Wastewater Microbiology

Sewage Properties
• Physical Properties
– Color
– Odour
– Turbidity
– Temperature
– Taste
• Chemical Properties
– Organic: carbonaceous matter &Nitrogenous matter
– Inorganic
• Biological Properties: Protista, plants &animals
 Micro-organisms in Wastewater

• Bacteria
• Viruses
• Protozoa
• Helminths
 Bacterial Growth Curve
• There are several distinct segments in the biomass curve.
The microorganisms must first become acclimated to their
surrounding environment and to the food provided. The
acclimation period called the lag phase, is represented by
segment 1on the curve and will vary in length depending
on the history of the seed organisms. If the organism have
been accustomed to a similar environment and similar
food, the lag phase will be brief.
• Once growth has been initiated, it will proceed quite
rapidly. Bacterial cells reproduced by binary fission i.e.
cells divide into segments that separate to become two
new independent cells. When maximum growth is
occurring, the rate of reproduction is exponential. So the
segment 2 is called the log growth phase .
• Cells that are unable to obtain food from external sources
begin endogenous catabolism or catabolizing of stored
protoplasm for maintenance energy. Segment 3of the curve
represents stationary phase where the production of new
cellular material is roughly offset by death and endogenous
respiration.
• Though Some reproduction continues beyond stationary
phase ,death and endogenous respiration predominate in
segment 4 of the curve. So , biomass slowly decreases
approaching zero asymptotically after a very long period.
Bacterial Growth Curve
 Indicator Micro-organisms
• The numbers of pathogenic organisms present in wastes
and polluted waters are usually few and difficult to isolate
and identify
• Micro-organisms, which are more numerous and more
easily tested for, are commonly used as surrogate (i.e., an
indicator) organisms for the target pathogen(s).
Characteristics of an Ideal Indicator Organism
1. The indicator organism must be present when fecal contamination is present.
2. The numbers of indicator organisms present should be equal to or greater than
those of the target pathogenic organism (e.g., pathogenic viruses).
3. The indicator organism must exhibit the same greater survival characteristics in
the environment as the target pathogen organism for which it is a surrogate.
4. The indicator organism must not reproduce outside of the host organism (i.e.,
the culturing procedure itself should not produce a serious health threat to
laboratory workers).
5. The isolation and quantification of the indicator organism must be faster than
that of the target pathogen (i.e, the procedure must be cheaper and easier for
cultivating the indicator organisms than for the target pathogen).
6. The organism should be a member of the intestinal micro-flora of warm-
blooded animals.
Identification of Indicator Microorganisms

• Most Probable Number Method

• Direct Counting Method/Membrane filter
(MF) method
 Multiple Tube Method/Most probable Number
(MPN) Method

• In the multiple tube method, different amounts of water are added to tubes
containing a suitable culture media. The bacteria present in the water
reproduce and in accordance with the number inoculated and the number of
tubes with a positive reaction, the most probable number (MPN) of bacteria
present in the original water sample can then be statistically determined. The
multiple tube method is applicable to all kinds of water samples. It can cope
with clean, colored, or turbid waters containing sewage or sewage sludge or
mud soils panicles provided that the bacteria are homogenously disturbed in
the prepared test samples. Theoretically the technique is sufficiently sensitive
to measure low levels of bacteria in water sample are needed to be used as
culture vessels. However for normal water analysis 10 ml is usually the largest
volume used. This method is often preferred by microbiologists.
 Membrane filter (MF) method
• In contrast to the multiple tube method, the membrane filter (MF)
method gives a direct count of total coli forms and fecal coli forms
present in a given sample of water. The method is based on the
filtration of a known volume of water through a membrane filter
consisting of a cellular compound with a uniform pore diameter of
0.45micrometer,the bacteria are retained on the surface of the
membrane filter. When the membrane containing the bacteria is
incubated in a sterile container at an appropriate temperature with
selective differential culture medium. characteristics colonies of
coliforms and fecal coliforms develop which can be counted directly.
This technique is popular with environmental engineers.
 Biochemical Oxygen Demand
(BOD)
• The amount of oxygen required by bacteria while stabilizing
decomposable organic matter under aerobic condition.
• When biodegradable organic matter/waste (the most common category
of pollutant affecting surface water) is released into a water body,
microorganisms (especially bacteria) feed on the wastes, breaking it
down to simple organic and inorganic substance. When this
decomposition takes place in an aerobic substance, it produces non –
objectionable, stable and products (e.g., CO2 SO4 PO4 and NO3) and in
the process draws down the dissolved oxygen (DO) content of water.
Organic matter + O2 = CO2 + H2O + New cells + Stable products
(Bacteria)
• When insufficient oxygen is available or when oxygen is
exhausted by the aerobic decomposition of waste. Different
set of microorganisms carry out the decomposition
anaerobically producing highly objectionable products
including H2S NH3 and CH4.
Organic matter = CO2 + CH4 + New cells + Unstable products
(Bacteria)
 Modeling of BOD

• It is often assumed that the rate of decomposition of

organic waste is proportional to the amount of waste
available. If Lt represent amount of oxygen demand left
after time t, then, assuming a first order reaction, we can
write, dLt/dt = -k Lt (1)
Where, k = BOD reaction rate constant (time-l).
Integrating, we get, Lt =L0e-kt (2)

Where, L0 is the ultimate carbonaceous oxygen demand,

which is the total amount of oxygen required by
microorganisms to oxidize the carbonaceous portion of
waste.
Now, L0 - Lt = L0 - L0e-kt
So, BODt = L0 (1- e-kt ) (3)
• Figure 1 shows the graphical representation of Eq. (2) and
Eq. (3) respectively.
• Besides the oxidation of carbonaceous organic, a significant
additional demand may be caused by the oxidation of
nitrogenous compounds. To distinguish this nitrogenous
biochemical oxygen demand from the carbonaceous
biochemical oxygen demand the two are sometimes referred
to as NBOD and CBOD respectively. Figure 2 illustrates
the two demands for a typical municipal waste. Note that
the NBOD does not being to exert itself for at least 5 to 8
days, so most 5 days BOD tests are not affected by the
nitrification process.
k is the reaction constant, which is temperature dependent
Problem:
BOD5 of wastewater was found 150mg/l at 20oC temperature. The k value is
0.23/day. What would the BOD8 be if the test was run at 15oC.

Solution:
BOD5 = L0 (1- e-kt )
L0
k value at 15 oC

BOD8 at 15 o C
BOD8 = L0 (1- e-k 15t )
BOD8 =220(1-e-0.18x8 )
=168 mg/L
 Chemical Oxygen Demand
(COD)
• COD is the amount of oxygen required to stabilized the organic
matter chemically, i.e. the COD is used as a measure of the oxygen
equivalent of the organic matter contents of a sample that is
susceptible to oxidation by a strong chemical oxidant.
• During determination of COD, organic matter is converted to
carbon dioxide and water
• The limitation of the COD test is its inability to differentiate
between biologically oxidizable and biologically inert organic matter