Components of a Stirred Tank Fermenter

A stirred tank fermenter (STR) is the most widely used bioreactor for both submerged fermentation and cell culture. It consists of a cylindrical vessel fitted with mechanical agitation and aeration systems that keep the microorganisms uniformly suspended and well supplied with nutrients and oxygen. The main components are described below.

1. Vessel (Body)

The vessel is normally a cylindrical tank made of stainless steel (grade 316 or 304) to withstand repeated steam sterilization (autoclaving at 121 °C) and to resist corrosion. The height-to-diameter (H/D) ratio is usually 2:1 to 3:1. The bottom is rounded (dish-shaped) to prevent dead zones and to help in draining the broth. A larger vessel is provided with a jacket or coils for temperature control. The top is closed with a flanged head plate carrying ports for the agitator shaft, inoculum, acid/base, antifoam, exhaust gas, and sensors.

2. Agitator (Impeller) and Shaft

The agitator provides mixing, mass transfer, and heat transfer. Common impeller types are:

• Rushton turbine (flat-bladed disc turbine) — used for high-shear, gas-dispersing duties in microbial fermentations.
• Marine propeller and pitched-blade turbine — used for low-viscosity, axial-flow mixing.
• Anchor, paddle, or helical ribbon — used for viscous broths and shear-sensitive cells.

The impeller is mounted on a central shaft driven by a motor through a gearbox. The shaft enters through a mechanical seal or stuffing box to prevent contamination. Baffles (usually 4, at 1/10 of the vessel diameter) are fixed to the wall to prevent vortex formation and improve mixing.

3. Aeration System (Sparger and Air Supply)

Sterile air is supplied through a sparger located below the impeller:

• Porous sparger — fine bubbles, high oxygen transfer, but prone to blockage.
• Orifice sparger — simple holes, moderate oxygen transfer.
• Nozzle sparger — used in large vessels.

Air is passed through a sterilizing filter (membrane or depth filter) before entering the vessel. The impeller breaks the air into fine bubbles, increasing the gas–liquid interfacial area for oxygen transfer. The exhaust gas leaves through a condenser and exhaust filter to prevent escape of microorganisms.

4. Temperature Control System

Temperature is maintained by circulating cooling or heating water through a jacket surrounding the vessel or through internal coils. A temperature sensor (RTD or thermocouple) linked to a controller regulates the flow. This removes the metabolic heat generated during fermentation.

5. pH and Foam Control

• pH control: A pH electrode monitors the broth; sterile acid or alkali is added through a peristaltic pump to maintain optimum pH.
• Foam control: Foaming is controlled by adding antifoam agents (e.g., silicone) through a pump, or by using a mechanical foam breaker at the top.

6. Sampling and Harvesting Ports

A sampling port with a steam-sterilizable valve allows aseptic withdrawal of broth samples for analysis. A bottom drain valve is used for harvesting the product at the end of fermentation.

7. Sensors and Control Panel

Sensors for temperature, pH, dissolved oxygen (DO), and foam are inserted through the head plate. Their signals go to a control panel that automatically regulates agitation speed, airflow rate, temperature, pH, and antifoam addition.

8. Inoculation Port

A dedicated inoculation port with a septum or steam valve allows introduction of the sterile inoculum without contaminating the system.

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Diagram

        (1) Motor + gearbox
               |
               | (2) Shaft
  (13) Inoculation   |      (7) Exhaust gas outlet (with filter)
       port ----+    |    +---- 
                |    |    |
      ----------+----+----+----------
     |  (9) pH probe  (10) DO probe  |
     |  (11) Temperature sensor      |
     |  (12) Acid/base/antifoam inlet|
     |                               |
     |   (4) Baffle           (4)    |  <- (8) Cooling jacket
     |      |                 |      |     (water in and out)
     |      |      |          |      |
     |      |   [=====]       |      |
     |      |   (3) Impeller  |      |
     |      |                 |      |
     |      |    o o o o      |      |
     |      |   (5) Sparger   |      |
      \                             /
       \___________________________/
                  |      |
   (6) Sterile air inlet   (15) Harvest valve
                  (14) Sampling port on the side wall

Labels: (1) Motor and gearbox, (2) Shaft, (3) Impeller, (4) Baffles, (5) Sparger, (6) Sterile air inlet, (7) Exhaust gas outlet, (8) Cooling jacket, (9) pH electrode, (10) DO probe, (11) Temperature sensor, (12) Antifoam/acid/base inlet, (13) Inoculation port, (14) Sampling port, (15) Harvest valve.

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Working (brief)

The vessel is sterilized, filled with sterile medium, and inoculated. The agitator and air supply are started; the impeller disperses air into fine bubbles and keeps cells suspended. Temperature, pH, DO, and foam are continuously monitored and controlled. After the fermentation period, the broth is harvested through the bottom valve.

Structures of Proteins

Proteins are linear polymers of amino acids joined by peptide bonds, but they fold into specific three-dimensional shapes to become functional. This organisation is described at four levels: primary, secondary, tertiary, and quaternary structure.

1. Primary Structure

The primary structure is the linear sequence of amino acids in a polypeptide chain, held together by peptide bonds (covalent bonds between the –COOH group of one amino acid and the –NH₂ group of the next).

• It is determined by the gene (DNA) sequence.
• It defines all higher levels of structure.
• Example: the sequence of 51 amino acids in insulin (A and B chains).

2. Secondary Structure

The secondary structure is the local, regular folding of the polypeptide chain, stabilised mainly by hydrogen bonds between the C=O of one peptide bond and the N–H of another.

Main types:

• α-Helix: The chain coils into a right-handed spiral, with hydrogen bonds formed between every fourth amino acid along the same chain. Example: α-keratin in hair and wool.
• β-Pleated sheet: The chain lies extended and adjacent segments line up side by side, linked by hydrogen bonds. The sheets may be parallel or antiparallel. Example: silk fibroin.
• Turns and loops: Short regions (e.g., β-turns) that connect helices and sheets, allowing the chain to reverse direction.

3. Tertiary Structure

The tertiary structure is the overall three-dimensional shape of a single polypeptide chain, produced by folding of the secondary structure elements.

It is stabilised by several bonds and interactions:

Two common overall shapes are globular (e.g., myoglobin, enzymes) and fibrous (e.g., collagen). The tertiary structure creates the active site of enzymes and the functional domains of proteins.

4. Quaternary Structure

The quaternary structure is the arrangement of two or more polypeptide chains (subunits) into a single functional protein. The subunits are held together by the same non-covalent forces as in tertiary structure, and sometimes by disulphide bonds.

• Homomeric: all subunits identical — e.g., lactate dehydrogenase (4 identical subunits).
• Heteromeric: different subunits — e.g., haemoglobin (2 α and 2 β chains) and insulin (A and B chains).

Summary

Thus, the four levels together determine the shape and function of a protein; loss of this native structure (denaturation) destroys its biological activity.

RNA (Ribonucleic acid) is a single-stranded polynucleotide made of ribonucleotides, each consisting of a nitrogenous base (adenine, guanine, cytosine, or uracil), the sugar ribose, and a phosphate group. It differs from DNA in having ribose instead of deoxyribose and uracil instead of thymine. RNA is the molecule that carries and translates genetic information in cells; its main types are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).

Transcription in Bacteria

Transcription is the process by which the genetic information in DNA is copied into a complementary RNA strand. In bacteria it is carried out by a single DNA-dependent RNA polymerase, which synthesizes RNA in the 5′ → 3′ direction using the template (antisense) strand of DNA. The process has three stages: initiation, elongation, and termination.

1. Initiation

• The core enzyme of RNA polymerase consists of four subunits: two α, one β, and one β′. For correct initiation it must bind the sigma (σ) factor to form the holoenzyme.
• The σ factor recognizes and binds the promoter region of the DNA, which lies upstream of the gene. Two important promoter sequences are the −10 sequence (Pribnow box, TATAAT) and the −35 sequence (TTGACA), counted from the transcription start site (+1).
• Binding of the holoenzyme to the promoter forms the closed complex. The DNA is then locally unwound over about 17 bp to form the open complex, exposing the template strand.
• The first ribonucleotide is placed opposite the start base; the σ factor is released after initiation, leaving the core enzyme to continue synthesis.

Elongation

• The core enzyme moves along the template strand in the 3′ → 5′ direction and adds ribonucleotides to the 3′ end of the growing RNA, so the RNA chain grows in the 5′ → 3′ direction.
• Base pairing is antiparallel and complementary: Each template base specifies its RNA partner: A pairs with U, T with A, G with C and C with G (uracil replaces thymine in RNA).
• A short DNA–RNA hybrid of about 8–9 bp is maintained at the growing point; behind the polymerase the DNA rewinds into its double helix. The enzyme has no proofreading like DNA polymerase, so the error rate is higher.
• The transcription bubble contains the enzyme, the DNA template, and the growing RNA chain.

Termination

Termination occurs at specific sequences and stops RNA synthesis, releasing the RNA transcript. Bacteria use two main mechanisms:

1. Rho-independent (intrinsic) termination: The newly made RNA contains a GC-rich palindromic sequence that folds back on itself to form a stem-loop (hairpin) structure, followed by a run of 6–8 uracil residues. The hairpin destabilizes the DNA–RNA hybrid, and the weak rU–dA base pairs at the run cause the polymerase to pause and dissociate, releasing the RNA.

2. Rho-dependent termination: Here a protein called the Rho factor binds the RNA at a rut (Rho utilization) site and moves along it using ATP. When it catches the paused RNA polymerase at the terminator, its helicase activity unwinds the DNA–RNA hybrid and releases the transcript.

Post-transcriptional features in bacteria

• The primary transcript is often polycistronic (one mRNA carries several genes) and is used directly, since bacteria lack a nucleus and transcription and translation are coupled.
• rRNA and tRNA are made as larger precursors and then processed by ribonucleases; mRNA generally needs no processing and, unlike eukaryotic mRNA, is not capped or spliced and has no poly(A) tail of the eukaryotic type.

In short, bacterial transcription is carried out by RNA polymerase holoenzyme, which binds the promoter via the σ factor, synthesizes RNA in the 5′ → 3′ direction using the template strand, and stops at Rho-independent or Rho-dependent terminators.

EMP Pathway (Embden–Meyerhof–Parnas Pathway) of Glucose Metabolism

The EMP pathway, commonly called glycolysis, is the universal cytoplasmic route for the breakdown of glucose. It occurs in the cytosol of almost all living cells, requires no oxygen (anaerobic), and converts one molecule of glucose into two molecules of pyruvate. It was worked out by Embden, Meyerhof and Parnas, hence the name.

Overall equation:

$\text{C}_6\text{H}_{12}\text{O}_6 + 2\text{NAD}^+ + 2\text{ADP} + 2\text{P}_i \rightarrow 2\text{CH}_3\text{COCOOH} + 2\text{NADH} + 2\text{H}^+ + 2\text{ATP} + 2\text{H}_2\text{O}$

The pathway has 10 enzymatic steps and is conveniently divided into two phases.

A. Preparatory (investment) phase — 5 steps, 2 ATP consumed

1. Phosphorylation of glucose: Glucose is phosphorylated at C-6 by hexokinase (glucokinase in liver) using one ATP → glucose-6-phosphate.
2. Isomerization: Glucose-6-phosphate is converted to fructose-6-phosphate by phosphoglucose isomerase.
3. Second phosphorylation: Fructose-6-phosphate is phosphorylated by phosphofructokinase-1 (PFK-1) using a second ATP → fructose-1,6-bisphosphate. This is the rate-limiting, committed step of glycolysis.
4. Cleavage: Aldolase splits fructose-1,6-bisphosphate into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P).
5. Isomerization: Triose phosphate isomerase converts DHAP into G-3-P, so that two molecules of G-3-P enter the next phase.

B. Pay-off (energy-yielding) phase — 5 steps, 4 ATP and 2 NADH produced

6. Oxidation and phosphorylation: Glyceraldehyde-3-phosphate dehydrogenase oxidizes G-3-P, reducing NAD⁺ to NADH and forming 1,3-bisphosphoglycerate (an acyl phosphate with high phosphoryl-transfer potential).
7. First substrate-level phosphorylation: Phosphoglycerate kinase transfers the phosphate of 1,3-bisphosphoglycerate to ADP → 3-phosphoglycerate + ATP.
8. Phosphate shift: Phosphoglycerate mutase moves the phosphate from C-3 to C-2 → 2-phosphoglycerate.
9. Dehydration: Enolase removes water, forming phosphoenolpyruvate (PEP), another high-energy phosphate compound.
10. Second substrate-level phosphorylation: Pyruvate kinase transfers the phosphate of PEP to ADP → pyruvate + ATP.

Energy account (per glucose):

Fate of the products: In the absence of oxygen, pyruvate is reduced to lactate (animals) or ethanol + CO₂ (yeast) by reoxidizing NADH, so that glycolysis can continue. In the presence of oxygen, pyruvate enters the mitochondrion and is oxidized to acetyl-CoA, while NADH is oxidized in the electron transport chain, yielding much more ATP.

Significance: Glycolysis is the only source of ATP in anaerobic organisms and in tissues such as RBCs (which lack mitochondria). It also supplies pyruvate for the Krebs cycle and provides intermediates for biosynthetic pathways.

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Role of ATP in Metabolism

ATP (adenosine triphosphate) is the universal energy currency of the cell. Its terminal phosphoanhydride bond has a high free energy of hydrolysis (≈ 7.3 kcal/mol), so ATP acts as the link between energy-releasing (catabolic) and energy-requiring (anabolic) reactions.

1. Energy donor in biosynthesis: ATP phosphorylates substrates and drives endergonic reactions — e.g., glucose → glucose-6-phosphate in glycolysis, and gluconeogenesis, fatty acid synthesis, and protein synthesis.
2. Energy source for mechanical work: Muscle contraction, ciliary and flagellar movement, and chromosome movement in cell division are powered by ATP hydrolysis.
3. Active transport: Na⁺–K⁺ ATPase, Ca²⁺ pumps, and proton pumps use ATP to move ions against concentration gradients.
4. Energy source for physiological processes: Nerve impulse conduction, bioluminescence, and secretion depend on ATP.
5. Phosphoryl-group transfer: ATP donates phosphate groups to proteins and metabolites (kinase reactions), regulating enzyme activity.
6. Formation of other nucleotides: ATP serves as a precursor for RNA and for coenzymes such as NAD⁺, FAD, and coenzyme A.
7. Coupling agent: ATP couples exergonic oxidation (in glycolysis, Krebs cycle, and oxidative phosphorylation) with endergonic cellular work, and its hydrolysis products (ADP, AMP, Pᵢ) act as allosteric regulators of metabolic pathways.

Thus ATP is the immediate, rechargeable energy carrier that keeps metabolism continuous and regulated.

Production of Pectinase Enzyme

Pectinase is a group of enzymes (polygalacturonase, pectin esterase, pectatelyase) that hydrolyse pectin, the polysaccharide that cements plant cells together. It is used in fruit juice clarification, wine making, textile retting, coffee/tea fermentation and animal feed. It is produced mainly by Aspergillus niger (also A. oryzae, Penicillium, Bacillus, Erwinia).

Steps of production

1. Selection of microorganism (strain) A high-yielding strain of Aspergillus niger is selected. Improved strains are obtained by mutation (UV, nitrosoguanidine) or recombinant DNA techniques. The strain must be non-toxic and should give high pectinase with low side activities.

2. Inoculum preparation The selected strain is grown on potato dextrose agar (PDA) slants at 28–30 °C for 5–7 days to obtain a heavy spore suspension. The spores are harvested in sterile distilled water containing 0.1% Tween-80 and used to inoculate the production medium.

3. Preparation of production medium A cheap medium is used, e.g. wheat bran, rice bran, citrus peel, apple pomace or sugar beet pulp (these are rich in pectin and act as both carbon source and inducer). Mineral salts such as (NH₄)₂SO₄, KH₂PO₄, MgSO₄, and trace elements are added. pH is adjusted to about 4.5–5.5. Pectin itself is the inducer of pectinase.

4. Fermentation Two methods are used:

• Solid-state fermentation (SSF): The moistened substrate (60–70% moisture) is spread in trays or drums and inoculated with spores. Incubated at 28–30 °C for 3–5 days. This is the most common industrial method — cheap, high yield, less downstream problem.
• Submerged fermentation (SmF): The organism is grown in a liquid medium in a stirred-tank fermenter at 28–30 °C, pH 4.5–5.0, with aeration and agitation for 2–4 days. Easier to control but costlier.

5. Extraction / harvesting After fermentation, the enzyme is extracted by adding water or buffer (0.1 M citrate buffer, pH 5) and pressing/filtering. The filtrate contains the crude enzyme. Cells and solid debris are removed by filtration or centrifugation.

6. Purification (downstream processing) The crude extract is concentrated by ultrafiltration or vacuum evaporation, then purified by salt precipitation (ammonium sulphate), dialysis, ion-exchange chromatography, gel filtration and finally lyophilization (freeze drying) to obtain a stable powder.

7. Quality control and formulation Activity is assayed (e.g. by measuring reducing sugar released from pectin using DNS reagent or by viscosity reduction). The product is standardized, stabilized with glycerol/sorbitol, packed and stored cool.

Flow chart

Strain selection (A. niger)
        ↓
Inoculum (spore suspension from PDA slant)
        ↓
Medium preparation (wheat bran / citrus peel + salts, pH 4.5–5.5)
        ↓
Fermentation (SSF 28–30 °C, 3–5 days  or  SmF 2–4 days)
        ↓
Extraction (water / citrate buffer + filtration)
        ↓
Purification (ultrafiltration → (NH₄)₂SO₄ precipitation → dialysis → chromatography)
        ↓
Lyophilization → Standardization → Packaging → Pectinase enzyme

Factors affecting production

• Temperature: 28–30 °C optimum; above 40 °C denatures enzyme.
• pH: 4.5–5.5 for fungal pectinase.
• Carbon source: Pectin-rich substrate induces maximum enzyme.
• Nitrogen source: (NH₄)₂SO₄ or peptone.
• Aeration and agitation: needed in SmF.
• Fermentation time: longer time may cause proteolytic degradation.

Conclusion: Pectinase is produced industrially by fermentation of pectin-rich agro-wastes using Aspergillus niger, followed by extraction, purification and lyophilization. SSF is preferred because it is economical and gives high enzyme yield.

Importance of Tissue Culture Techniques (3 marks)

1. Rapid multiplication — A single plant or even a small piece of tissue can produce thousands of genetically identical plantlets in a short time, far faster than conventional propagation.
2. Production of disease-free plants — Meristem tip culture eliminates viruses and other pathogens, giving healthy planting material (e.g. potato, banana, sugarcane, strawberry).
3. Clonal propagation of elite genotypes — Superior varieties, hybrids and sterile plants that cannot be propagated by seed can be multiplied vegetatively without genetic change.
4. Conservation of germplasm — Rare, endangered and recalcitrant species can be maintained in vitro in a small space, free from pests and climatic hazards.
5. Genetic improvement and biotechnology — Provides material for somaclonal variation, somatic hybridization, haploid production (anther culture), embryo rescue, genetic transformation and micropropagation of transgenic plants.
6. Year-round production and transport — Culture is independent of season and climate, and plantlets can be exchanged between countries free of quarantine problems.
7. Production of secondary metabolites — Useful compounds (alkaloids, steroids, dyes) can be produced in cell suspension cultures.

General Steps Involved in Tissue Culture (7 marks)

1. Selection of explant (source tissue) A healthy, disease-free mother plant is chosen. The explant may be shoot tip, axillary bud, leaf, stem, root, anther, embryo or meristem. Meristem tips are preferred for virus-free plants.

2. Sterilization (surface disinfection) The explant is washed with tap water and detergent, then treated with 70% ethanol for a few seconds followed by sodium hypochlorite (0.5–2%) or mercuric chloride (0.1%) for 5–15 minutes, and finally rinsed 3–4 times with sterile distilled water. All operations are carried out in a laminar flow cabinet.

3. Preparation of culture medium A nutrient medium (usually MS — Murashige and Skoog medium) is prepared containing:

• inorganic macro- and micro-elements,
• carbon source (sucrose 2–3%),
• vitamins and amino acids (glycine, inositol),
• growth regulators — auxins (IAA, NAA, 2,4-D) and cytokinins (BAP, kinetin),
• gelling agent (agar) and distilled water. The pH is adjusted to 5.6–5.8. The medium is poured into culture vessels and autoclaved at 121 °C, 15 psi for 15–20 minutes.

4. Inoculation The sterilized explant is cut into small pieces with a sterile scalpel and placed on the surface of the medium in the culture vessel under aseptic conditions in a laminar flow hood. The vessel is then sealed with cotton plug or aluminium foil.

5. Incubation The cultures are kept in a growth room or incubator at 25 ± 2 °C, with 16 hours light and 8 hours dark photoperiod, light intensity about 2000–3000 lux and 50–60% relative humidity. The explant absorbs nutrients and begins to grow.

6. Callus formation / differentiation (morphogenesis) The explant first forms an undifferentiated mass called callus. By adjusting the auxin–cytokinin ratio, the callus undergoes organogenesis (shoot and root formation) or somatic embryogenesis (embryo-like structures). A high cytokinin : auxin ratio favours shoot buds; a high auxin : cytokinin ratio favours roots.

7. Sub-culturing Every 3–4 weeks the growing tissue is transferred to fresh medium of the same or modified composition to prevent nutrient depletion and toxic metabolite accumulation. This also increases the number of propagules.

8. Rooting and hardening (acclimatization) Regenerated shoots are transferred to a rooting medium (high auxin, low cytokinin) to induce roots. Well-rooted plantlets are then taken out, washed free of agar, and transferred to sterile soil or a potting mixture. They are kept initially in high humidity and low light (hardening) and gradually exposed to the natural environment before being planted in the field.

9. Field transfer and evaluation The hardened plants are transplanted to the field, and their growth, uniformity, yield and genetic stability are checked before large-scale release.

Conclusion Tissue culture is thus a powerful tool of modern biotechnology that combines aseptic technique, nutrient media and controlled environment to produce large numbers of healthy, uniform plants and to support genetic improvement programmes.

Enzymes लाई तिनीहरूले गर्ने जैवरासायनिक कार्य (type of reaction catalysed) को आधारमा Enzyme Commission (EC) प्रणाली अनुसार छ मुख्य वर्ग मा वर्गीकरण गरिन्छ:

संक्षेपमा: (१) Oxidoreductases ले electron सार्छन्, (२) Transferases ले group सार्छन्, (३) Hydrolases ले पानीले तोड्छन्, (४) Lyases ले पानी/अक्सिडेसन बिना तोड्छन् वा दोहोरो बन्धनमा थप्छन्, (५) Isomerases ले संरचना पुनर्व्यवस्थित गर्छन्, (६) Ligases ले ATP खर्च गरी जोड्छन्।

General Properties of Amino Acids

1. Definition and structure. Amino acids are organic compounds that contain both an amino group (–NH₂) and a carboxyl group (–COOH) attached to the same carbon atom, called the α-carbon. The general formula is:

$\text{R}-\text{CH}(\text{NH}_2)-\text{COOH}$

where R is a variable side chain that distinguishes one amino acid from another. About 20 amino acids occur in proteins, and all are α-amino acids (except proline, which is a secondary amine).

2. Amphoteric nature. Because they carry both an acidic (–COOH) and a basic (–NH₂) group, amino acids are amphoteric — they can react with both acids and bases. In neutral aqueous solution they exist mainly as zwitterions (dipolar ions), with –NH₃⁺ and –COO⁻ groups. Hence they have high melting points, are soluble in water, and are insoluble in non-polar solvents.

3. Isoelectric point (pI). Each amino acid has a characteristic pH at which the net charge is zero and it does not migrate in an electric field. This pH is the isoelectric point. At pH below pI the molecule is positively charged; above pI it is negatively charged. This property is used in electrophoresis and ion-exchange chromatography.

4. Optical activity. Except glycine (R = H), all amino acids have a chiral α-carbon and are therefore optically active. Naturally occurring amino acids are of the L-configuration and rotate plane-polarised light (usually to the left).

5. Solubility and physical state. They are generally crystalline solids, non-volatile, and have high melting points (often above 200 °C) due to strong ionic interactions. They are readily soluble in water and polar solvents but insoluble in benzene, ether, and chloroform.

6. Chemical reactivity. The –NH₂ group gives reactions such as acylation, deamination, and transamination, while –COOH gives esterification, decarboxylation, and amide formation. The R group may also carry –OH, –SH, –NH₂, –COOH, or aromatic rings, giving each amino acid its specific chemical character.

7. Peptide bond formation. The –COOH of one amino acid condenses with the –NH₂ of another, with loss of water, to form a peptide bond (–CO–NH–). Chains of amino acids so joined form peptides and proteins.

8. Buffer action. Because they can donate or accept protons, amino acids and their solutions act as buffers, resisting changes in pH — an important property in biological systems.

Advantages of microbial enzymes in industries

Microbial enzymes are enzymes obtained from microorganisms such as bacteria, fungi and yeasts, either by fermentation or by recombinant DNA technology. Their industrial use has largely replaced chemical catalysts and enzymes from plant and animal sources because of the following advantages:

1. High catalytic efficiency and specificity — Microbial enzymes catalyse reactions rapidly and act only on a particular substrate, so side products and unwanted reactions are minimised. This gives higher yield and purer products.

2. Easy and cheap large-scale production — Microorganisms can be grown rapidly in fermenters on inexpensive media (molasses, starch, agricultural wastes) within a short generation time. Enzyme production is therefore continuous, unlimited and independent of season, climate or geographical location.

3. Stable and active under extreme conditions — Enzymes from extremophiles (thermophiles, alkaliphiles, halophiles) work at high temperature, extreme pH and high salt concentration. Such enzymes suit industrial processes that run under harsh conditions, where plant and animal enzymes would be denatured.

4. Genetic manipulation is possible — Microbial genes can be cloned, mutated and over-expressed by recombinant DNA technology. Strains can be engineered to produce enzymes in very large amounts, to secrete them outside the cell for easy recovery, or to give enzymes with improved properties (e.g. thermostable proteases, lipases).

5. Economical and eco-friendly — Enzymes are needed in small amounts, are biodegradable, non-toxic and work under mild conditions of temperature and pressure. This saves energy, reduces the use of corrosive chemicals and reduces pollution, so the process is "green".

6. Easily purified and standardized — Downstream processing (filtration, centrifugation, chromatography) gives enzymes of consistent quality and defined activity, which is essential for food, pharmaceutical and detergent industries.

7. Wide range of enzymes available — Microbes provide almost every type of enzyme — amylases, proteases, lipases, cellulases, pectinases, isomerases, etc. — so a suitable enzyme can be selected for each industrial need.

8. Immobilization is possible — Microbial enzymes can be immobilized on inert supports and reused many times, further lowering cost and allowing continuous operation.

Because of these advantages, microbial enzymes are widely used in detergents (proteases, lipases), starch and sugar industry (amylase, glucose isomerase), dairy and food processing (rennet, lactase, pectinase), textiles (cellulase, amylase), leather, paper, pharmaceuticals and bioremediation.

Trickling Generator Process in Vinegar Production

The trickling generator (quick vinegar process or German process) is an industrial method of producing vinegar by the rapid oxidation of dilute ethanol to acetic acid by Acetobacter bacteria. It replaced the slow Orleans (surface) method.

Construction:

• A tall cylindrical tank (wood or acid-resistant material), about 4-6 m high and 2-3 m in diameter.
• It is packed with an inert, high surface-area material such as beechwood shavings, corn cobs or coke, which supports the bacterial film.
• Near the top, a rotating sprinkler or perforated distributor spreads the alcoholic liquid evenly over the packing.
• Near the bottom, air inlets let air rise upward, and a collection trough at the base draws off the finished vinegar.
• Cooling coils or a jacket control the temperature, since the reaction is exothermic.

Process:

1. Starting (inoculation): The packing is first soaked with good vinegar containing active Acetobacter aceti to develop a slime layer (biofilm) on the shavings.
2. Trickling: Dilute alcoholic liquid (about 10-13% ethanol, with nutrients) is sprinkled from the top and trickles slowly down over the bacterial film.
3. Aeration: Air enters from the bottom and moves upward (counter-current). The bacteria oxidize ethanol to acetic acid: $\text{C}_2\text{H}_5\text{OH} + \text{O}_2 \xrightarrow{\text{Acetobacter}} \text{CH}_3\text{COOH} + \text{H}_2\text{O} + \text{heat}$
4. Collection and recirculation: The liquid collected at the bottom is partly converted vinegar; it is recirculated several times until the acetic acid reaches about 5-8%. A portion is drawn off as product.
5. Temperature control: The temperature is kept at about 25-30 °C by cooling coils, as excess heat kills the bacteria.

Advantages:

• Very fast: vinegar is produced in 2-3 days instead of weeks or months.
• High yield and continuous operation.
• Requires less floor space and labour.

Disadvantages:

• Risk of contamination and clogging of the packing.
• Careful control of temperature, airflow and alcohol concentration is needed.
• The final vinegar is less aromatic than that from the slow Orleans process.

Role of Microorganisms as Biofertilizers (5 marks)

Biofertilizers are preparations containing living microorganisms that, when applied to soil or seed, colonise the rhizosphere and increase the availability of nutrients to plants. They supplement chemical fertilizers, are eco-friendly, and improve soil health.

1. Nitrogen fixation (Rhizobium, Azotobacter, Anabaena):

• Rhizobium forms symbiotic root nodules in legumes and fixes atmospheric N₂ into ammonia using the nitrogenase enzyme. It can add 50–200 kg N/ha per season.
• Free-living Azotobacter (aerobic) and Clostridium (anaerobic) fix nitrogen non-symbiotically in non-legume fields.
• Cyanobacteria (Anabaena, Nostoc) fix nitrogen in rice paddies, also adding organic matter.

2. Phosphate solubilisation (Bacillus, Pseudomonas, Aspergillus):

• These solubilise insoluble soil phosphates (rock phosphate, tricalcium phosphate) by secreting organic acids, making P available to plants.
• Mycorrhiza (Glomus) extends the root surface area and mobilises P and other minerals.

3. Potassium and other nutrient mobilisation:

• Frateuria and silicate bacteria release K from silicate minerals.
• Some microbes produce siderophores that chelate iron and make it available.

4. Growth promotion and soil health:

• Azospirillum and Pseudomonas produce auxins, gibberellins and cytokinins that stimulate root growth.
• They suppress soil-borne pathogens (biocontrol) and improve soil structure, water-holding capacity, and organic matter.

Significance: Biofertilizers are cheap, renewable, reduce chemical fertilizer use and pollution, and are especially useful for sustainable and organic farming. Their main limitations are short shelf life, sensitivity to temperature and moisture, and slower response than chemical fertilizers.

Characteristics of vectors used in recombinant DNA technology

A vector is a self-replicating DNA molecule used to carry a foreign DNA fragment into a host cell. To be useful in recombinant DNA technology, a vector must have the following characteristics:

1. Ability to replicate autonomously (origin of replication): It must possess a functional ori so that it multiplies independently inside the host cell, producing many copies of the inserted DNA.

2. Selectable marker: It must carry a selectable marker gene (e.g., genes for antibiotic resistance such as ampicillin or tetracycline resistance) so that transformed cells can be easily identified and selected from non-transformed ones.

3. Unique restriction sites (multiple cloning site): It should have one or more unique recognition sites for restriction endonucleases, so that foreign DNA can be inserted at a specific point without disturbing essential vector functions.

4. Small size: It should be relatively small in size, which makes it easy to isolate, manipulate, and introduce into the host cell.

5. High copy number: It should be present in multiple copies per host cell so that a large amount of the cloned gene (and its product) can be obtained.

6. Ability to accept large foreign DNA inserts: It must be capable of accommodating a reasonably large foreign DNA fragment without losing its own stability.

7. Stability and compatibility with host: It must replicate stably in the host cell, not be lost during cell division, and be compatible with the host's enzymatic machinery.

8. Promoter and regulatory sequences (for expression vectors): For expression of the cloned gene, it should carry suitable promoter, terminator, and ribosome-binding sequences so the insert can be transcribed and translated.

9. Easy recovery of insert: It should allow easy re-isolation of the cloned DNA fragment by restriction digestion.

10. Non-pathogenic / safe: It should be harmless to the host and to the worker, and should not transfer undesirable traits.

Common examples of vectors satisfying these characteristics are plasmids (pBR322, pUC19), bacteriophages (λ phage), cosmids, BACs, YACs, and expression vectors.

Water is essential for cellular life for the following reasons:

1. Universal solvent — Water's polar nature allows it to dissolve ions, sugars, amino acids, gases and most metabolites. Almost all biochemical reactions occur in aqueous solution, so water provides the medium in which cellular chemistry takes place.

2. Medium for biochemical reactions — Metabolic pathways such as glycolysis, the Krebs cycle and photosynthesis occur only in water. Water also directly participates as a reactant in many reactions, e.g. hydrolysis of ATP, proteins, carbohydrates and fats.

3. Transport of materials — Because of its solvent property and fluidity, water transports nutrients, gases, hormones and waste products into, within and out of the cell (e.g. blood plasma, xylem and phloem sap).

4. High specific heat and thermal stability — Water absorbs or releases a large amount of heat with little change in its own temperature. This protects cells from sudden temperature fluctuations and keeps enzyme activity stable.

5. High latent heat of vaporisation — Evaporation of water (transpiration, sweating) removes excess heat, providing an effective cooling mechanism for cells and organisms.

6. Cohesion, adhesion and surface tension — Hydrogen bonding gives water high cohesion and adhesion, which maintain the structural integrity of protoplasm and help in the ascent of sap in plants.

7. Structural and turgidity role — Water constitutes 70–90% of cell mass and keeps the cell turgid, maintains cell shape and provides mechanical support to soft tissues.

8. Ionisation and pH maintenance — Water ionises slightly into H⁺ and OH⁻ ions, which help maintain pH and ionic balance essential for enzyme function and cellular processes.

9. Protection and lubrication — Water acts as a lubricant in joints, in mucous secretions and as a shock absorber (amniotic fluid), protecting delicate cellular structures.

10. Photosynthesis — In plants, water is the source of electrons and hydrogen, and the oxygen released in photosynthesis comes from the photolysis of water.

Because of these properties, no cell can survive without water; life on Earth is therefore impossible in its absence.

Biological functions of lipids:

1. Energy storage and supply — Triglycerides (fats and oils) are the most concentrated energy reserve, yielding about 9 kcal per gram, roughly twice that of carbohydrates or proteins. They are stored in adipose tissue and mobilised during fasting or starvation.

2. Structural component of membranes — Phospholipids and cholesterol form the lipid bilayer of cell membranes, providing the framework that separates the cell from its surroundings and compartmentalises organelles. Glycolipids contribute to membrane recognition.

3. Thermal insulation — Subcutaneous fat acts as a poor conductor of heat, reducing heat loss and helping endotherms maintain a constant body temperature.

4. Protection and padding — Fat deposits around the kidneys, heart, and other vital organs act as shock absorbers, cushioning them against mechanical injury.

5. Transport of fat-soluble substances — Lipids serve as carriers for the absorption and transport of fat-soluble vitamins (A, D, E, K) and of lipid-soluble drugs and hormones through the blood as lipoproteins.

6. Precursors of biologically active molecules — Cholesterol is the precursor of steroid hormones (cortisol, aldosterone, testosterone, oestrogen), vitamin D, and bile acids. Phospholipids give rise to signalling molecules such as prostaglandins and leukotrienes.

7. Cofactors and electron carriers — Certain lipids function as enzyme cofactors (e.g., vitamin K in blood clotting) and as electron carriers in the respiratory chain (ubiquinone/coenzyme Q).

8. Signalling and hormone action — Steroid hormones and lipid-derived messengers regulate gene expression, metabolism, inflammation, and reproduction.

9. Waterproofing and lubrication — Waxes and cutin on plant surfaces and sebum on skin and feathers prevent excessive water loss and keep surfaces supple and water-repellent.

10. Source of metabolic water — Oxidation of fats releases metabolic water, which is important for desert animals such as the camel and kangaroo rat.

Production of Yoghurt (5 marks)

Yoghurt is a fermented dairy product made from milk by the action of lactic acid bacteria. The standard method is as follows:

1. Selection and standardization of milk Fresh, clean cow or buffalo milk is taken. Its fat and solid-not-fat (SNF) content is standardized (usually 3–4% fat and 8.5–9% SNF) so that the product has uniform body and texture. The milk is filtered to remove dirt and foreign particles.

2. Pasteurization The standardized milk is heated to about 85–90 °C for 15–30 minutes (or 90–95 °C for 5 minutes). This kills pathogenic and spoilage microorganisms, inactivates enzymes, and denatures whey proteins, which helps in producing a firm curd with less whey separation.

3. Cooling and inoculation The pasteurized milk is cooled to 42–45 °C, the optimum temperature for the starter culture. It is then inoculated with a mixed starter culture of Lactobacillus bulgaricus and Streptococcus thermophilus (usually in the ratio 1:1) at the rate of about 1–2% of the milk volume. The culture is mixed thoroughly to distribute the organisms evenly.

4. Incubation (fermentation) The inoculated milk is filled into clean, pre-sterilized cups or containers and incubated at 42–45 °C for 3–4 hours. During this period, the bacteria ferment lactose into lactic acid. The pH falls from about 6.6 to 4.0–4.6, the milk proteins coagulate, and a firm gel-like curd with characteristic flavour and aroma develops. Incubation is stopped when the curd is set and the acidity reaches about 0.8–1.0% lactic acid.

5. Cooling and storage After incubation, the yoghurt is rapidly cooled to 4–5 °C to stop further acid production and prevent over-souring. It is then stored under refrigeration and marketed as plain yoghurt. Fruit pulp, sugar or flavours may be added before or after fermentation to make flavoured yoghurt.

Key points: Pasteurization at 85–90 °C → cooling to 42–45 °C → inoculation with L. bulgaricus and S. thermophilus → incubation at 42–45 °C for 3–4 hours → cooling to 4–5 °C → storage.

The lac operon is a classic example of an inducible operon in Escherichia coli that controls the metabolism of lactose. It consists of the following components:

1. Structural genes (three genes):

• lacZ — encodes β-galactosidase, which hydrolyses lactose into glucose and galactose.
• lacY — encodes β-galactoside permease, a membrane protein that facilitates the entry of lactose into the cell.
• lacA — encodes β-galactoside transacetylase, which transfers an acetyl group from acetyl-CoA to β-galactosides.

2. Regulatory components:

• Promoter (P) — the binding site for RNA polymerase, located upstream of the structural genes.
• Operator (O) — a short DNA sequence between the promoter and the structural genes; the repressor protein binds here to block transcription.
• Regulatory gene (lacI) — located upstream of the promoter; it encodes the repressor protein, which binds to the operator and prevents transcription in the absence of lactose.
• CAP binding site (catabolite activator protein site) — a site near the promoter where the CAP–cAMP complex binds to enhance transcription when glucose is absent.

Summary: The lac operon consists of three structural genes (lacZ, lacA, lacY), a promoter, an operator, a regulatory gene (lacI), and a CAP binding site. Together these allow the operon to be induced only when lactose is present and glucose is absent.

Very Short Answer Questions (Attempt any Eight)

a) What are the biomolecules?

Biomolecules are organic molecules synthesized by living cells that are essential for life processes. They include carbohydrates, proteins, lipids, nucleic acids, vitamins, and enzymes. They serve as structural components, energy sources, and regulators of metabolism.

b) Explain the importance of immobilization in enzyme technology.

Enzyme immobilization means fixing enzymes to a solid support so they can be reused. Its importance:

• Enzymes can be recovered and reused many times, reducing cost.
• Products are easily separated from the enzyme.
• Enzymes become more stable against heat, pH, and denaturation.
• Continuous industrial processes become possible (e.g., glucose isomerase for high-fructose syrup).

c) Write short notes on scope of microbial biotechnology.

Microbial biotechnology uses microorganisms (bacteria, fungi, algae) for useful products and services. Its scope includes:

• Production of antibiotics, vaccines, enzymes, and vitamins.
• Fermented foods and beverages (curd, cheese, beer, bread).
• Biofuels (ethanol, biogas) and bioplastics.
• Bioremediation of pollutants and waste treatment.
• Gene cloning, recombinant proteins (insulin, growth hormone), and biofertilizers.

d) Define coenzyme.

A coenzyme is a non-protein organic molecule that binds loosely to an enzyme and assists in catalysis by carrying chemical groups, electrons, or hydrogen between reactions. Examples: NAD⁺, NADP⁺, FAD, coenzyme A, and ATP.

e) Write down the fate of pyruvate in metabolism.

Pyruvate formed by glycolysis has three main fates:

• Aerobic respiration: converted to acetyl-CoA by pyruvate dehydrogenase, entering the Krebs cycle.
• Anaerobic respiration (alcoholic fermentation): converted to ethanol and CO₂ in yeast.
• Anaerobic respiration (lactic acid fermentation): converted to lactic acid in muscle cells and some bacteria.

f) Define the reducing sugars with examples.

Reducing sugars are carbohydrates that contain a free aldehyde or ketone group and can reduce oxidizing agents such as Fehling's solution, Benedict's solution, or Tollen's reagent. Examples: glucose, fructose, maltose, and lactose.

g) What is pasteurization of milk?

Pasteurization is a heat treatment in which milk is heated to about 63 °C for 30 minutes (holding method) or 72 °C for 15 seconds (flash method) and then cooled rapidly. It kills pathogenic microorganisms and extends shelf life without greatly changing taste or nutritive value.

h) Enlist enzymes involved in beta-oxidation of lipids.

The four main enzymes of β-oxidation are:

1. Acyl-CoA dehydrogenase (dehydrogenation)
2. Enoyl-CoA hydratase (hydration)
3. 3-Hydroxyacyl-CoA dehydrogenase (dehydrogenation)
4. Thiolase (thiolytic cleavage)

i) What is genetic code?

The genetic code is the set of rules by which the nucleotide sequence of DNA or mRNA is translated into the amino acid sequence of a protein. It is a triplet code of 64 codons, degenerate, non-overlapping, and nearly universal.

j) What is the functions of DNA polymerase?

DNA polymerase synthesizes a new DNA strand by adding deoxyribonucleotides to the 3′ end of a primer, using the parent strand as template. It also has 3′→5′ exonuclease activity for proofreading and error correction during DNA replication.