Env. Biotechnology - Scragg

Cheatsheet Content

### How to Use This Cheatsheet This cheatsheet summarizes key concepts, formulas, and reactions from "Environmental Biotechnology" by Alan Scragg. Each numbered section corresponds to a chapter in the book. #### 1. Navigate by Chapter Sections are numbered to match the book's chapters. Use the headings to quickly find relevant topics. #### 2. Focus on Key Information Definitions, core principles, formulas, equations, and important derivations are highlighted. #### 3. Review Notations and Constants A dedicated section at the end explains common notations and constants used throughout the cheatsheet. ### 1. Overview #### 1.1 Introduction to Biotechnology - **Definition (General):** Application of biological organisms, systems, or processes to manufacturing or services. - **Definition (European Federation of Biotechnology, 1988):** "The integrated use of biochemistry, microbiology, and engineering sciences in order to achieve applications of the capabilities of microorganisms, cultured animal or plant cells or parts thereof in industry, agriculture, health care and in environmental processes." - **Houwink (1989):** "The controlled use of biological information." - **Multidisciplinary Nature:** Combines microbiology, molecular biology, cell biology, engineering. Illustrated by the "hourglass model" (Fig. 1.1 in text). #### 1.1.1 Environmental Biotechnology - **Definition:** Specific application of biotechnology to environmental problems, including waste treatment, pollution control, and integration with non-biological technologies. - **Market:** Estimated $300 billion worldwide (1997), $84-94 billion in Europe (1995). - **Key Applications:** Monitoring pollution, treating waste, remediating polluted sites, preventing pollution. #### 1.1.2 Biotechnology Past (Historical Eras) - **Pre-Pasteur Era (before 1865):** Empirical use of microorganisms for fermented foods (alcoholic beverages, dairy products). - **Pasteur Era (1865-1940):** Controlled use of selected microorganisms for specific products (e.g., microbial production of lactic acid, acetone/butanol by *Clostridium acetobutylicum*). - **Acetone Production:** Used for smokeless explosive cordite in WWI. 12 tonnes acetone per 100 tonnes molasses. Replaced by petrochemicals in 1950s. - **Sewage Treatment:** Biological treatment started in 1914 (filter beds, activated sludge). - **Antibiotic Era (1940-1960):** Mass production of penicillin (*Penicillium chrysogenum*) using submerged deep fermentation. Led to production of other antibiotics, animal cell culture (virus vaccines), and microbial steroid transformations. - **Post-antibiotic Era (1960-1975):** Production of amino acids, single cell protein (SCP), enzymes (detergents), immobilized enzyme/cell technology, anaerobic wastewater treatment (biogas), bacterial polysaccharides (xanthan), gasohol. - **SCP (Single Cell Protein):** High-protein animal feed using microorganisms (e.g., ICI's Pruteen from *Methylophilus methylotrophus* on methanol). Declined due to rising methanol prices, increased competitor production, and the Green Revolution. *Quorn* (human food from *Fusarium graminarium*) is a successful SCP product. - **Immobilized Enzymes:** Extended active life, continuous processes (e.g., glucose isomerase for high-fructose syrups). - **Biofuels:** Ethanol (from sugarcane fermentation in Brazil) and methane (biogas from anaerobic digestion). #### 1.1.3 Biotechnology Present (New Biotechnology Era: 1975-) - **Key Developments:** Genetic engineering (recombinant DNA technology) and monoclonal antibodies. - **Genetic Engineering:** Isolation, manipulation, and expression of genes across species barriers. - **Types:** 1. Insertion of single gene for new characteristic (e.g., herbicide resistance). 2. Alteration of existing genes (e.g., delayed fruit ripening in Flavr Savr™ tomato). 3. Insertion of gene for specific product (biopharming, e.g., human insulin in *E. coli*). - **Applications:** Medical (human insulin, growth hormone), agriculture (herbicide/insect resistant crops), food (chymosin from transgenic yeast for cheese). - **Public Concern (GMOs):** Distrust due to profit motivation, perceived unnaturalness, unknown risks, "playing God" notion, and labeling issues (e.g., "frankenfood"). #### 1.2 Environmental Biotechnology - **Context:** Increased public awareness due to disasters (Chernobyl, Seveso, Bhopal, Exxon Valdez) and global issues (greenhouse gases, ozone depletion). - **Exxon Valdez (1989):** 11 million gallons crude oil spilled in Alaska. - **Seveso (1976):** Dioxin release from chemical factory. - **Bhopal (1984):** Methylisocyanate release, 3300 deaths. - **Global Warming:** Increase in greenhouse gases (CO₂, O₃, CFCs, CH₄, N₂O) trapping solar radiation. - **Ozone Depletion:** CFCs reducing ozone layer, increasing UV radiation. #### 1.2.1 Legislation - **UK:** Public Health Act (1848), Environmental Protection Act (1990), Environmental Act (1995). - **Definitions:** "Environment" (air, water, land), "Pollution" (release of harmful substances). - **USA:** Clean Air Act, Clean Water Act, Hazardous Waste Acts (1977-1982), Maritime Pollution Treaty (1994). - **International:** UN Conference on Environment and Development (UNCED), Rio (1992) – Conventions on Biodiversity and Climate Change. Montreal Protocol (1987) – phasing out CFCs. Kyoto (1997) – ratified agreements. - **Principles:** Access to environmental information, precautionary principle, "polluter pays." #### 1.2.2 Integrated Pollution Control - **Aim:** "To prevent, minimise or render harmless releases of prescribed substances using the best available technology not entailing excessive cost (BATNEEC)." - **Concept:** Pollutants transfer between air, land, and water; requires holistic assessment of environmental impact. #### 1.3 Pollution - **Diversity:** Organic, inorganic, biological, gaseous (Table 1.3 in text). - **EU "Black List":** Organohalogen, organophosphorus, organotin compounds, mercury, cadmium, persistent mineral oils/hydrocarbons, persistent synthetic substances (Table 2.1 in text). - **EU "Grey List":** Less dangerous chemicals (Table 2.1 in text). - **US EPA Priority List:** 129 pollutants (Table 2.2 in text). - **Fate:** Depends on properties and conditions (Fig. 1.3 in text). - **Organic Wastes:** Biodegradable (domestic, agricultural), generally do not accumulate. - **Inorganic Wastes:** Phosphate, nitrate (e.g., from agricultural run-off) can cause eutrophication. - **Eutrophication:** Nutrient enrichment causing increased algae/macrophyte production, water quality deterioration. #### 1.3.1 Industrial Pollution - **Sources:** Defunct industries, current waste releases (estuaries, rivers, lakes). - **Metals:** Not degraded, but bioaccumulated by organisms (Box 1.2 in text). - **Xenobiotic Compounds:** Synthetic chemicals not found in nature, often persistent (e.g., many on EPA list). - **Persistence Factors:** Structure (solubility, toxicity), halogen atoms (type, position, number). - **Examples:** Polychlorobiphenyls (PCBs), trichlorophenol, dichlorodiphenyltrichloroethane (DDT) (Fig. 1.4 in text). - **Solubility vs. Bioaccumulation:** Lower water solubility often means higher lipid solubility and accumulation in fatty tissues (Fig. 1.5 in text). - **Petrochemical Wastes:** Oil, petrol, diesel (tank leakage, marine spills). Degraded by natural microorganisms. #### 1.3.2 Gaseous Pollution - **Components:** Particulates, volatiles (VOCs), gases (CO₂, SO₂, NOₓ, CH₄). - **Sources:** Industrial processes, fossil fuel burning. - **CFCs:** Destroy ozone layer (Montreal Protocol 1987). - **SO₂ & NOₓ:** From power stations, cause acid rain (reduces pH of water/soil, kills plants, corrodes buildings). - **Reduction Strategies:** Less fossil fuel, low-sulfur fuels, improved combustion, flue gas desulfurization (e.g., with limestone). #### 1.3.3 Greenhouse Gases - **Sources:** Burning fossil fuels (oil, coal, gas). - **CO₂:** Released from fossil fuels (fixed millions of years ago). Oceans and plants act as sinks. Increased atmospheric CO₂ traps heat, causing global warming. - **Biological Fuels:** Carbon dioxide neutral alternative to fossil fuels. #### 1.4 Biotechnological Treatment of Pollution - **Role:** Part of an integrated approach. - **Areas:** - **Environmental Monitoring:** Detecting chemicals, concentrations, risks. - **Bioremediation:** Biological treatment/removal from environment (e.g., polluted sites). - **Reduction/Removal of Current Wastes:** "End-of-pipe" solutions (e.g., metal removal, reed beds, biofilters). - **Prevention of Pollution (Clean Technology):** Greener processes, replacing chemical synthesis with microorganisms/enzymes, biodegradable plastics, biofuels. ### 2. Environmental Monitoring #### 2.1 Introduction - **Definition of Pollution (Holdgate, 1979):** "Introduction into the environment of substances or energy liable to cause hazards to human health, harm to living resources and ecological damage, or interference with legitimate uses of the environment." - **Pollutant Types:** Inorganics (metals, nitrates), organics (sewage, petrochemicals, synthetics), biological (pathogens), gaseous (volatiles, gases, particulates). (Table 1.2, Chapter 1 in text). - **EU "Black" and "Grey" Lists, US EPA Priority List:** Categorize dangerous compounds (Table 2.1, 2.2 in text). - **Monitoring Need:** Essential for enforcing legislation and managing contamination levels, especially for very low levels due to biomagnification/bioaccumulation. - **Biotechnology's Role:** Offers accurate, reliable monitoring techniques, especially with recombinant DNA technology and biosensors. #### 2.2 Sampling - **Challenge:** Environmental samples (soil, air, water) are rarely homogeneous. Contamination can be localized, intermittent, or poorly mixed. #### 2.2.1 Land (Site) Sampling - **Method:** Surveys often required due to poor documentation of site history. - **Guidelines (BSI, 1988):** 17 samples for 5-hectare site (3 depths), 30 samples for ### 3. Sewage Treatment #### 3.1 Introduction - **History:** Developed 1910-1914 (Manchester, continental Europe). Earlier, sewage buried or discharged into rivers. Industrialization led to excessive pollution, disease (typhoid, cholera). - **Impact:** Sewage disposal crucial for public health, reducing waterborne diseases. - **Pollution Effects:** - **Organic Material Addition:** Increases aerobic microbial growth, depletes dissolved oxygen (BOD sag - Fig. 3.1 in text). If too high, leads to anaerobic conditions, decline of aerobes, increase of anaerobes. - **Anaerobic Conditions:** Slower degradation, organic material buildup, death of aquatic organisms, production of H₂S and CH₄. - **Stagnant Waters:** Lakes/ponds become anaerobic faster. - **Domestic Waste:** Liquid (sewage) or solid. - **Sewage Sources:** Private homes, commercial buildings, institutions (may include industrial wastewater). Industrial waste often pre-treated. - **Composition:** 99.9% water, dissolved organic material, suspended solids, microorganisms (pathogens), other components (Table 3.1 in text). - **Organic Content:** 75% of suspended solids, 40% of dissolved material. Proteins, carbohydrates, fats, detergents. - **Inorganic Components:** Na, Ca, Mg, Cl, sulfates, phosphates, bicarbonates, nitrates, ammonia, trace heavy metals. - **Strength:** Expressed as BOD₅ (200-600 mg/l). Industrial/agricultural wastes can be much higher (up to 50,000 mg/l). - **Chemical/Physical Pollution:** - **Chemical:** Industrial acids, alkalis, toxic compounds. - **Physical:** Warm water from power stations, changing water temperature, encouraging new/excessive organism growth. #### 3.2 Function of Waste Treatment Systems - **Main Function:** Reduce organic content for safe river/coastal discharge, prevent nutritional pollution (especially for drinking water sources). - **Additional Goals:** Remove suspended matter, reduce pathogens, remove nitrates, heavy metals, man-made chemicals. - **Effluent Quality:** Depends on receiving water volume/condition, dilution capacity, downstream abstraction. - **Standard:** 20:30 standard (20 mg BOD₅/l, 30 mg/l suspended solids) for 8:1 dilution. (Table 3.2 in text for drinking water standards). - **Volume (UK):** ~18 million m³/day (domestic, industrial, rainfall run-off). Each person ~230 liters/day. - **Challenge:** Very dilute growth medium, low value per tonne compared to other biotechnological processes. #### 3.3 Treatment (Four Stages - Fig. 3.2 in text) #### 3.3.1 Preliminary Treatment - **Purpose:** Removal of large debris and grit. - **Process:** Screens collect debris (often macerated/broken up and returned). Grit (from roads) removed in grit channel (can be recycled). #### 3.3.2 Primary Treatment - **Purpose:** Remove easily flocculated suspended solids. - **Process:** Sewage settles for 1.5-2.5 hours. - **Result:** Reduces BOD₅ load by 40-60%. #### 3.3.3 Secondary Treatment - **Purpose:** Remove dissolved organic materials and remaining suspended solids (40-50%). - **Mechanism:** Biological action (aerobic or anaerobic, aerobic is faster and more common). - **Processes:** Lagoons/ponds, trickling filters, activated sludge, rotating biological contactors, anaerobic digesters. #### 3.3.4 Tertiary Treatment - **Purpose:** Remove phosphates, nitrates, pathogenic microorganisms to produce potable water and prevent eutrophication. - **Processes:** Chemical precipitation, disinfection (chlorine), sand filtration, maturation ponds. #### 3.3.5 Lagoons or Ponds (Fig. 3.3 in text) - **Historical Use:** Before controlled microbiology. Popular where land/sunshine abundant. - **Operation:** Aerobic, anaerobic, or mixed. - **Facultative Ponds:** Shallow (1-2.5m). Combines aerobic (surface, oxygen from diffusion/algae) and anaerobic (bottom, where solids settle, broken down to CH₄, N₂, CO₂ – 30% BOD load). - **Parameters (Table 3.3 in text):** 7-50 days retention, 70-95% BOD removal. - **Aerobic Ponds:** Shallower ( 300 mg/l BOD). Deeper (1-7m), high BOD load. - **Retention:** 2-160 days, 70-80% BOD removal. - **Purification Lakes:** Large lakes for river pollution removal (large maturation ponds). - **Aeration Lagoons:** Primary treatment. Deeper (3-4m), mechanical oxygen supply (diffusers, surface aerators). - **Combined Use (Fig. 3.5 in text):** High BOD wastes (anaerobic lagoon → facultative pond → maturation pond). Settled sewage can go directly to facultative pond. #### 3.3.6 Trickling Filter (Fig. 3.6 in text) - **Principle:** Biofilm (mixed microbial culture) attached to solid medium (randomly packed). Metabolizes organic content. - **Design:** Bed (1-3m deep) of stones, clinker, slag (40-60mm diameter). Gaps allow air penetration. Rotating distributor applies wastewater. Treated water collected at base. - **Biofilm:** Complex mixture (bacteria, fungi, protozoa, algae, insects, worms). Dynamic equilibrium, affected by waste/season. Layers detach, collected in settling tank. - **Advantages:** Widely used, low maintenance, economical, tolerant to waste changes. - **Improvements:** Plastic solid phase (random/modular) increases surface area, allows higher BOD load (Table 3.4 in text). - **Modifications:** Recirculation, two-stage systems (first filter high-rate, second for final treatment), alternating filters. #### 3.3.7 Activated Sludge Process (Fig. 3.7 in text) - **Principle:** Waste contacted with high concentration of microorganisms (flocs) under aerobic conditions. - **Process:** Primary effluent into aerated tank (plug flow). Biomass metabolizes organics, forms more cells. Effluent to settling tank; biomass/solids settle. Clarified effluent discharged. - **Recycling:** Some settled biomass (20%) recycled to maintain high biomass concentration. - **Tank Design:** Rectangular, 6-10m wide, 30-100m long, 4-5m deep. #### 3.3.8 Mixed Liquor Suspended Solids (MLSS) and Sludge Residence Time - **Activated Sludge Population:** Less heterogeneous than trickling filter, but similar organisms. Mostly flocs (20-1000 µm). - **MLSS:** Biomass concentration in tank (1500-3500 mg/l), controlled by recycling. - **Performance Comparison:** - **Trickling Filters:** Hydraulic load (m³/m³/d), Organic load (kg BOD/m³/d). High load >3 m³/m³/d. BOD load 99% removal). #### 3.5 Removal of Nitrogen Compounds - **Sources:** Ammonia, proteins, amino acids (sewage), nitrates (agricultural run-off). - **Ammonia:** Offensive smell, poisonous to aquatic life (>0.5 mg/l), increases chlorine dosage for drinking water. EC limit 50 mg/l nitrate. - **Microbial Role:** Microorganisms use nitrogen compounds for protein synthesis (ammonia, nitrates, urea). 1/3 of total nitrogen removed by assimilation. #### 3.5.1 Nitrification - **Process:** Oxidation of ammonia to nitrate. - **Organisms:** Chemoautotrophic bacteria use ammonia oxidation for energy. - **Stage 1 (Ammonia to Nitrite):** *Nitrosomonas*, *Nitrosococcus*, *Nitrosospira*, *Nitrocystis*, *Nitrosogloea*. - **Reaction:** $2NH_4^+ + 3O_2 \rightarrow 2NO_2^- + 4H^+ + 2H_2O + \text{(energy 480-700 kJ)}$ - **Characteristics:** Releases H⁺ (drops pH), requires good O₂ supply, slow growth (µmax 0.46-2.2/d), low cell yield. - **Stage 2 (Nitrite to Nitrate):** *Nitrobacter*, *Nitrocystis*, *Nitrosococcus*, *Nitrosocystis*. - **Reaction:** $2NO_2^- + O_2 \rightarrow 2NO_3^- + \text{(energy 130-180 kJ)}$ - **Characteristics:** Less energy released, lower cell yield than Stage 1, slow growth (µmax 0.28-1.44/d). - **Wastewater Treatment Implications:** Organic load balanced for slow growth, significant O₂ requirement (4.2g O₂/g NH₄⁺), buffering needed due to acid production. #### 3.5.2 Denitrification - **Problem:** High nitrate levels (>50 mg/l) in waterways cause methaemoglobinaemia in infants, potential for carcinogenic nitrosamines. - **Methods:** Ion exchange (sulfate binds preferentially to nitrate, regenerated with NaCl, producing high-salt waste), biological processes. - **Biological Process:** Conversion of nitrate to nitrite then nitrogen gas. - **Conditions:** Low/absent oxygen (anaerobic), organic carbon source (electron donor, e.g., methanol), nitrate level ≥2 mg/l, pH 6.5-7.5. - **Organisms:** Facultative heterotrophic microorganisms in sewage. - **Methanol Reactions:** - $3NO_3^- + CH_3OH \rightarrow 3NO_2^- + CO_2 + 2H_2O$ - $2NO_2^- + CH_3OH \rightarrow N_2 + CO_2 + H_2O + 2OH^-$ #### 3.5.3 Nitrification and Denitrification Processes - **Integration:** - **Parallel:** Nitrification can occur with organic removal if HRT is adequate (Table 3.5 in text). - **Sequential:** Denitrification requires aerobic to anaerobic shift and carbon source. - **System Designs:** - **Single Vessel (Fig. 3.15 in text):** Anoxic zone at start of aeration tank (stop aeration, high carbon). - **Separate Reactors (Fig. 3.16 in text):** Easier control, suspended or fixed film cultures. - **Sequencing Batch Reactor (SBR):** Single vessel, programmed sequence (feeding, anaerobic, aerobic, settling, effluent removal). Used for agricultural run-off, landfill leachates, combining N/DN (Fig. 3.17 in text). #### 3.6 Sludge Treatment - **Problem:** Large volumes of primary sludge + excess secondary sludge (activated sludge) produced (e.g., ~1 million tonnes dry weight/year in UK). - **Activated Sludge:** Microbial biomass (~50% yield). 20% recycled, rest combined with primary sludge. - **Trickling Filters:** Less sludge but no recycle. - **Characteristics:** 1-4% solids content, mixture of organic material and microbial cells. - **Disposal Methods:** - **Dumping at Sea:** Banned in UK by 1998 (EC Urban Wastewater Treatment Directive 91/271/EEC). - **Landfill:** 16% in UK (co-disposed with domestic wastes), 62% in USA (1993). Many sites closing. - **Incineration:** 4% in UK. Expensive capital costs, ash disposal needed. Autothermic incineration (sludge generates heat - Fig. 3.18 in text) more attractive. - **Spray Irrigation/Agricultural Disposal:** 67% in UK. Requires biological/chemical treatment to reduce pathogens (pasteurization, anaerobic digestion, thermophilic aerobic digestion, composting, alkali stabilization, liquid storage, drying/storage). Restrictions on crops grown. - **Drying:** Sludge conditioned (Fe³⁺, Al³⁺, polyelectrolytes) to improve settling before dewatering (drying beds, filtration, centrifugation). - **Composting:** Covered in Chapter 4. - **Heavy Metals:** Sludge accumulates metals. Limits set for metals in sludge and soil (Table 3.6 in text). #### 3.7 Anaerobic Digestion - **Purpose:** Treat excess sludge, high BOD industrial wastes. - **Advantages:** Less biomass/sludge, produces methane (biogas), no aeration, enclosed (less smell). Methane used as fuel (1.16 x 10⁷ kJ/1000 tonnes COD removed). - **Disadvantages:** Requires good mixing, 37°C, high BOD (1.2-2 g/l), long retention (30-60 days). Not suitable for normal sewage. - **Stages (Fig. 3.19 in text):** Complex, involves three groups of organisms. 1. **Hydrolytic Phase:** Fats, proteins, carbohydrates hydrolyzed to fatty acids, alcohols, ketones (*Clostridium, Eubacterium, Bacterioides*). 2. **Acidifying Phase:** Fatty acids, amino acids, sugars converted to acetate, CO₂, H₂ (*Peptococcus, Propionibacterium*). 3. **Acetogenic Phase:** Organic acids converted to acetate, CO₂ (*Syntrophobacter, Desulfovibrio, Syntrophomonas*). 4. **Methanogenic Phase:** Methane formation by methanogenic bacteria (*Methanobacterium, Methanobacillus, Methanococcus, Methanosarcina*). Oxygen-sensitive. - **Reactions (Fig. 3.20 in text):** - $4H_2 + HCO_3^- + H^+ \rightarrow CH_4 + 3H_2O$ - $CH_3COO^- + H_2O \rightarrow CH_4 + HCO_3^-$ #### 3.7.1 The Anaerobic Process - **Early Digesters:** Simple sealed vessels, unmixed/unheated. - **Contact Process:** Waste mixed with recycled anaerobic sludge, sealed vessel (30-37°C). Digestion 30-60 days. Mixture separated, liquid for further treatment, biomass returned. - **Problem:** Methanogens (strict anaerobes) exposed to aerobic environment during discharge. - **Continuous Flow Systems:** Avoid disturbance. - **Upflow Sludge Blanket (Fig. 3.22a in text):** Waste introduced at bottom, contacts sludge, rapid reaction. Mixing by flow/gas. Biomass, liquid, gas separated at top. High loading rate, COD removal (Table 3.7 in text). - **Fixed-Film / Immobilized Biomass (Fig. 3.22b in text):** Porous material (plastics, gravel, sand) immobilizes microorganisms. Operated upflow/downflow. Fixed-bed reactor (waste up through coated particles) suffers from channelling/clogging. - **Fluidized Bed Reactor (Fig. 3.22c in text):** Cells immobilized on sand particles, fluidized by upflow of waste. - **Two-Stage Process:** Separate acidifying and methanogenesis stages (different conditions/flow rates). #### 3.8 Conclusions - **Aerobic Treatment:** Will continue, with engineering improvements for mixing/aeration. - **Recombinant DNA Technology:** Increased use to study population dynamics in digesters. - **Landfill:** Better management for gas production/leachate recycling. ### 4. Clean Technology, Domestic, Industrial and Agricultural Wastes #### 4.1 Introduction - **Pollutants:** Inorganic, organic, biological, gaseous (Table 1.3, Chapter 1). - **Organic Wastes:** - Biodegradable (biological sources). - From petrochemical industry. - Synthetic chemicals. - **Chapter Focus:** Treatment/disposal of biological organic wastes (domestic, agricultural, industrial), and waste reduction/elimination via clean technology. #### 4.2 Clean Technology - **Traditional Approach:** "End-of-pipe" treatment; often transfers pollution. - **Waste Management Hierarchy:** Waste minimization/pollution prevention (top) → Recycling → Treatment → Disposal. - **Clean Technology Forms:** - **Procedural Changes:** Loss prevention, materials handling, plant improvements (less affected by biotech). - **Technology Changes:** Process engineering alterations, operational settings, automation. - **Material Input Changes:** Less toxic chemicals, material recycling. - **Biotechnology Applications:** - **Process Changes:** Replacing chemical methods with microorganisms/enzymes (including genetically engineered organisms). - **Integrated Pest/Crop Management (IPM/ICM):** Reduce agrochemical use. - **Biological Control:** Use biological materials for pest/disease control. - **Biodegradable Plastics:** Production by microorganisms. - **Biological Desulfurization:** Coal and oil (Chapter 5). - **Biofuel Production:** (Chapter 6). #### 4.2.1 Process Changes - **Microorganisms Replacing Chemicals:** - **Acetone/Butanol Production:** Biological process (1914) replaced petrochemicals (1950s). Potential for reintroduction with recombinant technology (Girbal and Soucaille, 1998). - **Indigo Dye Production:** *Pseudomonas* enzyme (naphthalene dioxygenase) converted indole to indoxyl, then indigo. Gene cloned into *E. coli* (Bialy, 1997). Engineered *E. coli* with 15 new genes for high yields, cleaner process than chemical synthesis. - **Enzymes Replacing Chemicals:** Fewer side-products, lower energy (lower temperatures). - **Leather Processing:** Hair/fat removal. - **Textile Processing:** Starch removal, bleach neutralization. - **Paper Processing:** Enzymatic (xylanase) bleaching reduces chlorine use (Box 4.1 in text). - **Detergents:** Lipases, proteases (40°C wash), cellulases (restore cotton color). - **Food Processes:** Starch to sugar (amylase). - **Adhesives:** Phenol oxidase from mold (stronger, water-based). - **Animal Feed:** Phytase breaks down plant phytates (improves phosphorus/metal availability, reduces excretion). - **Extremophiles (Box 4.2, Fig. 4.2 in text):** - **Archaea:** Grow in extreme pH, salinity, pressure, temperature. - **Hyperthermophiles:** Produce thermostable enzymes (Table 4.1 in text) – extend activity, function at higher T, longer shelf-life. - **Applications:** PCR (temperature-tolerant DNA polymerase). - **Engineering:** Genes cloned into mesophiles, or used to alter mesophilic enzymes for stability. #### 4.2.2 Integrated Pest Management (IPM) - **Goal:** Reduce agrochemical use (pesticides, herbicides). Shift to less persistent chemicals. - **Strategy:** Combines agrochemicals with biological/cultural methods for crop protection. - **Factors:** Crop rotation, cultivation techniques, resistant crops, better prediction methods, accurate diagnosis, timing of agrochemical addition, specific agrochemicals (e.g., natural pyrethrins), biological control, semiochemicals. - **Resistant Crops:** Biotechnology can produce (Chapter 8). - **Biosensors/Detection Systems:** Recombinant technology for early detection. - **Semiochemicals:** Chemical signals modifying animal/plant behavior (e.g., aphid sex pheromones, antifeedants like ajugarin I - Fig. 4.3 in text). Biotechnology can produce complex molecules in bulk, or engineer plants to produce them (e.g., Dimboa in maize for corn borer resistance). #### 4.2.3 Biological Control - **Definition:** Use of biological material (natural predators, pheromones, resistant plants, sterile insects) for pest control instead of chemicals. - **Advantages:** Reduces agrochemicals, limits environmental effects. - **Limitations:** Slower than chemical treatments, limited success with commercial crops. - **Biotechnology's Role:** Insect-resistant plants (Chapter 8), biopesticides. - **Biopesticides:** No toxic residues, low pest resistance, reduced secondary pests/resurgence. - ***Bacillus thuringiensis* (Bt):** Gram-positive soil bacterium, produces endotoxin (protoxin) toxic to caterpillars (Table 4.2 in text). Toxins cleaved in insect gut, bind to epithelial cells, kill insect. Used commercially. - **Other Biopesticides:** Genetically engineered baculoviruses, fungi (for weeds), fungi/bacteria (for plant pathogens/nematodes, e.g., *Streptomyces lydicus* WYEC108 for chitinase activity). - **Concerns:** Risks to natural diversity, danger of exotic species introduction. FAO code of conduct for biological control agents. Overuse can lead to resistance. #### 4.2.4 Microbial Biopolymers - **Problem:** Plastics (up to 29% of solid domestic waste) are synthesized from petrochemicals, not biodegradable. MARPOL banned sea disposal. - **Solution:** Microorganisms produce polymers (storage materials) with plastic-like properties. - **Poly(R)-(3-hydroxybutyrate) (PHB):** Found in >50 bacterial species (e.g., *Acaligenes eutrophus*). Accumulated when deprived of N, P, Mg, S (up to 90% dry weight). - **Properties (Table 4.3 in text):** Similar to polypropylene (melting point, density, tensile strength), but biodegradable. - **Copolymer (PHB/3-hydroxyvalerate - PHB-V):** More amenable to processing, lower melting point. Marketed as Biopol (Zeneca). - **Cost:** $5-15/kg vs. $1/kg for petroleum plastics (due to medium, bioreactor, purification costs). - **Genetic Engineering:** Genes for PHB production cloned into *E. coli* (90% dry weight PHB) and *Arabidopsis thaliana*. - **Plants:** *A. thaliana* accumulated PHB but grew slowly (drain of acetyl CoA). Directing enzymes to specific tissues (e.g., plastids) could reduce this. Plant production cheaper than bioreactor. - **Other Biopolymers:** Lactic acid-based (from *Lactobacillus*), protein-based (e.g., spider silk). #### 4.3 Recycling - **Position:** Second option in waste reduction hierarchy. - **Involves:** Recycling materials produced during manufacturing or from products after use (Fig. 4.1 in text). - **Benefits:** Saves energy (e.g., 95% for metals/glass), reduces raw material use. - **Focus:** Metals, glass, paper (major component of solid domestic waste). #### 4.4 Domestic Wastes - **Definition:** Solid refuse from households/institutions (glass, metal, paper, organic material - Table 4.4 in text). - **Trends:** Shift from landfill to recycling/combustion/composting (USA 1985-1993). Landfill still dominant. - **Reduction:** Recycling glass, metal, paper; reduced packaging. #### 4.4.1 Landfill (Fig. 4.4 in text) - **Traditional:** Burial. Early sites unsealed, leachate contaminated groundwater. - **Modern:** Suitable sites (geology, non-porous substratum, away from habitation). Stricter regulations. - **Construction:** Lined with impermeable barrier (clay, plastics). Waste filled in cells/terraces (Fig. 4.5 in text). - **Compaction:** By vehicles; covers waste with soil/ash daily. Controls air/water, reduces volume, prevents spontaneous combustion. - **Gas/Leachate Collection:** Horizontal trenches/perforated pipes for gas extraction, leachate collection (Fig. 4.6 in text). - **Capping:** Top sealed with clay, impervious lining, drainage layer, soil. Prevents rainwater entry, reduces leachate. - **Post-Capping:** Organic content degrades anaerobically (like anaerobic digester), produces methane and leachate. - **Degradation Rate:** Dependent on moisture (may need water addition if capped). - **Leachate (Table 4.5 in text):** High BOD, may contain metals/toxic chemicals (co-disposal). Requires treatment (aerobic tanks/lagoons, SBRs, anaerobic treatment, spray irrigation, reed beds). - **Landfill Gas (Fig. 4.7, 4.8 in text):** Variable composition (CH₄, CO₂, H₂). Calorific value ~50% of natural gas. Wells inserted 1-2 years after capping. Yield ~100 m³ gas/tonne refuse (25% of possible). - **Management:** Landfills as anaerobic reactors, not inert dumps. Leachate recycling for faster degradation/gas production. Landfill tax (UK). - **Alternative:** Alternatives to landfill being investigated in Europe/USA. #### 4.4.2 Incineration - **Use:** Industrial, domestic, toxic wastes. - **Critical Factors:** Temperature, time at high temperature, effective mixing with air. - **Systems:** Rotary kilns, liquid injection, fluidized beds, multiple hearths (1500-3000°C). - **Advantages:** Volume reduction, treats toxic materials, can be constructed where landfill unavailable. Autocalorific (e.g., sewage sludge). - **Disadvantages:** Expensive to operate/construct, ash disposal (may contain metals), produces particulates/flue gases. - **Flue Gases:** HCl, SOₓ, NOₓ (removed by wet scrubbing), CO (incomplete combustion), CO₂ (greenhouse gas). - **Dioxins/Furans:** Formed from chlorinated compounds at low temperatures. - **Particulates:** Avoided by electrostatic precipitators. #### 4.4.3 Composting - **Purpose:** Disposal/reuse of organic waste (domestic, sewage sludge, straw, agricultural wastes). - **Definition:** "Biological stabilization of wastes of biological origin under controlled aerobic conditions." - **Process:** Organic components decomposed by microorganisms under warm, moist, aerobic conditions. - **Aerobic Metabolism:** Produces heat, raising compost temperature (50°C+). Inactivates pathogens. - **Microorganisms (Fig. 4.9 in text):** Mesophiles initiate, then thermophiles take over. - **Rate:** Rapid (1-6 weeks), low energy, hygienic end-product. - **Systems (Table 4.6 in text):** - **Open Systems:** - **Windrow (Fig. 4.10, 4.11 in text):** Piled in long heaps, turned periodically for aeration. - **Static Pile (Beltsville/Rutgers):** Aeration by blowing air in or suction from base. - **Closed Systems:** In buildings, mechanical aeration/mixing. - **Dano Biostabilizer:** Sloping plug-flow bioreactor, rotating cylinder mixes/aerates. Partial composting (1-5 days), then Windrow. - **Silo System:** Vertical plug-flow, air at base. Material pumped in at top. Residence 14 days, then curing vessel. - **Advantages:** Organic waste reuse, faster/cleaner than anaerobic landfill. - **UK:** Not popular for domestic waste, but increasing as landfill becomes difficult. Used for agricultural products (e.g., straw). #### 4.5 Artificial Wetlands - **Principle:** Plants take up metals, degrade organic materials. - **System:** Uses common reed (*Phragmites* sp.) in fresh/brackish water. Reed bed capped with clay/polypropylene to prevent leakage, surrounded by wall (Fig. 4.12 in text). - **Mechanism:** Reeds transfer oxygen from leaves to roots, encouraging large aerobic microbial population on roots (rhizosphere). Microbes sequester metals, degrade organics. - **Applications:** Mine leakage (heavy metals, e.g., Wheal Jane - Box 4.3 in text), industrial organic waste (methylmethacrylate plant), dairy waste. - **Aquatic Plants:** Water hyacinth (*Eichhornia crassipes*) for waste treatment – grows luxuriantly in sewage, concentrates metals, degrades phenols, treats tannery/dairy wastes. Disadvantage: shallow water, large ponds needed. #### 4.6 Agricultural Wastes - **Types:** Solid (chaff, straw), liquid (slurry). - **Solid:** 5-10% chaff/straw used for mushroom compost (Windrow system). - **Liquid:** Cattle, pig, chicken slurry (dung + urine). High BOD (10,000-25,000 mg/l). - **Disposal:** Spraying/spreading on land (limited for intensive farming). Anaerobic digesters or facultative pond systems common. - **Other:** Run-off of nitrates/phosphates, herbicides/pesticides. #### 4.7 Industrial Wastes - **Characteristics:** Liquid/solid, generally higher BOD than domestic wastes. - **Sources:** Food, drink, meat/vegetable processing, yeast, citric acid, antibiotic production. - **Disposal:** Can be discharged to sewage system (with charges). Biological methods for waste removal, upgrading waste to animal food. - **Anaerobic Digestion:** Suitable for high BOD industrial wastes. - **Example: Cheese Production Plant (Fig. 4.13 in text):** Anaerobic digestion of whey produced methane (fuel for boilers/electricity). 95% BOD reduction, high-quality effluent after denitrification. - **Novel Approaches (Historical):** - **Bassetts Confectionery (UK):** Continuous cultivation of *Candida utilis* on sugar waste (Fig. 4.14 in text). Yeast harvested/dried, sold as food grade. BOD reduction ~50%. - **Symba Process:** *Candida utilis* + *Endomycopsis fibriguler* cultivated on potato processing waste for animal feed. *E. fibriguler* produces amylase for starch breakdown, *C. utilis* grows on sugars. - **Pekilo Process (Finland):** Utilized wood pulp wastewater (leached material). *Paecilomyces* spp. grown continuously, biomass harvested (15 tonnes/hour), dried, sold as animal food (69% protein). #### 4.8 Conclusions - **Shift:** Waste management hierarchy reversed, more focus on prevention. - **Biotechnology Contributions:** - **Clean Technology:** Enzymes (especially hyperthermophiles) replacing chemical synthesis. - **Recombinant Technology:** Modifying microorganisms (GEMs) to produce chemically synthesized products. - **Biodegradable Plastics:** Economically viable production a high priority. - **Waste Upgrading:** SCP production uneconomic due to agricultural improvements. - **Recombinant Technology:** Analyzing degradation in landfill/disposal systems. - **Landfill:** Regarded as anaerobic digesters, leachate recycling/treatment. ### 5. Bioremediation #### 5.1 Introduction - **Pollutant Sources:** Industrial effluents, outfalls, mine waters, landfill run-offs, waste tips, accidental spillages (Table 5.1 in text). Found in marine, estuaries, lakes, soil. - **Bioremediation Definition:** Natural or managed biological degradation of environmental pollution. - **Mechanism:** Indigenous microorganisms (activity enhanced by nutrients) or bioaugmentation (enhancing microbial population). - **Chapter Focus:** Application of biotechnology to inorganic, synthetic organic (xenobiotic), petrochemical, and gaseous pollutants (clean-up and prevention). #### 5.2 Inorganic Waste - **Sources:** Mining, smelting, electroplating, farming. - **Toxicity:** Many essential metals become toxic at high concentrations (Table 5.2 in text). - **Example: Copper:** Micronutrient, but excess inhibits photosynthesis, damages membranes (generates superoxide, H₂O₂, hydroxyl radicals). - **Example: Mercury:** Minamata poisoning (Japan), biomagnification in food chain. - **Standards:** National/international drinking water standards (Table 5.3 in text). Industrial wastes often exceed these. - **Fate:** Metals cannot be degraded. Treatment must concentrate for containment or recycling. Bioaccumulation. #### 5.2.1 Biosorption - **Mechanism:** Microbial cells adsorb metals. - **Responses to High Metal Concentrations:** - Exclusion from cell. - Energy-dependent efflux. - Intracellular sequestration (e.g., metallothioneins). - Extracellular sequestration (cell wall, polysaccharides). - Chemical modification. - **Capacity:** Biological material can bind metals up to 30% dry weight (*Volesky and Holan, 1995*). - **Applications:** - **Detoxification:** Wastewater streams. - **Recovery:** Valuable metals (e.g., gold). - **Uptake:** - **Passive:** Independent of metabolism, rapid (5-10 min), affected by pH/ionic strength, reversible. Occurs with living/dead material (e.g., marine macroalgae - *Ecklonia radiata* for copper). - **Active:** Slower, metabolism-dependent, affected by inhibitors. Metals complexed with proteins (metallothioneins) or in vacuole. - **Commercial Products:** Immobilized non-living microalgae (AlgaSORB) for cadmium removal. - **Enhanced Binding:** Over-expression of metallothioneins. Engineered *E. coli* with hexahistidine clusters for increased cadmium binding (*Sousa et al., 1996*). - **Process Formats (Fig. 5.1 in text):** Immobilized material in packed-bed, fluidized-bed, rotating-disc reactors. Includes biomass regeneration. #### 5.2.2 Extracellular Precipitation - **Problem:** Industrial/mining effluents with metals and sulfates. - **Mechanism:** Anaerobic, sulfate-reducing bacteria (*Desulfovibrio, Desulfotomaculum*) use simple carbon sources (lactic acid) to generate H₂S from sulfate. - **Reaction:** $3SO_4^{2-} + 2 \text{lactic acid} \rightarrow 3H_2S + 6HCO_3^-$ - **Metal Precipitation:** H₂S reacts with metals to form insoluble metal sulfides ($H_2S + Cu^{2+} \rightarrow CuS + 2H^+$). - **pH Increase:** Bicarbonate breakdown increases pH, encouraging sulfide precipitation. - **Challenges:** Excess H₂S is poisonous/corrosive; can be burned or controlled by limiting organic carbon. - **Improvements:** H₂S oxidized to elemental sulfur by oxygen or sulfur bacteria (*Kolmert et al., 1997*). - **Process Formats:** Sludge blanket bioreactor (Fig. 5.2 in text), biomass immobilized in spent mushroom compost. Packed-bed system better than suspended carriers (*Kolmert et al., 1997*). #### 5.2.3 Other Inorganic Wastes - **Nitrates/Nitrites/Phosphates:** From sewage, agriculture, industry. High levels cause eutrophication in lakes. - **Removal:** Sewage system (Chapter 3). - **Biological Systems:** *Klebsiella oxytoca* tolerates/removes high nitrate (pilot-plant tested). Photosynthetic cyanobacterium *Phormidium bohneri* in photobioreactor for N/P removal (simple medium, sunlight energy). *Phormidium laminosum* (immobilized) for nitrate. *Chlamydomonas reinhartii* (immobilized) for nitrite. *Chlorella vulgaris* (immobilized) for nitrate/phosphate. - **Sulfide-Containing Waste:** Treated chemically, but biological method (colorless sulfur bacteria oxidize sulfide to elemental sulfur under aerobic conditions) is cheaper. - **Cyanide:** From gold extraction, other industries. Removed by oxidation (chlorine, peroxide). Biological methods: biosorption by mold *Fusarium lateritium*. #### 5.3 Petroleum-Based Wastes - **Crude Oil Composition:** Complex, variable mixture of hydrocarbons (aliphatic, aromatic, polycyclic aromatic hydrocarbons (PAHs) like naphthalene, anthracene, phenanthrene). Also contains S, N, O, heavy metals. - **Refining:** Converts PAHs to monocyclic aromatics. - **Oil Types:** Light oils (low molecular weight), heavy oils, bitumen, tars. #### 5.3.1 Crude Oil Leaks/Spills - **Origin:** Anaerobic degradation of biological material over long time. - **Tar Sands:** Derived from oil reaching surface, volatiles lost. Mined, tar stripped by hot water/alkali, upgraded by cracking. - **Underground Reservoirs:** Oil trapped in porous rock. Pressure (dissolved gas, overlying rock, aquifer) forces oil to surface. - **Leaks/Spills:** Leaking storage tanks (100,000-300,000 in USA), spillages, transport accidents (e.g., Torrey Canyon, Exxon Valdez). - **BTEX:** Petroleum can contain up to 20% BTEX (hazardous list), mobile, contaminates groundwater. - **Fate on Land (Fig. 5.3 in text):** Volatiles lost to atmosphere. Mobile components migrate through soil to water table. Water-soluble compounds contaminate groundwater. High molecular weight components remain on/near surface or adsorbed to soil. Water-immiscible components form layer on water table. #### 5.3.2 Bioremediation of Marine Oil Spills - **Mechanism:** Crude oil floats on surface, volatiles escape. Dispersed by waves. Hydrocarbon-degrading organisms break down oil at oil-water interface. - **Biodegradable (Box 5.1 in text):** Compound degraded by biological process. Mineralization = complete degradation to CO₂, H₂O, inorganics. Partial degradation = intermediate stages. Persistent = slow degradation. Recalcitrant = not degraded. - **Cleanup Stages:** 1. **Stop Release & Contain:** Mechanical removal (skimmers). 2. **Shore Cleanup:** Mechanical removal on sand, washing oil back to sea on rocks. 3. **Chemical Dispersants:** On floating oil/rocky shores (care needed, can be harmful). - **Bioremediation Role:** Remaining high molecular weight oil. - **Enhancement:** Indigenous microbial population (widely distributed). Limiting factors: phosphorus/nitrogen. - **Fértilizers:** Slow-release fertilizers (N:oil 1:100), N/P-containing dispersants (with surfactant to oil droplets). - **Exxon Valdez:** Alaskan shore lacked N/P, so fertilizer addition was effective. Oleophilic fertilizer best, cleaned rocks in 10 days (*Atlas, 1991*). #### 5.3.3 Bioremediation of Soils - **Microorganisms:** Large numbers (10⁴-10⁷ cells/g soil), including hydrocarbon-utilizing bacteria/fungi (1% of total). Cyanobacteria/algae also degrade. - **Contaminated Soils:** More microorganisms, but reduced diversity. - **Factors Affecting Fate of Organics:** - **Microbial Growth/Metabolism:** Presence of other biodegradable material, N/P compounds, oxygen levels, temperature, pH, water, microbial numbers/types, heavy metals. - **Compound Itself:** Chemical structure, availability/solubility, photochemistry. - **Degradation:** - **Aerobic:** Faster than anaerobic. Oxygen supply needed. - **Temperature:** Low T slows degradation. - **pH:** Affects growth/solubility. - **Seeding:** Not always needed, indigenous populations adapt. - **Heavy Metals:** High levels can inhibit microbial growth. - **Hydrocarbon Structure:** Simpler aliphatics/monocyclic aromatics readily degradable. Complex PAHs persist. - **Availability:** Affected by soil structure, porosity, composition, compound solubility. Adsorption to clays renders invulnerable. - **Enhancement:** Surfactants improve availability (*Mihelcic et al., 1993*). #### 5.3.4 Pathways of Degradation - **Hydrocarbons:** Used as energy/carbon source. Degradation by oxidation (aerobic/anaerobic). - **Monocyclic Aromatics (e.g., Benzene):** 1. Hydroxylated by dioxygenase to *cis*-1,2-dihydroxy-1,2-dihydrobenzene. 2. Converted to catechol (Fig. 5.4 in text). 3. **Ring Cleavage:** - **Ortho Cleavage:** Yields *cis,cis*-muconate. - **Meta Cleavage:** Yields 2-hydroxymuconic semialdehyde. 4. Both pathways lead to Krebs cycle intermediates. - **Polycyclic Aromatic Hydrocarbons (PAHs - Fig. 5.5 in text):** - **Initial Steps:** Similar to monocyclic aromatics. Aromatic ring hydroxylation followed by ring cleavage. - **Enzymes:** Oxygenases (dioxygenases for two O₂, monooxygenases for one O₂). - **Genetic Basis:** Many monocyclic aromatic-degrading bacteria have chromosomal genes. Plasmids (e.g., TOL plasmid in *Pseudomonas putida* mt-2 - Fig. 5.6 in text) code for degradation of camphor, octane, toluene, naphthalene, herbicides/pesticides. - **TOL Plasmid Genes (Table 5.4 in text):** *xyl* genes arranged in operons (e.g., *xylC,A,B* for degradation to benzoic acid; *xylX,Y,Z,L,E,G,F,J,I,H* for breakdown of benzoate to pyruvate/acetaldehyde). - **Anaerobic Degradation:** Slower than aerobic. Aliphatic, monocyclic, PAHs degraded if oxygen from water (methanogenic), nitrate (nitrifying), or sulfate (sulfur-reducing). - **Mechanism:** Hydration, dehydration, reductive dehydroxylation, nitroreduction, carboxylation to benzoyl CoA/resorcinol, then reduced/hydrolyzed to Krebs cycle intermediates (*Holliger and Zehnder, 1996*). - **Gratuitous Metabolism:** Enzymes acting on non-normal substrates (broad specificity). - **Co-metabolism:** Metabolism of substrate not required for growth, no apparent benefit (*Semprini, 1997*). Often due to imprecise specificity/induction. - **Example:** Trichloroethylene with phenol/toluene as co-metabolic substrates (*McCarty, 1993*). #### 5.4 Synthetic Organic Compounds - **Sources:** Pesticides, herbicides, preservatives (Table 5.5 in text). Polychlorobiphenyls (PCBs) from industrial uses. Chlorinated solvents (trichloroethene, carbon tetrachloride, tetrachloroethene) from disposal/spillage. Dibenzop-dioxins/dibenzofurans from PAH combustion. - **Scale:** Large-scale release (Table 5.6 in text for herbicides). - **Degradation:** Many microbially degradable, but some are persistent ("effectively permanent"). - **Persistence (Table 5.7 in text):** Long half-lives (e.g., DDT up to 10 years). - **Consequences of Persistence:** Protracted exposure, increased toxicity, biomagnification (compound accumulates in food chain, e.g., DDT in grebes). Lack of toxicity/long-term effect information. - **Factors Affecting Persistence:** Molecular structure/properties, toxicity to microorganisms, environmental conditions. - **Organohalogens:** Activity/persistence linked to halogens (number, position, type). Hydrophobic, disrupt membranes, inhibit oxidative phosphorylation. Solubility decreases with increasing chlorine (often increases toxicity). - **Example:** PCBs (mixture of isomers) slow degradation. - **Enzymatic Capability:** Degradation depends on microorganisms with enzymes. Xenobiotics are new, so degrading population may be low. Adaptation period often needed. - **Environmental Conditions:** Same as for hydrocarbons. #### 5.4.1 Pathways of Degradation - **Organisms:** Bacteria, fungi, algae from contaminated soil/sediment. - **Chloroaromatics:** Cleaved by monooxygenases/dioxygenases (similar to PAHs). Dehalogenation (four mechanisms): - **Oxidative Dehalogenation:** Halogen removed, replaced by two hydroxyl ions. - **Eliminative Dehalogenation:** Simultaneous removal of halogen and adjacent H⁺. - **Hydrolytic (Substitutive) Dehalogenation:** Halogen replaced by hydroxyl ion. - **Reductive Dehalogenation:** Halogen replaced by H⁺. - **Examples:** - **Pentachlorophenol (PCP - Fig. 5.7 in text):** Herbicide/fungicide. Degraded aerobically (reductive dehalogenation) and anaerobically by *Flavobacterium, Arthrobacter, Rhodococcus*, white rot fungus *Phanerochaete chryososporium*. Pathways involve dechlorination, hydroxylation, ring cleavage. - **Polychlorinated Biphenyls (PCBs - Fig. 5.8 in text):** Banned >20 years. Aerobically degraded, first step similar to monocyclic aromatics (dioxygenase). - **Atrazine (Fig. 5.9 in text):** Triazine herbicide. Degraded by pure cultures (*De Souza et al., 1998*) or consortia. Converted to cyanuric acid (three steps), then CO₂/NH₃. Three genes (often on plasmid in *Pseudomonas* spp.) involved. *Clavibacter* performs first two steps, *Pseudomonas* for remaining. - **Organophosphorus Compounds:** Pesticides. Detoxified by chemical treatment, landfill, incineration. Soil microorganisms (*Pseudomonas diminuta, Flavobacterium*) use hydrolase enzyme. - **Enhancement:** Hydrolase enzyme purified/immobilized (expensive). Cloning hydrolase gene and expressing on cell surface (*Chen and Mulchandani, 1998*) eliminates purification and transport issues. #### 5.4.2 Bioremediation Technology - **Goal:** Stimulate indigenous degrading population by providing optimum conditions. - **Methods (Fig. 5.10 in text):** - **Ex Situ:** Excavation for treatment/disposal. - **Land Treatment/Farming:** Tilling soil, fertilizer addition to increase aeration/microbial population. Lined/dammed areas. Degradation rate depends on microbes, contamination, soil type. Half-life for diesel/heavy oils ~54 days. - **Composting:** Contaminated soil mixed with organic material (straw, bark, wood chips), piled (Windrow). Rise in temperature (60°C+) by microbial activity. Faster than land farming. - **Biopile (Fig. 5.11 in text):** Soil heaped in lined area, covered with polythene. Liquid nutrients applied. Aeration (suction at base). Leachate collected/recycled. Used when space limited, vapor emissions restricted (biofilter needed). - **Bioreactors:** For solid waste (slurry) or liquid leachate. Control parameters (T, pH, mixing, O₂). Prepared bed, slurry, biofilter, anaerobic digesters. - **In Situ:** - **Bioventing (Fig. 5.12 in text):** Increased O₂ supply + vapor extraction. Vacuum applied, draws air into soil, sweeps out VOCs. Nutrient supplementation (trenches). Effective for volatile compounds, permeable soil. Extracted vapor may need biofilter. - **Biosparging:** Increases O₂ supply by sparging air/oxygen into soil. Pure O₂ for higher rates. Expensive, but on-site O₂ generation reduces cost. H₂O₂ also used (can be toxic). - **Extraction:** Contaminants extracted, treated in surface bioreactors. - **Stimulation:** Microbial growth stimulated by nutrients (N/P) or second carbon source (for co-metabolism). - **Bioaugmentation:** Adding microorganisms (selected or GEMs). Best to stimulate indigenous populations, but useful if degradation pathways produce toxic intermediates. Fungal inocula for PCP, *Methylosinus trichosporium* for TCE. - **Extraction Challenges:** Many contaminants insoluble. - **Biosurfactants:** Surface-active molecules (solubilize, emulsify, disperse). More complex than chemical surfactants. Increase degradation rate for oil, xenobiotics (PCBs, organophosphates). Produced in situ or added. - **Liquid CO₂:** For compound removal (e.g., diesel). - **Soil Washing:** Soil removed, scrubbed, separated. Contaminants partitioned to liquid phase, fine particles collected for biotreatment/disposal. Water treated in bioreactors. #### 5.5 Phytoremediation - **Definition:** Use of plants for contaminant/metal removal from soil. - **Processes:** - **Phytoextraction:** Removal of contaminants/metals, storage/degradation in plant. - **Hyperaccumulators:** Plants (e.g., *Thlaspi caerulescens*, *Cardaminopsis halleri* for Zn/Cd; *Alyssum lesbiacum* for Ni) accumulate 50-100x more metal. Tolerate high metal levels (concentration in vacuole, chelation). High uptake/translocation rates. - **Phytovolatilization:** Plants convert metal ions to more volatile species (e.g., selenium to dimethylselenide, methyl mercury to mercury vapor). - **Rhizofiltration:** Removal of contaminants from flowing water by plant or root-associated microorganisms (rhizosphere). Includes organic compounds/metals (e.g., reed bed systems). - **Phytostabilization:** Transformation of molecule to less toxic species. - **Degradation of Synthetics:** Poplar trees for trichloroethylene. Other plants for explosives. Plant cell cultures for nitroglycerin, PCBs (*Goel et al., 1997*; *Mackova et al., 1997*). - **Advantages:** Low-cost, easy to apply, minimal maintenance, public acceptance. #### 5.6 Gaseous Wastes - **Contaminants:** VOCs, SO₂, NOₓ, CFCs, greenhouse gases (CO₂, CH₄), particulates. - **Sources:** Hydrocarbon vaporization, combustion of sulfur-containing fuels, fossil fuels. - **Removal Methods:** - **Biofiltration:** Microorganisms degrade volatile compounds. - **Simple Design:** Soil bed, VOCs piped into base, degrade as they pass to surface. - **Sophisticated Biofilter (Fig. 5.13 in text):** Reactor with high surface area packing (peat, wood bark, plastic) supporting active microbial biofilm. Maintained by nutrients, high humidity (humidify inlet air, recirculate medium). Controls T, pH. Removes dichloromethane, styrene, toluene (*Cox et al., 1997*). - **Hydrogen Sulfide (H₂S):** Removed from flue gases. Physical processes exist. Biological: *Thiobacillus* oxidizes H₂S to sulfate (removed by neutralization). #### 5.7 Desulfurization of Coal and Oil - **Problem:** Coal/oil contain sulfur compounds, combustion yields SO₂. SO₂ causes acid rain, pulmonary irritation. - **Acid Rain:** Lowers pH of rain ( ### 6. Energy and Biofuel #### 6.1 Introduction - **Energy Demand:** Steadily increasing globally (5x from 1937-1988), correlated with living standards. - **Current Demand:** 398 EJ/year (10¹⁸ J). - **Sources (Fig. 6.1 in text):** Oil, gas, coal (main); biomass, hydroelectric, nuclear. - **Fossil Fuels:** 75% of world energy. Consumption expected to rise 50-60% by 2010. - **Reasons for Use:** Found worldwide, simple extraction, easy transport (liquid for automotive). - **Problems:** Finite supply, greenhouse gases (global warming), other pollutants (air pollution, acid rain). - **Coal:** Estimated to last until 2180. Replaced by natural gas for electricity (cheaper, cleaner). - **Natural Gas:** Less abundant, not evenly distributed. Lasts until 2047. - **Oil:** Supplies last until 2080. Demand increasing (Table 6.1 in text), extraction in hostile conditions, increasing costs. - **Alternatives:** Many not cost-competitive yet. #### Combustion Products (Fossil Fuels - Table 6.2 in text) - **Coal:** CO₂, SO₂ (from sulfur), NOₓ (from nitrogen), particulates. - **Oil/Gas:** Similar gases, CO. - **Greenhouse Gases (GHGs):** Water vapor (largest effect, not human-affected), O₃, CO₂, CH₄, N₂O, CFCs. - **Mechanism (Fig. 6.4 in text):** Absorb outgoing long-wave radiation from Earth, radiate back, warming surface. - **Human Activity:** Increased GHG concentrations (Table 6.3 in text). - **CO₂:** Increased 26% since Industrial Revolution (Fig. 6.6 in text). Annual emissions 7 billion tonnes, could rise to 20 billion by 2010. Global T increase of 2.5°C by 2100 (Houghton, 1996), rising sea levels (0.5m). - **CH₄:** Doubled since pre-industrial times (Table 6.3 in text). Sources: rice cultivation, natural gas leaks, coal mining (Table 6.4 in text). Principal removal: reaction with hydroxyl radicals. - **CFCs:** New to environment (last 30 years). Increased rapidly, deplete stratospheric ozone (UV filter). Montreal Protocol (1987) limited production. - **N₂O:** 8% above pre-industrial levels. Sources difficult to quantify. Loss: photochemical decomposition in stratosphere. - **Other Pollutants:** - **SO₂/NOₓ:** From fossil fuel burning (54% of atmospheric SO₂ - Table 6.5 in text). Cause acid rain, urban smog. - **Mitigation Strategies:** - Increase CO₂ sinks (forests). - Reduce GHG/pollutant emissions by increased energy efficiency. - Remove CO₂ from fossil fuel emissions. - Use alternative energy sources that do not produce GHGs. #### 6.1.1 Removal of Carbon Dioxide - **Forests:** To absorb CO₂. Requires vast areas (e.g., 23% of USA land area for USA emissions). Reafforestation helps. - **Separation/Storage:** From exhaust gases, piped to deep ocean or depleted gas reservoirs (expensive). - **Microalgae:** Remove CO₂ from exhaust gases. - **Mechanism:** Fix CO₂ via photosynthesis. Large-scale cultivation systems developed. - **Example:** Marine microalga *Tetraselmis suecica* (96% CO₂ utilization). *Chlorococcum littorale* fixes CO₂ at 4g/l/day from flue gases, tolerates high CO₂/low pH (*Kurano et al., 1995*). - **Challenge:** Algae degrade, return CO₂ to atmosphere unless fixed. #### 6.1.2 Increases in Efficiency of Existing Energy Generation - **Conventional Power Stations:** Coal burnt, steam drives turbine (37% efficiency). - **SO₂ Reduction:** Low-sulfur coal, pre-burning sulfur removal (Chapter 5). - **NOₓ Reduction:** Catalytic reduction with ammonia in flue gases. - **Advanced Combustion (e.g., AFBC/PFBC):** Burn coal with limestone (90% sulfur removal as slag). Lower T reduces NOₓ. - **Coal to Gas:** Sulfur removed before burning. - **Problem:** All these methods still produce CO₂. #### 6.2 Alternative Non-Fossil Energy Sources - **Need:** To reduce fossil fuel use, mitigate GHGs. - **Types:** Nuclear, hydroelectric, tidal, wave, wind, geothermal, solar, biological. #### 6.2.1 Nuclear Power - **Basis:** Fission of uranium. - **Advantages:** Large energy release (50 million x coal/weight), little fuel needed (reduces transport/storage), no combustion gases. - **Disadvantages:** - **Fuel:** Expensive uranium-235 enrichment (0.7% to 3%). - **Radioactive Waste:** Long half-lives, difficult to contain/dispose. - **Safety:** High radiation levels cause injury/death, cell transformation/mutation (cancers). Accidents (Three Mile Island, Chernobyl) create public distrust. - **Costs:** Reprocessing/disposal of spent fuel, leaks, decommissioning. #### 6.2.2 Hydroelectric Power - **Advantages:** Clean, non-polluting, long-lasting, renewable. Proven technology, no CO₂/gas emissions. - **Disadvantages:** - **Environmental Impact:** Site-specific. Flooded villages, changed river flow, fish/bird impacts. Dam collapse risk (earthquakes). - **Costs:** Capital intensive. - **Availability:** Limited suitable sites. #### 6.2.3 Tidal Power - **Advantages:** Clean, reliable, long-lasting, renewable. - **Mechanism:** Harnesses regular tide rise/fall. Traps tidal flow behind barrier/dam, releases water through turbines. - **Limitations:** Limited sites with sufficient tidal range/area. ~10% of hydroelectric potential. - **Examples:** Rance estuary (France - 240 kW), proposed Severn estuary (UK - 8.6 GW), Bay of Fundy (Canada). - **Disadvantages:** Huge projects (e.g., Severn estuary $8.28 billion). Enormous local ecological impact (tidal/current patterns, fish, birds). Siltation, turbine fouling. #### 6.2.4 Wave Power - **Research:** Devices for converting wave energy to shaft power/compression. - **Challenges:** Efficiency, construction strength (withstand winter), initial costs. - **Location:** Best offshore for steadier velocity. #### 6.2.5 Wind Power - **Advantages:** Promising alternative, substantial energy without pollution. Over 7000 MW installed globally. - **Applications:** Drive water pumps, charge batteries in remote regions, off-grid power. - **Turbines:** Horizontal-axis rotors (up to 66m diameter, 3 blades). Rotate ~25 rpm, generate 100-700 kW. - **Wind Farms:** 10-100 machines. - **Disadvantages:** - **Siting:** Exposed locations, can be intrusive (landscape, noise). - **Variability:** Reliant on wind. - **Offshore:** Reduces visual/noise impact, steadier wind (e.g., Vindely, Denmark). - **Economics:** Capital intensive. Price drop (Denmark 8%/year). Dependent on local conditions, fossil fuel prices. #### 6.2.6 Geothermal Energy - **Source:** Earth's internal heat (4000°C core, radioactive decay). - **Mechanism (Fig. 6.8 in text):** Heat conducted to surface. In volcanic areas, high-grade heat in molten/hot rocks (2-10km deep). - **Natural Extraction:** Hot rocks contact groundwater, forming hot springs/geysers (up to 300°C). - **Electricity:** If water >150-170°C, runs steam turbines directly. Or heats second liquid (lower vaporization T) to drive turbines. - **Heating:** If water 96% purity. Azeotropic distillation with benzene for water-free ethanol. #### 6.7.2 Production of Ethanol in Brazil - **Motivation:** Reduce petrol imports, open up land for cultivation, employment, industrial base, ethanol exports. - **Source:** Sugarcane (readily available, no processing, high yield - Table 6.8 in text). - **Process:** Sugar extracted, fermented in 100-200 m³ bioreactors (8-12% ethanol). Bagasse (fiber/cellulose) burnt for distillation heat. - **Scale:** 95% of cars on ethanol/gasohol by 1984/85, declined to 50% by 1990 due to cheap petroleum. #### 6.7.3 Economics of Ethanol Production - **Energy Balance (Table 6.16 in text):** - **Maize:** Energy input to grow/process > energy output as ethanol. - **Sugarcane:** Similar. - **Improvement:** If fossil fuel replaced by bagasse for distillation (maize ratio 1.29, sugarcane 2.03). - **Costs:** Fixed costs. Disagreement on Brazilian ethanol economics (some say competitive with fossil fuels at $0.185/liter). #### 6.7.4 Future Developments - **Microorganisms (Table 6.14 in text):** - **Ideal Characteristics:** Rapid fermentation of diverse carbohydrates, ethanol tolerance, high ethanol production, low by-products, osmotolerance, high cell viability (for recycling), good flocculation/sedimentation. - ***Zymomonas mobilis*:** Faster growth/ethanol production than *Saccharomyces*, but limited sugar use. Tolerates high sugar/ethanol. - **Pathway (Fig. 6.14 in text):** Entner-Doudoroff pathway (2 NADH, 1 ATP vs. 2 ATP in glycolysis). - **Problem:** Produces by-products (dihydroxyacetone, mannitol, glycerol from fructose; levans from sucrose). - ***Clostridial Strains* (*C. thermocellum, C. thermosaccharolyticum, C. thermohydrosulfuricum*):** Gram-positive thermophilic anaerobes. Degrade cellulose to ethanol. - **Challenges:** Lignocellulose/cellulose need physical disruption, slow breakdown. Less ethanol tolerant than *S. cerevisiae*. Produce organic acid by-products. - **Ideal Scenario:** Thermophilic bacterium converting cellulose to ethanol, with ethanol distilling off at reaction temperature. #### 6.7.5 Other Alcohols - **Butanediol:** Similar heating value to ethanol, but more expensive. Dehydrated to MEK or condensed for aviation fuels. - **Production:** By *Bacillus polymyxa, Klebsiella pneumoniae, Bacillus subtilis, Serratia marcescens*. - **Challenges:** Low yields ( ### 7. Natural Resource Recovery #### 7.1 Introduction - **Biotechnology's Role:** Reduce/eliminate pollution, extract oil/metals. - **Resources:** Metals and crude oil are non-renewable. Demand requires new sources or improved extraction. - **Microorganisms:** - **Metal Extraction:** From ores (since 1950s for copper). Used industrially for copper, uranium, gold (bioleaching). - **Oil Extraction:** Microbially Enhanced Oil Recovery (MEOR) after primary flow ceases. Not commercial due to high costs, but future demand may require it. - **Chapter Focus:** Microbial use for crude oil extraction (after primary flow), and metal recovery from mining wastes, low-grade ores, worked-out mines. #### 7.2 Oil Recovery - **Primary Recovery:** 0-50% of Original Oil In Place (OOIP). - **Crude Oil Nature:** Diverse states (sand coated with viscous tars to light oils in reservoirs). Originates from anaerobic degradation of biological material at high T/P. Hydrocarbons: aliphatic, aromatic, polycyclic. High molecular weight: bitumen, tars. - **Properties:** Affects oil type (light, heavy, bitumen, tars). - **Tar Sands:** Derived from surface oil, volatiles lost. Mined, tar stripped by hot water/alkali, upgraded by cracking. - **Underground Reservoirs:** Oil in porous rock (sandstone, limestone, chalk). Less dense than water, forced up by rising water, trapped by impervious rock (dome - Fig. 7.1 in text). Often under pressure (dissolved gas, overlying rock, aquifer). Gas collects at top. - **Extraction:** 1. **Seismic Investigation:** Find dome. 2. **Drilling:** Into dome. 3. **Primary Extraction:** Reservoir pressure forces lighter crude oils to surface ("gusher"). Well capped, oil to storage. Accounts for 10-15% OOIP. 4. **Pumping:** As pressure drops. Mechanical/electrical pumps at well base. - **Coning (Fig. 7.2 in text):** Suction at well base causes aquifer water to rise, entering pump. Major problem, pump stopped. 5. **Yield:** Reduced to uneconomic levels (15-20% total OOIP). - **Secondary Production:** Extract more oil. - **Water/Gas Injection:** Into well to force oil out. Water common (gas sold). Seawater needs sulfate removal (prevents sulfate-reducing bacteria degrading oil). - **Water Flooding:** Drill new wells (up to five) some distance from production well. Water injected below oil layers. - **Problems (Fig. 7.3 in text):** - **Fingering:** Rock not homogeneous, larger channels allow water to flow easily (higher mobility than oil), bypasses oil. - **Clogging:** Oil droplets clog small pores in low-porosity rocks. - **Thief Zones:** Fractures/channels allow water to pass without displacing oil. - **Yield:** ~35% OOIP. #### 7.2.1 Enhanced Oil Recovery (EOR) - **Goal:** Influence crude oil flow characteristics. - **Flow Properties:** Mobility ratio, capillary number. - **Mobility Ratio (M):** Ratio of displacing fluid mobility (water) to oil mobility (M = γ_displacing / γ_oil). Mobility (γ) = k/μ (k=permeability, μ=viscosity). Ideal M≈1. If M >1, viscous liquid, flows in wide channels, leaves oil in small pores (viscous fingering). Can be reduced by increasing waterflood viscosity (polymers). - **Capillary Number (N_c):** Measure of oil permeability. $N_c = (\sigma \cdot k) / (\mu \cdot L)$. $\sigma$=interfacial tension, k=effective permeability, $\mu$=viscosity, L=length. Increased N_c reduces residual oil saturation. Increased by reducing oil viscosity, increasing pressure gradient, decreasing interfacial tension. - **EOR Techniques (Fig. 7.4 in text):** - **Non-Thermal:** - **Chemical Floods:** High molecular weight polymers, surfactants, alkalis, emulsions. Polymer flood is commercial. Polymers (polyacrylamides, biopolymers xanthan, curdlan, scleroglucan) increase water viscosity, reduce fingering. Problems: high cost, degradation in well, adsorption to rock. - **Miscible Floods:** Inject solvent (hydrocarbon gas, liquid CO₂, liquid hydrocarbons, alcohols). Miscible with oil, reduces interfacial tension to zero. Commercial: liquid CO₂, enriched gas drive. - **Thermal (for heavy viscous oils):** Reduces oil viscosity. - **Steam Injection:** Cyclic steam stimulation (steam then oil removal), continuous steam flooding. Steam Assisted Gravity Drainage (SAGD) for tar sands (horizontal steam injection, oil collected by lower horizontal well). - **In Situ Combustion:** Burn ~10% residual oil. Compressed air/oxygen pumped in, oil ignites. Combustion front sustained, heat reduces viscosity, cracks oil to lighter fractions. Successful in Russia, Romania, California, Texas, Canada. - **Cost:** All techniques expensive (chemicals, energy for steam). #### 7.2.2 Microbial Enhanced Oil Recovery (MEOR) - **Principle:** Introduce/stimulate existing microorganisms in well to produce polymers/gas in situ. More economic than ex situ preparation. - **Challenges:** Extreme well conditions (Table 7.1 in text): anaerobic, high T/P/salinity. - **Microorganisms:** *Clostridium* (anaerobic, 45°C, 20,000 tonnes/year. Pseudoplastic. - **Curdulan:** Neutral gel-forming 1,3-β-D-glucan. From *Agrobacterium, Rhizobium*. Elastic, heat-stable gel. Gel formation upon acidification for oil wells. - **Scleroglucan:** Glucose homopolymer (*Sclerotium glucanium*). Commercial, alternative to xanthan (Elf Aquitaine). Lower yields, longer process, lower final concentration. - **Production Challenges:** Viscous culture requires impeller design changes in stirred-tank bioreactors. Poor mixing/aeration reduces growth/yield. Helical ribbon-screw design improved it (*McNeil and Harvey, 1993*). - **Market:** Established own market, advantages over conventional rivals. #### 7.3 Biorecovery of Metals - **Biotechnology's Role:** Extract metals from ores (solubilize insoluble deposits, usually sulfides). - **"Bioleaching":** Used industrially since 1950s (copper). Commercial for copper, uranium, gold. - **Advantages:** Low-energy process, not affected by metal content (unlike traditional methods). Reworks mine waste/tailings. - **Problem:** Uncontrolled natural bioleaching produces acid leachate, pollutes environment. Controlled bioleaching can eliminate this. #### 7.3.1 Recovery of Metals from Mining Wastes - **History:** Research on iron/sulfur-oxidizing bacteria (1920s-30s) laid foundation. *Bryner et al. (1954)* described oxidation of iron pyrites/copper sulfide by acid *Thiobacillus*. First patent 1958. - **Sources:** Tailings, low-grade ores (too low for conventional extraction). Significant metal loss. - **Metals Extracted:** Cobalt, nickel, cadmium, antimony, zinc, lead, gallium, indium, manganese, copper, tin (from sulfur-based ores). Gold from pyritic ores. - **Chemolithotrophs (Box 7.1 in text):** Obtain energy by oxidizing inorganic compounds. Carbon from CO₂ fixation. Examples: *Thiobacillus, Sulfolobus, Beggiatoa*. - **Bacteria (Table 7.3 in text):** Isolated from natural/commercial bioleaching. Classified by optimum T. - **Mesophiles (25-35°C):** Chemolithotrophic, highly acidophilic (pH 1.5-2.0). *Thiobacillus ferrooxidans* (oxidizes Fe, S, S₂O₃, metal sulfides), *T. thiooxidans* (oxidizes S, S₂O₃), *Leptospirillum ferrooxidans* (oxidizes Fe). Together, rapidly degrade pyrites. - **Thermophiles:** *Thiobacillus* TH-1, *Sulfolobus brierleyi* (grow on chalcopyrite). *S. brierleyi* (extreme thermophile, 70°C) metabolizes pyrite, chalcopyrite, pyrrhotite. - **Mechanism:** Direct or indirect leaching of metals from sulfur-based ores. #### 7.3.2 Indirect Leaching - **Principle:** Chemolithotrophs generate ferric ions by oxidizing soluble ferrous ions. Ferric ions are oxidizing agents, oxidize metal sulfite, release metal as soluble sulfate. - **Reactions (Pyrites):** - $2FeS_2 + 7O_2 + 2H_2O \rightarrow 2FeSO_4 + 2H_2SO_4$ (*T. ferrooxidans* catalyzed). - $4FeSO_4 + O_2 + 2H_2SO_4 \rightarrow 2Fe_2(SO_4)_3 + 2H_2O$ (*T. ferrooxidans* catalyzed). - $FeS_2 + Fe_2(SO_4)_3 \rightarrow 3FeSO_4 + 2S$ (Ferric ion oxidizes sulfite). - $2S + 3O_2 + 2H_2O \rightarrow 2H_2SO_4$ (Elemental sulfur oxidized). - **Metal Sulfides:** Ferric ion reacts with other sulfides (chalcopyrite, chalcocite, bornite, covellite). H₂SO₄ keeps pH low, leaches other copper minerals. - **Full Cycle:** Fig. 7.5 in text. #### 7.3.3 Direct Leaching - **Principle:** *T. ferrooxidans* attach to mineral particles. Direct enzymatic reactions. - **Reaction:** $4FeS_2 + 15O_2 + 2H_2O \rightarrow 2Fe_2(SO_4)_3 + 2H_2SO_4$ - **Temperature:** Some bacteria grow up to 70°C. Leaching systems can reach 60-80°C due to biological activity. #### 7.3.4 Bioleaching Processes - **Methods:** In situ treatment, heaps/dumps, bioreactors. - **In Situ Bioleaching (Fig. 7.6 in text):** For mines at end of life with remaining ores. Leaching solution (*T. ferrooxidans*) pumped into ore, recovered from below. Metal recovered at surface, suspension aerated, returned to mine. - **Heaps/Dumps (Fig. 7.7 in text):** Most common for low-grade ores/tailings. - **Design:** On slope, 7-20m deep crushed ore. Sprayed with H₂SO₄ (pH 1.5-3) to encourage *Thiobacillus* (10⁸ cells/g rock). - **Process:** Leaching solution collected at base, recirculated. *Thiobacillus* leaches metal into solution. Metal collected by precipitation (scrap iron) or solvent extraction/electrowinning. Excess iron removed in oxidation pond. - **Improvements:** More even particle size, open for O₂ diffusion. Avoid acid-consuming rock (calcium chloride) or toxic compounds (molybdate). - **Problems:** Galena (PbS) forms insoluble PbSO₄, coats ore, prevents leaching. Anaerobes (*Desulfovibrio*) in anoxic zones form metal sulfides, reduce leaching. Process needs to be aerobic. - **Microbial Community:** Diverse, dynamic (10⁸ cells/g rock). Difficult to follow dynamics (sampling, special media). PCR/DNA probes help. - **Bioreactors:** For uranium, gold, silver, copper (due to costs). Stirred tank, airlift, immobilized bed (pilot-plant scale). - **Advantages:** Close control of T, pH, aeration. Airlift design more efficient (*Ferraiolo and Del Borghi, 1987*). #### 7.3.5 Extraction of Copper - **Scale:** Biological recovery >$1000 million, 25% of copper production (1991). - **Advantages:** No adverse side-effects (apart from groundwater leaching), cheap. Utilizes waste ore (0.1-0.5% metal). - **Method:** Waste ore in terraced dumps (100m wide, 10m deep) with impermeable base. Dilute H₂SO₄ sprinkled to reduce pH (2-3), promotes *T. ferrooxidans*. - **Metal Recovery:** Copper dissolved in dilute acid, collected at bottom. Precipitated onto scrap iron. Dilute acid recycled (but ferric salts coat ore). - **Examples (Table 7.4 in text):** Dump leaching operations. #### 7.3.6 Extraction of Uranium - **Process:** Bioleaching from uranium oxides. - **Direct Leaching (*T. ferrooxidans*):** $2UO_2 + O_2 + 2H_2SO_4 \rightarrow 2UO_2SO_4 + 2H_2O$ - **Indirect Bioleaching:** Pyrite (associated with uranium ores). *T. ferrooxidans* produces ferric ion, reacts with uranium ore. - **Example:** Dennison mine (Canada) extracted 300 tons uranium (worth $25 million) in 1988. #### 7.3.7 Extraction of Gold - **Process:** Bioleaching removes arsenopyrites and pyrite to expose gold for cyanide extraction. - **Problem:** High pyrites react with cyanide, forming thiocyanate, uses large quantities of cyanide. - **Mechanism:** *T. ferrooxidans* degrades arsenopyrite: $2FeAsS + 7O_2 + 2H_2O \rightarrow 2FeAsO_4 + 2H_2SO_4$ #### 7.3.8 Recent Developments - **Microbial Strains:** - Isolate new strains from extreme environments (mine drainage, hot springs, waste sites) to seed bioleaching. - Improve existing strains by mutation/selection or genetic engineering. - Introduce arsenic resistance into bioleaching organisms for gold leaching. - **Population Dynamics:** Better understanding needed. PCR/small-subunit rRNA sequences replace plating techniques (*De Wulf-Durand et al., 1997*). #### 7.4 Conclusions - **Polysaccharides:** Microorganisms produce polysaccharides for drilling muds. - **MEOR:** Potential for enhanced oil recovery by in situ growth if crude oil prices rise. Requires further research. - **Bioleaching:** Extracts metals (directly/indirectly) from low-grade ores, tailings, low-yielding mines. - **Current Use:** Extensive for copper, increasing for uranium/gold. ### 8. Agrobiotechnology #### 8.1 Introduction - **Agriculture:** Wide spectrum of activities affected by biotech. - **Biotechnological Influence (mainly genetic engineering):** - Improved plants. - Improved animals. - Diagnostics for plant and animal diseases. - Animal vaccines. - Biological control (instead of chemicals). - Biodiversity. - Waste reduction and disposal. - **Concerns:** Release of genetically engineered plants. - **Benefits:** Reduction in pesticide/herbicide use. #### 8.2 Improved Plants - **Conventional Breeding:** Successful, but slow. - **Biotechnology Advantages:** Genetic engineering produces transgenic plants with properties not possible conventionally. Faster development of new varieties. - **Transgenic Plant Definition:** "Plants with unique gene combinations that do not naturally occur and are produced by using either recombinant DNA technology or protoplast fusion technology" (*Ratledge, 1993*). - **Commercialization:** Flavr Savr™ tomato, herbicide-resistant soya plant (*Senior and Dale, 1996*). - **Traits Transferred:** Mainly single genes (e.g., herbicide resistance). Complex traits (e.g., drought tolerance) harder (multiple genes, biochemical basis not fully understood). - **Requirements for Gene Transfer:** 1. Identify gene, pathway, controls. 2. Isolate gene (from related plant, bacterium, animal). 3. Transfer gene to target plant, ensure correct control/expression. 4. Regenerate transformed plant material, test gene activity, multiply, field test. #### 8.2.1 Gene Isolation (Fig. 8.1 in text) - **Methods:** - **From Total DNA:** Cloning restriction fragments. - **Chemical Synthesis:** If protein sequence known. - **From mRNA:** Production of complementary DNA (cDNA). - **PCR:** Using primers from related genes. - **Steps:** 1. **Isolate DNA:** From donor cells/tissue. 2. **Restriction Fragments:** Cut DNA with restriction enzymes (recognize specific sequences). 3. **Ligation:** Insert fragments into vector (cut with same enzyme), anneal with DNA ligase. 4. **Transformation:** Circular DNA taken up by bacterium (*E. coli*). 5. **Selection:** If vector confers antibiotic resistance, transformed cells grow on antibiotics. 6. **Library:** Collection of transformed colonies (represents original DNA). 7. **cDNA:** From mRNA (using reverse transcriptase). Represents expressed proteins. 8. **PCR:** Amplify specific genes from isolated DNA/cDNA. Needs primers. Detects pathogens, assesses genome relationships. #### 8.2.2 Vectors - **Bacterial Systems:** Plasmids, bacteriophages. - **Plasmids:** Extrachromosomal, circular DNA, multiple copies. Modified for: small size, autonomous replication, single restriction sites, selectable marker (e.g., antibiotic resistance). - **Example (Fig. 8.2 in text):** pBR322 (ampicillin/tetracycline resistance, restriction sites). - **Ligation:** Cut vector/DNA with same restriction enzyme, anneal, DNA ligase. - **Transformation:** Bacteria take up DNA (e.g., *E. coli*). - **Selection:** Antibiotic resistance. #### 8.2.3 Selection of Transformants - **Detection:** - **Nucleic Acid Probe:** Hybridize to plasmid DNA from bacterial colonies (if gene sequence known). Labeled with radioactivity, biotin, fluorescent protein. - **Functional Protein:** Enzyme activity or reaction with antibodies. - **Manipulation:** Once cloned, gene isolated, placed under strong promoter (e.g., CaMV 35S RNA). Transferred to eukaryotic vector with marker gene. - **Marker Genes (Table 8.1 in text):** - **Antibiotic Resistance:** Kanamycin (nptII), geneticin, hygromycin. - **Herbicide Resistance:** Phosphinothricin (bar), glyphosate (EPSPS), sulfonylurea (csr-1). - **Non-Invasive:** *gus* (β-glucuronidase, blue color with X-Gluc), *gfp* (green fluorescent protein, light emission - Chapter 2). #### 8.2.4 Transformation of Plants - **Challenge:** Plant cell walls, bark, waxy cuticles. - **Techniques (Fig. 8.3 in text):** - **Direct Gene Transfer to Protoplasts:** Using PEG, liposomes, electroporation. - **Direct Gene Transfer to Intact Tissue/Cell Cultures:** Electroporation, microinjection, silicone carbide whiskers, biolistics. - **Agrobacterium Infection:** Wounding or co-cultivation. - **Vacuum Infiltration:** Plant tissues. #### 8.2.5 Protoplasts - **Isolation:** Plant material + fungal enzymes (cellulases) remove cell wall. Spherical cell bounded by membrane. Stabilized by osmoticum (e.g., mannitol). - **Protocol (Box 8.1 in text):** Young leaves, cut strips, enzyme incubation, filter, centrifuge, resuspend in sucrose (protoplasts float at interface). - **Regeneration:** Transformed protoplast must regenerate whole plant. Difficult: sensitive to shear, cultural conditions, media. Growth requirements change. Often sterile, phenotypically abnormal (*Datta et al., 1994*). - **DNA Transfer Methods:** - **PEG:** High concentrations (up to 28%) in high MgCl₂ medium. Widely used, easy, higher survival, but introduces multiple/fragmented copies. - **Liposomes:** DNA-containing liposomes with PEG. - **Electroporation:** High voltage DC pulses open cell membrane. DNA enters. Reduces viability. - **Transformation Frequency:** ~0.46% (1400/3x10⁵) for electroporation/PEG. #### 8.2.6 Direct DNA Transfer - **Advantages:** Avoids cell wall removal/regeneration, not limited by protoplast production. - **Methods:** - **Microinjection:** Physical injection. Difficult, laborious. Controls DNA molecules transferred, co-transfer possible. Limited use. - **Electroporation:** Similar to protoplasts. Transformed maize, rice, sugarcane, cassava. - **Silicone Carbide Whiskers:** Whiskers + DNA + cell suspensions. Agitation penetrates cells, transfers DNA. Applied to sunflower, maize. - **Particle Bombardment (Biolistics - Fig. 8.4 in text):** Most widely used. Microscopic particles coated with DNA. - **Advantages:** Mechanical, not limited by *Agrobacterium* host range/protoplast restrictions. No plasmid manipulation. Simple, large pieces of tissue. - **Designs:** Tungsten particles propelled by gunpowder (Sanford, 1988), helium gas burst (Kikkert, 1993), airgun (Oard, 1993), electric discharge (ACCELL™ - McCabe and Christou, 1993). - **Low-Pressure Helium:** Macrocarrier eliminated, particles accelerated directly (Gray et al., 1994). - **Mechanism:** Gold/tungsten particles (0.1-2 µm) coated with DNA, fired into tissue. Particles penetrate, transformation occurs. Used for diverse plants, especially those difficult for *Agrobacterium*. #### 8.2.7 Agrobacterium - **Mechanism:** Gram-negative soil bacterium forms crown gall on dicotyledonous plants. Ti (tumor-inducing) plasmid integrates part of its DNA (T-DNA) into plant genome. - **T-DNA Genes:** Code for auxins/cytokinins (onc), opine synthesis (amino acid/sugar derivatives). Opines consumed by *Agrobacterium*. - **vir (virulence) region:** Required for infection. - **Vector Modification:** Ti plasmid modified (most genes removed between border sequences) to avoid overproduction of growth regulators/callus formation. More room for added DNA (e.g., antibiotic resistance gene). - **Advantages:** Precise integration of multiple copies. - **Limitations:** Size of DNA T-DNA can contain, host range. - **Host Range:** Thought limited to monocots (Liliales, Arales), but wheat/maize transformed. - **Methods:** Co-cultivation (with/without wounding), sonnication before treatment. Vacuum infiltration for Arabidopsis. - **Choice:** Method of choice (simple, extended host range). Disadvantage: depends on regenerating whole plant from transformed tissue. - **T-DNA Size:** Was thought limited to 25kb, but new binary vectors with enhanced *vir* genes transfer up to 150kb. - **Precision:** More precise than random DNA introduction. - **Regulatory Concern:** DNA sequences outside border sequences can be transferred (*Kononov et al., 1997*). Requires full description of transferred DNA. #### 8.2.8 Regeneration (Fig. 8.6 in text) - **Need:** Transformed cells/parts regenerate into whole plants. - **Process:** Controlled by growth regulators. - **Callus/Suspension Cultures (Fig. 8.6a):** Explant on solid medium with growth regulators forms undifferentiated callus. - **Organogenesis (Fig. 8.6b):** Correct auxin:cytokinin ratio induces roots or shoots. High auxin:cytokinin (4:1) for shoots, (100:1) for roots. Shoots excised, rooted, form plantlet. - **Embryogenesis:** Somatic embryos form, cultured into whole plants. - **Challenges:** Exact conditions specie-specific, determined experimentally. Growth requirements change as protoplasts divide. #### 8.2.9 Plant Improvement via Plant Cell Culture - **Benefits:** Rapid production of plants, selection for variants. - **Somaclonal Variation:** High level of variation in regenerated plants (highest in protoplasts). Genetic changes: point mutations, chromosome changes, cryptic changes, copy number changes, transposable elements, somatic crossing over, sister chromatid exchange, DNA amplification/deletion (*Karp, 1995*). - **Application:** 1. Induce/grow callus/cell suspensions. 2. Regenerate many plants. 3. Screen for desirable traits. 4. Test selected variants for stability. 5. Multiply selected variant. - **Examples:** Variants in maize, tomato, spruce, ornamentals. Tomato line with higher solids content (commercial for soup). #### 8.2.10 Protoplast Fusion - **Methods:** Chemical (PEG, dextran, PVA + high pH/Ca²⁺), electrical. - **Electrical Fusion (Fig. 8.7 in text):** AC aligns protoplasts, DC pulse induces membrane breakdown/fusion. - **Commercialization:** Limited despite research. #### 8.2.11 Transgenic Plants - **Traits:** Herbicide resistance, insect resistance, product quality, viral resistance, agronomic traits, fungal resistance, bioreactors. - **Approval:** Approved for sale/consumption in Europe/USA (Tables 8.2, 8.3 in text). Table 8.4 shows EU/UK approvals. - **Most Frequent Trait (Fig. 8.8 in text):** Herbicide resistance. - **Dominant Crops (Fig. 8.9 in text):** Soybean, cotton, corn (maize), rapeseed, tomato, potato. - **Regulation:** Advisory Committee on Novel Foods and Processes (ACNFP) in UK, FDA in USA. Deliberate Release Directive (90/220/EEC). - **Concerns:** - **Risk Assessment:** Genes transferred, not technique. Compared to weed water hyacinth. - **Human Health:** Introduced gene product, marker genes (e.g., nptII for kanamycin resistance). Transfer of antibiotic resistance to environmental microbes. Allergenic pollen. - **Gene Transfer to Environment:** - **Microorganisms:** Bacteria transfer DNA by transformation, conjugation, transduction. - **Wild Populations:** Cross-pollination (if compatible, same location, flower time). - **Strategies to Avoid Transfer:** Male sterility, lethal gene in pollen, flower removal, buffer plants. Direct gene to chloroplast (not transferred in pollen). Growth regulators to alter flowering time. - **Ecological Impact:** Selective advantage for transgenic plant (new pest/weed), gene transfer to weeds, competition with beneficial plants, upset plant communities. - **Release Considerations (Table 8.5 in text):** Case-by-case basis. - Kanamycin resistance gene (nptII) tested, but non-antibiotic markers preferred. - Field trials for gene transfer strategies. - Field evaluation for expression levels. #### 8.2.12 Tolerance to Pesticides and Herbicides - **Goal:** Reduce herbicide use. - **Resistance Mechanisms:** - Increase affected enzyme. - Express mutant enzyme. - Express enzyme that detoxifies herbicide. - **Example: Glyphosate (Fig. 8.10 in text):** Inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in chloroplasts (aromatic amino acid pathway). - **Tolerance:** Overexpression of EPSPS gene (from petunia) or expression of mutant bacterial EPSPS (less susceptible). - **Commercial Use:** Glyphosate-resistant cotton (1997), soybean. Cotton growers reported premature boll drop/deformation. - **Transgenic Soybean:** Transformed with *Agrobacterium* gene (CP4) for glyphosate-insensitive EPSPS. Also contains *Petunia hybrida* EPSPS gene with chloroplast transit peptide (CTP). Transformed by particle gun. PCR/Southern blot confirmed only EPSPS/CTP genes (no GUS/nptII). #### 8.2.13 Resistance to Pests - **Problem:** Insects, nematodes, molluscs affect crop yield. - **Solution:** Genetic engineering for pest-resistant plants. - **Example: *Bacillus thuringiensis* (Bt):** Gene for endotoxin (toxic to caterpillars, harmless to animals/other insects - Section 4.2.3). - **Mechanism:** Inactive protoxin (1200 amino acids) in spores. Cleaved by proteases in caterpillar gut to active toxin (68,000 daltons). Binds to midgut cells, kills caterpillar. - **Transgenic Plants:** Poplar, elm, spruce, maize, cotton. Maize resistant to European corn borer. Cotton with truncated/modified toxin gene for 100x increase in toxin. Trees resistant to gypsy moth (*Podila and Karnosky, 1996*). - **Other Possibilities:** Cholesterase oxidase (Vip3A), amylase inhibitor, systemic wound-response proteins (*Dempsey et al., 1998*). #### 8.2.14 Resistance to Pathogens - **Pathogens:** Viruses, bacteria, fungi. - **Viral Infections:** - **Pathogen-Derived Resistance (PDR):** Transform plant cells with viral genes/sequences. Plants resistant to viruses. - **Examples:** Coat protein of tobacco mosaic virus (TMV) in tobacco plants. Transgenic squash/papaya approved. - **Mutant Viral Movement Protein:** Expressed for resistance. - **Ribosome-Inactivation Protein (RIP):** Pokeweed antiviral protein (antiviral activity). Transgenic plants with RIP were stunted. - **Double-Stranded RNA-Specific Ribonuclease:** *Sano et al. (1997)*: Transgenic potato with yeast ribonuclease resistant to potato spindle tuber viroid (digested double-stranded RNA). - **Bacterial Diseases (Table 8.6 in text):** - **Antibacterial Proteins:** Lytic peptides (cecropins from giant silk moth in potato/tobacco), lysozymes (degrade peptidoglycan), lactoferrin, toxins. - **Enhanced Natural Defenses:** - **Pathogen Recognition Gene (R):** Cloning R gene from resistant line into susceptible plant (e.g., rice transformed with R gene for resistance to *Xanthomonas orzyae*). - **Reactive Oxygen Species:** Glucose oxidase gene from *Aspergillus niger* in tobacco for increased H₂O₂ production, resistance to *E. carotovora*. - **Rapid Localized Cell Death:** Limits infection spread. - **Phytoalexins:** Small antimicrobial compounds. Target for genetic engineering, but complex pathways, toxicity to plant itself. #### 8.2.15 Fruit Quality - **Example: Flavr Savr™ Tomato (Calgene):** Increased shelf-life by antisense silencing of polygalacturonase (involved in cell wall degradation during ripening). - **Antisense Technology (Fig. 8.11 in text):** Reverse coding region of gene, transcribes antisense DNA, forms duplex with normal mRNA, reduces enzyme. - **Zeneca Tomato:** Truncated sense polygalacturonase gene for longer shelf-life, higher solids. Sold processed (paste). - **Testing:** Extensive testing for food safety (effect on tomato as food, naturally occurring toxins, gene copy number). FDA/ACNFP approved. #### 8.2.16 Agronomic Traits - **Potential:** Change color, nitrogen fixation, starch synthesis, stress resistance, oil modification. - **Starch Synthesis:** - **Composition (Fig. 8.12 in text):** Amylose (linear), amylopectin (branched). Most starches are amylopectin. - **Pathway (Fig. 8.13 in text):** Sucrose to starch in amyloplasts. Key enzymes: ADP-glucose pyrophosphorylase, starch synthase (elongase), branching starch synthase. - **Genetic Engineering:** Increase starch in potatoes with mutated bacterial gene (g/gC16) for ADP-glucose pyrophorylase (60% increase). Reduce amylose in potato by antisense of granule-bound starch synthase. - **Oil Modification:** - **Major Oils:** Soybean, oil palm, rapeseed, sunflower (72% world vegetable oil). - **Rapeseed:** Good transformation system. Herbicide-resistant lines approved. Focus on altering oil composition (Table 8.7 in text). - **Example:** Transgenic rapeseed forms 40% lauric acid (C12) with California Bay plant's ACP thioesterase. Accumulates 40% stearic acid by antisense of *Brassica* stearate desaturase. - **Flower Color:** Inactivate endogenous enzymes or introduce new genes (e.g., reduce CHS chalcone synthase for red/blue pigments). - **Nitrogen Fixation:** Challenging (bacterial process, O₂ sensitive). - **Stress Tolerance (Table 8.8 in text):** Multifaceted, many genes. Attempts: osmoregulatory products, membrane-modifying enzymes, radical-scavenging enzymes, stress-induced proteins. #### 8.2.17 Plants as Bioreactors - **Purpose:** Modify plants to produce foreign compounds with commercial value. - **Advantages:** Versatile, renewable, low-cost source of carbohydrates, fatty acids, proteins, enzymes, pharmaceuticals, biodegradable plastics. - **Examples (Table 8.9 in text):** - **Poly-3-hydroxybutyrate (PHB - Fig. 8.14 in text):** Biodegradable polyester. Synthesized in *Arabidopsis* by introducing genes for acetoacetyl-CoA reductase and poly(3-hydroxybutyrate) synthase. - **Enhancement:** Redirecting enzymes to plastids (site of fatty acid synthesis, high acetyl-CoA) increased PHB accumulation 100-fold. #### 8.3 Diagnostics - **Importance:** Disease detection in animals/plants (health, commerce). - **Biotechnology Role:** - **Pure Antigens (Genetic Engineering):** Allows formation of specific monoclonal antibodies. - **Monoclonal Antibodies:** Distinguish closely related species, accurate detection of infections. - **Techniques:** Radioimmune assay (RIA), fluorescence immunoassay (FIA), enzyme-linked immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA). - **PCR:** Detects very low levels of organisms (Chapter 2). #### 8.4 Improved Animals - **Conventional Breeding:** Slow process. - **Transgenic Animals (Two Main Areas):** 1. **Biopharming:** Animals produce foreign proteins for medical use (high-value pharmaceuticals). - **Examples:** Blood factors VII/IX in sheep, lactoferrin in cattle. - **Extraction:** Link gene to milk-produced protein for extraction from milk. 2. **Breeding Scheme:** Improve growth. - **Growth Hormone:** Animals/fish engineered for more growth hormone, faster growth. - **Non-Genetic Manipulation:** - **Embryo Cloning:** 16-cell embryo (from artificial insemination) as source of nuclei. Nuclei transplanted into enucleated eggs, cultured, implanted into cows. 16 identical calves. - **Somatic Cell Nuclear Transfer:** Nuclei from embryo-derived mouse cell line transplanted into unfertilized eggs. *Dolly* (lamb from sheep mammary cells). Public concern ("playing God"). - **Bovine Somatotrophin (BST):** Hormone for growth promotion, increased lactation. - **Mechanism:** Administered to lactating cows, increases milk by 10-25%. - **Production:** Gene cloned/expressed in *E. coli* for bulk production. - **Controversy:** Approved for use, but resisted. Imposes stress on cows, increases feed, adverse effects (mastitis, reduced pregnancy, knee lesions). Milk surplus argument. Public acceptance (natural products). #### 8.5 Animal Vaccines - **Traditional Vaccines:** Live (attenuated viral/bacterial), killed (dead whole cells/viruses/inactivated toxins). - **Genetic Engineering:** Develop vaccines against animal diseases (rabies, Newcastle disease, foot and mouth, swine pseudorabies, fowl plague, bacterial infections like colibacillosis, pasteurellosis). - **Subunit Vaccines:** - **Mechanism:** Identify, clone, express antigenic molecules in bacteria. Easy, inexpensive, devoid of other material, no live organisms. - **Problems:** Low expression of cloned gene, improper protein folding (e.g., animal protein in bacterium with different post-translational processing). - **Solution:** Expression in animal cell cultures (expensive, requires removal of animal DNA). - **Limitations:** Weak response (improved by concentrated antigen + bacterial cell wall material). Short life. Limited use (e.g., human hepatitis B subunit vaccine). - **Live Recombinant Vaccines:** Incorporate subunit vaccine gene into live, attenuated virus/bacteria. Stronger response, no injection needed. - **Example: Vaccinia Virus:** Subunit gene cloned between vaccinia sequences in *E. coli* plasmid, recombines on infection. - **Rabies Vaccine:** Glycoprotein from virus as antigen. Eradicated rabies in field trials (RaboraR for wild animals in France). - **Other Examples:** Newcastle disease, fowl pox in poultry. - **Synthetic Peptide Vaccines:** Use only part of antigen molecule (epitopes). Chemically synthesized, eliminates purification. - **Example: Foot and Mouth Virus:** Traditional vaccine (killed virus) loses activity. VP1 peptide (140-160) linked to carrier protein, expressed in *E. coli*. Successful in guinea pigs, ineffective in cattle. #### 8.6 Biodiversity - **Problem:** Loss of plant/animal species at increasing rate (due to agricultural practices). Irreplaceable genetic material. - **Convention on Biological Diversity (UNEP, 1995):** "Variability among living organisms from all sources... includes diversity within species, between species and of ecosystems." - **Reasons for Maintenance:** - **Intrinsic Value:** All species have value. - **Human Welfare:** Potential value (e.g., new drugs from plants). - **Breeding:** Required for high variation. - **Ecological Communities:** Nutrient recycling, oxygen/CO₂ balance. - **Biotechnology Role:** - **Rescue Endangered Species:** Plant/animal cell culture, cryopreservation. #### 8.7 Conclusions - **Agriculture:** Biotechnology (genetic engineering) has and will continue to have major impact. - **Environmental Effects:** - **Antibiotic Use:** New subunit vaccines reduce antibiotic use in animals, combating resistance. - **Diagnostics:** Increased use of engineered products/monoclonal antibodies for plant/animal disease diagnosis. - **Pesticides/Herbicides:** Transgenic plants reduce use. Public concern over antibiotic resistance genes in GMOs. Labeling may reduce fears. Market forces may limit introduction. - **Transgenic Animals:** Continued introduction, but public concerns may limit. - **Other Products:** Microbial inoculants, biopesticides, anaerobic digestion of wastes. - **Biodiversity:** PCR for analyzing microbial populations (only 1% identified). - **Biofuels:** Needed eventually. Biotechnology provides oils/alcohols, potentially hydrogen. - **Biocontrol:** Depends on public debate. Plants with insect/pathogen control reduce biocide use. - **Transgenic Plants:** Alterations to flavor, color, resistance. Significant debate. - **Animal Cloning:** Public concerns, but may become more common for high-grade animals. - **Vaccines/Growth Hormones/Transgenic Animals:** Animal vaccines will continue. BST/transgenic salmon/animals' acceptance depends on public. - **Clean Technology:** Reduce/eliminate pollution by applying biotech. IPM for reduced pesticide use. Enzymes from extremophiles reduce industrial pollution. - **Overall Goal:** Sustainable future ("meets the needs of the present without compromising the ability of future generations to meet their own needs"). ### 9. Future Prospects #### Rachel Carson's "Silent Spring" (1962) - Signaled change in public view of pesticides, initiated anti-pollution legislation. - Focused on organophosphate pesticides (use reduced). - Highlighted environmental issues: climate change (GHGs), ozone depletion (CFCs), acid rain (H₂S), genetically engineered organisms. #### Biotechnology's Public Perception - **Traditional Technologies (Waste Treatment, Biofuels):** Viewed as benign. - **Genetic Engineering (Transgenic Plants/Animals):** Viewed with suspicion ("against nature," compared to thalidomide). - **"Playing God":** Especially with transgenic humans/animals, though disease treatment accepted. - **Agrochemical Companies:** Motives perceived as profit-driven, risks not fully considered (e.g., bovine somatotropin (BST)). - **BST Example:** Growth hormone increases milk yield (15-20%). Gene cloned in *E. coli* for bulk production. Arguments against: stress on cows, milk surplus, labeling. #### Risk and Communication - **Technology Risk:** All technology involves risk. Risks and advantages must be fully explained. - **Public Ignorance:** Lack of knowledge about biotechnology ("fish genes transferred to strawberries"). Remedy: education (but seen as indoctrination). - **Public Confidence:** Influenced by shift to organic products, distrust of corporate/government institutions (e.g., BSE outbreak). - **Transgenic Crops in Developing Nations:** Accepted if relieving famine. Resistance in developed nations where good quality food is available. - **Human Genome Project:** Included ethical, legal, social implications from start. #### Concerns Regarding Genetically Engineered Organisms (GMOs) - **Scientific Community Concerns:** - **Gene Transfer:** From released organisms to environmental organisms (conjugation, transformation, transduction in microbes; cross-pollination in plants). - **Marker/Selectable Genes:** Transfer of antibiotic resistance. Safety of marker gene product (e.g., nptII for kanamycin). - **Selective Advantage:** New transgenic plant becomes dominant species. - **Pest Enhancement:** - **Harm to Non-Target Species:** - **Disruptive Ecological Effects:** (e.g., pollination, competition). - **Risk Assessment:** - **Consensus:** Risk is from the recombinant organism/product, and the environment of release, not the techniques. - **Qualitative Measure:** Probability of harm from known hazard. Information often insufficient for GMOs. - **Regulation Rationale:** Science-based judgment replaces vague fears. - **Case-by-Case Basis:** Risks to humans (allergenic pollen), plant origin, colonization potential, ecological relationships (pollination disruption, pest enhancement, competition). - **Conclusion (National Academy of Sciences, USA):** No evidence of unique hazards from gene transfer between unrelated species (*Miller, 1994*). Risks similar to unmodified organisms (*Sawahel, 1994*). - **Current Status:** Many field-tested crops (maize, squash, canola, cotton, soybean, tomato, rapeseed) for herbicide resistance, insect resistance, fruit quality. - **Tomatoes (Calgene, Zeneca):** Antisense technology for delayed ripening. Approved for public consumption (fresh fruit, canned paste). Public acceptance will determine future. - **Overall:** Genetic manipulation will profoundly influence agriculture and environment. #### Environmental Biotechnology's Future Development - **Three Sections of Application:** 1. Reduce/recycle waste ("end-of-pipe" treatments). 2. Eliminate waste production ("green technology"). 3. Clean-up contaminated sites ("bioremediation"). - **Key Topics:** - **Diagnosis/Bioindicators/Biosensors:** Estimate pollution, microbial populations, real-time analysis. Engineered biomarkers for in situ pollution. - **Bioremediation:** Continued expansion for metals, organics, xenobiotics. In situ/ex situ enhancement of natural microbial populations. - **Bioleaching:** Continued use for low-grade ores. Metal removal from mine leachate. - **Biopharming:** Crop plants for pharmaceuticals. Depends on public acceptance of transgenic plants. - **Biodiversity Preservation:** Analysis of microbial populations (e.g., PCR). - **Biofuels:** Eventual need for alternative fuels. Biotech provides oils/alcohol, potentially hydrogen. - **Biocontrol:** Depends on public debate on transgenic plants. Reduces biocide use. - **Transgenic Plants:** Flavor, color, resistance alterations. Significant future debate. - **Animal Cloning:** Public concerns, but may become more used for high-grade animals. - **Vaccines/Growth Hormones/Transgenic Animals:** Animal vaccines will continue. Acceptance of BST/transgenic salmon/animals depends on public need/acceptance. - **Clean Technology:** Applies biotech to reduce/eliminate pollution. IPM reduces pesticide use. Enzymes from extremophiles reduce industrial pollution. #### Sustainable Future - "Development that meets the needs of the present without compromising the ability of future generations to meet their own needs." ### Constants and Notations #### SI Units (Common Prefixes) - **exa (E):** 10¹⁸ - **peta (P):** 10¹⁵ - **tera (T):** 10¹² - **giga (G):** 10⁹ - **mega (M):** 10⁶ - **kilo (k):** 10³ - **hecto (h):** 10² - **deka (da):** 10¹ - **deci (d):** 10⁻¹ - **centi (c):** 10⁻² - **milli (m):** 10⁻³ - **micro (µ):** 10⁻⁶ - **nano (n):** 10⁻⁹ - **pico (p):** 10⁻¹² - **femto (f):** 10⁻¹⁵ - **atto (a):** 10⁻¹⁸ #### Common Notations and Abbreviations - **ADP:** Adenosine Diphosphate - **ATP:** Adenosine Triphosphate - **BATNEEC:** Best Available Technology Not Entailing Excessive Cost - **BCF:** Bioconcentration Factor - **BOD:** Biological Oxygen Demand - **BOD₅:** Biological Oxygen Demand over 5 days - **BST:** Bovine Somatotrophin - **BTEX:** Benzene, Toluene, Ethylbenzene, Xylenes - **CaMV:** Cauliflower Mosaic Virus - **cDNA:** Complementary DNA - **CFCs:** Chlorofluorocarbons - **CH₄:** Methane - **CLSM:** Confocal Scanning Laser Microscopy - **CO:** Carbon Monoxide - **CO₂:** Carbon Dioxide - **COD:** Chemical Oxygen Demand - **CTAB:** Cetyl-Trimethylammonium Bromide - **CTP:** Chloroplast Transit Peptide - **DBT:** Dibenzothiophene - **DDT:** Dichlorodiphenyltrichloroethane - **DGGE:** Denaturing Gradient Gel Electrophoresis - **DNA:** Deoxyribonucleic Acid - **EIA:** Enzyme Immunoassay - **EIFAC:** European Inland Fisheries Advisory Commission - **ELISA:** Enzyme-Linked Immunosorbent Assay - **EOR:** Enhanced Oil Recovery - **EPA:** Environmental Protection Agency (USA) - **EPSPS:** 5-Enolpyruvylshikimate-3-phosphate synthase - **EU:** European Union - **FGD:** Flue Gas Desulfurization - **FIA:** Fluorescence Immunoassay - ***gfp*:** Green Fluorescent Protein gene - **GHG:** Greenhouse Gas - **GMO:** Genetically Manipulated Organism - **GUS:** β-Glucuronidase gene - **HCl:** Hydrochloric Acid - **HF:** Hydrofluoric Acid - **H₂O:** Water - **H₂O₂:** Hydrogen Peroxide - HRT: Hydraulic Retention Time - **H₂S:** Hydrogen Sulfide - **ICM:** Integrated Crop Management - **IPM:** Integrated Pest Management - ***lacZ*:** β-Galactosidase gene - ***luxAB*:** Luciferase gene - **MEK:** Methyl Ethyl Ketone - **MEOR:** Microbially Enhanced Oil Recovery - **MLSS:** Mixed Liquor Suspended Solids - **MLVSS:** Mixed Liquor Volatile Suspended Solids - **mRNA:** Messenger Ribonucleic Acid - **NADH:** Nicotinamide Adenine Dinucleotide (reduced form) - **N₂O:** Nitrous Oxide - **NO:** Nitric Oxide - **NOₓ:** Nitrogen Oxides - **nptII:** Neomycin Phosphotransferase II gene - **OOIP:** Original Oil In Place - **O₃:** Ozone - **PAHs:** Polyaromatic Hydrocarbons - **PCBs:** Polychlorobiphenyls - **PCR:** Polymerase Chain Reaction - **PCP:** Pentachlorophenol - **PDR:** Pathogen-Derived Resistance - **PEG:** Polyethylene Glycol - **PHB:** Poly(R)-(3-hydroxybutyrate) - **PHB-V:** Poly(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) - **PLFA:** Phospholipid Fatty Acids - **PLFAME:** Phospholipid Ester-Linked Fatty Acids - **ppm:** Parts per Million - **ppb:** Parts per Billion - **ppt:** Parts per Trillion - **PVA:** Polyvinyl Alcohol - **RFLP:** Restriction Fragment Length Polymorphism - **RIA:** Radioimmunoassay - **RIP:** Ribosome-Inactivation Protein - **RNA:** Ribonucleic Acid - **SAGD:** Steam Assisted Gravity Drainage - **SBR:** Sequencing Batch Reactor - **SCP:** Single Cell Protein - **SDS:** Sodium Dodecyl Sulfate - **SO₂:** Sulfur Dioxide - **SO₃:** Sulfur Trioxide - **ssrRNA:** Small Subunit Ribosomal RNA - **SS:** Suspended Solids - **TCDD:** 2,3,7,8-Tetrachlorodibenzo-p-dioxin - **Ti plasmid:** Tumor-inducing plasmid - **TMV:** Tobacco Mosaic Virus - **TOC:** Total Organic Carbon - **TOL plasmid:** Toluene degradation plasmid - **µmax:** Maximum Specific Growth Rate - **UNCED:** United Nations Conference on Environment and Development - **UNEP:** United Nations Environment Programme - **VOCs:** Volatile Organic Compounds - **WWI:** World War I - **X-Gluc:** 5-bromo-4-chloro-3-indolyl-glucuronide