases act on the cotton fabric
and break off the small fiber ends on the yarn surface, thereby loosening the dye, which is
easily removed by mechanical abrasion in the wash cycle. The advantages in the replacement
of pumice stones by a cellulose-based treatment include less damage of fibers, increased
productivity of the machines, and less work-intensive and environment benign.
While the biopolishing is usually carried out during the wet processing stages, which
include desizing, scouring, bleaching, dyeing, and finishing. The acidic cellulases improve
softness and water absorbance property of fibres, strongly reduce the tendency for pill
formation, and provide a cleaner surface structure with less fuzz. Cellulase preparations rich
in endoglucanases are best suited for biopolishing enhancing fabric look, feel, and colour
without needing any chemical coating of fibers. The action of cellulases removes short fibers,
surface fuzziness, creates a smooth and glossy appearance, and improves colour brightness,
hydrophilicity and moisture absorbance, and environmentally friendly process.
Similarly, endoglucanase activity-rich cellulase is also proved better for biofinishing.
Most cotton or cotton-blended garments, during repeated washing, tend to become fluffy and
dull, which is mainly due to the presence of partially detached microfibrils on the surface of
garments. The use of cellulases can remove these microfibrils and restore a smooth surface
and original colour to the garments. The use of cellulase also helps in softening the garments
and in removal of dirt particles trapped within the microfibril network.
alkaline hydrolysis because enzyme hydrolysis is usually conducted at mild conditions (pH 4
6 and temperature 4550C) and does not have corrosion issues (Ramesh et al., 2011).
hemicellulases are also used for improved extraction of olive oil. Use of macerating enzymes
not only improves the cloud stability and texture of nectars and purees, but also decreases
their viscosity rapidly. Thus, the macerating enzymes, composed of mainly cellulase and
pectinase, play a key role in food biotechnology, and their demand will likely increase for
extraction of juice from a wide range of fruits and vegetables. Furthermore, infusion of
pectinases and -glucosidases has also shown to alter the texture, flavor, and other sensory
properties such as aroma and volatile characteristics of fruits and vegetables.
exoglucanase promoters of Trichoderma have been used for the expression of chymosin and
other proteins such as glucoamylase, lignin peroxidase, and laccase.
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Table 3: The processing of oil palm fresh fruit bunches (FFB) primarily for palm oil
also results in the production of wastes, in the form of palm oil mill effluent
(POME), empty fruit bunches, mesocarp fibre and shell (MPOB, 2012)
microorganisms in liquid nutrient broth. Most of the industrial enzymes can be produced
using this process. This involves growing carefully selected microorganisms (bacteria and
fungi) in closed vessels containing a rich broth of nutrients (the fermentation medium) and a
high concentration of oxygen. As the microorganisms break down the nutrients, they release
the desired enzymes into solution.
When compared with the enzyme market price (from $90/kg to $180/kg), Monte Carlo
analysis results showed that the SmF method was profitable with 85.8% certainty, which
implied the probability to achieve a profit (greater than or equal to the lower bound of market
price, $90/kg) was 85.8% (Demain et al., 2005). The mean unit cost for enzyme production
using the SmF method was $57.2/kg (Demain et al., 2005).
Figure 5: Frequency of simulated unit costs using submerged fermentation method (Demain et
al., 2005)
Table 4: Itemized unit costs for enzyme production (year 2004 prices) (Demain et al., 2005)
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Bioreactor
System
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of one or more CBH components, one or more EG components and possibly BG. The
complete cellulose system comprising CBH, EG and BG components synergistically act to
convert crystalline cellulose to glucose. The exocellobiohydrolases and endoglucanases act
together to hydrolyze cellulose to small cellooligosaccharides. The oligosaccharides (mainly
cellobiose) are subsequently hydrolysed to glucose by a major BG.
The T. reesei complex is a true cellulase in the most rigid sense, being able to convert
crystalline, amorphous, and chemically derived celluloses quantitatively to glucose. It has
been established that:
a) The system is multi-enzymatic
b) At least three enzyme components are both physically and enzymatically distinct
c) All three components play essential roles in the overall process of converting
cellulose to glucose
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Figure 7: Schematic in context cellulases production. Dotted arrows and gray squared text
indicates potential areas of research for enhancement of cellulase secretion in T.
reesei and other organisms.
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During this biosynthesis of cellulase from T. reesei, two phases are noticeable:
primary and secondary (Gaden, 1955). In the primary phase, biomass accumulation and
normal metabolic activities reach their maximum, then in the secondary, later phase, product
accumulation and formation rate reach their maximum values.
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Bio-Separation
Engineering
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3.1 Type of Product and Rules of Thumb for the Process of Bio-separation
Based on Figure 7, it can be determined that the cellulase produced from T. reesei is an
extracellular cellulase.
Rule 3: Choose those process that will exploit the differences in the physic-chemical
properties of the product and impurities in the most efficient manner.
The process that follows the third rule is the centrifugation. The centrifuge works
using the sedimentation principle, where the centripetal acceleration causes denser substances
and particles to move outward in the radial direction. At the same time, objects that are less
dense are displaced and move to the center. The radial acceleration causes denser particles to
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settle to the bottom of the tube, while low-density substances rise to the top. Disk-stack
centrifuge, C-100 is operates at higher rotational speeds and remove smaller and lighter
microorganisms. It separates particles by size using centrifugal sedimentation in a liquid
medium. Centrifugation does not require filter aid, which is a significant advantage compared
to rotary vacuum filtration. Besides, more-dense cell debris or can be separate out, while lessdense product is flow to the next steps for further purification.
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Industries
Agriculture
Bioconversion
Applications
Detergents
Fermentation
Cellulase-based detergents
Food
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Biomechanical pulping
Improved draining
Textile
Others
Biostoning of jeans
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Objective
of insoluble biomass
Remove or collect
Isolation of cellulase
Reduce volume
Remove materials
Precipitation, diafilter
having properties
widely different
from desired
product - Cellulase
Purification of cellulase
Reduce volume
Remove remaining
Ion exchange
impurities such as
chromatography
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The scheme of downstream process can be illustrated base on the block flow diagram:
Bioreactor
Extracellular Product
Biomass Removal
-Rotary vacuum filtration
-Centrifugation
Recovery Stage
Concentration
-Precipitation
Isolation Stage
Final Purification
-Diafiltration
Purification Stage
-Ion exchange
chromatography
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Process Control
and Dynamics
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4.1 Parameters to be Controlled in each Unit Operation Unit and their Control
Objective
i.
Unit operation
Parameter
Control objective
manipulating
Manipulate variable : The inlet flow rate of the POME to the tank
Disturbance variable : The outlet flow rate of the POME from the tank
ii.
Unit operation
Parameters
: Fermenter, R-100
: Level of medium in R-100, temperature in R-100, pH of the
medium in R-100
nutrient
Controlled variable
iii.
Control objective
Controlled variable
Manipulate variable : The outlet flow rate of the solution from F-100
Disturbance variable : The inlet flow rate of ammonium sulphate, (NH4)2S04
iv.
Controlled variable
Manipulate variable : The outlet flow rate of the solution from M-100
Disturbance variable : The outlet flow rate of supernatant into drain
v.
Controlled variable
Manipulate variable : The outlet flow rate of the solution from C-100
Disturbance variable : The inlet flow rate of citrate buffer
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vi.
vi.
Controlled variable
Manipulate variable : The outlet flow rate of the solution from I-100
Disturbance variable : The outlet flow rate of the solution from TK-100
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Simulation for
Bioprocess
Engineering
35
To study the flow production process of the cellulose enzyme from T. reesei sp.
To identify the throughput analysis of the cellulose enzyme production.
To identify the economic analysis of the production process.
To discuss the debottlenecking to increase the yield production of cellulose enzyme.
To understand the using of SuperPro software in designing the project.
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References
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Demain, A. L., M. Newcomb, and J. H. D. Wu. (2005). Cellulase, clostridia, and ethanol,
Microbiology and Molecular Biology Reviews, 69(1): 124154.
Gaden, E.L. (1955) Fermentation kinetics and productivity. Chem. Ind., 154159.
Man Kee Lam & Keat Teong Lee. (2011). Renewable and sustainable bioenergies production
from palm oil mill effluent (POME). Biotechnology Advances 29, 124141
Malaysian Palm Oil Board (MPOB). Overview of the Malaysian oil palm industry 2012.
Ramesh Chander Kuhad, Rishi Gupta, and Ajay Singh. (2011). Microbial Cellulases and
Their Industrial Applications. Enzyme Research, vol. 2011, Article ID 280696, 10
pages, 2011. doi:10.4061/2011/280696
Rashid, S. S., Alam, M. Z*., Karim, M. I. A., & Salleh, M. H. (2009). Optimization of the
Nutrient Supplients for Cellulase Production with the Basal Medium. World Academy
of Science, Engineering and Technology.
Rjeev, K. S., Reeta, R. S., & Ashok, P. (2005). Microbial cellulases Production, applications
and challenges. Journal of Scientific & Industrial Research. 64(1), 821-844.
Shah Samiur Rashid, Md. Zahangir Alam, M. Ismail A. Karim & M. Hamzah Salleh. (2009).
Management of palm oil mill effluent through production of cellulose by filamentous
fungi. World J Microbial Biotechnol, 25(1), 2219-2226.
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