Components of a Stirred Tank Fermenter

A stirred tank fermenter (STR) is the most widely used bioreactor for submerged fermentation. It is a cylindrical vessel with mechanical agitation and forced aeration, used for antibiotics, enzymes, organic acids and alcohol.

1. Main Components

(a) Vessel / body

• Cylindrical tank of stainless steel (glass for laboratory scale) with a height-to-diameter ratio of about 2:1 to 3:1.
• Fitted with a jacket or internal coil for heating and cooling, and ports for inoculum, acid/base, antifoam and sampling.

(b) Agitator (impeller)

• Mixes the broth and breaks air into fine bubbles, raising oxygen transfer.
• Rushton turbine (disc with 6 flat blades) gives high shear and gas dispersion; marine propeller or pitched-blade turbine gives axial flow and gentle mixing.
• Tall vessels carry two or more impellers on one shaft.

(c) Baffles

• 4 vertical strips (width about 1/10 of tank diameter) fixed to the wall; they stop vortex formation and promote turbulence.

(d) Sparger

• A perforated ring, pipe or porous disc below the lowest impeller that releases sterile air as fine bubbles.

(e) Shaft, seal and drive

• A variable-speed motor turns the shaft through a mechanical seal, which prevents contamination.

(f) Temperature control

• Jacket or coil with cooling or heating water, regulated by a temperature sensor and controller.

(g) pH and foam control

• A pH electrode triggers automatic addition of acid or alkali; a foam probe triggers antifoam addition.

(h) Ports and valves

• Inoculation port, sampling port, sterile air inlet filter, exhaust gas outlet with filter, and harvest valve at the bottom.

(i) Sensors

• Probes for temperature, pH, dissolved oxygen (DO) and foam, connected to a control panel.

2. Labelled Diagram

                 [ Motor ]
                     |
          Sterile     |            Exhaust gas
          air filter  |            outlet + filter
                      |                 ^
   Acid/Base --->     |                 |
   Antifoam  --->  ===+=== seal ========+===
   Inoculum  --->  /  |                  \
   pH probe  --->  |  |                  |   <- Cooling water out
   DO probe  --->  |  |                  |
                   |  |  <- Shaft        |
        Baffle --> |  |  |               | <-- Baffle
                   | -+--+- Impeller 2   |
                   |   (Rushton turbine)  |
                   |  |                  |   Jacket
                   | -+--+- Impeller 1   |
                   |   (Rushton turbine)  |
                   |  o o o o o          |   <- Cooling water in
                   | (Sparger ring)      |
                   \__________|_________/
                              ^
               Sterile air in |          Harvest valve (bottom)

Figure: Stirred tank fermenter with its main parts.

3. Functions (Summary)

Thus the stirred tank fermenter combines mechanical agitation with forced aeration to give uniform mixing, efficient oxygen transfer and controlled conditions for microbial growth and product formation.

Structures of Proteins

Proteins exhibit four levels of structural organization. Each higher level is built upon the one below it.

1. Primary Structure

The primary structure is the linear sequence of amino acids joined by peptide bonds in a polypeptide chain. It is determined by the gene and is stabilized by covalent peptide bonds (and disulfide bridges between cysteine residues). The sequence dictates all higher levels of folding. Example: the sequence of 51 amino acids in insulin.

2. Secondary Structure

This refers to the local, regular folding of the polypeptide backbone, stabilized mainly by hydrogen bonds between the C=O of one peptide bond and the N–H of another. The main types are:

• α-Helix: A right-handed coiled structure with about 3.6 amino acid residues per turn, hydrogen bonds running parallel to the helix axis. Example: keratin in hair.
• β-Pleated Sheet: Extended, zig-zag chains lying side by side, hydrogen bonds running perpendicular to the chain direction. It may be parallel or antiparallel. Example: silk fibroin.
• Turns and loops: β-turns connect the strands and reverse the chain direction.

3. Tertiary Structure

This is the overall three-dimensional shape of a single polypeptide chain, formed by folding of secondary structural elements. It is stabilized by:

• Hydrogen bonds (between polar side chains)
• Disulfide bridges (covalent, between cysteine residues)
• Hydrophobic interactions (between nonpolar side chains, buried in the interior)
• Ionic (electrostatic) bonds (between oppositely charged side chains)
• Van der Waals forces

Two common motifs are the fibrous (elongated, e.g., collagen) and globular (compact, e.g., myoglobin) forms. The tertiary structure creates the active site of enzymes.

4. Quaternary Structure

This is the arrangement of two or more polypeptide subunits (each with its own tertiary structure) into a functional multimeric protein. Subunits are held by the same non-covalent forces as in tertiary structure. It may be homomeric (identical subunits) or heteromeric (different subunits). Examples: hemoglobin (α₂β₂ tetramer), immunoglobulin G, and lactate dehydrogenase.

Summary Table

Note: Loss of native three-dimensional structure (denaturation) by heat, pH, or chemicals destroys biological activity, showing that higher-order structure is essential for protein function.

RNA (Ribonucleic acid)

RNA is a single-stranded polynucleotide made of ribonucleotide monomers. Each ribonucleotide consists of a nitrogenous base (purine: adenine, guanine; pyrimidine: cytosine, uracil), the pentose sugar ribose (having a 2′–OH group), and a phosphate group. The bases are joined to ribose by $N$-glycosidic bonds and successive nucleotides are linked by $3'\to5'$ phosphodiester bonds. In the cell, RNA occurs mainly in the cytoplasm and is of three main types — messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) — and it functions chiefly in protein synthesis (mRNA carries the genetic message, tRNA carries amino acids, rRNA forms ribosomes); some RNAs are also catalytic (ribozymes) and act as the genetic material in some viruses. Unlike DNA, RNA contains uracil in place of thymine and ribose in place of deoxyribose, and it is usually single-stranded.

Transcription in bacteria

Transcription is the process of synthesis of an RNA strand from a DNA template strand, catalysed by DNA-dependent RNA polymerase. In bacteria (E. coli) it occurs in the cytoplasm and involves the following steps.

1. Components required. The DNA template (template/antisense strand read in the $3'\to5'$ direction), the four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) as substrates, $\mathrm{Mg^{2+}}$ as cofactor, and the holoenzyme RNA polymerase. The bacterial RNA polymerase holoenzyme has the core enzyme ($\alpha_2\beta\beta'$) plus the sigma ($\sigma$) factor; the $\sigma$ factor is needed only for correct initiation.

2. Initiation. The $\sigma$ factor recognises and binds the holoenzyme to the promoter region of the DNA, i.e. the specific start site. In bacteria the promoter has two consensus sequences: the $-10$ sequence (Pribnow box, TATAAT) and the $-35$ sequence (TTGACA), counted upstream from the transcription start point ($+1$). Binding causes local unwinding of the DNA double helix over about 17 bp, forming the "open promoter complex" and exposing the template strand. The first nucleotide is placed opposite the $+1$ base; the $\sigma$ factor is released after the first few nucleotides are added, leaving the core enzyme to continue.

3. Elongation. The core enzyme moves along the template strand in the $3'\to5$ direction, and the new RNA is built in the $5'\to3$ direction by adding ribonucleotides complementary to the template (A–U, T–A, G–C, C–G). A short transient DNA–RNA hybrid (about 8–9 bp) is maintained at the growing point, while the DNA behind is rewound into a double helix. The enzyme has no proofreading like DNA polymerase, so error rate is about $10^{-4}$. A moving transcription bubble (about 17 bp of unwound DNA) travels with the polymerase.

4. Termination. Elongation stops at the terminator sequence, and the RNA transcript, the polymerase and the DNA template are separated. Two mechanisms occur in bacteria:

• Rho-independent (intrinsic) termination: the terminator contains a GC-rich palindromic sequence followed by a run of A's on the template. The RNA transcript folds into a stable GC-rich hairpin (stem–loop) followed by a run of U's. The hairpin destabilises the polymerase–RNA–DNA complex, and the weak rU–dA hybrid causes the transcript to be released.
• Rho-dependent termination: the terminator lacks the hairpin, and the protein Rho ($\rho$) factor binds the rut site on the nascent RNA, moves along it using ATP, and pulls the RNA away from the polymerase at the termination site.

5. Post-transcriptional processing. In bacteria the primary transcript is often polycistronic (one mRNA for several genes) and is used almost directly; rRNA and tRNA are cut from larger precursors by ribonucleases. There is no capping or poly-A tailing as in eukaryotes. Transcription and translation are coupled — ribosomes begin translating the mRNA while it is still being transcribed.

EMP Pathway (Embden–Meyerhof–Parnas Pathway / Glycolysis)

The EMP pathway is the universal cytoplasmic route for the breakdown of glucose to pyruvate. It occurs in almost all living cells, needs no oxygen, and takes place in the cytosol. Net result per glucose molecule:

$\text{Glucose} + 2\text{NAD}^+ + 2\text{ADP} + 2\text{P}_i \rightarrow 2\text{Pyruvate} + 2\text{NADH} + 2\text{H}^+ + 2\text{ATP} + 2\text{H}_2\text{O}$

The pathway has 10 enzymatic steps, conventionally divided into three phases.

Phase I — Preparatory (investment) phase

1. Phosphorylation of glucose: Glucose is phosphorylated at C-6 by hexokinase (glucokinase in liver) using ATP → glucose-6-phosphate. This traps glucose inside the cell.
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 ATP → fructose-1,6-bisphosphate. This is the committed, rate-limiting step of glycolysis.
4. Cleavage: Aldolase splits fructose-1,6-bisphosphate into two triose phosphates — dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P).
5. Isomerization: Triose phosphate isomerase converts DHAP into G3P, so that two molecules of G3P enter the next phase.

Thus 2 ATP are consumed in this phase (one each in steps 1 and 3).

Phase II — Oxidation (pay-off) phase

6. Oxidation and phosphorylation: Glyceraldehyde-3-phosphate dehydrogenase oxidises G3P, reducing NAD⁺ to NADH and attaching inorganic phosphate → 1,3-bisphosphoglycerate. (Two NADH formed per glucose.)
7. First substrate-level phosphorylation: Phosphoglycerate kinase transfers the high-energy phosphate of 1,3-bisphosphoglycerate to ADP → 3-phosphoglycerate + ATP. (Two ATP formed.)
8. Phosphate shift: Phosphoglycerate mutase moves the phosphate from C-3 to C-2 → 2-phosphoglycerate.
9. Dehydration: Enolase removes water, forming phosphoenolpyruvate (PEP), which has a very high phosphate-transfer potential.
10. Second substrate-level phosphorylation: Pyruvate kinase transfers the phosphate of PEP to ADP → pyruvate + ATP. (Two ATP formed.)

Energy yield (per glucose)

So the net gain is 2 ATP and 2 NADH per glucose. In aerobic cells the NADH is reoxidised in the mitochondrion (glycerol-phosphate or malate–aspartate shuttle) and yields about 5 ATP, while pyruvate enters the Krebs cycle; in anaerobic cells NADH is reoxidised by lactate or ethanol fermentation, regenerating NAD⁺ so that glycolysis can continue.

Significance: It is the only source of ATP in cells without mitochondria (e.g. mature RBCs), the main energy source in anaerobic conditions, and it supplies pyruvate, NADH and carbon skeletons for the Krebs cycle and 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 is high in energy, so hydrolysis of ATP to ADP + Pᵢ releases about 7.3 kcal/mol under cellular conditions.

1. Energy donor: ATP drives all endergonic (energy-requiring) reactions — biosynthesis of proteins, nucleic acids, lipids and carbohydrates, and active transport across membranes.
2. Energy carrier: Energy released by catabolism (glycolysis, Krebs cycle, oxidative phosphorylation) is trapped in ATP and delivered to sites of energy need, linking catabolism with anabolism.
3. Phosphoryl-group transfer: ATP acts as a phosphate donor in phosphorylation reactions, e.g. glucose → glucose-6-phosphate in glycolysis, activating substrates for further reaction.
4. Muscle contraction, nerve conduction and other cell work: mechanical, osmotic and electrical work of the cell are all powered by ATP.
5. Regulation: ATP acts as an allosteric inhibitor of PFK-1 and of phosphofructokinase-type regulatory enzymes, so a high ATP level slows down the energy-yielding pathways and prevents waste.
6. Cyclic regeneration: ATP is continuously regenerated from ADP and Pᵢ during respiration (and by creatine phosphate in muscle), so it works as a rechargeable carrier rather than a stored fuel.

Production of Pectinase Enzyme

Pectinase is an industrial enzyme that hydrolyses pectin, the polysaccharide that cements plant cells together. It is produced chiefly by submerged fermentation using fungal and bacterial strains, followed by recovery and purification. The process is described below.

1. Selection of Microorganism

Suitable pectinase-producing strains are selected and improved:

• Fungi: Aspergillus niger, Aspergillus oryzae, Penicillium spp., Rhizopus spp. (most widely used, since they secrete pectinase extracellularly and grow well on cheap agricultural residues).
• Bacteria: Bacillus subtilis, Erwinia spp.
• Yeasts: Saccharomyces spp.

The selected strain is maintained on potato dextrose agar (PDA) slants at 4 °C and improved by mutation or recombinant DNA techniques to obtain high-yielding, stable strains.

2. Preparation of Inoculum

A pure culture from the slant is transferred to a seed medium (e.g., potato dextrose broth) and incubated at 28–30 °C for 24–48 hours with shaking. This actively growing culture (about 5–10 % v/v) is used as the inoculum for the production fermenter.

3. Preparation of Production Medium

A cheap medium containing pectin-rich carbon sources is formulated, for example:

The medium is sterilised at 121 °C, 15 psi for 15–20 minutes and cooled to 30 °C.

4. Fermentation

Submerged fermentation is the method of choice for large-scale production:

• The sterilised medium is charged into a stirred-tank bioreactor.
• It is inoculated with the seed culture.
• Conditions are maintained at pH 4.5–5.5, temperature 28–30 °C, aeration 0.5–1 vvm, agitation 150–200 rpm for 3–7 days.
• Pectin in the medium acts as an inducer, so pectinase is secreted mainly in the late log and stationary phase.
• Antifoam is added as required, and samples are withdrawn periodically to monitor enzyme activity.

(Solid-state fermentation on wheat bran or citrus peel is also used, especially in developing countries, because it is cheap and gives high yields.)

4.1 Downstream Processing

1. Separation of biomass: The fermented broth is filtered or centrifuged to remove fungal mycelium and cell debris.
2. Clarification: The cell-free filtrate is clarified by filtration or centrifugation.
3. Concentration: The filtrate is concentrated by ultrafiltration or vacuum evaporation.
4. Precipitation: The enzyme is precipitated by adding ammonium sulphate (salt precipitation) or chilled ethanol/acetone.
5. Purification: Dialysis, ion-exchange chromatography, gel filtration, and if needed affinity chromatography are used to obtain a pure enzyme.
6. Formulation and packaging: Stabilisers (glycerol, sorbitol) and preservatives (sodium benzoate) are added; the preparation is freeze-dried or kept as a liquid concentrate and packed in sterile containers.

5. Quality Control

The final product is tested for:

• Pectinase activity (assay by measuring reducing sugar released from pectin, expressed in IU/mL or IU/mg protein).
• Protein content (Lowry/Bradford method).
• Purity (SDS-PAGE).
• Microbial contamination and stability at storage temperature.

6. Uses of Pectinase

• Clarification of fruit juices and wines
• Extraction of fruit juices and vegetable oils
• Degumming of plant fibres (retting of flax, hemp, jute)
• Coffee and tea fermentation
• Animal feed supplementation
• Wastewater treatment of pectin-rich effluents

Conclusion: Pectinase is produced industrially by submerged (or solid-state) fermentation of selected Aspergillus niger strains on pectin-rich agro-wastes, followed by filtration, concentration, precipitation, purification, and formulation. The process is economical because cheap agricultural residues serve as both carbon source and inducer.

Importance of Tissue Culture Techniques [3]

1. Rapid multiplication — A single plant or even a small explant can produce thousands of genetically identical plantlets in a short time, independent of season.
2. Production of disease-free plants — Meristem/shoot-tip culture eliminates viruses and other pathogens, giving healthy planting material (e.g., potato, banana, sugarcane, strawberry).
3. Conservation of germplasm — Rare, endangered and elite genotypes can be maintained in a small space, free from pests and pathogens, and exchanged between countries without quarantine risk.
4. Genetic improvement — Enables somaclonal variation, haploid production (anther/pollen culture), embryo rescue, protoplast fusion and genetic transformation (transgenic plants).
5. Clonal propagation of sterile/hybrid plants — Plants that do not set viable seed or are heterozygous (e.g., banana, sugarcane, orchids) can be multiplied true-to-type.
6. Secondary metabolite production — Hairy root and cell suspension cultures yield pharmaceuticals (alkaloids, taxol, shikonin) and other useful compounds.
7. Synthetic seed production — Somatic embryos can be encapsulated to give artificial seeds for storage and transport.

General Steps Involved in Tissue Culture [7]

1. Selection of explant (source tissue) A healthy, disease-free mother plant is selected. The explant may be shoot tip, axillary bud, leaf, stem, root, anther, pollen, embryo or protoplast. Meristematic tissue is preferred because it is free from viruses and divides actively.

2. Sterilization (surface disinfection) The explant and all glassware, media and instruments are sterilized to avoid microbial contamination.

• Explant: washed with detergent, then treated with 70% ethanol (30 s) followed by 0.1–1% mercuric chloride (HgCl₂) or sodium hypochlorite (5–10 min), then rinsed 3–4 times with sterile distilled water.
• Media and glassware: autoclaved at 121 °C, 15 psi for 15–20 min.
• Instruments: flame-sterilized or autoclaved. All work is done aseptically in a laminar air-flow cabinet.

3. Preparation of culture medium A nutrient medium is prepared containing:

• Inorganic nutrients — macroelements (N, P, K, Ca, Mg, S) and microelements (Fe, Mn, Zn, Cu, B, Mo, I).
• Organic nutrients — carbon source (sucrose 2–3%), vitamins (thiamine, nicotinic acid, pyridoxine, myo-inositol), amino acids (glycine).
• Growth regulators — auxins (IAA, NAA, 2,4-D) and cytokinins (kinetin, BAP) in definite proportions; auxin : cytokinin ratio decides root or shoot formation.
• Gelling agent — agar (0.8–1%) for solid medium; pH adjusted to 5.6–5.8. Murashige and Skoog (MS) medium is the most widely used. The medium is dispensed into culture vessels and autoclaved.

4. Inoculation The sterilized explant is cut into suitable pieces and transferred to the culture medium under aseptic conditions in a laminar air-flow cabinet. The vessels are then sealed with cotton plugs or cling film.

5. Incubation (callus induction and growth) The cultures are kept in a growth chamber at 25 ± 2 °C, 16 h light/8 h dark photoperiod and 50–60% relative humidity. The explant dedifferentiates to form an undifferentiated mass called callus, or directly gives shoots/buds.

6. Sub-culturing (subculture) After 3–4 weeks, growing tissues are transferred to fresh medium of the same or modified composition to maintain growth and to induce organogenesis (shoot and root formation). Repeated sub-culturing gives multiple shoots.

7. Rooting and hardening (acclimatization) Regenerated shoots are transferred to a rooting medium (high auxin, low cytokinin) to form adventitious roots. The plantlets are then removed from the culture vessels and kept in a greenhouse/glasshouse at high humidity, gradually exposed to the external environment (hardening) so they can survive field conditions.

8. Field transfer Hardened plantlets are transplanted to the field or nursery for normal growth and development.

Conclusion Tissue culture is thus a powerful biotechnological tool for rapid, disease-free clonal propagation and crop improvement, and its success depends on strict asepsis and correct hormonal balance at each step.

Enzymes लाई तिनीहरूले गर्ने जैवरासायनिक अभिक्रियाको प्रकार (biochemical function) का आधारमा IUBMB ले ६ प्रमुख वर्ग मा वर्गीकरण गरेको छ:

१. ऑक्सीडोरेडक्टेज (Oxidoreductases): एक अणुबाट अर्कोमा इलेक्ट्रोन/हाइड्रोजन सार्ने (ऑक्सीकरण–अपचयन)। उदाहरण: डिहाइड्रोजनेज, अक्सिडेज, क्याटालेज।

२. ट्रान्सफरेज (Transferases): कार्यात्मक समूह (मिथाइल, अमिनो, फस्फेट आदि) एक अणुबाट अर्कोमा सार्ने। उदाहरण: किनेज, ट्रान्सएमिनेज।

३. हाइड्रोलेज (Hydrolases): पानीको प्रयोगले बन्धन तोड्ने (जल-अपघटन)। उदाहरण: एमाइलेज, लाइपेज, प्रोटिएज।

४. लायेज (Lyases): पानी वा अक्सीकरण बिना, विलोपन (elimination) वा योग (addition) द्वारा C–C, C–O, C–N बन्धन तोड्ने वा द्वि-बन्धन बनाउने। उदाहरण: डिकार्बोक्सिलेज, एल्डोलेज, फ्युमारेज।

५. आइसोमरेज (Isomerases): एउटै अणुभित्र परमाणुहरूको पुनर्व्यवस्था गरी आइसोमर बनाउने। उदाहरण: फस्फोग्लुकोज आइसोमरेज, म्युटेज, रेसिमेज।

६. लिगेज (Ligases / Synthetases): ATP जस्ता उच्च-ऊर्जा अणुको ऊर्जा प्रयोग गरी दुई अणुलाई जोड्ने। उदाहरण: DNA लिगेज, पाइरुभेट कार्बोक्सिलेज, एमिनोएसाइल-tRNA सिन्थेटेज।

General properties of amino acids

1. Building blocks of proteins — about 20 standard α-amino acids occur in proteins, all of the general formula R–CH(NH₂)–COOH, i.e. an amino group and a carboxyl group attached to the same (α) carbon.

2. Amphoteric nature — they contain both a basic –NH₂ group and an acidic –COOH group, so they can donate as well as accept protons. Hence they act as ampholytes and show buffering action.

3. Zwitterion formation and isoelectric point — in neutral aqueous solution the –COOH loses a proton and the –NH₂ gains one, giving a dipolar ion (zwitterion), ⁺NH₃–CHR–COO⁻. At a definite pH called the isoelectric point (pI), the net charge is zero; at pH below pI the cation predominates and at pH above pI the anion predominates. At pI the amino acid has minimum solubility and does not migrate in an electric field.

4. Solubility — soluble in water and in polar solvents, but insoluble in non-polar organic solvents (benzene, ether, chloroform), because of the ionic nature of the zwitterion.

5. High melting point — they are crystalline solids with high melting (decomposition) points, again due to strong electrostatic forces between zwitterions.

6. Optical activity — except glycine (R = H), all amino acids have a chiral α-carbon and exist as D- and L-forms; natural proteins contain only L-amino acids. They are therefore optically active.

7. Chemical reactions — the –COOH group forms salts, esters and acid chlorides; the –NH₂ group undergoes acylation, alkylation and reaction with ninhydrin (giving a blue-violet colour used for detection). The two groups together give the ninhydrin test, xanthoproteic test (aromatic amino acids) and biuret test (peptides).

8. 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–); repeated condensation gives polypeptides and proteins.

9. Amphiprotic / dipolar nature in solution — they do not conduct electricity in the solid state but conduct in aqueous solution, and their melting points are not sharp because they decompose before melting.

Advantages of the Use of Microbial Enzymes in Industries

Microbial enzymes are enzymes obtained from microorganisms such as bacteria, fungi and yeasts, produced on a large scale by fermentation. They have largely replaced plant and animal enzymes in industry because of the following advantages:

1. High yield and easy mass production. Microorganisms multiply very rapidly and can be grown in cheap media inside fermenters, so enzymes can be produced in bulk within a short time, independent of season, climate or geographical location.

2. Low cost of production. Cheap raw materials such as molasses, starch and agricultural wastes are used as substrates, which reduces the cost of the enzyme and hence of the final product.

3. Easy genetic improvement. Microbes are genetically simple and can be improved by mutation, recombinant DNA technology and strain selection to obtain enzymes with higher activity, better stability and desired specificity. Example: genetically engineered Bacillus strains for detergent proteases.

4. Extracellular secretion. Many microbial enzymes (e.g., amylases, proteases, cellulases) are secreted outside the cell, so they can be recovered easily from the culture medium without breaking the cells, making downstream processing simple and cheap.

5. High stability and wide working range. Microbial enzymes are thermostable and can work over a wide range of temperature, pH and salt concentration. Example: Taq polymerase from Thermus aquaticus works at high temperature; enzymes from alkaliphiles work in alkaline detergents.

6. High specificity and purity. They act on specific substrates only, so side reactions and unwanted by-products are minimized, giving products of high purity and uniform quality.

7. Immobilization and reuse. Microbial enzymes can be immobilized on solid supports and reused many times, which further lowers cost and allows continuous industrial processes. Example: immobilized glucose isomerase in high-fructose syrup production.

8. Environment friendly. They are biodegradable, work under mild conditions of temperature and pressure, and reduce the use of harsh chemicals, thus saving energy and reducing pollution. Example: use of microbial lipases and proteases in eco-friendly detergents and in leather and paper processing.

9. Wide industrial application. Microbial enzymes are used in food and beverage, textile, leather, detergent, paper, pharmaceutical and biofuel industries — e.g., amylase in starch hydrolysis, pectinase in fruit juice clarification, cellulase in bioethanol production and penicillin acylase in antibiotic preparation.

Conclusion: Because of high yield, low cost, easy genetic manipulation, stability, specificity and eco-friendly nature, microbial enzymes are the preferred biocatalysts in modern industries.

Trickling Generator Process in Vinegar Production

The trickling generator (also called the quick vinegar process or German generator) is an industrial method for producing vinegar by the rapid oxidation of dilute ethanol to acetic acid using Acetobacter bacteria. It replaces the slow Orleans (open-vat) method and completes the conversion in a few days instead of weeks.

Construction

• A tall cylindrical wooden (or plastic/stainless steel) tank, about 2–4 m high and 1–2 m in diameter, open at the top and fitted with an air inlet at the base.
• The tank is packed with an inert, high-surface-area support material such as beechwood shavings, corn cobs, coke, or ceramic Raschig rings. This packing provides a large surface for the bacterial film and for gas–liquid contact.
• Near the top, a rotating or fixed perforated distributor (sprinkler) spreads the feed liquid evenly over the packing.
• Near the bottom, a collecting tray with an outlet draws off the finished vinegar; a side tube allows part of it to be recirculated.
• Air holes at the bottom let air rise upward (counter-current to the trickling liquid), supplying the oxygen needed for oxidation.

Working / Process

1. Preparation of feed (mash): A dilute alcoholic solution containing about 10–13 % ethanol is prepared from fruit juice, molasses, malt, or diluted distilled spirit. Nutrients (ammonium phosphate, etc.) may be added.
2. Starting culture: The generator is charged with good "mother vinegar" containing active Acetobacter aceti (or A. pasteurianus), which forms a slimy film over the wood shavings.
3. Trickling: The alcoholic mash is sprinkled from the top and trickles slowly downward over the bacterial film on the shavings.
4. Oxidation: As the liquid flows down, the bacteria oxidize ethanol to acetic acid: $\text{C}_2\text{H}_5\text{OH} + \text{O}_2 \xrightarrow{\text{Acetobacter}} \text{CH}_3\text{COOH} + \text{H}_2\text{O} + \text{heat}$ Air drawn in from the bottom rises through the packing and supplies oxygen; the large surface area and abundant air make the reaction very fast.
5. Collection and recirculation: The partially oxidized liquid collected at the bottom is recirculated (trickled again) until the alcohol is almost completely converted (residual ethanol < 0.5 %). A portion is drawn off as finished vinegar.
6. Temperature control: The reaction is exothermic, so the temperature is kept at about 25–30 °C by controlling the flow rate and air supply; overheating kills the bacteria and volatilizes the acetic acid.

Advantages

• Very rapid oxidation — vinegar is produced in 2–3 days.
• High yield and high acetic acid content (up to 10–12 %).
• Continuous, large-scale operation with low labour cost.
• Occupies less floor space than the Orleans method.

Limitations

• Requires careful control of temperature, aeration, and flow rate.
• The product is turbid and needs filtration/clarification.
• The generator is bulky and the wood shavings must be replaced periodically.

Role of Microorganisms as Biofertilizers

Biofertilizers are preparations containing living microorganisms that, when applied to soil or seed, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply or availability of primary nutrients. They are eco-friendly, renewable, and reduce dependence on chemical fertilizers. The major groups and their roles are discussed below.

1. Nitrogen-fixing microorganisms

• Symbiotic bacteria — Rhizobium: Forms root nodules in legumes (pea, bean, gram, soybean). Inside the nodule, the bacterium fixes atmospheric nitrogen (N₂) into ammonia using the enzyme nitrogenase and supplies it to the host plant in exchange for carbohydrates. It can add 40–200 kg N/ha per season.
• Symbiotic actinomycete — Frankia: Forms nodules in non-leguminous plants such as Casuarina and Alnus, fixing nitrogen in forest and wasteland soils.
• Free-living bacteria — Azotobacter, Clostridium, Beijerinckia: Fix nitrogen non-symbiotically in the soil (about 10–25 kg N/ha/year) and also produce growth-promoting substances like auxins and vitamins.
• Cyanobacteria (blue-green algae) — Anabaena, Nostoc, Oscillatoria: Fix nitrogen in flooded paddy fields, either free-living or in symbiosis (e.g., Anabaena azollae in the fern Azolla). Azolla is widely used as a green manure in rice cultivation.

2. Phosphate-solubilizing microorganisms

• Bacteria — Bacillus, Pseudomonas, Rhizobium; Fungi — Aspergillus, Penicillium: Secrete organic acids (citric, oxalic, gluconic) that dissolve insoluble soil phosphates and rock phosphate, making soluble phosphorus available to plants. They also release phosphatases that mineralize organic phosphorus.

3. Phosphate-mobilizing microorganisms

• Mycorrhizal fungi — VAM (vesicular-arbuscular mycorrhiza) and ectomycorrhiza: The fungal hyphae extend far into the soil, absorb phosphate, zinc, copper, and water, and translocate them to the host root. In return, the fungus receives carbohydrates. VAM inoculation improves phosphorus uptake, especially in phosphorus-deficient soils.

4. Other beneficial roles

• Potassium-solubilizing bacteria (Bacillus mucilaginosus) release potassium from silicate minerals.
• Plant growth-promoting rhizobacteria (PGPR) such as Azospirillum produce auxins, gibberellins, and cytokinins, and suppress root pathogens by producing antibiotics and siderophores.
• Sulphur-oxidizing bacteria (Thiobacillus) oxidize elemental sulphur to sulphate, making it available to plants.
• Organic matter decomposers (Aspergillus, Trichoderma, earthworms associated microbes) mineralize crop residues, releasing N, P, K in available forms.

Advantages: cheap, renewable, non-polluting, improve soil health and fertility, enhance crop yield by 10–20%, and reduce dependence on chemical fertilizers.

Limitations: strain specificity, short shelf life, sensitivity to high temperature, drought, and chemical fertilizers/pesticides, and slow response compared to chemical fertilizers.

Conclusion: Microorganisms thus serve as living nutrient factories — fixing nitrogen, solubilizing phosphorus and potassium, and mobilizing nutrients through mycorrhizae — making them indispensable components of sustainable and organic agriculture.

Characteristics of Vectors Used in Recombinant DNA Technology

A vector is a carrier DNA molecule into which foreign (target) DNA is inserted and which can replicate autonomously inside a host cell. The main characteristics required of a good vector are:

1. Ability to replicate autonomously (origin of replication): The vector must possess a functional ori so that it multiplies independently of the host chromosome, producing many copies per cell (high copy number is preferred).

2. Selectable marker: It must carry a selectable gene (e.g., antibiotic resistance such as $amp^R$, $tet^R$, or a nutritional marker) so that transformed cells containing the vector can be easily identified and selected.

3. Unique restriction sites (multiple cloning site): It should have single, unique recognition sites for restriction enzymes in a polylinker region, so that foreign DNA can be inserted without disrupting essential vector functions.

4. Small size: A small molecular weight makes the vector easy to isolate, manipulate, and take up efficiently by host cells; it also allows insertion of larger foreign DNA fragments.

5. Ability to accept foreign DNA: It must be able to accommodate a reasonably large insert without losing its replicative ability.

6. Suitable for screening (insertional inactivation): Insertion of foreign DNA should be detectable — e.g., loss of a marker gene function (blue–white screening using lacZ, or loss of antibiotic resistance).

7. Compatibility with the host: It must be able to enter and replicate in the chosen host (e.g., E. coli) without being degraded by host nucleases; often a shuttle vector is used for more than one host.

8. Non-transferability / stability: It should not readily transfer itself to other cells and must remain stable through generations without rearrangement or loss.

9. Promoter and regulatory sequences (for expression vectors): For protein production, the vector should contain a strong promoter, ribosome-binding site, and terminator for efficient transcription and translation of the insert.

In short: a good vector must be small, self-replicating, selectable, have unique restriction sites, accept foreign DNA, and be stable and compatible with the host.

Why water is essential for cellular life

Water makes up 70–90% of the mass of a living cell and is the medium in which almost all cellular reactions occur. Its importance follows directly from its structure: the oxygen atom is strongly electronegative, so the O–H bonds are polar and the molecule is bent (bond angle about 104.5°). This gives water a partial negative charge on oxygen and partial positive charges on the two hydrogens. The consequences are:

1. Universal solvent. Because water is polar, it dissolves ions, sugars, amino acids, nucleotides and most metabolites. Polar and ionic substances (hydrophilic) disperse in it, so reactants meet and react. Non-polar molecules (lipids, fats) are excluded and cluster together — this hydrophobic effect drives membrane formation and protein folding.

2. Medium for biochemical reactions. All metabolic reactions — glycolysis, Krebs cycle, photosynthesis, hydrolysis — take place in an aqueous cytoplasm. Water itself takes part in many reactions: hydrolysis (digestion of starch, proteins, fats) and photosynthesis, where it is the electron donor: $2H_2O \rightarrow 4H^+ + 4e^- + O_2$

3. High specific heat capacity. Water absorbs or releases a large amount of heat for a small change in temperature (about 4.2 J g⁻¹ °C⁻¹). This keeps cell and body temperature stable and protects enzymes from denaturation.

4. High latent heat of vaporisation. A large amount of heat is lost when water evaporates (about 2260 J g⁻¹ at 100 °C). This makes sweating an effective cooling mechanism in warm-blooded animals and helps leaves cool in transpiration.

5. Transport medium. Because water is a fluid solvent, it carries nutrients, gases, hormones and waste products in blood plasma, lymph, xylem and phloem.

6. Cohesion, adhesion and surface tension. Hydrogen bonding between water molecules (cohesion) and with other surfaces (adhesion) produces capillary action, which pulls water up the plant stem and helps in the surface film of water in cells.

7. Ionisation and pH buffering. Water dissociates slightly: $H_2O \rightleftharpoons H^+ + OH^-$ This gives the H⁺ and OH⁻ ions needed for acid–base balance and for reactions such as the ionisation of weak acids and bases in the cell.

8. Turgidity and structure. Water in the vacuole maintains turgor pressure, which keeps plant cells firm, supports herbaceous plants and drives cell enlargement.

9. Protection and lubrication. Water acts as a shock absorber (amniotic fluid, cerebrospinal fluid) and as a lubricant in joints and mucous membranes.

Conclusion. Water is essential because its polar structure and hydrogen bonding make it an excellent solvent, a stable temperature buffer, a reactant and a transport medium — without it, no cellular reaction, structure or homeostasis would be possible.

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 starvation or prolonged exercise.

2. Structural component of membranes — Phospholipids and cholesterol form the lipid bilayer of cell membranes, providing the permeability barrier, fluidity, and matrix for membrane proteins.

3. Thermal insulation and protection — Subcutaneous fat insulates the body against heat loss, while fat pads around kidneys, heart, and eyes act as shock absorbers and cushion vital organs.

4. Transport of fat-soluble vitamins — Lipids act as carriers and solvents for vitamins A, D, E, and K, enabling their absorption from the intestine and transport in blood via lipoproteins.

5. Source of essential fatty acids — Linoleic and linolenic acids must be supplied in the diet; they are precursors of prostaglandins, thromboxanes, and leukotrienes, and are needed for growth and skin integrity.

6. Hormonal and signalling role — Steroid hormones (cortisol, aldosterone, testosterone, estradiol) and vitamin D are synthesised from cholesterol; phosphatidylinositol derivatives serve as second messengers in cell signalling.

7. Bile salts and emulsification — Cholesterol-derived bile acids emulsify dietary fats in the small intestine and aid absorption of lipids and fat-soluble vitamins.

8. Lipoprotein and lipid transport — Chylomicrons, VLDL, LDL, and HDL transport lipids between intestine, liver, and tissues; HDL also participates in reverse cholesterol transport.

9. Cofactors and electron carriers — Lipid-derived molecules such as ubiquinone (coenzyme Q) and vitamin K function in the electron transport chain and in blood clotting respectively.

10. Waterproofing and surface protection — Waxes and cutin in plants and sebum in animals reduce water loss and protect skin, hair, and feathers.

Production of Yoghurt

Yoghurt is a fermented dairy product made from milk by the action of lactic acid bacteria. The two organisms normally used are Streptococcus thermophilus and Lactobacillus bulgaricus, often in a 1:1 ratio. The process 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 are adjusted (standardized) so that the final product has the desired body and texture. Usually the SNF is raised to about 11–12% by adding skim milk powder.

2. Clarification and homogenization. The milk is clarified/filtered to remove dirt and foreign particles. It is then homogenized at about 60–70 °C and 150–200 kg/cm² pressure so that the fat globules are broken up and the yoghurt does not show a cream layer on standing.

3. Heat treatment (pasteurization). The milk is heated to about 85–90 °C for 15–30 minutes (or 90–95 °C for a few minutes). This kills pathogens and spoilage organisms, denatures the whey proteins (which helps in water binding and gives a firm curd), and drives off dissolved oxygen, creating favourable conditions for the starter culture.

4. Cooling. The heated milk is cooled to the incubation temperature, about 42–45 °C. This is the optimum temperature for the growth of the thermophilic starter organisms.

5. Inoculation (adding starter culture). The cooled milk is inoculated with the active mixed starter culture of S. thermophilus and L. bulgaricus at about 1–2% level. The culture may be added as a bulk liquid starter or as a freeze-dried powder. The milk is mixed thoroughly so that the organisms are uniformly distributed.

6. Incubation (fermentation). The inoculated milk is filled into clean, pre-sterilized cups or containers and kept undisturbed in an incubator at 42–45 °C for about 3–4 hours. During this period the bacteria ferment lactose to lactic acid. The pH falls from about 6.6 to about 4.5, the casein micelles lose their charge and coagulate, and a firm gel or curd is formed. S. thermophilus grows first and produces acid and formic acid; L. bulgaricus then produces acetaldehyde and other carbonyl compounds that give the characteristic flavour. The two organisms thus act symbiotically.

7. Cooling and ripening. When the curd is firm and the acidity reaches about 0.8–1.0% lactic acid, the containers are removed and cooled to 4–5 °C to stop further fermentation. The product is held at this low temperature for a few hours (ripening) to develop flavour and firmness.

8. Packaging, storage and marketing. The cups are sealed, labelled with date of manufacture and expiry, and stored at refrigerated temperature (4–7 °C) until sale. Cold storage is essential, otherwise the yoghurt becomes sour and whey separates.

Flow chart:

Fresh milk → Standardization → Clarification → Homogenization → Heat treatment (85–90 °C) → Cooling (42–45 °C) → Inoculation with starter culture → Incubation (42–45 °C, 3–4 h) → Cooling (4–5 °C) → Packaging → Refrigerated storage → Marketing

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

2. Regulatory components:

• Promoter (lacP) — the site where RNA polymerase binds to initiate transcription of the structural genes.
• Operator (lacO) — a short DNA sequence between the promoter and structural genes to which the repressor protein binds and blocks transcription.
• Regulatory gene (lacI) — codes for the repressor protein, which binds to the operator in the absence of lactose (inducer) and prevents transcription. It is located upstream of the promoter and is expressed constitutively.

2. Inducer:

• Allolactose (formed from lactose) acts as the inducer. It binds to the repressor, causing a conformational change that inactivates it, thereby allowing RNA polymerase to transcribe the structural genes.

Summary: The Lac operon comprises three structural genes (lacZ, lacY, lacA), a promoter, an operator, and a regulatory gene (lacI), and it is regulated by the inducer allolactose.

(a) Biomolecules

Biomolecules are the organic molecules synthesised by living cells that are essential for the structure and functioning of life. They include carbohydrates, proteins, lipids, nucleic acids, vitamins and enzymes. They are mainly composed of carbon, hydrogen, oxygen, nitrogen, phosphorus and sulphur.

(b) Importance of immobilization in enzyme technology

Immobilization is the technique of fixing enzymes to an inert support so that they remain confined during the reaction. Its importance:

• The enzyme can be recovered and reused many times, reducing cost.
• The product is easily separated from the enzyme, giving a pure product.
• The enzyme becomes more stable against heat, pH and denaturing agents.
• Continuous operation of the bioreactor becomes possible.
• It prevents contamination of the product by the enzyme.

(c) Scope of microbial biotechnology

Microbial biotechnology uses microorganisms or their components to make useful products and services. Its scope includes:

• Production of fermented foods and beverages (curd, cheese, bread, beer, alcohol).
• Production of antibiotics, vaccines, vitamins, enzymes and organic acids.
• Production of single-cell protein and biofuel (biogas, bioethanol).
• Bioremediation of pollutants and treatment of sewage and industrial waste.
• Use in genetic engineering as hosts for recombinant DNA and gene cloning.
• Bioleaching of metals and production of biopesticides and biofertilizers.

(d) Coenzyme

A coenzyme is a non-protein organic molecule that binds loosely to an enzyme and is essential for its catalytic activity. It acts as a carrier of chemical groups, electrons or hydrogen between reactions. Examples: NAD⁺, NADP⁺, FAD, coenzyme A and ATP.

(e) Fate of pyruvate in metabolism

Pyruvate formed by glycolysis is channelled into different pathways depending on the organism and oxygen supply:

• Aerobic respiration: Pyruvate enters the mitochondrion and is oxidatively decarboxylated to acetyl-CoA, which enters the Krebs cycle and finally gives CO₂, H₂O and ATP.
• Alcoholic fermentation (yeast): Pyruvate is decarboxylated to acetaldehyde and reduced to ethanol, with regeneration of NAD⁺.
• Lactic acid fermentation (muscle, lactic acid bacteria): Pyruvate is reduced to lactic acid, regenerating NAD⁺.
• Gluconeogenesis: Pyruvate is carboxylated to oxaloacetate and used to synthesise glucose.

(f) Reducing sugars

Reducing sugars are carbohydrates that contain a free aldehyde or ketone group and can reduce mild oxidising agents such as Fehling's solution, Benedict's solution and Tollen's reagent, giving a red precipitate or silver mirror. Examples: glucose, fructose, maltose, lactose and galactose. Sucrose is a non-reducing sugar.

(g) Pasteurization of milk

Pasteurization is the process of heating milk to a high temperature for a short time and then cooling it quickly, in order to kill pathogenic microorganisms and increase the keeping quality of milk. In the holding (batch) method milk is heated at 63 °C for 30 minutes; in the HTST (flash) method it is heated at 72 °C for 15 seconds. It does not sterilise the milk, but it destroys most harmful microbes while retaining the nutritive value.

(h) Enzymes involved in beta-oxidation of lipids

• Acyl-CoA synthetase (thiokinase) — activation of fatty acid to acyl-CoA
• Acyl-CoA dehydrogenase — first dehydrogenation (FAD)
• Enoyl-CoA hydratase — hydration of the double bond
• 3-Hydroxyacyl-CoA dehydrogenase — second dehydrogenation (NAD⁺)
• Thiolase (acyl-CoA acetyltransferase) — cleavage to acetyl-CoA and shortened acyl-CoA

(i) Genetic code

The genetic code is the set of rules by which the nucleotide sequence of mRNA is translated into the amino acid sequence of a protein. A sequence of three nucleotides (a codon) specifies one amino acid. The code is triplet, degenerate, non-overlapping, comma-less and nearly universal; AUG is the start codon and UAA, UAG and UGA are stop codons.

(j) Functions of DNA polymerase

DNA polymerase is the enzyme that synthesises a new DNA strand using a DNA template.

• It adds deoxyribonucleotides to the 3′-OH end of the growing chain, so synthesis proceeds in the 5′→3′ direction.
• It selects nucleotides by complementary base pairing with the template.
• It has 3′→5′ exonuclease (proof-reading) activity, removing wrongly paired nucleotides.
• In E. coli, DNA polymerase I also has 5′→3′ exonuclease activity and removes the RNA primer, while DNA polymerase III is the main replicating enzyme.