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Covenant Journal of Physical and Life Sciences (CJPL) Vol.

4 No 1 June, 2016

Biochemical Oxygen Demand and Carbonaceous

Oxygen Demand of the Covenant University Sewage
Oxidation Pond
Ajayi A.A.1, Peter-Albert C.F.1, Ajojesu T.P.1, Bishop S.A.3,
Olasehinde G.I.1, Siyanbola T.O.2
Department of Biological Sciences, College of Science and Technology,
Covenant University, Ota, Nigeria
Department of Chemistry, College of Science and Technology,
Covenant University, Ota, Nigeria
Department of Mathematics, College of Science and Technology,
Covenant University, Ota, Nigeria
Corresponding Author: Ajayi, Adesola Adetutu
E mail: adesola.ajayi@covenantuniversity.edu.ng
Abstract: Biochemical Oxygen Demand (BOD) is a measure of the dissolved oxygen
consumed by microorganisms during the oxidation of reduced substances in waters and
wastewaters. It is often used ambiguously in relation to Carbonaceous Oxygen Demand
(CBOD) which is the oxygen consumed during the oxidation of carbonaceous compounds
to carbon dioxide (CO2) and other oxidized end product. BOD is actually the sum of CBOD
and NBOD where NBOD is the Nitrogenous Oxygen Demand which is the oxygen
consumed during the oxidation of nitrogenous compounds (mainly NH3) to nitrates with
nitrites being an unstable intermediate. The major difference between CBOD and NBOD is
that there are two classes of bacteria believed to be responsible for the oxidation of reduced
nitrogen. The BOD5 value of Sewage samples collected from Covenant University
oxidation pond was therefore measured and the samples examined for the presence of
Escherichia coli. The sewage samples collected from four points (starting point (A), two
middle points (B, C), and end point (D) were inoculated on an Eosin Methylene Blue agar
plates and the presence of E. coli was confirmed by the appearance of greenish metallic
sheen colonies on the agar plates and biochemical Tests. The BOD of the effluent at the
different points (A, B, C, D) respectively showed a reduction in microbial load. The
ultimate CBOD was also estimated based on the BOD5 value which is based upon the
exponential (first-order) nature of oxygen demand. This research describes the formulations
of CBOD breakdown using simplified oxidation kinetics.
Keywords: Biochemical Oxygen Demand; Carbonaceous Oxygen Demand; Escherichia
coli; Wastewater
Introduction in a given water sample at certain
Biochemical oxygen demand (BOD) temperature over a specific time
is the amount of dissolved oxygen period (Manyuchi and Ketiwa , 2013
needed by aerobic biological ). The BOD value is most commonly
organisms in a body of water to expressed in milligrams of oxygen
break down organic material present consumed per litre of sample over

Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

five days of incubation at 20 °C and for various wastewater treatment

it is often used as a robust surrogate plants to help in the design and
of the degree of organic pollution of operation of treatment plants
water (Virendra et al., 2013). BOD (Abdalla and Hamman, 2014).
can be used to gauge the However, BOD is often used
effectiveness of wastewater ambiguously in relation to
treatment plants (Penn et al., 2013). Carbonaceous Oxygen Demand
Chemical Oxygen Demand (COD) is (CBOD) which is the oxygen
a measurement of the oxygen consumed during the oxidation of
depletion capacity of a water sample carbonaceous compounds to carbon
contaminated with organic waste dioxide (CO2) and other oxidized
matter. It is similar in function to end product (Penn et al., 2009).
Biochemical Oxygen Demand BOD is actually the sum of CBOD
(BOD) because they both measure and NBOD where NBOD is the
the amount of organic compounds in Nitrogenous Oxygen Demand which
water and they are the most is the oxygen consumed during the
commonly used parameters for the oxidation of nitrogenous compounds
characterization of wastewaters (mainly NH3) to nitrates with nitrites
(Abdalla and Hamman, 2014). COD being an unstable intermediate
also used to estimate BOD because a (Yudianto and Yuebo, 2008).
strong correlation exists between Escherichia coli is an index
them, however COD is a much faster organism used for the determination
and more accurate test but it is less of faecal contamination it can be
specific, since it measures everything used to measure the effectiveness of
that can be chemically oxidized, the disposal mechanisms or
rather than just levels of biologically treatment plants in ensuring that the
active organic matter (Sawyer et al., effluents are environmental friendly
2003). The conventional standard (Naidoo and Olaniran, 2014). This
method for the determination of research work was therefore carried
BOD measures the microorganisms’ out to isolate Escherichia coli from
oxygen consumption or respiration the Covenant University Oxidation
over a period of 5 days and it is pond, evaluate the BOD of the
reported as BOD5 (Liu et al., 2014). oxidation pond and determine the
The BOD measurement is a good CBOD based on the BOD5 values.
indicator of the concentration of Materials and Methods
organic pollutants in water but it is Collection of Samples
extremely slow hence not suitable Four sewage water samples were
for process control (Chen et al., obtained from four different point
2002) but it is essential to obtain a
correlation between BOD5 and COD
of the Covenant University collected with the aid of sterile
Oxidation pond. The samples were sampling bottles and a long rope tied

Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

around the neck of each bottle was The dissolved oxygen of the samples
allowed to gradually sink into the collected at the four points was
sewage to fill the bottles. The bottles measured using the MW600
were covered aseptically and Dissolved Oxygen Meter. The device
transported to the Microbiology was calibrated according to
Laboratory of the Department of manufacturer’s specification. The
Biological Sciences, Covenant probe was verified to be polarized
University, Ota. The samples were and probe meter calibrated. The tip
analyzed immediately. of the probe was immersed in the
Cultivation of Escherichia coli samples (A, B, C, D) respectively.
Ten milliliter (10ml) of water sample For accurate Dissolved Oxygen (DO)
was dispensed into three test tubes measurements a minimal water
containing ten milliliters of double movement of 0.3m/sec was required
strength McConkey broth (10ml), and each sample was dispensed into
one milliliter of the water sample a sterile beaker and placed upon a
was dispensed into single strength stirrer. To check if the water speed
McConkey broth (10ml) in each of was sufficient, a waiting period was
three test tubes and 0.1ml of the observed for the reading to stabilize
water sample into another set of and move the DO probe.
three test tubes containing single Measurement of Biochemical
strength McConkey broth (10ml). Oxygen Demand of the Covenant
The inoculated broths were University Oxidation Pond
incubated at 37 C for 24 - 48h and The Biochemical Oxygen Demand of
they were monitored for acid and gas the samples was carried out
production. The pour plate method according to the methods described
was used for the presence of E.coli. in UGA extension (2013) whereby a
One milliliter of each sample was DO meter was used to measure the
aseptically transferred into a sterile initial dissolved oxygen
petridish to which about fifteen concentration in the sample bottle
milliliter of cooled molten agar was collected from point D of the
poured. The organisms were oxidation pond and the bottle was
subcultured on EMB to obtain pure placed in a dark incubator at 20oC
cultures and they were thereafter for five days. After five days, the DO
streaked on nutrient agar slant and meter was used to measure a final
incubated at 37oC for 24h and stored dissolved oxygen concentration. The
as stock cultures. Final DO reading is then subtracted
Measurement of Dissolved Oxygen from the initial DO reading and the
of the Covenant University result is the BOD concentration.
Oxidation Pond [BOD5] = [DO] Final – [DO] Initial

Mathematical Determination of This was determined according to

CBOD the method described by Penn et al.

Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

(2009) whereby the equation for the Blue agar further confirms the
determination of CBOD is: presence of E.coli (Table. 2). The
C6H12O6 + 6O2 = 6CO2 + 6H2O (1) biochemical characteristics of the E.
coli isolates (Table. 3 ) revealed that
2H3+ + O2- = 2H2O + 2H+ (2) the E.coli were Indole positive,
[BOD5] = [DO] Final – [DO] Initial (3) Methyl red positive, Catalase
positive and Voges Proskauer
d [DO]/dt = d [CBOD]/dt = -K[CBOD] (4) negative, Starch hydrolysis negative,
The BOD exerted (Oxygen Demand) Urease negative and Citrate negative.
increases with time, therefore, They appeared as Gram-negative
rods under the microscope.
[CBOD] = [CBOD] o x e-kt (5) Determination of Dissolved
Where K = First-order reaction rate Oxygen at points of collection
constant The Dissolved oxygen measurement
for samples taken at four random
T = Time in days
points along the oxidation pond
[CBOD]o = initial CBOD decreased from a value of 10.1 mg/l
concentration to 7.9 mg/l from point A to point D
respectively (Fig. 1).
Ultimate CBOD using the
approximation of the BOD5 which is Determination of Biochemical
based on using the exponential (first- Oxygen Demand
order) nature of oxygen demand is The BOD values obtained is as
therefore, follows:
[BOD5] = [DO] Final – [DO] Initial
Ultimate-CBOD = BOD5 x (1-ekt)-1 (6) [BOD5] = 39.5mg/l – 7.9mg/l
Ultimate – [CBOD] = [BOD5] x (1–ekt)-1 [BOD5] = 31.6 mg/l
Where (BOD5) = the Biochemical Mathematical Determination of
Oxygen Demand exerted over the Ultimate Carbonaceous
five day period Biochemical Oxygen Demand
Results This was determined using the
Escherichia coli strains obtained following:
from the Covenant University DO final =39.5 mg/l
Sewage pond DO Initial =7.9 mg/l
All the samples from the sewage Time in Days = Five Days; 5 x
oxidation pond investigated revealed 24=120h
the presence of E.coli as shown by K = ranging from 0.3 to 0.7
the Most Probable Number (MPN) Ultimate [CBOD] = [BOD5] x (1–e-
test whereby all the samples showed kt)-1
gas production (Table. 1). The Ultimate CBOD = 31.6mg/1 for K=
appearance of greenish metallic 0.3 and K= 0.7
sheen colonies on Eosin Methylene

Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

Table 1: The Most Probable Number of Organisms from the

Covenant University Oxidation Pond
Sample Zones Combinations MPN Index per g/ml
A 3-3-1 4.6
B 3-2-2 2.1
C 3-3-3 >11
D 3-2-2 2.1

Table 2: Growth of Escherichia coli on Eosin Methylene Blue Agar

Samples Growth on EMB Presence of E.coli
A + +
B + +
C + +
D + +
+ indicates a positive test
- Indicates a negative test

Table 3: Biochemical Characterization of Escherichia coli strains

isolated from Covenant University Oxidation Pond
Sample Oxidase Catalase Urease Citrate Voges Methyl Indole Starch Gram Staining
Test Test test Test Proskauer Red Test Hydrolysis Reaction
A - + - - - + + - Negative Rods
B - + - - - + + - Negative Rods
C - + - - - + + - Negative Rods
D - + - - - + + - Negative Rods
+ indicates a positive result
- indicates a negative result

Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

Fig 1: Dissolved Oxygen at the various points (A-D) of the

Covenant University Oxidation Pond
The results of this investigation amount of total suspended solids and
revealed that the Biochemical Oygen biochemical oxygen demand
Demand was employed as a removed from wastewater is used to
parameter to define the strenght and gauge the effectiveness of
examine the efficiency of the wastewater treatment plants (Penn et
Covenant University Oxidation al., 2009).
Pond. The goal of wastewater The Dissolved oxygen values of the
treatment is to protect and maintain water samples obtained from the
healthy rivers and oceans which is Covenant University oxidation pond
the aim of evaluating the BOD5 and decreased in value from the first
COD of waste water (Abdalla and point of collection from 10.1mg/l to
Hamman, 2014). If pollutants in 7.9mg/l respectively for four random
wastewater are not removed, they collection points. This shows that the
flow directly into our waterways and water is in a healthy condition and is
this can threaten public health, fit for aquatic life. It also reveals that
fisheries, wildlife habitat, recreation the Covenant University treatment
opportunities and ultimately, our plant is effective. Nester et al. (2001)
quality of life (Metro Vancouver, reported that when there is excessive
2013). Two of the important BOD, there will be deficiency of DO
components of wastewater addressed and water will be in anaerobic
through treatment are: Total condition resulting in mortality of
Suspended Solids (TSS) and the living aquatic organisms; release of
Biochemical Oxygen Demand. The

Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

ammonia, methane, CO2 in the from being an indicator of faecal

absence of oxygen, anaerobic pollution of water, E.coli has been
bacteria becomes active. CBOD is a implicated in diseases, although most
method defined test which is strains are harmless (Nataro et al.,
measured by the depletion of 1998). The Ultimate CBOD has the
dissolved oxygen by biological same value as BOD5 for K = 0.3 and
organisms in a body of H2O in which K = 0.7 when calculated
the contribution from nitrogenous mathematically with the time for five
bacteria has been suppressed (Penn days recorded in hours but an
et al., 2009). It is used as an undefined result was obtained for the
indicator of the pollutant removal time calculated in seconds. This is
from wastewater. probably due to the exponential
The results of this investigation function. The importance of the
revealed the presence of Escherichia oxygen demand of wastewater for a
coli in the four sewage samples healthy living condition cannot be
obtained. The strains of E.coli overemphasized for two major
isolated were found to be urease related purposes which are: To
positive with the exception of the provide an indirect measure of the
strains isolated from the last total amount of organic matter in the
collection point (D). Also, the strains wastewater and to provide a basis for
were found to be indole positive, assessing the effects of the natural
Methyl red positive, Catalase water receiving it. CBOD is
positive fermenting Glucose, sometimes advantageous when
Lactose, Maltose and Sucrose, with compared with BOD because it
exception of the strain from the third measures just the oxygen demand
collection point(C). E.coli has been exerted by organic (carbonaceous)
used as an indicator for water compounds, excluding the oxygen
pollution since it is entirely foreign demand exerted by the nitrogenous
to water (Akande et al., 2011; Health compounds. The CBOD
Canada, 2012). E.coli is a facultative accomplishes this by inhibiting the
anaerobe, mixed acid fermenter, able nitrifying organisms from using
to convert formic acid to hydrogen oxygen by the addition of a
and carbondioxide, lactose fermenter nitrification inhibitor to the samples
and unable to utilize citrate as the (Acton, 2012).
sole carbon source (Holt et al.,
1994). The presence of E.coli in
water samples such as sewage
implies faecal contamination and
strongly suggests the possible
presence of enteric pathogenic
bacteria, enteric viruses and
protozoans (Feng et al., 2002). Apart
Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016

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