Bioreactor

System

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 enzyme is important in
bioconversion of the most abundant cellulosic wastes into the simplest carbohydrate
monomer, glucose.
Cellulases are the enzymes that hydrolyze -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 -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 2.1: Enzymatic Reaction of Cellulase

There are 3 major types of cellulose enzymes which are cellobiohydrolase (CBH),
Endo--1,4-glucanase (EG) and -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.

Figure 2.2: Cellulase from T. reesei

The cellulolytic system of T. reesei can be divided into three major enzyme classes:
(i) exoglucanases - in the case of T. reesei cellobiohydrolases (CBHs) which liberate the Dglucose dimer cellobiose consecutively from the ends of the cellulose chain (ii)
endoglucanases (EGs) randomly cut within the cellulose chain and (iii) -glucosidases
release D-glucose from the soluble oligomeric breakdown products, thereby preventing
cellobiose inhibition of the other enzymes.
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
Cellulase enzymes are used in food, brewery and wine, animal feed, textile and
laundry, pulp and paper industries, as well as in agriculture and for research purposes. Indeed,
the demand for these enzymes is growing more rapidly than ever before, and this demand has
become the driving force for research on cellulases and related enzymes.

Figure 2.3: Degradation of cellulose by cellulases and non-enzymatic proteins of T. reesei.

Multiple members of the different types of cellulase enzymes - CBHs, EGs and glucosidases (BGLs) - degrade the crystalline cellulose synergistically to glucose.

Figure 2.4: Schematic representations depicting the general morphology of T. reesei (top)
and proposed pathways of protein synthesis and secretion (enlarged hyphal tip, below).
Proteins are synthesized in the endoplasmic reticulum (ER) then travel in secretory vesicles
(sv) to the Golgi for further post-translational modification. Secretory vesicles then carry the
modified proteins to the hyphal tip for apical secretion, or possibly to the septa in an
alternative secretory pathway.

2.2 Biological System

In the production of cellulose from T. reesei, the submerged culture was run for 6 days
and the optimum condition for fermentation are temperature of 28oC and pH 3.5 (Shah
Samiur Rashid et al., 2009). POME was used as sole source of carbon and nitrogen and the
fermentation. The carbon course is important in synthesis of cell material, maintenance
function such as turnover of cell material, osmotic work to maintain concentration gradients
and cell motility. (NH4)2SO4 is added as nitrogen source for T. reesei fermentation. In reactor
R-100, HCl and NaOH were added to control the pH of the medium. Besides, KH 2PO4, Urea,
CaCl2, MgSO4.7H2O, FeSO4.7H2O, MnSO4.H2O and CoCl2 are used to promote cell growth. In
addition, microcrystalline cellulose, Difco Peptone and Tween 80 (Polyoxyethylene sorbitan
molooleate) were added to the medium to induce cellulase production (Shah Samiur Rashid
et al., 2009).
T. reesei produce large amounts of extracellular cellulolytic enzymes. Based in Figure
2.5, 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 by active
transport and this time it is called extracellular cellulase.

Figure 2.5: 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.

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
(iii)

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

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

Figure 2.6: Primary mycelium of T. reesei

Figure 2.7: Secondary mycelium of T. reesei

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 2.8: Cellulase Production and Specific Rates (Gaden, 1955)

2.3 Media Formulation for Growth and Product Formation

In the bioreactor, nutrients are required for the growth T. reesei of and cellulase
formation. Hence, basal or complex media is used because it is suitable for the growth of
most heterotrophic organisms such as T. reesei and complex media are rich in nutrients.
For 1 000 L media,
Nutrient
Macroelemen

Component /

Function or Constituent of

Concentration

Amount

(g/L)
10

(g)
10,000

1.4

1400

2.0

2000

0.3

300

CaCl2

polymer
Ca: Enzyme cofactor

0.3

300

MgSO4.7H2O

Mg: Ribosomes, enzyme

0.3

300

0.0050

0.0014

1.4

Element
Cellulose

C: Carbon (C) and energy

(NH4)2SO4

source
N: Protein, nucleic acids, cell

wall polymer
S: Sulphur amino acids,
KH2PO4

biotin, coenzyme A
K: RNA, enzyme cofactor,
principle cation
P: Nucleic acids,
phospholipid, cell wall

Microelement

Urea

polymer
N: Protein, nucleic acids, cell
wall polymer
S: Sulphur amino acids,
biotin, coenzyme A
P: Nucleic acids,
phospholipid, cell wall

cofactor
S: Sulphur amino acids,
FeSO4.7H2O

biotin, coenzyme A
Fe: Cytochromes, enzyme
cofactor
S: Sulphur amino acids,

MnSO4.H2O

biotin, coenzyme A
Mn: Enzyme cofactor
S: Sulphur amino acids,

CoCl2
Microcrystalli
ne cellulose
Difco Peptone
Tween 80

biotin, coenzyme A
Enzyme cofactor

0.0020

0.2

Induce cellulase production

0.0010

0.1

Induce cellulase production

Induce cellulase production

0.0001
0.0001

0.1
0.1

(Polyoxyethyl
ene sorbitan
molooleate
Then, each component is added accordingly and water is added up to 1000L.

2.4 Propose the Most Suitable Bioreactor for the Desired Product Formation
In production of cellulase from POME by using T. reesei, stirred tank bioreactor is
being chosen because it is the most common reactor used for biological reactions. Single
stage bioreactor with batch mode of operation is being chosen because it only involves only
one stage of fermentation that can reduce the chances of contamination. Besides, single-use
bioreactors provide maximum savings on the time spent to prepare the bioreactor for the next
batch. In a batch reactor, the reagents are added together and allowed to react for a given

amount of time. The compositions change with time, but there is no flow through the process.
Additional reagents may be added as the reaction proceeds, and changes in temperature may
also be made. Products are removed from the reactor after the reaction has proceeded to
completion. The batch mode of fermentation also allows the cleaning process after every bath
of fermentation. In addition, stainless steel bioreactor is being used because resist it stains and
corrosion, heat damage and chemical damage.
Stirred tank bioreactor is most suitable to be used due to the features below:
i.

Agitation (Impeller)
Mixing is conducted by an impeller mounted on a shaft driven by a motor. The
impeller is designed to homogeneously mix cells, gases, and nutrients throughout the
culture vessel. The mixing action evenly distributes oxygen and nutrients to cells for
healthy growth, keeps them from settling to the bottom of the vessel and helps to
maintain a uniform culture temperature.
Rushton turbine is being chosen. The impeller has blades which are parallel to
the vertical axis of the stirrer shaft and tank. The flow pattern of this type turbine is
radial flow. The medium is driven radially from the impeller against the walls of the
tank where it divides into two streams, one flowing up to the top of the tank and
another one flowing down to bottom. These streams eventually reach the central axis
of the tank and are draw back to the impeller. Radial flow impeller also set up circular
flow which must be reduced by baffles.

ii.

Foam Reduction
Mechanical foam breaker is used to remove foam that build up in the head
space of bioreactor and evenly cause the contamination of culture broth.

iii.

Baffles
Induce turbulence and prevent the contents from swirling and creating a vortex.

iv.

Aeration System
T. reesei is an aerobe and require oxygen for growth. Oxygen gas is supply by
sparger in order to maintain proper conditions for cell metabolism. However, it will tend
to produce spore when there is depletion of nutrient. Hence, bubbles form from sparger
will increase the oxygen transfer rate in the vessel and improved concentration of
dissolved gas which further uptake by microbes. Bubbling of air through the sparger not
only provide the adequate oxygen to the growing cells but also helps in the mixing of the

reactor contents thereby reducing the power consumed to achieve a particular level of
(mixing) homogeneity in the culture.
v.

Temperature control
Both microbial activity and agitation will generate heat. Hence, the temperature
of culture medium must be controlled because high temperature will cause the denatured
of biological product and low temperature will cause the inactivation of the
microorganism.
The jacket provides the annular area for circulation of constant temperature water
which keeps the temperature of the bioreactor at a constant value. The desired
temperature of the circulating water is maintained in a separate chilled water circulator
which has the provision for the maintenance of low or high temperature in a reservoir.
The contact area of jacket provides adequate heat transfer area wherein desired
temperature water is constantly circulated to maintain a particular temperature in the
bioreactor.
In case of larger bioreactor beyond a certain size, excess heat is generated, and
the bioreactor surface becomes inadequate for heat removal. Hence, internal coils are
used to circulate cold water through them for removing the excess heat.

Besides, the bioreactor has few control systems that is suitable for biological
process:

i. Temperature measurement and control

The measurement of the temperature of the bioreactor is done by a
thermocouple which essentially sends the signal to the temperature controller. The
set point is entered in the controller which then compares the set point with the
measured value and depending on the error, either the heating or cooling finger of
the bioreactor is activated to slowly decrease the error and essentially bring the
measured temperature value close to the set point.

ii. pH measurement and control

The measurement of pH in the bioreactor is done by the autoclavable pH
probe. The measured signal is compared with the set point in the controller unit
which then activates the acid or alkali to bring the measured value close to the set
point. However before the pH probe is used, it needs to be calibrated with two
buffers usually in the pH range which is to be used in the bioreactor cultivation
experiment.

iii. Dissolved oxygen controller

The dissolved oxygen in the bioreactor broth is measured by a dissolved
oxygen probe which basically generates some potential corresponding to the
dissolved oxygen diffused in the probe. Before the measurement can be done by the
probe, it is to be calibrated for its zero and hundred percent values. After calibration,
the instrument is ready for the measurement of the dissolved oxygen in the broth. In
the event of low oxygen in the fermentation broth, more oxygen can be purged in the
bioreactor or stirrer speed can be increased to enhance the beating of the bubbles
which essentially enhances the oxygen transfer area and net availability of oxygen in
the fermentation broth.

iv. Foam control

The fermentation broth contains a number of organic compounds and the
broth is vigorously agitated to keep the cells in suspension and ensure efficient
nutrient transfer from the dissolved nutrients and oxygen. This invariably gives rise
to lot of foam. It is essential that control of the foam is done as soon as possible.

Figure 2.9: Stirred Tank Bioreactor

Figure 2.9: Rushton Turbine (6-flat-blade disc turbine)

Figure 2.10: Mixing Pattern of Rushton Turbine (6-flat-blade disc turbine)

2.5 Design the Bioreactor

Based on Figure 2.9, stirred tank bioreactor is cylindrical in shape with the base of the
tank is rounded at the edges. The rounded edge eliminates the sharp corners and pockets into
which fluid currents may not penetrate and discourages formation of stagnant region.

Volume of tank, Vt
80% of the total tank volume is filled with liquid, 20% is for gas space.
Working volume of liquid, Vw = 1000 L = 1 m3
Volume of tank , V t =

Vw 1
= =1.25m3
0.8 0.8

Hence, the volume of tank is 1.25 m3.

Diameter of tank, Dt
The bioreactor is assumed to be cylindrical in shape.
The ratio of height of tank (Ht) to tank diameter (Dt) is 2 : 1.
H t : Dt =2:1
Ht 2
=
Dt 1
H t =2 Dt

2
V t = Dt H t
4

V t = Dt2 2 D t
4

V t = Dt3
2

1.25 m3 = Dt3
2

D t =0.80 m
D t =0.93 m

Hence, the diameter of tank is 0.93 m.

Height of tank, Ht
H t =2 Dt
= 2 0.93 m
= 1.86 m
Hence, the height of tank is 1.86 m.
Height of liquid in tank, HL
H L =0.8 H t
= 0.8 1.86 m
= 1.49 m
Hence, the height of liquid in tank is 1.49 m.
Diameter of impeller, Di
Diameter of impeller is set to one third of tank diameter.
Di=

Dt
3

0.93 m
3

= 0.47 m
Hence, the diameter of impeller is 0.47 m.
Space between impeller, Di
Since Ht = 2Dt, additional set of impeller is added.
'

Di < Di <2 Di

'

0.47 m< Di < 0.94 m

D i' =0.5 m
Hence, the space between impeller is 0.5 m.
Space between sparger and impeller, Ds
Ds=

Di 0.47
=
=0.24 m
2
2

Hence, the space between sparger and impeller is 0.24 m.

Width of baffle, DB
Width of baffle is

DB =

Dt
10

Dt
10

0.93 m
10

0.093 m
9.3 cm

Hence, the width of baffle is 9.3 cm.

In conclusion, the dimension of stirred tank bioreactor is as below:

Figure 2.11: Top View of Stirred Tank Bioreactor with Dimension

Ds =
0.24m

Figure 2.12: Side View of Stirred Tank Bioreactor with Dimension

2.6 Discussion
In this project, T. reesei is fungi that used to produce cellulase from POME. The
cellulase produced is an enzyme that is used to hydrolyze -1,4 linkages in cellulose chains.
In other words, it is used to break down cellulase into glucose monomer. This hydrolysis is
facilitate by the presence of three enzyme classes in the T. reesei system which are: (i)
exoglucanases - in the case of T. reesei cellobiohydrolases (CBHs) which liberate the Dglucose dimer cellobiose consecutively from the ends of the cellulose chain (ii)
endoglucanases (EGs) randomly cut within the cellulose chain and (iii) -glucosidases
release D-glucose from the soluble oligomeric breakdown products, thereby preventing
cellobiose inhibition of the other enzymes.
However, the cellulase produced in an extracellular product where proteins are
synthesized in the endoplasmic reticulum then travel in secretory vesicles to the golgi for
further post-translational modification. Secretory vesicles then carry the modified proteins to
the hyphal tip for apical secretion, or possibly to the septa in an alternative secretory
pathway.
The basal medium for the growth of T. reesei and production of cellulase is as follows
(g/l): cellulase: 10g/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
o

NaOH. The submerged culture was run for 6 days at 28 C and at pH 3.5 in bioreactor (Shah
Samiur Rashid et al., 2009).
The bioreactor that being chosen in this project is stirred tank bioreactor due its few
features such as the presence of the agitation and aeration system and baffle. The agitation,
aeration system and baffle will improve mixing process and allowed the oxygen and nutrient
to be thoroughly distributed to cells. Besides, stirred tank bioreactor has few control system
such as temperature measurement and control, pH measurement and control, dissolved
oxygen control and foam control. All this control system is suitable to maintain the optimum
condition for biological reaction such as fermentation.

From the design of bioreactor, cylindrical and stainless steel tank bioreactor is used.
The impeller being chosen is Rushton turbine because it is commonly used in fermentations
of cell lines that not considered shear sensitive including fungi. This type of 6-flat blade disc
turbine will produce radial mixing pattern. The working volume of liquid in bioreactor tank is
1000L. Since 80% of the total tank volume is filled with liquid and 20% is for gas space, the
volume of bioreactor tank is determined to be 1.25 m 3. Since the ratio of tank height to tank
diameter is 2 to 1, the diameter of tank is 0.93 m; the height of tank is 1.86 m; the height of
liquid in tank is 1.49 m. The diameter of impeller is 0.47 m which is one-third to the diameter
on tank. Furthermore, 2 set of impeller is used and the space between impeller is 0.5 m. The
width of baffle used is 9.3 cm which is one-tenth to the diameter of tank.
Since the raw material, POME is aerated non-Newtonian liquid, it is not required to
determine the Reynold numbers, power input and oxygen transfer rate. Liquid into which gas
is sparged have reduced power requirements. The gas bubbles decrease the density of the
liquid and affect the hydrodynamic behaviour of liquid around the impeller. Large gas-filled
cavities develop behind the stirrer blades in aerated liquids; these cavities reduce the
resistance to fluid flow and decrease the drag coefficient of impeller. Besides, the estimation
of non-Newtonian liquid is more difficult. The non-Newtonian liquidis so viscous that flow
of liquid is very low in the tank. It may be impossible with highly viscous liquid to achieve
fully-developed turbulence so that power number is always independent on Reynold number.
In addition, since the viscosity of non-Newtonian liquids varies with shear conditions, the
impeller Reynold number used to correlate power required must be re-defined.