2.0 bioreactor

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Design of bioreactor

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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 nutr