LAT 401 Microbiology: Microbial Ecology, Sewage Treatment, and Water Microbiology Study Notes

Introduction to Microbial Ecology

• Microbial Ecology Definition: This is the study of the relationships of microorganisms to each other and to their environment. It focuses on the activities of microorganisms that help to maintain, sustain, and control the life support system on Earth.

• Scope of Study: This field involves:

  • Natural Roles: Examining the roles microbes play in the environment and their contributions to the ecological balance, including soil, water, and mineral cycles.

  • Artificial Applications: The use of microbes in food, medicinal, biochemical, drug, and agricultural industries.

Role of Microbes in the Environment

• General Contributions: Microbes contribute profoundly to Earth's structure and function and are essential for the survival of other life forms.

• Primary Application Areas:

  • Agriculture.

  • Food production.

  • Industrial processes.

  • Water treatment.

  • Waste (sewage) treatment.

  • Bioremediation of polluted environments.

• Ecological Functions:

  • Habitat Adaptation: Microbes adapt to specific habitats and niches to obtain food, energy, shelter, and other essential components of the biosphere.

  • Recycling: Microbes act as decomposers, mineralizers, and recyclers to maintain and cycle biologically important elements (e.g., $C$, $N$, and $P$) that exist only in certain reservoirs.

Microbiology of Sewage Treatment

• Role of Microbes: Microbes are central to sewage treatment. Through biological digestion, contaminated, infectious liquid is converted into a relatively stable, inert sludge and a harmless effluent.

• Sewage Definition: The used water supply containing domestic waste, human excrement, wash water, and industrial waste (acids, greases, oils, animal and vegetable matter) and storm waters.

• Components of Sewage:

  • Waste Water: Material from household plumbing (washing/bath water and toilet waste).

  • Municipal Waste Water: Includes industrial wastes like acids, greases, and oils.

• Basic Principles of Treatment:

  1. Water is separated from the waste.

  2. Solid organic matter is biodegraded (decomposed) by microorganisms into simple compounds such as nitrates ($NO_3^-$), sulphates ($SO_4^{2-}$), carbonates ($CO_3^{2-}$), carbon dioxide ($CO_2$), and methane ($CH_4$).

• Management Scales: Small scale (individual homes), rural areas, and large scale (towns/municipalities).

• Rationale for Treatment: Sewage contains large amounts of solid waste, dissolved organic matter, and toxic chemicals. If untreated, it leads to:

  • Accumulation of pathogenic microbes and toxic substances.

  • Harm to flora and fauna in water bodies.

  • Spread of waterborne diseases.

  • Eutrophication: An increase in nutrient content leading to Algal Bloom.

  • Algal Bloom Consequences: Green scum or mats of filamentous algae form. When algae die, aerobic decomposers break them down using available oxygen ($O_2$) for respiration, which depletes oxygen levels and causes the death of fish and other aquatic organisms.

Biochemical Oxygen Demand (BOD)

• Definition: The amount of oxygen required for the microbial decomposition of organic matter in a water sample.

• Significance: BOD is roughly proportional to the amount of degradable organic material present. High BOD values indicate high pollution and high utilization of dissolved oxygen by microbes for respiration.

• BOD Reduction: One of the primary goals of sewage treatment is to reduce BOD.

• Determination of BOD (Step-by-Step):

  1. Measure the oxygen level of a well-aerated sample ($O_1$).

  2. Incubate the sample in a sealed container in the dark under standard conditions: $5 \text { days}$ at $20^{\circ}C$.

  3. Measure the oxygen level again ($O_2$).

  4. Formula: $O_1 - O_2 = BOD$.

• Reference Values:

  • Raw Sewage BOD: Approximately $300 \text { to } 400\,mg/litre$.

  • Natural Water Dissolved Oxygen: $5 \text { to } 10\,mg/litre$ (Source: Nester et. al., 2005).

Methods for Reducing BOD

• Aerobic Treatment: Microbes oxidize organic compounds in the presence of large amounts of oxygen, yielding $CO_2$ and inorganic compounds. This is relatively expensive due to the engineering required for oxygenation.

• Anaerobic Treatment: This involves a succession of microbial populations.

  • Anaerobic microbes ferment organic compounds.

  • Methanogens: These generate methane ($CH_4$) by oxidizing hydrogen gas ($H_2$) as an energy source, using $CO_2$ as a terminal electron acceptor.

  • The resulting methane can be discarded, used for fuel, or oxidized back to $CO_2$ by methane-oxidizing bacteria.

Municipal Sewage Treatment Phases

• Phase 1: Primary Treatment (Physical Process):

  • Designed to remove materials that settle or sediment out.

  • Removes approximately $50\%$ of solids and $25\%$ of BOD.

  • Process: Pass influent through screens for large objects (sticks, rags, plastic), use skimmers for scum, and use sedimentation tanks where gravity facilitates solid removal over $2 \text { to } 10 \text { hours}$.

  • Output: Sludge and supernatant (rich in organic matter).

• Phase 2: Secondary Treatment (Biological Process):

  • Involves active microbial decomposition (biodegradation) in large digester tanks by bacteria, algae, and protozoa.

  • Activation: Air is introduced to speed up aerobic decomposition. Organic matter is converted to sulphates, nitrates, phosphates, $CO_2$, and water ($H_2O$).

  • Volatile Gases Released: Hydrogen sulfide ($H_2S$), Ammonia ($NH_3$), Nitrogen ($N_2$), and Methane ($CH_4$).

  • Inoculum: A portion of the settled sludge is reintroduced into new wastewater loads.

  • Complication (Bulking): Occurs when filamentous bacteria, such as $Thriothrix \text { sp.}$, overgrow and create a buoyant mass that does not settle, interfering with sludge separation.

• Phase 3: Tertiary Treatment (Chemical Process/Disinfection):

  • Involves further filtering and chlorination.

  • Disinfection Methods: Chlorine, ozone ($O_3$), or UV light to target microorganisms and viruses.

  • De-chlorination: May be performed to prevent toxic chemical release.

  • Nutrient Removal: Specifically designed to remove nitrates and phosphates to prevent algal bloom in receiving waters.

  • Sludge Reuse: Remaining sludge is rich in nitrogen ($N$), potassium ($K$), and phosphorus ($P$), making it a useful fertilizer.

Water Microbiology

• Definition: The study of microorganisms living in or transported by water.

• Advantages: Certain yeast strains provide beer and bread; some bacteria digest poisons in contaminated water.

• Disadvantages (Pathogens):

  • Bacteria: $Escherichia \text { coli}$, $Salmonella$, $Shigella$, and $Vibrio$ from fecal contamination.

  • E. coli O157:H7: Can be fatal; in Summer 2000 (Canada), this strain sickened $2,000$ people and killed $7$.

  • Viruses: Rotavirus, enteroviruses, and coxsackievirus.

  • Protozoa: $Giardia$ and $Cryptosporidium$ (lived in intestines of beaver, deer, humans). These form cysts, which are resistant to chlorine and standard filters, causing prolonged diarrhea.

• Natural Flora: Bacteria, cyanobacteria, protozoa, algae, and tiny animals like rotifers. Cyanobacteria and algae are vital food sources in the water food chain.

• Freshwater Zones:

  • Littoral Zone: Shoreline, shallow, well-lighted, and warmer. Home to photosynthetic algae and bacteria.

  • Limnetic Zone: Open water further from shore; also contains photosynthetic microbes.

  • Benthic Zone: Bottom of the water body. Low oxygen and light. Dominated by bacteria that survive without oxygen, such as methane-producing bacteria. Purple and green sulfur bacteria dominate intermediate depths.

• Saltwater Environment: Higher salt concentration, higher $pH$, and lower nutrients compared to freshwater. Characterized by:

  • Halophilic bacteria: Salt-loving bacteria near the surface.

  • Other species: $Pseudomonas$ and $Vibrio$.

  • Archaebacteria: Demonstrated in 2001 to be a dominant life form in the ocean.

  • Dinoflagellates: Growth can cause "red tide," depleting nutrients/oxygen and killing fish.

• Transport by Boats: Ballast water in the hulls of ships stabilizes vessels but transports microbes globally (e.g., $Vibrio \text { cholerae}$).

Drinking Water Treatment and Testing

• Treatment Process (Stepwise):

  1. Reservoirs/Sedimentation: Large materials sediment; Alum (Aluminum/Potassium sulphate) is added for flocculation.

  2. Filtration: Passed through sand beds (removes bacteria) and activated charcoal (removes toxic organics).

  3. Disinfection/Fluoridation: Use of chlorine, UV, or ozone. Fluoride may be added for dental protection.

• Testing Methods:

  • Turbidity: Meaures suspended material (soil/microbes) to indicate water quality.

  • Indicator Organisms: Surrogates used because testing for every pathogen is unfeasible. High organic matter and bacteria correlate with lower dissolved oxygen.

Coliform Enumeration and Testing Methods

• Definition of Coliforms: Gram-negative, non-spore-forming rods that are facultative anaerobes and ferment lactose to produce gas (e.g., $E. \text { coli}$, $Enterobacter \text { aerogenes}$).

• Fecal vs. Facultative: $E. \text { coli}$ is a true fecal coliform (human/animal intestines) and the best indicator of recent contamination. $Enterobacter$ is facultative and found in soil/water.

• Multiple Tube Method (Most Probable Number/MPN):

  1. Presumptive Test: Inoculate $9 \text { or } 12$ tubes of lactose/laurel tryptose broth. Check for gas production after $24 \text { hours}$. Numbers are applied to a statistical table for MPN per $100\,ml$.

  2. Confirmatory Test: Inoculate positive samples onto Levine EMB or Endo agar. EMB produces small colonies with dark centers (nucleated); Endo produces reddish colonies. Both inhibit Gram-positive bacteria.

  3. Complete Test: Uses nutrient agar slant and a Durham tube of lactose broth. Positive if gas is produced and a slide shows Gram-negative, non-spore-forming rods.

• Membrane Filter Method: An alternative routine testing method for coliforms.

Bioremediation

• Definition: Use of microorganisms or plants to biodegrade or remove toxic waste in water and soil. Derived from "bio" (biological) and "remediation" (to remedy).

• Key Microbes: Natural residents like $Pseudomonas$, $Bacillus$ (denitrifying), and toxin-eating fungi.

• Engineering: Approximately $35$ recombinant microbes exist. $Rhodococcus$ decomposes industrial hydrocarbon waste ($PCB's$). Engineered $Pseudomonas$ detoxify heavy metals, $CCl_4$, and naphthalene.

• Influencing Factors: Adequate nutrients, near-neutral $pH$, raised temperature, and optimum moisture.

• Types of Bioremediation:

  • Biostimulation: Adding nutrients ($N$, $P$) and oxygen to encourage existing microbial growth. (e.g., oil spill fertilizers).

  • Bioaugmentation: Adding specific microbes to the site, such as using activated sludge in wastewater treatment.

  • Intrinsic Bioremediation (Natural Attenuation): Cleanup by nature without human help.

• Location of Treatment:

  • In situ: Treatment at the site of contamination.

  • Ex situ: Removal of material to be treated elsewhere.