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 fromT. 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 D-glucose 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, KH2PO4, 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. reeseiproduce 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 Component / ElementFunction or Constituent ofConcentration (g/L)Amount (g)

MacroelementCellulose C: Carbon (C) and energy source1010,000

(NH4)2SO4 N: Protein, nucleic acids, cell wall polymerS: Sulphur amino acids, biotin, coenzyme A1.41400

KH2PO4 K: RNA, enzyme cofactor, principle cationP: Nucleic acids, phospholipid, cell wall polymer2.02000

MicroelementUreaN: Protein, nucleic acids, cell wall polymerS: Sulphur amino acids, biotin, coenzyme AP: Nucleic acids, phospholipid, cell wall polymer0.3300

CaCl2Ca: Enzyme cofactor0.3300

MgSO4.7H2OMg: Ribosomes, enzyme cofactorS: Sulphur amino acids, biotin, coenzyme A0.3300

FeSO4.7H2OFe: Cytochromes, enzyme cofactorS: Sulphur amino acids, biotin, coenzyme A0.00505

MnSO4.H2OMn: Enzyme cofactorS: Sulphur amino acids, biotin, coenzyme A0.00141.4

CoCl2Enzyme cofactor0.00200.2

Microcrystalline celluloseInduce cellulase production0.00100.1

Difco PeptoneInduce cellulase production0.00010.1

Tween 80 (Polyoxyethylene sorbitan molooleateInduce cellulase production0.00010.1

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, singleuse 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 ReductionMechanical 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 controlBoth 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 controlThe 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 controlThe 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 controllerThe 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 controlThe 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, Vt80% of the total tank volume is filled with liquid, 20% is for gas space.Working volume of liquid, Vw = 1000 L = 1 m3

Hence, the volume of tank is 1.25 m3. Diameter of tank, DtThe bioreactor is assumed to be cylindrical in shape.The ratio of height of tank (Ht) to tank diameter (Dt) is 2 : 1.

Hence, the diameter of tank is 0.93 m.

Height of tank, Ht

= 2 0.93 m= 1.86 mHence, the height of tank is 1.86 m.

Height of liquid in tank, HL

= 0.8 1.86 m= 1.49 mHence, the height of liquid in tank is 1.49 m.

Diameter of impeller, DiDiameter of impeller is set to one third of tank diameter.

= 0.47 mHence, the diameter of impeller is 0.47 m.

Space between impeller, DiSince Ht = 2Dt, additional set of impeller is added.

Hence, the space between impeller is 0.5 m.

Space between sparger and impeller, Ds

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

Width of baffle, DB

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 D-glucose 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 NaOH. The submerged culture was run for 6 days at 28oC 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 m3. 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.