ITU, Environmental Engineering Undergraduate Program

Fall Semester
CEV213E ENVIRONMENTAL MICROBIOLOGY Lab Session

ACTIVATED SLUDGE MICROBIOLOGY

Examination of activated sludge flocks / Macro structures

AIM and SIGNIFICANCE:

WHAT is Biological Wastewater Treatment (BioWWT)?
conversion of (colloidal / dissolved) organic matter (C) and/or nutrients (N, P) in polluted water
to CO2, NH3, N2 (gases) + H2O (now treated) + Biomass (solids) via microbial activity

WHAT are the Two Main Targets of BioWWT to obtain good effluent quality?
1. Remove C, N, P from wastewater : BIOCONVERSIONS
2. Separate the biomass from treated wastewater : SETTLING

To ensure good effluent quality, the activated sludge (biomass) should also have:
a “healthy AS structure” and “good settling properties”.

Activated Sludge Components in suspended growth systems:

Activated sludge (AS) : overall gathering of all microorganisms (bacteria+archaea+eukarya)
present in SUSPENSION in a BioWWTP
Granules (200 - 3500 μm) : EXTREMELY COMPACT and dense aggregates of microorganisms;
a type of biofilm formation without an inert attachment surface. They
have excellent settling properties (SVI < 50 mL/g)
Flocks (25 - 250 μm) : FIRM gatherings of microorganisms
Filaments : HAIRY microorganisms which do not settle and thus may cause
sludge bulking/foaming problems if they are excess in numbers
Single cells (dispersed) (1 - 20 μm): pure cultures and single cells do not settle, but remain in
suspension
Healthy Activated Sludge (AS)

Dense and irregularly shaped aggregates facilitating attachment of more microorganisms and
containing a few filamentous organisms, which might serve as a backbone structure.
There must be a good proportion between the flocs and filamentous organisms. The more the
numbers of filaments, the more deteriorates the settling properties of the biomass.
How to evaluate?
1. Microscopic examination (wet-mounting, dry sampling and staining)
2. Subjective Scoring for Filament Abundance (Jenkins et. al., 1993)
0  none
1  few
2  some
3  common
4  very common
5  abundant
6  excessive

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 ITU, Environmental Engineering Undergraduate Program
Fall Semester
CEV213E ENVIRONMENTAL MICROBIOLOGY Lab Session

Good Settling Properties

Run Sludge Volume Index (SVI) experiment

Sludge Volume Index (SVI): SVI is used to monitor the settling characteristics of activated
sludge and is used in routine process control. It is defined as “the volume (mL) occupied by 1 g
of settled sludge after 30 minutes of settling. SVI is calculated by the following equation and
expressed in mL per gram:

settled sludgevolume mL / L 

SVI mL / g  
mixed liquor suspendedsolidsg / L 
A mixed liquor sample with an SVI value between 50 and 100 mL/g is considered as a good
settling sludge, whereas SVI values above 150 mL/g are typically associated with excess growth
and abundance of filamentous microorganisms with poor settling properties. An SVI value less
than 50 mL/g indicates excellent settling properties, as in granular sludge systems. On the other
hand, if the SVI value is less than 50 mL/g, and the supernatant is highly turbid, then this indicates
the predominance of pin point flocks, hence poor effluent quality (Tchobanoglous et al., 2003;
Bitton, 2005).
List of Materials / Equipment / Instruments Used
1 L of graduated cylinder (or Imhoff cone) , clock or stopwatch, light microscope, microscope slide,
cover slip, loop, Bunsen burner, alcohol, beaker, forceps, Pasteur pipette, paper towel, distilled
water
Sample: Mixed liquor activated sludge sample

EXPERIMENTAL PROCEDURE:
Sludge Volume Index
1. Pour 1 L of mixed liquor into a 1 L graduated cylinder.
2. Allow to settle for 30 minutes.
3. Record the settled sludge volume in milliliters after 30 min.
4. Analyze the suspended solids (MLSS*) concentration of the mixed liquor.
5. Calculate the SVI value.
Wet mount
1. Using a Pasteur pipette place a drop of well mixed sample on a clean slide.
2. Place carefully a cover slip on the sample to exclude air bubbles (place one end of the
cover slip on the slide and slowly lower the other end to prevent air bubbles).
3. Remove the excess with a piece of paper towel.
4. Examine under microscope using 10x and 100x objectives.
*The MLSS is determined by filtering an aliquot of mixed liquor, drying the filter at 105 C, and determining
the weight of solids in the sample.
REFERENCES
APHA, AWWA and WEF, (2005). “Standard Methods for the Examination of Water and Wastewater”,
21th edn, American Public Health Association/ American Water Works Association / Water Environment
Federation, Washington, DC, USA.
Bitton, G. (2005). “Wastewater Microbiology”, John Wiley & Sons, New Jersey, USA.
Tchobanoglous, G., Burton, F.L. and Stensel, H.D. (2003). “Wastewater Engineering, Treatment and
Reuse / Metcalf&Eddy”, McGraw Hill, NY, USA.
Jenkins, D., Richard, M.G., Daigger, G.T. (1993). “Manual on the Causes and Control of Activated
Sludge Bulking, Foaming, and Other Solids Separation Problems”, 3rd edn., Lewis Publishers, Inc.,
Boca-Rota, USA, ISBN: 9781566706476. [Mustafa Inan Main Library: TD756 .J46 2004]