Biotechnology Production Processes

Cheatsheet Content

Industrial Amino Acids History of L-Glutamate Discovered by Japanese chemist Dr. K. Ikeda in 1908 as a flavor enhancer (monosodium glutamate, MSG). Commercialized by Ajinomoto Co. Ltd. from vegetable proteins. Uses of Amino Acids L-Glutamate: Flavor enhancer. Glycine: Sweetener in juices. Feed Additives: Supplement essential amino acids (e.g., methionine, lysine) in animal feed to increase effectiveness and reduce environmental pollution. L-Aspartate & L-Phenylalanine: Required for Aspartame synthesis. L-Glutamate Production Key Facts First amino acid produced industrially. Uses Corynebacterium glutamicum . Wild-type strains exhibit feedback inhibition at 5% glutamic acid concentration. Production strains (mutagenesis & selection) accumulate ~30% L-glutamic acid, yielding 1 mol glutamate per 1.4 mol glucose, and are phage resistant. Biochemical Pathway Oxoglutaric acid + $NH_4^+$ + NAD(P)H + $H^+$ $\xrightarrow{\text{Glutamate dehydrogenase}}$ L-glutamic acid + NAD(P)$^+$ + $H_2O$ Control Parameters Ammonium Concentration: High concentration inhibits growth and production. Added continuously at low concentration. Oxygen Concentration: Insufficient oxygen: Poor L-glutamate production, lactic and succinic acid accumulation. Excess oxygen: $\alpha$-ketoglutarate accumulates as a by-product. pH: Maintained at 7-8 by alkali addition. Process Description Fermenters: Stainless steel stirred tank reactors (up to 500 $m^3$), aerobic operation at 30-37°C. Growth Medium: Carbon sources: Carbohydrates (glucose, sucrose). Nitrogen source: Ammonium salts, urea, ammonia (fed slowly). Inorganic salts: Mg, Mn, P, K. Trace elements: Biotin, other vitamins, amino acids, purines, pyrimidines for Corynebacteria . Yields: 60-70% L-glutamate. End Product: L-glutamate as ammonium salt in broth. Downstream Processing Cell separation, spent medium processed for product recovery. Broth passed through basic anion exchange resin; L-Glutamate binds, ammonia released (recovered via distillation). Elution with NaOH forms MSG directly. MSG crystallized, followed by decolorization and sieving for food-grade quality. Citric Acid Production Introduction to Organic Acids Various organic acids (Citric, Gluconic, Lactic, L-Ascorbic) accumulated by eukaryotic and prokaryotic microorganisms. Citric Acid Overview First discovered in lemons; intermediate of TCA cycle. Formula: 2-hydroxy-propane-1,2,3-tricarboxylic acid. Historically produced from lemons; now primarily by fermentation using Aspergillus niger . Microbial Strains & Pathways Most citric acid produced by A. niger . Mutants show tolerance to high sugar concentrations, inhibitors, low pH. Pathways involve glycolytic catabolism of glucose to pyruvate, then conversion to citrate precursors (oxaloacetate, acetyl-CoA). Regulation by Nutrient Parameters Controlled Nutrient Conditions: Excessive carbon source, $H^+$, dissolved $O_2$ are undesirable. Sub-optimal trace metals (Mn, Fe, Zn) and phosphate are critical for high yields. Sugar Type & Concentration: Crucial parameter; rapidly catabolized sugars (sucrose, maltose, glucose) yield high rates. Beet and cane molasses are common raw materials. Yields decrease below 100 g/L carbon source. Trace Metal Ions: Mn$^{2+}$, Fe$^{3+}$, Zn$^{2+}$ need to be growth-limiting for high citric acid yields. Even 2 $\mu g/L$ Mn$^{2+}$ can decrease accumulation by 20%. pH: Accumulation significant only below pH 2.5. Low pH prevents production of gluconic and oxalic acid. Dissolved $O_2$ Tension: High $O_2$ tension is essential for strong aeration and induction of alternative respiratory pathway. Nitrogen: Sources (ammonia salts, nitrates, urea) used; effect mainly in chemically defined media. Phosphate: Kept low; balance with nitrogen and trace metals important for batch cultures. Production Processes Surface Fermentation: Older, labor-intensive but still used due to lower power requirements and higher reproducibility. Carried out in aluminum trays. Submerged Fermentation: Higher efficacy, more susceptible to trace metal ions and $O_2$ variations. Uses stirred tanks or aerated tower fermenters. Koji Process: Solid-state fermentation (e.g., Japanese wheat bran process, 20% annual production). Uses potato starch or wheat bran solids. Downstream Processing for Citric Acid Filtration of culture broth, washing of mycelial cake. Lime (CaO) added to clarified solution to form calcium citrate precipitate. Precipitate filtered, treated with sulfuric acid to form citric acid and calcium sulfate (gypsum). Dilute citric acid solution decolorized with activated carbon. Ion exchange for metal ions, nitrates removal. Crystallization in vacuum crystallizers, centrifugation, drying, packaging. Citric acid monohydrate (<36°C), citric acid anhydride (>36°C). Applications of Citric Acid Food & Sugar Confectionery (21%), Beverages (45%): Pleasant taste, low toxicity. Pharmaceutical & Detergent/Cleaning Industry (8%, 19%). Stabilization: Complexes heavy metal ions (Fe, Cu) to prevent oxidation in oils, fats, ascorbic acid. Buffer: Effective over a broad pH range. Plasticizers: Citric acid esters. Salts: Trisodium citrate as blood preservative (complexes Ca), emulsion stabilizer in cheese. Lactic Acid Production Lactic Acid Isomers First isolated from sour milk in 1798. Occurs in L(+) and D(-) isomeric forms. Capital letters (L, D) indicate configuration related to glyceraldehyde. $(+)$ and $(-)$ symbols indicate direction of polarized light rotation. Racemic mixture is called D,L-lactic acid. Organisms & Biochemical Pathways First organic acid produced industrially by fermentation. Uses acid-tolerant, facultative anaerobic lactic acid bacteria. Homofermentative bacteria: Produce almost exclusively lactic acid from glucose. Used for bulk production. Heterofermentative bacteria: Produce lactic acid, acetic acid, formic acid, ethanol. Involved in food/feed preservation. Yields: Theoretical 2 mol lactic acid from 1 mol hexose (1 kg lactic acid/kg hexose). Practical yields 90-92%. Most produce only one isomer. Production Processes for Lactic Acid Microbial Strain Selection: High yields, acid tolerance, phage insensitivity. Homolactic species: Lactobacillus , Streptococcus . Rhizopus : Low nutritional requirements, produces stereochemically pure L(+) lactic acid from starch. Raw Materials: Purity is important for final purification. Low selling price necessitates careful carbon source selection. Common carbon sources: Glucose syrups, maltose-containing materials, sucrose (molasses), lactose (whey). Growth Medium: Carbon source: 120-180 g/L. Nitrogen- and phosphate-containing salts. Micronutrients, B-vitamins, amino acids (e.g., malt sprouts). Fermenter: Stainless steel (historically wood/concrete), up to 100 $m^3$. Batch operation, 45°C. Gentle stirring. pH 5.5-6.0, maintained by sterile calcium carbonate. Ammonia can also be used. Conversion Yields: 85-95% of theoretical maximum, obtained in 4-6 days. Downstream Processing for Lactic Acid First Step: Increase temperature (80-100°C) and pH (10-11) to kill organisms, coagulate proteins, solubilize calcium lactate, degrade residual sugars. Filtration: Biomass removal. Decomposition: Sulfuric acid added to obtain lactic acid. Filtration: Calcium sulfate removal. Lactic Acid Concentration. Purification: Bleaching with activated carbon, ion exchange, electrodialysis, solvent extraction, esterification. Product Quality: Edible lactic acid is colorless (50-65% lactic acid); pharmaceutical requires >90% purity. Applications of Lactic Acid Quality Property Application Technical grade Light brown, iron-free 20-80% lactic acid Deliming hides, textile industry, ester manufacture Food grade Colourless, odourless, >80% lactic acid Food additive, acidulant, sour flour/dough production Pharmacopoeia grade Colourless, odourless, >90% lactic acid, <0.1% ash Intestine treatment, hygienic preparations, metal ion lactates Plastic grade Colourless, <0.01% ash Lacquers, varnishes, biodegradable polymers Biomass as Product (Baker's Yeast) Why Baker's Yeast? Commercial bakeries, daily consumption of baked goods, flavoring industry, ethanol production (million tons/year). Saccharomyces cerevisiae : Ethanol under anaerobic conditions; baker's yeast under aerobic growth. Growth Medium Carbon source: Molasses, carbohydrates (glucose, sucrose, fructose, hydrolyzed starch). Nitrogen source: Aqueous ammonium ($NH_4OH$), ammonium salts, urea. pH control: $H_3PO_4$. Stoichiometry Material Balance: Sucrose (200g) + NH (10.3g) + $O_2$ (100.5g) + Minerals (7.5g) $\longrightarrow$ Biomass (100g) + $CO_2$ (140g) + $H_2O$ (78g) Typical Yields: $Y_{X/S} = 0.5 \ g/g$, $Y_{X/O2} = 1 \ g/g$. Max. specific growth rate: 0.6 $h^{-1}$ (doubling time 1.2 h). Optimal Conditions: 30°C, pH 6-7, high dissolved oxygen (>2 mg/L). High carbohydrate levels lead to ethanol production (inhibits growth). Reactor for Baker's Yeast Production Highly viscous broth, mechanically agitated. Fermenter volume: 50-350 $m^3$ with H/D = 3. Vigorous aeration & agitation; $O_2$ 1 mol/L-h, $k_L a$ of 600 $h^{-1}$. Fed batch mode for 20-30 hours. Centrifuge to remove cells, wash repeatedly. Filter press/rotary vacuum filters to concentrate. Dry at 45°C, 1-4 rpm, 10-15 hours. Pack 95% yeast solids. Summary of Baker's Yeast Production Seed Yeast, Nutrients, Sterilize carbon source. Seed & semi Seed Fermenter, Trade Fermenter. Wash and Cool, Cream Tank. Rotary vacuum filter, Extrude and Cut the Cake. Fluid bed dry for Instant Yeast, Pack and Distribute. Algae as Bioproduct Open System vs. Closed System (for Algae Cultivation) Open System Closed System 1000 $m^2$ open pond: 500 kg/d Plate / tubular / vertical column 0.5 m depth 1 kg needs 1000 L water High water loss 30 L batch reactor: 2.1 g/d Lower biomass yield High cost, not yet cost effective Easy bacterial contamination Fouling in walls Day-night temperature cycle Algae Harvesting Techniques Physical: Centrifugation, Gravity sedimentation, Filtration, Flotation. Chemical: Flocculation & pH. Biological: Flocculation (flocculating algae, bacteria/fungi, pH change). Cell Disruption Methods (for Microalgae) Mechanical: Shear force (bead milling, high-pressure homogenization, hydrodynamic cavitation), Wave energy (ultrasonication, microwave), Current (pulsed electric field). Non-mechanical: Heat (steam explosion, hydrothermal liquefaction, freeze drying), Biological (algicidal treatment, enzymatic lysis), Chemical (acid, ionic liquid, nanoparticles, oxidation, osmotic shock, surfactant). Uses of Algae Primary Products: Lipids, Carbohydrates, Proteins, Vitamins/Carotenoids. Secondary Products: From Lipids: Foods and Nutrient Supplements, Bio-diesel. From Carbohydrates: Foods and Nutrient Supplements, Bio-ethanol. From Proteins: Foods and Nutrient Supplements, Cosmetics. From Vitamins/Carotenoids: Medicine, Cosmetics. Antibiotics Introduction to Antibiotics Antibacterial drugs. Most important group of compounds synthesized by industrial microorganisms. Secondary metabolites produced by filamentous fungi and bacteria, especially actinomycetes. $\beta$-Lactams Contain a $\beta$-lactam ring, responsible for biological activity. Examples: Penicillins (7%), Cephalosporins (30%), Other $\beta$-lactams (15%). Other Major Classes Fluoroquinolones (24%). Macrolides (20%). Oxazolidinones, streptogramins, tetracyclines, aminoglycosides, carbapenems (4%). Classification of Antibiotics (by Biosynthetic Routes) Amino Acid Precursors: Non-ribosome synthetic pathway: $\beta$-lactams, cyclic peptides, lipopeptides. Ribosome-based synthetic route: Nisins. Sugars: Aminoglycosides. Fatty Acid Precursors (Polyketide Antibiotics): Tetracyclines, macrolides, polyenes. Penicillin Production Penicillins First microbially produced antibiotics. Submerged fermentation developed for penicillin G and V. Share a common bicyclic ring structure; $\beta$-lactam portion is responsible for biological activity. R group variations determine antimicrobial spectrum and pharmacokinetic properties. Penicillin G: Acid labile, given by injection. Penicillin V: Acid stable, given orally. Microbe Used Different penicillins by different strains of Penicillium . Fleming used Penicillium notatum . Penicillium chrysogenum : Most widely used in industry, utilizes various carbohydrates and oils. Fermenter Modern fermentations are highly efficient processes. Continuous feeding of glucose/sucrose mixtures: Promotes growth, sustains production. Continuous sterilization of media. Semicontinuous operational mode: Part of broth drawn off and processed. Fermentation variables (pH, aeration) computer-controlled. Growth in pellets; downstream processing based on whole broth recovery. Tank volumes: 100-200 $m^3$; titres >40 g/L; efficiencies >90%; costs $15-20/kg. Control Parameters for Penicillin Production Growth Medium: Designed for fast growth in batch mode with minimal pH changes. Readily available carbohydrate (glucose, sucrose), soluble nitrogen (corn steep liquor, yeast extract). Calcium carbonate or phosphates for buffering. Ammonium sulfate for additional nitrogen. Production Media: Proprietary, fine-tuned, compromise between cost and performance. Inexpensive raw materials for maximal productivity. Fed-batch fermentations to optimize cell growth and biosynthesis. Foam Control: Proteinaceous nutrients cause foaming. Oils (triacylglycerol, lard, soy, palm, peanut, rape seed) used as antifoam agents and alternative carbon sources. Automated feedback control. pH: Maintained at 6.5. Controlled within 0.1 pH units by acid (sulfuric, phosphoric) or base (caustic, ammonia gas). Can also be controlled by culture's metabolism of sugar. Dissolved Oxygen: Controlled at >2 mg/L. High viscosity of broth makes oxygen transfer difficult. Critical for maximum antibiotic production and culture viability. Ambient air used as oxygen source. Maintain >20% saturation at 1.5-2 atm pressure, high air flow to sweep out $CO_2$. $CO_2$ build-up adversely affects microorganism. Penicillin Production Process Flow Sheet A diagram illustrating the process from seed fermenter to final product, including: Rotary filter, centrifugal extractors, evaporator, precipitation, filter, screen, freeze drier. Penicillin Production Process Description Seed Fermenter: Inoculum (5x$10^3$ spores/ml) grown in stages. Vegetative growth phase for biomass development (doubles every 6 h for 2 days). Mycelium forms loose pellets for optimum yield. Production Fermenter: Carbon source fed at low rate for 6-8 days. Penicillin excreted and recovered. Rotary Vacuum Filter: Removes mycelium; efficiency affected by culture media composition. Washing: Recovered mycelium washed to remove residual penicillin. Solvent Extraction: Antibiotic recovery from cell-free medium (up to 90% yield). pH reduced to 2.0-2.5 with sulfuric/phosphoric acid. Continuous countercurrent extraction with amyl acetate, butyl acetate, or methyl isobutyl ketone at 0-3°C. Low temperature reduces damage. Ion-pair extraction at pH 5-7 (penicillin is stable). Adsorption: Pigments and trace impurities removed by activated charcoal. Precipitation: Penicillin retrieved from solvent by adding sodium or potassium acetate, precipitating as salt. Filtration: Potassium or sodium penicillin crystals separated by rotary vacuum filtration. Solvent Recovery: Solvents and other materials (charcoal) recovered for economic reasons. Purification: Penicillin crystals mixed with volatile solvent (anhydrous ethanol, butanol, isopropanol) to remove impurities. Further Processing: Mixed with procaine hydrochloride to form procaine penicillin product. Separation & Drying: Crystals collected by centrifugation, screened, and freeze-dried. Biofuels (Bioethanol & Biogas) Bioethanol Production from Molasses Molasses Storage Tank: Molasses (by-product of sugar industries) is a viscous material containing sucrose, fructose, glucose. Sterilization Tank: Molasses sterilized under pressure, then cooled. Yeast Cultivation Tank: Yeast grows in presence of oxygen, cultivated in advance. Yeast Storage Tank: Yeast (unicellular, oval, 0.004-0.010mm). pH 4.8-5, temp 32°C. Fermentation Tank: Chemical changes by invertase and zymase enzymes from yeast. Anaerobic process. Heat evolved, removed by cooling coils. Residence Time: 30-70 hours, temp 20-30°C. 8-10% alcohol (beer) produced. HCl or sulfuric acid added to obtain pH 4.5. Diluter: Molasses diluted to 10-15% sugar solution. Scrubber: By-product $CO_2$ contains ethanol vapor, recovered by water scrubbing. $CO_2$ released and utilized. Water sent back to diluter stream. Beer Still: Produces 50-60% concentrated alcohol and aldehyde. Slops removed as bottom product, concentrated by evaporation for cattle feed or waste. Slop contains proteins, sugar, vitamins. Aldehyde Still: Undesirable volatile aldehyde taken off top. Alcohol fed to decanter from side stream. Extractive distillation column (0.6-0.7 MPa). Decanter: Fusel oil (high molecular weight alcohol) recovered by decantation, fractionated to produce amyl alcohol or sold. Rectifying Column: Azeotropic alcohol-water mixture (95% ethanol) withdrawn as side product, condensed, stored. Side stream sent to decanter. Water discharged at bottom. Alcohol-water mixtures rectified to increase strength. Storage Tank: Direct sale (portable), industrial use, or to anhydrous still for 100% ethanol. Mix Tank: Denatured alcohol produced by mixing 95% ethanol with denaturant (10 vol% methanol). Ternary Azeotropic Distillation: Product from rectifying column is ternary minimum boiling azeotrope of ethanol, water, benzene (azeotropic agent). Produces anhydrous motor fuel grade ethanol (100%). Heat integration vital for energy reduction. Microbes and Substrates for Ethanol Production Feedstock Sugarcane Molasses Cassava Corn Ethanol/ton of biomass (litres/ton) 70 270 180 370 Biomass/ha of land (tons/ha) 50 12 6 Ethanol/ha of land (litres/ha) 3500 2160 2220 Comparison of fermentations (Initial sugar concentration = 100 g glucose/l) Zymomonas mobilis Saccharomyces carlsbergensis Specific growth rate $\mu \ (h^{-1})$ 0.276 0.123 Specific ethanol productivity 5.44 0.82 Cell yield 0.03 0.04 Ethanol yield (%)* (*100% = 0.511 g.g$^{-1}$) 95.00 90.00 Biogas Production Biogas Production Process Organic Matter Input: Organic waste collected and prepared for digestion. Anaerobic Digestion: Microorganisms break down organic matter in absence of oxygen. Biogas Production: Biogas (methane and carbon dioxide) produced. Digestate Formation: Nutrient-rich residue formed as a byproduct. Anaerobic Digestion (AD) Steps Hydrolysis: Breakdown of complex organic matter into simpler compounds by hydrolytic bacteria. Acidogenesis: Conversion of simpler compounds into volatile fatty acids by fermentative bacteria. Acetogenesis: Transformation of volatile fatty acids into acetic acid by acetogenic bacteria. Methanogenesis: Production of methane from acetic acid by methanogenic bacteria. Feedstock for Biogas Production Feedstock Biogas yield ($m^3/t$) Cattle slurry 15-25 (10% DM) Grass silage 160-200 (28% DM) Wheat grain 610 (85% DM) Sunflower 154-400 Crude glycerine 580-1000 (80% DM) Fats up to 1200 Potatoes 276-400 Sorghum 295-372 Barley 353-658 Peas 390 Rye grain 283-492 Wheat grain 384-426 Pretreatment Technologies for Biogas Production Pretreatment method Pros Cons Milling No inhibitors, increased methane yield (25%) Energy intensive, high maintenance cost Extrusion Increased surface area of pretreated substrate Energy intensive, high maintenance cost Steam explosion Effective hemicellulose removal, fast Inhibitors, high capital investment, lignin relocation Liquid hot water High cellulose retention, hemicellulose solubilization with low inhibitors Effectiveness, inhibitors generation Microwave Effective, reduces time, high biogas production Scale up challenges, safety concerns Acid pretreatment Effective hemicellulose solubilization, leaves cellulose and lignin Inhibitors, equipment corrosion Alkaline delignification Effective lignin solubilization, high cellulosic and hemicellulosic material Hemicellulose loss, solubilized lignin as inhibitors, corrosiveness Biogas Upgrading Technologies Biogas Composition: $CH_4$, $CO_2$, $H_2S$, $NH_3$, $H_2O$, $O_2$, $N_2$, CO, Siloxanes, Halogens. Upgrading Methods: High pressure water scrubbing, organic physical scrubbing, chemical scrubbing, membrane separation, cryogenic separation, biological technologies. Applications of Upgraded Biogas: Heat and electricity production, vehicle fuel, combined heat and power production. Microbial Polysaccharides and Lipids Introduction to Microbial Polysaccharides Microorganisms produce substantial polysaccharides with surplus carbon source. Some accumulate intracellularly as storage (e.g., glycogen). Others produce exopolysaccharides (EPS), excreted by cell, commercially valuable. Modify rheology (flow characteristics), increase viscosity, used as thickening, gelling, suspending agents. Examples: Dextran, Alginate, Xanthan gum, Gellan gum. Polysaccharides Produced by Microbes Microbe Polysaccharide Use Streptococcus mutans Dextran Wound dressing Azotobacter vinelandii , Pseudomonas Alginate Gels and encapsulation Xanthomonas campestris Xanthan gum Food Sphingomonas paucimobilis Gellan gum Food Brown algae Alginate Saccharomyces cerevisiae (Yeast) $\beta$-glucan, mannan, chitin (cell wall), glycogen Production of Microbial Polysaccharides Fermenter: Batch culture in aerated stirred tank reactors. Polysaccharide excretion increases viscosity, limiting attainable concentration and $O_2$ transfer. High power required for stirring, high heat removal cost. Medium: Polysaccharide synthesis starts during growth and continues after. Favored by high carbon/nitrogen ratio. Nitrogen source is growth-limiting, sets biomass concentration. Additional carbon source added after growth cessation. Alginate Extraction process: Chopped algae $\to$ Drying $\to$ Washing $\to$ Drying $\to$ Soaking (formaldehyde) $\to$ Soaking (HCl) $\to$ Washing $\to$ Extraction ($Na_2CO_3$) $\to$ Dissolving (water) $\to$ Filtration $\to$ Precipitation (ethanol) $\to$ Drying. Uses: Gels, encapsulation. Xanthan Gum Outer layer of inactive Xanthomonas campestris . Holds food particles together, affects viscosity (rheology). Used in gluten-free baking. Pentasaccharide repeating unit. Production process: Fermentation (growth, production), ultrafiltration, alcohol precipitation, centrifugation/filtration, drying. Operating conditions: 20,000 tons/year, 50-200 $m^3$ fed batch, 28-30°C, aerobic, pH 7 (if acidic, decreases). Exponential growth and stationary phase. 100-110°C for 10 min kills. Carbon sources: Starch, starch hydrolysates, corn syrup, molasses, glucose, sucrose (10 C : 1 N). Low cost: cereal grain, dry milled corn starch. Nitrogen sources: Ammonium salts, urea, corn steep liquor, $MgCl_2$. Microbial Lipids – Single Cell Oils (SCOs) What are SCOs? Lipids defined by solubility in non-polar organic solvents (hexane, diethyl ether) and insolubility in water. Microbial lipids produced by microorganisms via fermentation (fatty acyl lipids, triacylglycerol lipids - TAG). Also called single cell oils or microbial oils. Used for human consumption. Fatty acid synthesis is fundamental to living cells; obligate parasites are exceptions. Conditions of Production Significant TAG accumulation does not occur during active growth. Occurs after depletion of critical nutrients (usually nitrogen), while carbon remains available. SCO fermentations include active growth phase for biomass, followed by restricted growth (nutrient depletion, C source available) for TAG production. Lipid Extraction Methods Physical/Mechanical: Decompression, bead milling, supercritical fluids, ultrasound, high-pressure homogenization, osmotic shock, microwave treatment, pulsed electric field (PEF), supercritical fluid extraction. Chemical: Bligh-Dyer, Folch, Soxhlet. Enzymatic: Cell wall lysis. Methods vary for intracellular lipid-rich biomass, extracellular lipid-rich biomass, and low-lipid biomass.