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Introduction

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1.1 Project Background

1.1-1 Raw Material – Palm Oil Mill Effluent (POME)

Malaysia produces and exports the highest quantity of palm oil products in the world.

Malaysia produced 15.8 million tons of palm oil in the year 2006 alone (Rashid et al., 2009).

It is estimated that 0.5 – 0.7 tons of POME is discharged from the mill for every tons of oil

palm fresh fruit bunches in which about 40 million tons of POME from 372 mills was

discharged in the year of 2004 (Rashid et al., 2009).

Large quantities of solid and liquid wastes are generated in the form of empty fruit

bunches, pericarp fibres, palm shells, palm kernel cake and palm oil mill effluent (POME)

during the processing of oil palm fruits to produce oil. Microorganisms from relevant

environments are capable of degradation and utilization of organic waste during their

metabolism as well as secretion of secondary metabolites such as bio-products. Therefore,

bioconversion of POME is a useful measure to produce the natural product like - cellulase

enzyme as POME consists of favourable nutrient composition such as water (95-96%), oil

(0.6-0.7%), total suspended solids (4- 5%) and considerable amount of minerals (Rashid et al.,

2009). The proximate analysis of POME was determined in order to utilize it as a carbon

source as well as trace elements source prior to the fermentation process.

Table 1: Proximate composition of POME and PPF (Rashid et al., 2009)

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.

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1.1-1 Raw Material – Palm Oil Mill Effluent (POME)

The wet palm oil milling process is the most common way of extracting palm oil from

fresh fruit bunches (FFB), typically in Malaysia. It involves several stages in which huge

amount of water and steam are required for washing and sterilizing. Thus, this has resulted to

huge amount of wastewater generated from palm oil mill or better known as POME. Since

large amount of POME can be produced from palm oil, it is another reason to which it is

selected as raw material for the production of cellulose. Figure 1 shows a simplified process

flow diagram to produce palm oil.

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)

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1.1-2 Microorganism – Trichoderma reesei (T. reesei)

Trichoderma reesei (T. reesei) is a mesophilic and filamentous fungus. Fungi strains

of T. reesei used as an activator for the biodegradation process. The best characterized and

most widely studied cellulase system is that of the soft rot fungus trichoderma, particularly T.

reesei. T. reesei can metabolize cellulose as an energy source, secreting large amounts of

extracellular cellulolytic enzymes which are cellulases and hemicellulases. Microbial

cellulases have industrial application in the conversion of cellulose, a major component of

plant biomass, into glucose. Application of fungi into compost resulted in higher xylanase and

cellulase activity hence leads to rapid degradation of cellulose and hemicellulose.

Figure 2: Scientific classification and binomial name of T. reesei

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1.1-2 Microorganism – Trichoderma reesei (T. reesei)

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.

Table 2: Major microorganism employed in cellulose production (Rjeev et al., 2005)

However, T. reesei is an efficient producer of cellulases and industrial production

exceeds 100 g cellulases per liter (Rjeev et al., 2005). The quest of T. reesei to most

efficiently detect cellulose in the environment, to degrade the insoluble substrate by producing

different cellulases, transport the soluble break-down products from the surrounding through

the cytoplasmic membrane into the cell and subsequently assimilate these sugars, is an

essential process to survive. T. reesei has flexible and immediately respond and adapt to

changes in the nutrient composition of the environment. When exposed to a mixture of carbon

sources, T. reesei will utilize the best carbon and energy source available and down regulate

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the expression of genes which are involved in the degradation of less favourable and complex

carbon sources such as cellulose.

1.1-3 Product - Cellulase

Cellulase enzymes provide a tremendous benefit of biomass utilization through the

bioconversion of the most abundant cellulosic wastes into the simplest carbohydrate monomer,

glucose. Cellulases are the enzymes that hydrolyze β-1,4 linkages in cellulose chains. They

are produced by fungi, bacteria, protozoans, plants, and animals.

There are 3 major types of cellulose enzymes which are cellobiohydrolase (CBH),

Endo-β-1,4-glucanase (EG) and β-glucosidase (BG). Enzymes within these classifications can

be separated into individual components such as microbial cellulase compositions may consist

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.

Cellulase enzyme production with expensive media constituents such as celluclast,

glucose, yeast extract, peptone, urea, KH2PO4, (NH4)2SO4, MgSO4, FeSO4, MnSO4, CoCl2,

CaCl2 have been reported by many researchers (Rjeev et al., 2005). Apart from the extensive

use in textile industry, cellulase enzymes are used to convert non-food biomasses to

fermentable sugar, and ultimately to sustainable products including bio-fuel and bio-based

performance ingredients in household goods such as laundry detergents and shampoos. With

the increasing population and expanding consumption of textile products, detergent and

shampoos, demand for cellulase enzyme is expected to be on the rise.

Cellulose is the most abundant renewable biological resource and a low-cost energy

source based on energy content ($3–4/GJ). The production of bio-based products and

bioenergy from less costly renewable lignocellulosic materials would bring benefits to the

local economy, environment, and national energy security.

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1.2 Process Description

1.2-1 Raw Material Collection

The substrate which is Palm Oil Mill Effluent (POME) was collected from Seri Ulu

Langat Palm Oil Mill Sdn. Bhd., Dengkil, Selangor, Malaysia (Shah Samiur Rashid et al.,

2009). POME was collected into 20 L washed and cleaned container. The POME was

sterilized in sterilisation tank, T-100 to kill any microbial contamination and to break down

lignin into cellulose and hemicellulose. The POME contains 90-95% water and only about 4-5%

suspended solid (Shah Samiur Rashid et al., 2009). The samples were stored at 4oC for further

use (Shah Samiur Rashid et al., 2009).

1.2-2 Cellulase Production

The basal medium for the growth of T. reesei and production of cellulase is as follows

(g/l): (NH4)2SO4: 1.4, KH2PO4: 2.0, Urea: 0.3, CaCl2: 0.3, MgSO4.7H2O: 0.3 and (mg/l):

FeSO4.7H2O: 5.0, MnSO4.H2O: 1.4, CoCl2: 2.0 (Shah Samiur Rashid et al., 2009). In addition,

microcrystalline cellulose (1%), Difco Peptone (0.1%) and Tween 80 (Polyoxyethylene

sorbitan molooleate, 0.1%) were added to the medium to induce cellulase production (Shah

Samiur Rashid et al., 2009). pH was controlled using 2N HCl and 2N NaOH. The medium

was autoclaved for 30 min and seeded with a suspension of T. reesei spores, to a final

concentration of 2 x 105 spores/ml (Shah Samiur Rashid et al., 2009). The submerged culture

was run for 6 days at 28oC and at pH 3.5 in bioreactor, R-100 (Shah Samiur Rashid et al.,

2009).

1.2-3 Purification of Cellulase

The mixture from the R-100 was filtered in rotary vacuum filter, F-100 to remove the

biomass. The culture filtrate was then concentrated by mixing with 70% ammonium sulphate

(NH4)2SO4 saturation to form precipitate in mixer, M-101 (Shah Samiur Rashid et al., 2009).

The precipitates were then separated by centrifugation C-100 and dissolved in citrate buffer

(0.05 M) at pH 4.8 and water by using mixer M-102 (Shah Samiur Rashid et al., 2009).

This mixture was then filtered using a diafilter, D-100. Diafiltration is a technique that

uses ultrafiltration membranes to completely remove, replace, or lower the concentration of

salts or solvents from the solution. The process selectively utilizes permeable membrane

filters to separate the components of solutions and suspensions based on their molecular size.

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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 TK-

100. The concentrated enzyme solution was filled into 1L bottles and packaged into boxes.

Each box comprises of 12 bottles containing cellulase enzyme solutions.

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POME

Nutrient

Sterilisation Tank

Fermentor Tank

Filtration

Mixing TankAmmonium

Sulphide

CO2

Water

Water

Centrifuge

Tricoderma

Reesei

Water

Air

Storage Tank

Purification

Filtrate

Mixing TankBuffer

Product

Figure 3: Block Flow Diagram of Production of Cellulase from POME by using Trichoderma

Reesei species.

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Figure 4: Process Flow Diagram of Production of Cellulase from POME by using

Trichoderma Reesei species.

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1.3 Marketability and Economic Analysis

1.3-1 Application of Cellulases in Various Industries

1.3.1-1 Pulp and Paper Industry

The mechanical pulping processes such as refining and grinding of the woody raw

material lead to pulps with high content of fines, bulk, and stiffness. While biomechanical

pulping using cellulases resulted in substantial energy savings (20–40%) during refining and

improvements in hand-sheet strength properties (Ramesh et al., 2011).

Mixtures of cellulases (endoglucanases I and II) and hemicellulases have also been

used for biomodification of fiber properties with the aim of improving drainage and

beatability in the paper mills before or after beating of pulp. Endoglucanases have the ability

to decrease the pulp viscosity with a lower degree of hydrolysis, cellulases have also been

reported to enhance the bleachability of softwood kraft pulp producing a final brightness score

comparable to that of xylanase treatment.

Cellulases alone, or used in combination with xylanases, are beneficial for deinking of

different types of paper wastes. Cellulases and hemicellulases are used for the release of ink

from the fiber surface by partial hydrolysis of carbohydrate molecules. The main advantages

of enzymatic deinking are reduced or eliminated alkali usage, improved fiber brightness,

enhanced strength properties, higher pulp freeness and cleanliness, and reduced fine particles

in the pulp. Moreover, deinking using enzymes at acidic pH also prevents the alkaline

yellowing, simplifies the deinking process, changes the ink particle size distribution, and

reduces the environmental pollution.

Enzyme treatments remove some of the fines or peel off fibrils on the fiber surface and

dissolved and colloidal substances, which often cause severe drainage problems in paper mills.

These enzymes are used in preparation of easily biodegradable cardboard, manufacturing of

soft paper including paper towels and sanitary paper, and removal of adhered paper.

1.3.1-2 Textile Industry

Cellulases are the most successful enzymes used in textile wet processing, especially

finishing of cellulose-based textiles, with the goal of improved hand and appearance.

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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.

1.3.1-3 Bioethanol Industry

Enzymatic saccharification of lignocellulosic materials such as sugarcane bagasse,

corncob, rice straw, Prosopis juliflora, Lantana camara, switch grass, saw dust, and forest

residues by cellulases for biofuel production is the most popular application currently being

investigated. Bioconversion of lignocellulosic materials into useful and higher value products

normally requires multistep processes. These processes include pretreatment (mechanical,

chemical, or biological), hydrolysis of the polymers to produce readily metabolizable

molecules (hexose and pentose sugars), bioconversion of these smaller molecules to support

microbial growth and/or produce chemical products, and the separation and purification of the

desired products. The utility cost of enzymatic hydrolysis may be low compared with acid or

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alkaline hydrolysis because enzyme hydrolysis is usually conducted at mild conditions (pH 4–

6 and temperature 45–50°C) and does not have corrosion issues (Ramesh et al., 2011).

1.3.1-4 Wine and Brewery Industry

In wine production, enzymes such as pectinases, glucanases, and hemicellulases play

an important role by improving colour extraction, skin maceration, must clarification,

filtration, and finally the wine quality and stability. β-Glucosidases can improve the aroma of

wines by modifying glycosylated precursors. Macerating enzymes also improve pressability,

settling, and juice yields of grapes used for wine fermentation. The main benefits of using

these enzymes during wine making include better maceration, improved colour extraction,

easy clarification, easy filtration, improved wine quality, and improved stability.

Beer brewing is based on the action of enzymes activated during malting and

fermentation. Malting of barley depends on seed germination, which initiates the biosynthesis

and activation of α- and β-amylases, carboxypeptidase, and β-glucanase that hydrolyze the

seed reserves. In an earlier study, endoglucanase II and exoglucanase II of the Trichoderma

cellulase system were responsible for a maximum reduction in the degree of polymerization

and wort viscosity.

1.3.1-5 Food Processing Industry

Cellulases have a wide range of potential applications in food biotechnology as well.

The production of fruit and vegetable juices requires improved methods for extraction,

clarification, and stabilization. Cellulases also have an important application as a part of

macerating enzymes complex (cellulases, xylanases, and pectinases) used for extraction and

clarification of fruit and vegetable juices to increase the yield of juices. The use of macerating

enzymes increases both yield and process performance without additional capital investment.

The macerating enzymes are used to improve cloud stability and texture and decrease

viscosity of the nectars and purees from tropical fruits such as mango, peach, papaya, plum,

apricot, and pear. Texture, flavor, and aroma properties of fruits and vegetables can be

improved by reducing excessive bitterness of citrus fruits by infusion of enzymes such as

pectinases and β-glucosidases. Enzyme mixtures containing pectinases, cellulases, and

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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 β-glucosidases has also shown to alter the texture, flavor, and other sensory

properties such as aroma and volatile characteristics of fruits and vegetables.

1.3.1-6 Animal Feed Industry

Applications of cellulases and hemicellulases in the feed industry have received

considerable attention because of their potential to improve feed value and performance of

animals. Pretreatment of agricultural silage and grain feed by cellulases or xylanases can

improve its nutritional value. The enzymes can also eliminate antinutritional factors present in

the feed grains, degrade certain feed constituents to improve the nutritional value, and provide

supplementary digestive enzymes such as proteases, amylases, and glucanases. For instance,

the dietary fiber consists of non-starch polysaccharides such as arabinoxylans, cellulose, and

many other plant components including resistant dextrins, inulin, lignin, waxes, chitins,

pectins, β-glucan, and oligosaccharides, which can act as anti-nutritional factor for several

animals such as swine In this case, the cellulases effectively hydrolyse the anti-nutritional

factor, cellulose, in the feed materials into easily absorbent ingredient thus improve animal

health and performance.

1.3.1-7 Agricultural Industries

Cellulases produced from fungi are capable of degrading the cell wall of plant

pathogens in controlling the plant disease. Fungal β-glucanases are capable of controlling

diseases by degrading cell walls of plant pathogens. Cellulolytic fungi including Trichoderma

species is used to enhance seed germination, rapid plant growth and flowering, improved root

system and increased crop yields. These fungi have both direct (probably through growth-

promoting diffusible factor) and indirect (by controlling the plant disease and pathogens)

effects on plants. Moreover, the exoglucanase promoters of Trichoderma are used for the

expression of the different proteins, enzymes, and antibodies in large amount. The

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exoglucanase promoters of Trichoderma have been used for the expression of chymosin and

other proteins such as glucoamylase, lignin peroxidase, and laccase.

1.3.1-8 Detergent Industry

Cellulase preparation capable of modifying cellulose fibrils can improve colour

brightness, feel, and dirt removal from the cotton blend garments. The industrial application

of alkaline cellulases as a potential detergent additive is being actively pursued with a view to

selectively contact the cellulose within the interior of fibers and remove soil in the interfibril

spaces in the presence of the more conventional detergent ingredients. Nowadays, liquid

laundry detergent containing anionic or non-ionic surfactant, citric acid or a water-soluble salt,

protease, cellulose, and a mixture of propanediol and boric acid or its derivative has been used

to improve the stability of cellulases. The cellulases are applied to remove the rough

protuberances for a smoother, glossier, and brighter-coloured fabric.

1.3.1-9 Waste Management

The wastes generated from forests, agricultural fields, and agro-industries contain a

large amount of unutilized or underutilized cellulose, causing environmental pollution.

Nowadays, these so-called wastes are judiciously utilized to produce valuable products such

as enzymes, sugars, biofuels, chemicals, cheap energy sources for fermentation, improved

animal feeds, and human nutrients.

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1.3-2 Economic Analysis

1.3.2-1 Economic Analysis of POME

Palm oil industry plays an important role in Malaysian economy by contributing a

significant portion of the export earnings and acts as a prime driver of the global vegetable oil

business. In May 2011, the palm oil and palm oil-based products remained as the second

largest export with a total combined value of about MYR 7 billion, equivalent to more than 12%

of the total exports (Shah Samiur Rashid et al., 2009). Malaysian government has strategically

made significant efforts to expand this sector by facilitating various means including

allocation of more areas for palm-oil cultivation. However, with the expansion of this sector,

the production of non–toxic waste water product of palm oil known as palm oil mill effluent

(POME) is also to increase. According to the statistics provided by Malaysian Palm Oil Board

(MPOB), the quantity of POME produced is about 55–60 % for every ton of the extraction

process (Shah Samiur Rashid et al., 2009). This waste product is currently used as live

feedstock and organic fertilizer.

Prospect of POME as fertilizer in the form a bioconversion product has been widely

acknowledged until recently research studies in lab and pilot-scale have indicated that POME

can be a valuable raw material in the production of cellulase enzyme and bio-ethanol. Apart

from the extensive use in textile industry, cellulase enzymes are used to convert non-food

biomasses to fermentable sugar, and ultimately to sustainable products including bio-fuel and

bio-based performance ingredients in household goods such as laundry detergents and

shampoos. With the increasing population and expanding consumption of textile products,

detergent and shampoos, demand for cellulase enzyme is expected to be on the rise.

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 prospective

alternative raw material for the production of cellulase enzyme leaving an attractive

opportunity for potential investment.

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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)

1.3.2-2 Economic Analysis of Cellulases

Results from an economic analysis indicated that the unit costs for cellulase enzyme

production were $40.36 per kilogram ($/kg) for the submerged fermentation (SmF) methods,

while the corresponding market price was over $90.00/kg (Demain et al., 2005). A sensitivity

analysis conducted using Monte Carlo simulation suggests that the unit cost of production

using the solid state fermentation (SSC) method is lower than the unit cost of production

using SmF with a certainty of 99.6% (9,959 out of 10,000 cases) (Demain et al., 2005).

Although there are potential advantages of the SSC method, there are technical problems

limiting its large‐scale implementation. For instance, heat and mass transfer is more difficult

in SSC than in SmF because of limited diffusion through the solid substrate. If left

uncontrolled, heat accumulation and decline in available oxygen could result in the cessation

of mesophilic aerobic microbial activity and the consequential cessation of enzyme

production. Hence, SmF method is being chosen because it is the cultivation of

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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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2.1 Type of Product – Cellulase

Cellulase is an enzyme which is used to hydrolyze cellulose. They are produced by

fungi, bacteria, protozoans, plants, and animals. Cellulase enzymes is important in

bioconversion of the most abundant cellulosic wastes into the simplest carbohydrate monomer,

glucose.

Cellulases are the enzymes that hydrolyze β-1,4 linkages in cellulose chains. They are

produced by fungi, bacteria, protozoans, plants, and animals. Cellulose is a linear

polysaccharide of glucose residues connected by β-1,4 linkages. It is not cross-linked. Native

crystalline cellulose is insoluble and occurs as fibers of densely packed, hydrogen bonded,

anhydroglucose chains of 15 to 10,000 glucose units. Its density and complexity make it very

resistant to hydrolysis without preliminary chemical or mechanical degradation or swelling.

Cellulose is usually associated with other polysaccharides such as xylan or lignin. It is the

skeletal basis of plant cell walls. Cellulose is the most abundant organic source of food, fuel

and chemicals. However, its usefulness is dependent upon its hydrolysis to glucose. Acid and

high temperature degradation are unsatisfactory in that the resulting sugars are decomposed;

enzymatic degradation (cellulase) is the most effective means of degrading cellulose into

useful components. Although cellulases are distributed throughout the biosphere, they are

most prevalent in fungal and microbial sources.

Figure 6: Enzymatic Reaction of Cellulase

There are 3 major types of cellulose enzymes which are cellobiohydrolase (CBH),

Endo-β-1,4-glucanase (EG) and β-glucosidase (BG). Enzymes within these classifications can

be separated into individual components such as microbial cellulase compositions may consist

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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

2.2 Biological System

The basal medium for the growth of T. reesei and production of cellulase is as follows

(g/l): (NH4)2SO4: 1.4, KH2PO4: 2.0, Urea: 0.3, CaCl2: 0.3, MgSO4.7H2O: 0.3 and (mg/l):

FeSO4.7H2O: 5.0, MnSO4.H2O: 1.4, CoCl2: 2.0 (Shah Samiur Rashid et al., 2009). In addition,

microcrystalline cellulose (1%), Difco Peptone (0.1%) and Tween 80 (Polyoxyethylene

sorbitan molooleate, 0.1%) were added to the medium to induce cellulase production (Shah

Samiur Rashid et al., 2009). pH was controlled using 2N HCl and 2N NaOH. The medium

was autoclaved for 30 min and seeded with a suspension of T. reesei spores, to a final

concentration of 2 x 105 spores/ml (Shah Samiur Rashid et al., 2009). The submerged culture

was run for 6 days at 28oC and at pH 3.5 in bioreactor, R-100 (Shah Samiur Rashid et al.,

2009).

The model for batch cellulase enzyme production by T. reesei from cellulose substrate

from POME has four key concepts included:

(i) Existence of primary and secondary mycelia

(ii) Cellulase production by secondary mycelia only

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(iii) The adsorption of cellulase (catalyst) on the particulate cellulose (substrate)

(iv) The decline of cellulose reactivity with extent of conversion.

Cellulose which is substrate from cellulase production is transport from medium

through cell membrane to cytoplasm of T. reesei to produce intracellular cellulose.

Meanwhile in the cytoplasm, the intracellular cellulose will undergo repression and then

transcription and translation process will take place. Thus, the cell-bound cellulase is

produced. Cell-bound cellulase will be released from cytoplasm to cell membrane and then

to the medium and this time it is called extracellular cellulase.

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.

Figure 8: Cellulase Production and Specific Rates (Gaden, 1955)

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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 1: Choose separation processes based on different physical, chemical or

biochemical properties.

In this rule, physical, chemical or biochemical properties of effluent are determined to

decide the equipment that used in the process. The first step of downstream processing is

filtration. This equipment is used in order to separate the product from the broth solution. The

physical properties that influenced the choice of the separation processes are temperature, pH,

and shear sensitivity of the product.

Rule 2: Separate the most plentiful impurities first.

For the design of the downstream processes of the cellulase enzyme production, the

most abundance impurities are biomass. Biomass removal is usually the first step of

downstream processing of extracellular product. This is the reason for the design processes

start with the filtration.

The first step of downstream processing is filtration which is removed the easiest-to-

remove impurities first. This step is accomplished by using rotary vacuum filtration, F-100.

Rotary vacuum filtration with precoat, is for removal of excess or residual T. reesei fungi.

Rotary vacuum filters can operate continuously for long periods of time. In addition, the

filtrate flux in these units is usually higher than 200 L/m2-h and may reach 1,000 L/ m

2-h.

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 less-

dense product is flow to the next steps for further purification.

Rule 4: Use a high-resolution step as soon as possible.

Based on this rule, diafilter, D-100 and ion exchange chromatography, I-100 is used

as the final unit operation to get the pure product which is cellulase enzyme.

The mixture which contains cellulase is filtered using a diafilter that uses

ultrafiltration membranes to completely remove, replace, or lower the concentration of salts or

solvents from the solution. The process selectively utilizes permeable membrane filters to

separate the components of solutions and suspensions based on their molecular size by 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.

Ion exchange chromatography is then 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.

Rule 5: Do the most hardous step last.

The fifth rule implied that the hardest and most expensive step must be the last one.

Hence, the purification or ion exchange chromatography process carried out in the last steps

since there is no polishing process due to product is in liquid form.

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3.2 Applications of Cellulase in Various Industries

Industries Applications

Agriculture Plant pathogen and disease control

Generation of plant and fungal protoplasts

Enhanced seed germination and improved root system

Enhanced plant growth and flowering

Improved soil quality

Reduced dependence on mineral fertilizers

Bioconversion Conversion of cellulosic materials to ethanol, other

solvents, organic acids and single cell protein, and lipids

Production of energy-rich animal feed

Improved nutritional quality of animal feed

Improved ruminant performance

Improved feed digestion and absorption

Preservation of high quality fodder

Detergents Cellulase-based detergents

Superior cleaning action without damaging fibers

Improved color brightness and dirt removal

Remove of rough protuberances in cotton fabrics

Anti redeposition of ink particles

Fermentation Improved malting and mashing

Improved pressing and color extraction of grapes and

aroma of wines

Improved primary fermentation and quality of beer

Improved viscosity and filterability of wort

Improved must clarification in wine production

Improved filtration rate and wine stability

Food Release of the antioxidants from fruit and vegetable

pomace

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Improvement of yields in starch and protein extraction

Improved maceration, pressing, and color extraction of

fruits and vegetables

Clarification of fruit juices

Improved texture and quality of bakery products

Improved viscosity fruit purees

Improved texture, flavor, aroma, and volatile properties of

fruits and vegetables

Controlled bitterness of citrus fruits

Pulp and Paper Coadditive in pulp bleaching

Biomechanical pulping

Improved draining

Enzymatic deinking; reduced energy requirement

Improved fiber brightness, strength properties, and pulp

freeness and cleanliness

Improved drainage in paper mills

Production of biodegradable cardboard, paper towels, and

sanitary paper

Textile Biostoning of jeans

Biopolishing of textile fibers

Improved fabrics quality

Improved stability of cellulosic fabrics

Removal of excess dye from fabrics

Restoration of colour brightness

Others Improved carotenoids extraction

Improved oxidation and colour stability of carotenoids

Improved olive oil extraction

Improved malaxation of olive paste

Improved quality of olive oil

Reduced risk of biomass waste

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3.3 RIPP scheme for the Desired Product.

The block and flow diagram of downstream processing of cellulase production are as below:

1) Recovery stage

2) Isolation stage

3) Purification stage

4) Polishing stage

Since the product is in the form of liquid, polishing steps are not required.

Table 5: RIPP Scheme and typical unit operations categorized.

Stage Objective Typical unit operations

Recovery or separation

of insoluble biomass

Remove or collect

cells and cell

debris

Reduce volume

Filtration, centrifugation

Isolation of cellulase Remove materials

having properties

widely different

from desired

product - Cellulase

Reduce volume

Precipitation, diafilter

Purification of cellulase Remove remaining

impurities such as

cations and anions

Ion exchange

chromatography

30

The scheme of downstream process can be illustrated base on the block flow diagram:

Bioreactor

Biomass Removal

-Rotary vacuum filtration

-Centrifugation

Concentration

-Precipitation

Final Purification

-Diafiltration

-Ion exchange

chromatography

Extracellular Product

Recovery Stage

Isolation Stage

Purification Stage

31

Process Control

and Dynamics

32

4.1 Parameters to be Controlled in each Unit Operation Unit and their Control

Objective

i. Unit operation : Sterilisation Tank, T-100

Parameter : Level of POME solution in the tank.

Control objective : To control the level of POME in T-100 at 5 m by manipulating

the inlet flow rate of POME, via control valve.

Controlled variable : The level of POME in T-100

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 : Fermenter, R-100

Parameters : Level of medium in R-100, temperature in R-100, pH of the

medium in R-100

First control objectives: To control the level of medium in R-100 at 7.5 m by

manipulating the outlet flow rate of the solution from T-100,

via control valve.

Controlled variable : The level of medium in R-100

Manipulate variable : The outlet flow rate of POME in T-100

Disturbance variables: The inlet flow rate of cold water, inoculum and nutrient

Second control objective: To control the temperature in R-100 at 28oC by

manipulating the inlet flow rate of the cold water, via

control valve.

Controlled variable : The temperature of the R-100

Manipulate variable : The inlet flow rate of the cold water

Disturbance variables : The outlet flow rate of POME from T-100, inlet flow rate of

inoculum and nutrients

Third control objective: To control the pH of the medium in R-100 at pH 3.5 by

manipulating the inlet flowrate of the hydrochloric acid

(HCl) and sodium hydroxide (NaOH), via control valve.

33

Controlled variable : The pH of the medium in R-100

Manipulate variable : The inlet flowrate of 2N NaOH and 2N HCl

Disturbance variable : The outlet flow rate of POME from T-100, inlet flow rate of

inoculum and nutrients

iii. Unit operation: Mixer, M-100

Parameter : Level of solution in M-100

Control objective : To control the level of the solution in M-100 at 6 m by

manipulating the outlet flow rate of the solution from rotary

vacuum filter, F-100, via control valve.

Controlled variable : The level of solution in M-100

Manipulate variable : The outlet flow rate of the solution from F-100

Disturbance variable : The inlet flow rate of ammonium sulphate, (NH4)2S04

iv. Unit operation: C-100 Centrifuge

Parameter : Level of solution in C-100

Control objective : To control the level of solution in C-100 at 6 m by manipulating

the outlet flow rate of the solution from M-100, via control

valve.

Controlled variable : The level of solution in C-100

Manipulate variable : The outlet flow rate of the solution from M-100

Disturbance variable : The outlet flow rate of supernatant into drain

v. Unit operation: Mixer, M-101

Parameter : Level of solution in M-101

Control objective : To control the level of the solution in M-101 at 7 m by

manipulating the outlet flow rate of solution from C-100 via

control valve.

Controlled variable : The level of the solution in M-101

Manipulate variable : The outlet flow rate of the solution from C-100

Disturbance variable : The inlet flow rate of citrate buffer

34

vi. Unit operation: I-100 Ion Exchange Chromatography

Parameter : Level of solution in ion exchange chromatography, I-100

Control objective: To control the level of solution in I-100 at 4 m by manipulating the

outlet flow rate of solution from diafilter, D-100, via control valve.

Controlled variable : The level of solution in the I-100

Manipulate variable : The outlet flowrate of solution from D-100

Disturbance variable : The outlet flowrate of solution from I-100

vi. Unit operation: TK-100 Storage Tank

Parameter : Level of product in the Storage Tank, TK-100

Control objective : To control the level of the product in TK-100 at 4.5 m by

manipulating the outlet flow rate of the solution from I-100,

via control valve.

Controlled variable : The level of solution in the TK-100

Manipulate variable : The outlet flow rate of the solution from I-100

Disturbance variable : The outlet flow rate of the solution from TK-100

35

Simulation for

Bioprocess

Engineering

36

5.1 0bjective of Case Design

i. To study the flow production process of the cellulose enzyme from T. reesei sp.

ii. To identify the throughput analysis of the cellulose enzyme production.

iii. To identify the economic analysis of the production process.

iv. To discuss the debottlenecking to increase the yield production of cellulose enzyme.

v. To understand the using of SuperPro software in designing the project.

37

References

38

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Gaden, E.L. (1955) Fermentation kinetics and productivity. Chem. Ind., 154–159.

Man Kee Lam & Keat Teong Lee. (2011). Renewable and sustainable bioenergies production

from palm oil mill effluent (POME). Biotechnology Advances 29, 124–141

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

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