9.1 Activated Sludge Fundamentals & Microbiology

9.1 Activated Sludge Fundamentals & Microbiology

Secondary biological wastewater treatment is designed to remove dissolved, non-settleable, and colloidal organic pollutants that escape primary sedimentation. The activated sludge process is the most widely applied suspended-growth secondary treatment technology in municipal wastewater treatment. In this process, a dense, multi-species culture of microorganisms is continuously mixed and aerated in an aeration basin with incoming settled wastewater. The biological mass—termed Mixed Liquor Suspended Solids (MLSS)—metabolizes organic matter, converting it into cellular mass, carbon dioxide, water, and stable inorganic byproducts.

The mixed liquor then flows into a secondary clarifier, where biological solids separate from the treated liquid via gravity sedimentation. A portion of the concentrated biological solids is recycled back to the aeration basin as Return Activated Sludge (RAS) to maintain the required microbial population, while the excess biological growth is removed from the system as Waste Activated Sludge (WAS).

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Principles of Suspended-Growth Biological Oxidation

Biological wastewater treatment mimics and accelerates natural aquatic purification processes within engineered reactor basins. The primary objective is the biological conversion of Biochemical Oxygen Demand ($\text{BOD}_5$) and Chemical Oxygen Demand (COD) into settleable biological flocs:

  Soluble / Colloidal Organics + Dissolved Oxygen + Nutrients (N, P)
                      │
                      ▼  [ Microorganisms / Heterotrophs ]
  New Microbial Biomass (Floc) + Carbon Dioxide (CO2) + Water (H2O) + Energy

Bioflocculation & Extracellular Polymeric Substances

Individual bacterial cells range in size from $0.5$ to $2.0\text{ }\mu\text{m}$, making them too small to settle by gravity. Successful activated sludge operation depends upon bioflocculation—the process by which bacteria aggregate into dense, rapidly settling clusters called flocs ($50 - 500\text{ }\mu\text{m}$ in diameter).

Bioflocculation is mediated by Extracellular Polymeric Substances (EPS), which consist of polysaccharides, proteins, nucleic acids, and lipids secreted by bacteria during the declining growth and endogenous respiration phases. EPS forms a sticky, negatively charged glycocalyx slime matrix that bridges adjacent bacterial cells, encapsulating suspended particles, colloidal matter, and inert solids. Divalent cations, particularly calcium ($\text{Ca}^{2+}$) and magnesium ($\text{Mg}^{2+}$), act as ionic bridges between negatively charged EPS functional groups, strengthening the floc matrix against hydrodynamic shear.

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Microbial Ecology of Activated Sludge

The activated sludge ecosystem comprises a complex biological consortium of heterotrophic bacteria, autotrophic nitrifiers, protozoa, fungi, and metazoa. Each group performs specific biochemical functions essential for pollutant removal and solids separation.

1. Heterotrophic Bacteria (Carbonaceous BOD Removal)

Heterotrophs represent the vast majority ($> 90%$) of the active biomass in activated sludge. They obtain cellular carbon and metabolic energy from the oxidation of organic carbon compounds (sugars, amino acids, volatile fatty acids, lipids). Common genera include Pseudomonas, Zoogloea, Acinetobacter, Flavobacterium, Alcaligenes, and Bacillus.

Zoogloea ramigera is historically recognized for producing voluminous gelatinous finger-like zoogloeal matrices that accelerate floc formation. Heterotrophic bacteria exhibit rapid maximum specific growth rates (doubling times of $0.5 - 2.0\text{ hours}$ under optimal conditions) and rapidly assimilate soluble carbonaceous $\text{BOD}_5$.

2. Autotrophic Nitrifying Bacteria (Nitrification)

Autotrophs derive cellular carbon from inorganic carbon dioxide ($\text{CO}_2$) or bicarbonate alkalinity ($\text{HCO}_3^-$) and obtain metabolic energy from the oxidation of inorganic nitrogen compounds. Biological nitrification is an obligate aerobic, two-stage biochemical pathway carried out by two distinct groups of autotrophic bacteria:

+-----------------------------------------------------------------------------------------+
|                              TWO-STAGE BIOLOGICAL NITRIFICATION                         |
+-----------------------------------------------------------------------------------------+
| Stage 1: Ammonia Oxidation (Ammonia-Oxidizing Bacteria - AOB):                          |
| Genera: Nitrosomonas, Nitrosococcus                                                     |
| Reaction: 2 NH4+ + 3 O2  ──►  2 NO2- + 4 H+ + 2 H2O + Energy                            |
|                                                                                         |
| Stage 2: Nitrite Oxidation (Nitrite-Oxidizing Bacteria - NOB):                          |
| Genera: Nitrobacter, Nitrospira                                                         |
| Reaction: 2 NO2- + O2    ──►  2 NO3- + Energy                                           |
|                                                                                         |
| Overall Stoichiometric Reaction:                                                        |
| NH4+ + 2 O2 + 2 HCO3-   ──►  NO3- + 2 CO2 + 3 H2O + (Biomass)                           |
+-----------------------------------------------------------------------------------------+

Key Stoichiometric & Environmental Requirements for Nitrification:

1. Oxygen Demand: Oxidation of $1.0\text{ mg of Ammonia-Nitrogen (NH}_4^+\text{-N)}$ requires $4.57\text{ mg of Dissolved Oxygen (O}_2)$ ($3.43\text{ mg O}_2/\text{mg NH}_4^+$-N for AOB stage + $1.14\text{ mg O}_2/\text{mg NO}_2^-$-N for NOB stage).
2. Alkalinity Consumption: Nitrification generates hydrogen ions ($\text{H}^+$), destroying $7.14\text{ mg of total alkalinity as CaCO}_3$ for every $1.0\text{ mg of NH}_4^+\text{-N}$ oxidized to nitrate. If raw wastewater alkalinity is insufficient ($< 50 - 100\text{ mg/L as CaCO}_3$ residual), mixed liquor pH will drop precipitously, inhibiting nitrifiers.
3. pH Sensitivity: Optimal nitrification occurs between $\text{pH } 7.5 - 8.5$. Nitrification rates decline sharply below $\text{pH } 7.0$ and virtually cease below $\text{pH } 6.5$.
4. Temperature Sensitivity: Nitrifiers are highly sensitive to cold wastewater. Biological activity drops by approximately $50%$ for every $10^\circ\text{C}$ decrease. In Missouri winter conditions ($< 10^\circ\text{C}$), plants must significantly increase sludge age (MCRT) to maintain nitrification.
5. Growth Rate & MCRT: Nitrifiers grow much slower than heterotrophs (doubling times of $12 - 36\text{ hours}$). To prevent washing nitrifiers out of the system, the Mean Cell Residence Time (MCRT) must exceed the minimum nitrifier generation time (typically $\text{MCRT} \ge 8 - 15\text{ days}$).

Protozoan & Metazoan Indicator Succession

While bacteria perform the bulk of organic degradation, microscopic protozoa and metazoa consume dispersed, un-flocculated free bacteria, polishing the final effluent to a crystal-clear state. Because protozoa and metazoa are sensitive to physical-chemical conditions and possess distinct growth kinetics, their relative abundance provides an immediate biological indicator of sludge age (MCRT), food-to-microorganism ratio (F/M), dissolved oxygen status, and system stability.

+---------------------------------------------------------------------------------------------------------+
|                                 PROTOZOAN & METAZOAN SUCCESSION STAGES                                  |
+---------------------------------------------------------------------------------------------------------+
| 1. AMOEBAS (Sarcodina):                                                                                 |
|    - Morphology: Single-celled, amorphous, creeping protoplasm with pseudopodia (false feet).           |
|    - Operational Significance: Dominant in young sludge (MCRT < 2 days), high F/M (> 0.6), low DO,     |
|      or following severe toxic shock / organic overloading. Indicates poor settling and cloudy effluent. |
+---------------------------------------------------------------------------------------------------------+
| 2. FLAGELLATES (Mastigophora):                                                                          |
|    - Morphology: Small, oval cells propelled by 1 to 4 whip-like flagella in erratic spiral motion.      |
|    - Examples: Bodo, Euglena, Oikomonas.                                                                |
|    - Operational Significance: Characteristic of high organic loading, transitioning from young sludge |
|      (MCRT 2 - 4 days). High food supply allows flagellates to outcompete larger ciliates.              |
+---------------------------------------------------------------------------------------------------------+
| 3. FREE-SWIMMING CILIATES:                                                                              |
|    - Morphology: Covered entirely with synchronized cilia for rapid directional swimming.              |
|    - Examples: Paramecium, Colpidium, Lionotus, Tetrahymena.                                            |
|    - Operational Significance: Intermediate sludge age (MCRT 4 - 7 days). Rapidly graze on free-swimming|
|      bacteria, drastically reducing effluent turbidity.                                                 |
+---------------------------------------------------------------------------------------------------------+
| 4. CRAWLING CILIATES:                                                                                   |
|    - Morphology: Flattened bodies with specialized ventral ciliary tufts (cirri) for crawling on flocs. |
|    - Examples: Aspidisca costata, Euplotes patella.                                                     |
|    - Operational Significance: Indicates robust floc development, good settling, and stable operation. |
+---------------------------------------------------------------------------------------------------------+
| 5. STALKED CILIATES:                                                                                    |
|    - Morphology: Bell-shaped heads attached to floc particles via contractile or non-contractile stalks;|
|      cilia create localized vortex currents drawing free bacteria into oral grooves.                   |
|    - Examples: Vorticella (solitary contractile stalk), Epistylis (branched non-contractile colonies),  |
|      Opercularia, Carchesium.                                                                           |
|    - Operational Significance: Primary hallmark of mature, healthy, high-performing activated sludge   |
|      (MCRT 6 - 15 days, F/M 0.2 - 0.5). Correlates with SVI 80 - 150 mL/g and low effluent BOD/TSS.   |
+---------------------------------------------------------------------------------------------------------+
| 6. ROTIFERS & NEMATODES (Metazoa):                                                                      |
|    - Morphology: Multi-cellular organisms with rotating coronal cilia (rotifers) or microscopic         |
|      unsegmented roundworms with writhing movement (nematodes).                                         |
|    - Examples: Philodina, Rotaria, Nematoda.                                                            |
|    - Operational Significance: Indicator of old sludge (MCRT > 15 - 20 days), low F/M (< 0.1), and high|
|      nitrification. Capable of shearing and ingesting mature floc particles. Excessive numbers signal   |
|      floc disintegration and pin floc carryover.                                                        |
+---------------------------------------------------------------------------------------------------------+

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Filamentous Bacteria: Function, Overgrowth & Bulking

Filamentous bacteria are thread-like microorganisms that grow in long chains or trichomes. In balanced activated sludge, a small population of filaments serves a vital structural role by acting as a physical backbone onto which floc-forming bacteria attach, creating firm, compact flocs with good shear resistance.

However, when environmental conditions favor filamentous growth over floc-forming bacteria, filaments extend outward from the floc interior into the bulk liquid (bridging) or create loose, highly porous three-dimensional networks (open floc structure). This prevents gravity compaction in the secondary clarifier, resulting in filamentous sludge bulking ($\text{SVI} > 150 - 200\text{ mL/g}$) and sludge blanket washout.

  Balanced Floc (Optimal SVI: 80 - 150 mL/g)       Filamentous Bulking (Bulked SVI: > 200 mL/g)
  ┌────────────────────────────────────────┐       ┌────────────────────────────────────────┐
  │        ·····  ·  ·····                 │       │   \  /    ·····   /  /      \          │
  │      ·····  Floc  ·····                │       │ ───╳─── ····· Floc ───╳──── ───        │
  │     ··  (Filament Core) ··             │       │   /  \   ·····        /  \   /         │
  │        ·····  ·  ·····                 │       │  /      (Filament Bridging)            │
  │  (Dense, compact, fast-settling)       │       │  (Open, buoyant, porous, non-settling) │
  └────────────────────────────────────────┘       └────────────────────────────────────────┘

Common Filamentous Microorganisms & Root Causes

Primary Drivers of Filamentous Bulking

1. Low Dissolved Oxygen: When DO falls below $1.0 - 1.5\text{ mg/L}$, filaments outcompete floc-formers due to their higher surface-area-to-volume ratio.
2. Low F/M Ratio (Food Starvation): Prolonged low food availability favors slow-growing, scavenging filaments over floc-forming bacteria.
3. Nutrient Deficiency: Heterotrophic metabolism requires a minimum stoichiometric ratio of $\text{BOD}_5 : \text{Nitrogen (N)} : \text{Phosphorus (P)} = 100 : 5 : 1$. If nitrogen or phosphorus is deficient, bacteria produce soluble extracellular slime, causing non-filamentous "viscous/zoogloeal bulking" and promoting Type 021N and Thiothrix.
4. Septicity & Sulfides: Septic collection systems generate volatile organic acids and $\text{H}_2\text{S}$, promoting sulfur-oxidizing filaments (Thiothrix, Type 021N, Beggiatoa).
5. Low pH ($< 6.5$): Acidic conditions inhibit floc-formers and nitrifiers while promoting acid-tolerant true fungi and filaments.

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Microscopic Evaluation & Wet Mount Procedures

Daily or weekly microscopic examination of mixed liquor is an indispensable process control practice for wastewater operators. Microscopic wet mounts allow operators to track protozoan succession, evaluate floc size and density, and detect filamentous overgrowth several days before physical sludge bulking appears in the clarifier.

Standard Wet Mount Procedure

1. Sample Collection: Collect a fresh, representative sample of mixed liquor from the aeration basin discharge weir (before secondary clarification). Examine the sample within $15 - 30\text{ minutes}$ while organisms are active.
2. Slide Preparation: Using a clean Pasteur pipette, place one drop ($0.05\text{ mL}$) of mixed liquor onto a clean glass microscope slide. Carefully lower a $22 \times 22\text{ mm}$ cover slip at a $45^\circ$ angle to prevent air bubbles.
3. Low-Power Examination ($100\times$ Magnification):
   • Scan the entire slide to assess overall floc distribution, floc size (small $< 50\text{ }\mu\text{m}$, medium $50 - 150\text{ }\mu\text{m}$, large $> 150\text{ }\mu\text{m}$), and floc density (compact vs. open/straggly).
   • Count active protozoa and metazoa across 10 random fields to establish relative abundance.
4. High-Power Examination ($400\times$ to $1000\times$ Phase Contrast / Brightfield):
   • Identify dominant protozoan species (stalked ciliates, rotifers, flagellates).
   • Rate filamentous abundance using the Jenkins Filament Index (from 0 = None to 6 = Excessive/Bulking).
   • Check for internal sulfur granules in filaments using the Neisser or sulfur test.
   • Perform Gram Staining and Neisser Staining to differentiate Nocardia and Microthrix (Gram-positive) from Sphaerotilus and Type 021N (Gram-negative).