Wastewater Microbiology and Bacterial Development

Wastewater Treatment Fundamentals and Microbiology

• West Point Treatment Plant (Seattle, WA):

  • Largest facility of its type on the West Coast.

  • Serves over $700,000$ people.

  • 2017 Flood Incident: Resulted in the discharge of approximately $250$ million gallons of untreated sewage into Puget Sound.

• Objectives of Wastewater Treatment (Removal Targets):

  • Sand, grit, and debris: Inorganic materials that can damage equipment.

  • Suspended solids: Organic and inorganic particles.

  • Biochemical Oxygen Demand (BOD): Defined as the milligrams of oxygen consumed per liter of sample during $5$ days of incubation at $20^{\circ}C$. This serves as a primary indicator of organic contamination levels.

  • Pathogens: Disease-causing agents (bacteria, viruses, protozoa).

  • Nutrients: Primarily Nitrogen (ammonia, nitrate) and Phosphorus.

  • Nitrification: The process of converting ammonia to nitrate ($NH_3 + O_2 \rightarrow NO_3^-$).

  • Denitrification: The process of converting nitrate to nitrogen gas ($NO_3^- \rightarrow N_2$).

• Wastewater Treatment Process Flow:

  • Preliminary Treatment (Physical): Includes Bar Screening (removing coarse solids/rags), Grit Chambers (settling heavy solids like sand and glass), and Comminution (grinding solids). Does not significantly decrease BOD.

  • Primary Treatment (Physical): Uses primary clarifiers/settling tanks (retention time: $2-3$ hours). Removes $45-50\%$ of suspended solids and $25-30\%$ of BOD. Skimmer booms remove floating materials like grease/oil. Resulting "Primary Sludge" is sent to thickeners.

  • Secondary Treatment (Biological): Focuses on soluble BOD removal. Uses microorganisms to convert organic waste to $CO_2$ and $H_2O$. Methods include:

    • Trickling Filter: A biofilm process where sewage is sprayed over media (rocks/plastic). Space between media allows air circulation.

    • Activated Sludge: A suspended growth process utilizing aeration basins ($6-8$ hour residence time) and secondary clarifiers. Biomass is recycled as Return Activated Sludge (RAS) to maintain high concentrations.

  • Disinfection: Final treatment using chlorine or chloramines. Water is often dechlorinated with sodium bisulfite ($NaHSO_3$) before discharge: $NaHSO_3 + Cl_2 + H_2O \rightarrow NaHSO_4 + 2HCl$.

  • Sludge Treatment: Includes thickening, Anaerobic Digestion (converting organic sludge to $CO_2$ and $CH_4$ over $~30$ days), and drying beds. Digested sludge can be used as an agricultural amendment if it meets heavy metal and pathogen standards.

• Microbiology of Activated Sludge:

  • Flocs: Suspended aggregates of microbes. Composition typically includes rod-shaped and filamentous species.

  • Nocardia spp.: Gram-positive, non-motile actinomycetes. High concentrations (often due to high fats, oils, and greases/FOG) cause problematic foaming.

  • Zoogloea spp.: Gram-negative, motile rods known as "living glue." Produce exopolysaccharides (EPS) that hold flocs together, trap nutrients, and remove BOD. Can produce Poly-hydroxybutyrate.

• Wastewater Pathogens:

  • Bacteria: Salmonella typhi (typhoid), Vibrio cholerae (cholera), Shigella dysenteriae (dysentery).

  • Viruses: Rotaviruses (diarrhea).

  • Protozoa: Giardia lamblia and Cryptosporidium parvum (cause waterborne dysentery/diarrhea). These form resistant cysts.

  • Case Study: Milwaukee, WI, 1993. Cryptosporidium outbreak sickened $400,000$ people and killed more than $50$. The organism is resistant to chlorine disinfection.

The Nitrogen Cycle and Human Impact

• Fundamentals:

  • Nitrogen makes up $78\%$ of the atmosphere.

  • Nitrogen Fixation: Conversion of $N_2$ gas to ammonia ($NH_3$) by lightning or specialized bacteria.

  • Nitrification: Done by chemolithotrophs; ammonia is converted to nitrite, then nitrate.

  • Denitrification: Anaerobic respiration where nitrates are converted back to gaseous $N_2$.

• Anthropogenic Effects:

  • Haber-Bosch Process: Industrial synthesis of ammonia for fertilizers.

  • Consequences: Increased greenhouse gases, soil acidification, leaching of calcium/potassium, and reduction of plant diversity.

  • Gulf of Mexico Dead Zone: A nutrient-rich (N and P) runoff causing seasonal hypoxia, resulting in approximately $100$ million dollars in losses; the zone is roughly the size of Delaware.

• Experimental Study of N-Cycle in Biofilms (Okabe et al., 1999):

  • Used Fluorescence In Situ Hybridization (FISH) to map bacterial groups. Red = Ammonia-oxidizing bacteria; Green = Nitrite-oxidizing bacteria; Green = Heterotrophic Eubacteria.

  • Relatively localized production and consumption of $NH_4^+$, $NO_2^-$, and $NO_3^-$ were measured along chemical depth gradients ($0-2000\,\mu m$).

Hydrothermal Vent Biology and Chemosynthesis

• Discovery and Environment:

  • Discovered in $1977$ near the Galapagos Rift using the Alvin submersible (Woods Hole Oceanographic Institute).

  • Estimated ocean volume cycles through these vents every $100,000$ years.

  • Fluid temperature can reach $400^{\circ}C$. Fluid is acidic, anaerobic, and rich in dissolved metals ($Fe, Cu, Zn$) and $H_2S$.

  • Black Smokers: Hottest vents, spewing iron and sulfide which precipitate into black chimneys.

  • White Smokers: Cooler vents rich in barium, calcium, and silica.

• Foundation of Life:

  • Chemosynthesis: Primary production driven by thermophilic chemolithoautotrophs ($45-122^{\circ}C$).

  • Energy Generation: Microbes oxidize reduced compounds ($H_2, Fe^{2+}, Mn^{2+}, H_2S$) using $O_2$ or $NO_3^-$ as electron acceptors.

  • Carbon Fixation Pathways:

    • Calvin-Benson Cycle: Found at cooler temperatures (< 20^{\circ}C); uses RubisCO.

    • Reductive TCA Cycle: Energy-efficient but oxygen-sensitive ($20-90^{\circ}C$).

    • Dicarboxylate-4-hydroxybutyrate pathway: Used by hyperthermophilic archaea (> 90^{\circ}C).

• Symbiotic Relationships:

  • Riftia pachyptila (Tubeworms): Up to $2$ meters long. No mouth or stomach. Possess a "trophosome" housing symbiotic sulfur-oxidizing bacteria. Hemoglobin transports $H_2S$ and $O_2$ to the tissues.

  • Vent Clams (Calyptogena): House symbionts in thick gills.

  • Vent Shrimp: Some species feature light organs on their backs to detect faint vent glows.

Sociomicrobiology and Quorum Sensing

• Bacterial Sociality: Concepts introduced by Ed Wilson (sociobiology). Bacteria use communication to coordinate group behaviors like swarming, biofilm formation, and fruiting bodies.

• Quorum Sensing (QS) Mechanisms:

  • Scheme #1: Acyl-HSL (Gram-negative): Paradigm is Vibrio fischeri. Uses LuxI (synthase) to create Acyl-Homoserine Lactone (AHL) and LuxR (regulator) to detect it. Regulates luminescence in Bobtail squid for "counterillumination" camouflage.

  • Scheme #2: Peptide Signaling (Gram-positive): Uses a precursor peptide cleaved into an Autoinducing Peptide (AIP). Signal sensed via a two-component Histidine Kinase/Response Regulator system (e.g., S. aureus agr system).

  • Scheme #3: Volatile Signaling: Ralstonia solanacearum uses $3$-OH palmitic acid methyl ester.

• Myxobacteria (Myxococcus xanthus):

  • Large genome ($9.1\,Mbp$). Gliding bacteria that form fruiting bodies under starvation.

  • A-signaling: Amino acid sensing (Stringent Response) to gauge density.

  • C-signaling: Cell-to-cell contact signaling that coordinates aggregation.

  • Altruism: Only $0.1-10\%$ of cells survive as myxospores.

Motility and Specialized Structures

• Modes of Motility:

  • Twitching: Driven by Type IV pili (ATP-powered extension/retraction).

  • Gliding: Involves focal adhesins and motor proteins without flagella.

  • Swarming: Group flagellar motility across surfaces, often requiring surfactants.

• Vibrio parahaemolyticus Dual Flagellar System:

  • Polar Flagellum: $Na^+$-driven, used for swimming in low viscosity.

  • Lateral Flagella: $H^+$-driven, induced by high viscosity or slowing of the polar motor (sensed via sodium channel blockers like Phenamil).

• Internal Structures:

  • Magnetosomes: Membrane-bound chains of magnetite ($Fe_3O_4$) or greigite ($Fe_3S_4$). Facilitate magnetotaxis. Organized by the actin homolog MamK.

  • Gas Vesicles: Protein shells (GvpA, GvpC) providing buoyancy in aquatic phototrophs (e.g., Microcystis). Impermeable to water but permeable to gas.

Biofilms, Chronic Infections, and CF

• Biofilms: Aggregates encased in a self-produced matrix (EPS).

  • Resistance Mechanisms: Matrix diffusion barrier, slow-growing subpopulations (persisters), and unique biofilm phenotypes.

  • Cystic Fibrosis (CF): Autosomal recessive defect in the CFTR chloride transporter. Leads to thick mucus in lungs, providing a niche for Pseudomonas aeruginosa.

• P. aeruginosa Adaptations in CF:

  • Mucoidy: Overproduction of alginate due to mutations in mucA (anti-sigma factor).

  • RSCV (Rugose Small Colony Variants): Linked to high levels of cyclic-di-GMP, causing increased adherence and Pel/Psl exopolysaccharide production.

  • Biofilm Development: Regulated stages including attachment, microcolony formation, maturation (maintained by rhamnolipid surfactants), and dispersal.

Bacterial Organelles and Segregation

• Nucleoid: compacted DNA region; compacted by Nucleoid-Associated Proteins (NAPs).

• Chromosome Segregation:

  • ParABS/SMC: ParB binds to parS sites near the origin, recruiting SMC (Structural Maintenance of Chromosomes) rings to resolve and condense DNA.

  • FtsK: A DNA-pumping machine at the septum that resolves chromosome dimers via recombinases (XerC/D) and concatenated DNA via topoisomerases.

• Storage Inclusions:

  • Polyphosphate granules: Phosphate and energy storage.

  • Polyhydroxybutyrate (PHB): Carbon storage.

  • Ferrosomes: Lipid-bound iron ($Fe^{2+}$) storage regulated by fez genes.

• Metabolic Organelles:

  • Carboxysomes: Protein shells concentrating RubisCO and trapping $CO_2$; segregated by the ParA-like protein McdA.

  • Anammoxosomes: Site of anaerobic ammonia oxidation.

  • Thylakoids: Membranous structures for photosynthesis in cyanobacteria.

  • Phage "Nucleus": Jumbophages build a protein shell to protect their DNA from CRISPR/RM defenses.

Bacteriophages and Viral Ecology

• Virion Components: Head (capsid), tail, and tail fibers. Diverse genomes (dsDNA, ssDNA, RNA) ranging from $3\,kb$ to $500\,kb$.

• Lifestyles:

  • Lytic: Immediate replication and host lysis.

  • Lysogenic: Integration into the host genome as a prophage. Switch is often dictated by prey availability/QS.

• Bacterial Defenses:

  • Restriction-Modification (RM): Nucleases cut specific motifs; host methylates its own DNA for protection. Phages counter with DNA mimics (Ocr) or modified nucleotides (HMC).

  • CRISPR-Cas: Adaptive immunity that stores memory in spacers. Phages counter with Anti-CRISPR (ACR) proteins.

• Viral Shunt: Phage-mediated lysis recycles half of bacterial biomass daily, returning dissolved organic matter (DOM) to the environment instead of moving it up the food chain.

• Transduction:

  • Generalized: Accidental packaging of host DNA.

  • Specialized: Imprecise excision of a prophage, carrying adjacent host genes.

Bacterial Development: Sporulation

• Bacillus subtilis Endospores: Highly resistant dormant cells ($121^{\circ}C$ resistant).

• The Decision: Sensed via Histidine Kinases (KinA-E) triggered by P, N, or C starvation. Phosphorelay: $KinA\text{-}P \rightarrow Spo0F\text{-}P \rightarrow Spo0B\text{-}P \rightarrow Spo0A\text{-}P$ (Master Regulator).

• Sigma Factor Cascade:

  1. Predivisional: $\sigma^H$.

  2. Asymmetric Division: $\sigma^F$ (Forespore) and $\sigma^E$ (Mother Cell).

  3. Late Development: $\sigma^G$ (Forespore) and $\sigma^K$ (Mother Cell).

• Crisscross Regulation: The forespore signals the mother cell to activate $\sigma^E$ via the SpoIIR protein and SpoIIGA protease.

• Spore Composition: DNA is protected by Small Acid-Soluble Proteins (SASPs) and dehydrated by Calcium-Dipicolinic Acid ($Ca^{2+}:DPA$), which accounts for $10-15\%$ of dry weight.

• Germination: Triggered by nutrient-gated ion channels (germinant receptors) sensing molecules like L-alanine.