Biotechnology: Class 12 — Chapter 11: Bioremediation

Ananda Mohan Chakrabarty (4 April 1938 – 10 July 2020) was an Indian-American microbiologist pioneer in environmental microbiology and genetic engineering.

• Pseudomonas putida: In 1971, Chakrabarty developed a genetically engineered strain capable of digesting multiple hydrocarbons found in oil spills.
• Multi-plasmid Hydrocarbon-degrading Pseudomonas: Created by transferring plasmids carrying genes for degrading different hydrocarbons into a single bacterial cell.
• Diamond v. Chakrabarty (1980): Landmark US Supreme Court trial that established the legal right to patent genetically engineered living organisms.
• Padma Shri: Awarded by the Indian government in 2007 in recognition of his scientific contributions.

Wild-type Pseudomonas strains possessed individual plasmids capable of degrading only single hydrocarbon components (e.g., CAM plasmid for camphor, OCT for octane, XYL for xylol, NAH for naphthalene), making them inefficient for complex oil spill mixtures.

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Engineering Methodology & Mechanism: Multi-plasmid transfer via hybridization and recombination. Plasmids from four different strains of Pseudomonas were transferred into a single strain of Pseudomonas putida. The resulting "Superbug" stably carries CAM/OCT and XYL/NAH plasmids, enabling simultaneous degradation of camphor, octane, xylol, and naphthalene in oil spills.

Municipal sewage consists of approximately 99% water and 1% suspended and soluble organic/inorganic matter.

Biochemical Oxygen Demand (BOD) is the quantity of oxygen required by aerobic microorganisms to stabilize decomposable organic matter in water over a specific incubation period (typically 5 days at 20°C).

• Higher BOD indicates a higher concentration of oxidizable organic matter (strong sewage).
• BOD is calculated using the dilution method:

$$\text{BOD } (\text{O}_2 \text{ mg/L}) = \frac{D_1 - D_2}{\% \text{ dilution}} \times 100 \quad \text{or} \quad (D_1 - D_2) \times \text{Dilution Factor}$$

Where $D_1$ is the dissolved oxygen (DO) of the sample before incubation, and $D_2$ is the DO after incubation.

1. Primary (Physical) Treatment: Physical removal of coarse solids.
   • Screening: Removes large floating objects (bottles, paper, wood).
   • Grit Chamber: Filters out sand, grit, and small stones.
   • Sedimentation: Sewage is held in tanks for 2–10 hours; suspended organic solids settle to form primary sludge. Removes 30–40% of BOD.
2. Secondary (Biological) Treatment: Aerobic and anaerobic microbial degradation of dissolved organic matter.
   • Activated Sludge Process: Wastewater is pumped into an aeration tank with compressed air. Aerobic microbes form flocs that oxidize organic matter to $\text{CO}_2$ and $\text{H}_2\text{O}$. Settled biomass is recycled as activated sludge.
   • Trickling Filter: Wastewater is sprayed over a bed of crushed stones/gravel coated with a microbial slime layer (bacteria, fungi, algae) that degrades organic compounds.
   • Sewage Oxidation Ponds (Lagoons): Shallow ponds (< 5 feet) where natural wind action and algal photosynthesis provide oxygen for aerobic microbial digestion. Reduces BOD by 75–95%.
   • Anaerobic Sludge Digestion: Primary and secondary sludge is digested by anaerobic microbes in closed tanks for 30–40 days. Produces biogas containing 60–70% $\text{CH}_4$, 20–30% $\text{CO}_2$, $\text{H}_2\text{S}$, $\text{H}_2$, and $\text{N}_2$. Dewatered sludge is processed into fertilizer (e.g., Milorganite).
3. Tertiary (Advanced) Treatment: Chemical and physical processes to remove remaining nutrients and non-biodegradable pollutants to prevent eutrophication.
   • Phosphorus Removal: Precipitation using lime (calcium hydroxide) to form insoluble calcium phosphate:$$3\text{Ca}^{2+} + 2\text{PO}_4^{3-} \rightarrow \text{Ca}_3(\text{PO}_4)_2 \downarrow$$
   • Nitrogen Removal: Volatilization (air stripping) of ammonia gas ($\text{NH}_3$) at high pH in stripping towers.
   • Carbon Adsorption: Passing effluent through activated carbon filters to remove dissolved organic compounds (carbon polishing).
   • Disinfection: Chlorination using sodium hypochlorite ($\text{NaOCl}$), calcium hypochlorite ($\text{CaOCl}_2$), or chlorine gas to kill pathogens.

Composting is the biological degradation of organic waste by diverse microbial communities under controlled conditions (optimum moisture: 45–65%).

1. Mesophilic Phase: Moderate-temperature phase (up to 40°C) lasting a few days; rapid breakdown of soluble, easily degradable compounds by mesophilic microbes.
2. Thermophilic Phase: High-temperature phase (40°C to >65°C) lasting from days to months. High temperatures accelerate the degradation of proteins, fats, and complex structural carbohydrates (cellulose, hemicellulose). Pathogens are destroyed at $\ge 55^\circ\text{C}$. Dominant bacteria include the genus Thermus and actinomycetes.
3. Maturation (Cooling) Phase: Temperature gradually decreases; mesophilic microbes recolonize to cure the remaining organic matter over several months, producing stable humus.

Biomedical waste is categorized into four major groups based on segregation pathways and standard color-coded containers.

• Risk Group 1: Low individual and community risk (unlikely to cause human or animal disease).
• Risk Group 2: Moderate individual risk, low community risk (can cause disease but effective treatment and preventive measures are available).
• Risk Group 3: High individual risk, low community risk (causes serious disease but does not ordinarily spread from person to person).
• Risk Group 4: High individual and community risk (causes severe disease, easily transmitted, no effective treatment available).

The Atomic Energy Regulatory Board (AERB) is the competent authority in India governing the safe disposal of radioactive wastes under the Atomic Energy Rules, 1987.

• Radioactive waste must be stored in lead-shielded containers and disposed of in designated deep geological repositories.
• Mayapuri Radiological Accident (2010): A severe radiation exposure incident in Delhi caused by the improper dismantling and auction of an industrial Cobalt-60 source to scrap dealers.

The Atz gene family (AtzA to AtzF) in Pseudomonas species encodes enzymes that sequentially degrade the herbicide Atrazine into non-toxic end products:

$$\text{Atrazine} \xrightarrow{\text{AtzA}} \text{Hydroxyatrazine} \xrightarrow{\text{AtzB}} \text{N-isopropylammelide} \xrightarrow{\text{AtzC}} \text{Cyanuric acid} \xrightarrow{\text{AtzD}} \text{Biuret} \xrightarrow{\text{AtzE}} \text{Allophanate} \xrightarrow{\text{AtzF}} 2\text{NH}_4^+ + 2\text{CO}_2$$

• In Situ Bioremediation: Treatment of contaminated material directly at the site.
  • Intrinsic In Situ: Natural degradation by indigenous microflora without human intervention.
  • Engineered In Situ: Enhancing microbial activity by injecting oxygen, nitrogen, and nutrients via injection wells.
• Ex Situ Bioremediation: Excavation and physical removal of contaminated soil/water for treatment elsewhere.
  • Slurry Bioreactors: Mixing contaminated soil with water and nutrients in a bioreactor vessel under vigorous agitation.
  • Land Farming: Spreading excavated contaminated soil in thin layers on treatment beds and applying nutrients and moisture.
  • Biopiles: Piling excavated soil and piping air and nutrients through it to stimulate microbial growth.
  • Oilzapper: A biotechnology product developed by TERI consisting of a consortium of five non-pathogenic bacterial strains immobilized on a powdered corncob carrier, used to degrade crude oil sludge.

Phytoremediation is the use of plants to clean up, accumulate, or neutralize hazardous environmental contaminants.

• Brassica napus and Helianthus annuus: Accumulate heavy metals like lead and mercury from contaminated soils.
• Hydrilla verticillata and Nymphaea alba: Reduce toxic chromium levels in polluted water bodies.