Chapter-2

Biochemical Oxygen Demand (BOD): The Concept

2.1 Oxidation of Organic Matter and Theoretical Oxygen Demand (ThOD)

Oxidation of organic matter in the presence of oxygen or oxygen supplying oxidizing

agent results in the generation of carbon dioxide, water and other inorganic end products
(such as ammonia, hydrogen sulfide etc.). The end products generated depends on the
elemental composition of the organic matter. Through stoichiometric calculations, it is
possible to theoretically estimate the amount of oxygen demanded by the sample for the
complete oxidation of the organic matter present into inorganic end products. Oxygen
demand stoichiometrically estimated for a sample is known as its Theoretical Oxygen
Demand (ThOD).

Stoichiometric equation for the oxidation of organic matter can be written as

 a b 3c   a 3c 
C n H a Ob N c +  n + − − O2 → nCO 2 +  − H 2 O + cNH 3
 4 2 4  2 2 

For glucose it can be written as

C6H12O6 + 6O2 à 6CO2 + 6H2O

This stoichiometric equation indicates that six moles or 192 grams of oxygen is required
for the complete oxidation of one mole or 180 grams of glucose into inorganic end
products (water and carbon dioxide). From the above equation, one can deduce that a
sample having x mg/l of glucose theoretically has 1.067x mg/l of ThOD. Organic matter
of a sample is rarely completely oxidized. Hence, oxygen demand actually experienced in
the water bodies or measured by BOD or COD tests for water and wastewater samples is
usually lesser than the ThOD estimated.

In both BOD & COD tests, organic matter of the sample is not completely oxidized.
Hence, BOD and COD values of a sample are always lesser than the ThOD.
Solved problem-1:

Estimate Theoretical Oxygen Demand (ThOD) for the following three following samples:

a. Sample containing 500 mg/L of sucrose (C12H22O11)

b. Sample containing 500 mg/L of ethanol (C2H6O)
c. Sample containing 500 mg/L of oxalic acid (C2H2O4)
d. Sample of mixed liquor from an activated sludge treatment plant with 3000 mg/L
of microbial biomass (C60H87O23N12P)
e. Sample of treated effluent from an algal pond having 125 mg/L of algal biomass
(C106H180O45N16P)

a. ThOD of the sample containing sucrose

Stoicheometric equation for the oxidation of sucrose is

C12H22O11 + 12O2 à 12CO2 + 11H2O

12 moles (364 grams) of oxygen is required for oxidizing one mole (342 grams)
of sucrose

Sucrose concentration of the sample = 500 mg/L

500 mg / l X 364 grams

ThOD of the sample = = 532 mg / L
342 grams

b. ThOD of the sample containing ethanol

Stoicheometric equation for the oxidation of ethanol is

C2H6O + 3O2 à 2CO2 + 3H2O

3 moles (96 grams) of oxygen is required for oxidizing one mole (46 grams) of
ethanol

Concentration of ethanol = 500 mg/l

500 mg / l X 96 grams
ThOD of the sample = =1043 .5 mg / L
46 grams

c. ThOD of the sample containing oxalic acid

Stoicheometric equation for the oxidation of oxalic acid is

2C2H2O4 + O2 à 4CO2 + 2H2O

 One mole (32 grams) of oxygen is required for oxidizing two moles (180 grams)
of oxalic acid

Concentration of oxalic acid = 500 mg/L

500 mg / L X 32 grams
ThOD of the sample = = 88 .9 mg / L
180 grams

d. ThOD of the mixed liquor sample with microbial biomass

Stoicheometric equation for the oxidation of microbial biomass with

C60H87O23N12P empirical formula is

C60H87O23N12 + 61.25O2 à 60CO2 + 25.5H2O + 12NH3

245 moles (7840 grams) of oxygen is required for oxidizing 4 empirical moles
(5496 grams) of microbial biomass.

Concentration of microbial biomass = 3000 mg/L

3000 mg / L X 7840 grams

ThOD of the sample = = 4279 mg / L
5496 grams

e. ThOD of the treated effluent sample with algal biomass

Stoicheometric equation for the oxidation of algal biomass with C106H180O45N16P

empirical formula is

C106H180O45N16 + 116.5O2 à 106CO2 + 78H2O + 16NH3

233 moles (7456 grams) of oxygen is required for oxidizing 2 empirical moles
(4790 grams) of algal biomass.

Concentration of algal biomass = 125 mg/L

125 mg / L X 7456 grams

ThOD of the sample = =195 mg / L
4790 grams

2.2 Chemical Oxygen Demand (COD) Test

All organic compounds, with a few exceptions, can be oxidized by the action of strong
oxidizing agents under acidic conditions at elevated temperature. Potassium dichromate,
potassium permanganate, cerium sulfate, potassium iodide etc., can be used as the
oxidizing agents. The oxidizing agent used supplies the oxygen required for the oxidation
of the organic matter. The oxidation process, when potassium dichromate is used as the
oxidizing agent, can be shown by:

CnHaObNc + dCr2O7-2 + (8d+c)H+ à nCO2 + [(a +8d - 3c)/2] H2O + cNH4+ + 2dCr+3

Here, d = 2n/3 + a/6 - b/3 - c/2

Amount of the oxidizing agent consumed in the oxidation process is measured and used
for the estimation of COD of the sample. Annexure-1 provides details on the COD test.

During the chemical oxidation process some fraction of the organic matter escapes
chemical oxidation mainly because of the following two reasons:

1. The sample may contain such organic substances which are resistant to the
chemical oxidation process (aromatic hydrocarbon compounds and pyridine are
resistant to chemical oxidation)
2. At the elevated temperature, volatile organic matter, originally present and/or
formed during the oxidation, may escape the chemical oxidation process
3. Certain organic compounds, specially low molecular weight fatty acids, even in
the presence of silver ions as catalyst, may escape chemical oxidation

Hence, COD is always lesser than ThOD. COD test fails to differentiate biologically
oxidizable organic matter from the biologically inert organic matter. It results in the
oxidation of almost all the organic substances of the sample. Hence, unless the aromatic
hydrocarbons and pyridine content of the sample is significantly high, COD is greater
than BOD.

2.3 Biochemical Oxygen Demand (BOD) Test

In the BOD test, the sample is incubated for a fixed duration (5 days by convention), with
right kind of microorganisms (acclimatized microorganisms, which are capable of
utilizing the organic matter present in the sample as food), under favorable nutrients, pH,
temperature and osmotic conditions. During the incubation, organic matter of the sample
is taken up as food by the microorganisms (bacteria) and aerobically oxidized (bio-
oxidized) into inorganic end products. End products formed from the oxidation depend
on the elemental composition of the organic matter being bio-oxidized. Carbon dioxide,
water, ammonia and hydrogen sulfide are important among the end products formed.
Amount of oxygen utilized for the bio-oxidation during the incubation is measured and
used for the estimation of BOD of the sample.

Complete bio-oxidation of the organic matter present may require incubation of the
sample for an infinite time interval (over 60 days). Since it is not practically feasible and
acceptable to incubate for such a long time, the sample is incubated only for a shorter
duration, 5 days by convention (Central Pollution Control Board, CPCB, has
recommended just 3 days of incubation but at 27ºC rather than at 20ºC). Oxygen
utilization in the bio-oxidation process, during the incubation period is measured and
BOD of the sample is estimated by extrapolation through use of mathematical models
(BOD kinetics models).

2.4 Fate of the Organic Matter Present in the Sample

In the BOD test, microorganisms (bacteria) utilize biodegradable fraction of organic

matter of the sample as food. The microorganisms used may not be efficient in utilizing
the organic matter, specially, when its concentration is very low or drops below certain
threshold value. Because of this reason, some fraction of biodegradable organic matter
along with the non-biodegradable organic matter is left behind in the sample as residue.
Utilization of the organic matter by the microorganisms may include the following two
steps: biosorption and bio-oxidation. When organic polymers are biosorbed, they are first
hydrolyzed by extra-cellular enzymes and then taken inside the cell for bio-oxidation.

Bio-oxidation of organic matter may involve the following two routes:

1. Aerobic oxidation (respiration) of some fraction of the taken up organic matter

into inorganic end products (CO2, H2O, etc.) and generation of metabolic energy
required by the microorganisms.
2. Utilization of rest of the organic matter as building blocks in the synthesis of
cellular material or new microbial biomass. This synthesis utilizes the metabolic
energy generated during the aerobic oxidation

Due to the utilization of organic matter, during the initial period of incubation, the
sample’s biodegradable organic matter concentration decreases. Simultaneously, due to
the synthesis of new microbial biomass, microbial biomass concentration of the incubated
sample increases. This initial period of incubation during which organic matter
concentration decreases and microbial biomass concentration increases, is often called as
a synthesis phase.

Increase in the biomass concentration can not continue for long. As the organic matter
concentration decreases and biomass concentration increases, the former gradually
becomes limiting and the microorganisms will be subjected to starving. Under such
starving conditions, the microorganisms are forced to utilize their own cellular material
as food and oxidize it into inorganic end products. Because of this reason, concentration
of the microbial biomass, after reaching a peak value, will start gradually declining.
Figure-1 shows dynamics of the organic matter concentration and the microbial biomass
concentration of an incubated sample. This phase of incubation, during which
concentration of the microbial biomass declines, is known as auto-oxidation/ auto-lysis/
decay phase.
 Figure-1: Dynamics of
biomass and

All the cellular material synthesized during the synthesis phase may not get completely
auto-oxidized even when the sample is incubated for indefinite period. Some fraction of it
will be left behind in the sample as residual biomass.

Both synthesis and auto-oxidation phases of microbial growth consume oxygen. During
the synthesis phase, oxidation of organic matter into inorganic end products, and, during
the auto-oxidation phase, lysis of microbial biomass into inorganic end products both
require oxygen. In BOD test, oxygen utilized in both the synthesis and auto-oxidation
phases is together measured and recorded as BOD of the sample for the period of
incubation. This BOD value is commonly denoted as ‘BODt’ (Oxygen demanded by the
sample during the ‘t’ period of incubation).
mg/L

Microorganisms utilize only biodegradable organic matter of the sample. Further, a

significant fraction of the newly synthesized microbial biomass is not auto-oxidized even
when the sample is incubated for indefinite period. Work done on glucose solution of 300
mg/l (ref) has indicated that its oxygen demand, when incubated for over 20 days, is 250
to 285 mg/l (just about 85% of the ThOD, which is 320 mg/l). This indicates that, though
biologically assimilable, all the glucose present is not getting oxidized into inorganic end
products. That is, the fraction of organic matter oxidized in the BOD test is generally
lesser than that oxidized in the COD test. Hence, BOD is generally lesser than COD and
always lesser than ThOD. However, for certain industrial effluents (dairy effluents), BOD
can be greater than COD. These effluents have significant levels of nitrogen containing
heterocyclic compounds which resist oxidation in the COD test. But these will easily get
bio-oxidized in the BOD test.

Time (days)
Elemental composition of microbial biomass can be indicated by the approximate
formula C5H7O2N. During auto-oxidation, nitrogen present in the microbial biomass is
released as ammonia. The released ammonical nitrogen can be oxidized by certain class
of microorganisms (nitrifying bacteria: Nitrosomonas, Nitrobactor, etc.) into nitrate. This
oxidation process (usually known as nitrification) also demands oxygen. Hence, in the
BOD test, oxygen consumption by the sample during incubation, may actually include
three components, namely, oxygen consumption during the synthesis phase; oxygen
consumption during auto-oxidation phase; and oxygen consumption in the nitrification of
the ammonia produced. Oxygen consumption for the nitrification is usually known as
nitrogenous BOD (N-BOD), and the rest of the oxygen consumption is known as
carbonaceous BOD (C-BOD).

Fate of organic matter of the sample during incubation and oxygen demand in the BOD
test are schematically shown in Figure-2.

Suspended
organic matter

Hydrolysis
Nb. suspended

da
xi
organic matter

o
o-
Soluble organic
matter Bi
Bi
o
-s
yn
Nb soluble
th
organic matter Residual
biodegradable
organic matter
Nitrogenous BOD exertion is not very significant during the first few days of incubation.
Auto-oxidation of cellular material (microbial biomass) and bio-oxidation of nitrogen
containing organic matter produce ammonia. This is then nitrified first into nitrite and
then into nitrate by nitrifying bacteria. Population size of the nitrifying bacteria in the
incubated sample becomes large enough to affect nitrification usually after 5 to 7 days of
incubation. Nitrification rate is also affected by DO concentration of the incubated
sample. DO levels of above 2 mg/l favor the nitrification process. If the interest is to
measure only the C-BOD of a sample, one should avoid nitrogenous BOD exertion. For
this, one can limit the incubation period to less than 5 to 7 days, or use nitrification
inhibitors, such as methylene blue, thio-urea and allyl-thio-urea, 2-chloro-6-(tri-chloro-
methyl) pyridine (TCMP), etc.

2.5 Alternative schemes for measuring the BOD of a sample

Many alternative schemes are available for incubating the sample with the acclimatized
microbial seed under sufficient oxygen concentration conditions and measuring its BOD.
Important among them are:

1. Saturating the sample with DO and incubating in air tight vessel (BOD bottle)
2. Incubating the sample along with known volume of air in an air tight vessel
3. Incubating the sample added with seed while keeping in contact with unconfined
air

Saturating the sample with DO and incubating in air tight vessel (BOD bottle): In
this scheme, the sample is saturated with DO and then incubated in an air tight vessel
(BOD bottle). The sample is tested both before and at the end of the incubation period for
the amount of DO present. Difference between the initial and the final DO of the sample
is taken as the oxygen consumed by the incubated sample for bio-oxidizing the organic
matter present in it. In this scheme, DO concentration should not be allowed to become
limiting during the incubation period. Since solubility of oxygen is very low (about 9
mg/l at 20ºC), this scheme requires dilution of the sample whenever its BOD is expected
to be higher than that can be satisfied by the DO tat can be made available in the sample.
Such dilution of the sample can introduce error into the BOD measurement. BOD bottle
method (ref) is an example for this scheme. Accuracy level of BOD measurement by this
method is ± 15% (at 68% confidence level).

Incubating the sample along with known volume of air in an air tight vessel: In this
scheme, the sample is added with seed and incubated along with known volume of air in
an air tight vessel. Oxygen required for the bio-oxidation of the organic matter present in
the sample is supplied by the air which is in contact with the incubated sample. Carbon
dioxide released during the bio-oxidation process is simultaneously removed from this air
through absorbing into a carbon dioxide absorbing solution, like potassium hydroxide. As
a consequence of the utilization of oxygen, by the bio-oxidizing organic matter, there will
be changes in the volume or pressure of the confined air. These changes in the pressure or
volume of this confined air are measured and used for estimating the oxygen consumed
by the sample for the bio-oxidation of the organic matter. In this scheme, the need for
diluting the sample is very much reduced or in some cases even eliminated. Warberg’s
respirometer (ref) is an example for this scheme.

Incubating the sample added with seed while keeping in contact with unconfined
air: In this scheme, the sample is added with seed and incubated while keeping in contact
with unconfined air. During the incubation, the oxygen required (for the bio-oxidation of
the organic matter present in the sample) is obtained from the surrounding unconfined air.
Oxygen transfer from the unconfined air into the sample, the rate of which mainly
depends on the turbulence level in the incubated sample, surface area of the incubated
sample exposed to the unconfined air and dissolved oxygen concentration in the
incubated sample, is quantified and used in the BOD measurement. Accuracy with which
one can measure the oxygen transfer rate will depend on the extent to which the
turbulence level and the exposed area can be maintained constant. One novel method for
achieving this has been confinement of the incubated sample to an oxygen permeable
membrane chamber and controlling the level of mixing with the help of a (electronic)
magnetic stirrer. In this scheme also, the need for diluting the sample is very much
minimized. Garg’s respirometer (ref) is an example for this scheme.