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

Ajayi, A. A.

Ajojesu, T.P.

Bishop, S.A.

Olasehinde, G. I

Peter-Albert, C. F.

Siyanbola, T. O.

Covenant University Repository

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  1Department of Biological Sciences, College of Science and Technology, Covenant University, Ota, Nigeria 2Department of Chemistry, College of Science and Technology, Covenant University, Ota, Nigeria 3 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 Biochemical oxygen demand (BOD) is the amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down organic material present in a given water sample at certain temperature over a specific time period (Manyuchi and Ketiwa , 2013 ). The BOD value is most commonly expressed in milligrams of oxygen consumed per litre of sample over   11 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016   five days of incubation at 20 °C and it is often used as a robust surrogate of the degree of organic pollution of water (Virendra et al., 2013). BOD can be used to gauge the effectiveness of wastewater treatment plants (Penn et al., 2013). Chemical Oxygen Demand (COD) is a measurement of the oxygen depletion capacity of a water sample contaminated with organic waste matter. It is similar in function to Biochemical Oxygen Demand (BOD) because they both measure the amount of organic compounds in water and they are the most commonly used parameters for the characterization of wastewaters (Abdalla and Hamman, 2014). COD also used to estimate BOD because a strong correlation exists between them, however COD is a much faster and more accurate test but it is less specific, since it measures everything that can be chemically oxidized, rather than just levels of biologically active organic matter (Sawyer et al., 2003). The conventional standard method for the determination of BOD measures the microorganisms’ oxygen consumption or respiration over a  period of 5 days and it is reported as BOD5 (Liu et al., 2014). The BOD measurement is a good indicator of the concentration of organic pollutants in water but it is extremely slow hence not suitable for process control (Chen et al., 2002) but it is essential to obtain a correlation between BOD5 and COD for various wastewater treatment plants to help in the design and operation of treatment plants (Abdalla and Hamman, 2014).  However, BOD 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 (Penn et al., 2009). 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 (Yudianto and Yuebo, 2008). Escherichia coli is an index organism used for the determination of faecal contamination it can be used to measure the effectiveness of the disposal mechanisms or treatment plants in ensuring that the effluents are environmental friendly (Naidoo and Olaniran, 2014). This research work was therefore carried out to isolate Escherichia coli from the Covenant University Oxidation pond, evaluate the BOD of the oxidation pond and determine the CBOD based on the BOD5 values.  Materials and Methods Collection of Samples Four sewage water samples were obtained from four different pointof the Covenant University Oxidation pond. The samples were collected with the aid of sterile sampling bottles and a long rope tied  12  Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016   around the neck of each bottle was allowed to gradually sink into the sewage to fill the bottles. The bottles were covered aseptically and transported to the Microbiology Laboratory of the Department of Biological Sciences, Covenant University, Ota. The samples were analyzed immediately. Cultivation of Escherichia coli Ten milliliter (10ml) of water sample was dispensed into three test tubes containing ten milliliters of double strength McConkey broth (10ml), one milliliter of the water sample was dispensed into single strength McConkey broth (10ml) in each of three test tubes and 0.1ml of the water sample into another set of three test tubes containing single strength McConkey broth (10ml). The inoculated broths were incubated at 37oC for 24 - 48h and they were monitored for acid and gas production. The pour plate method was used for the presence of E.coli. One milliliter of each sample was aseptically transferred into a sterile petridish to which about fifteen milliliter of cooled molten agar was poured. The organisms were subcultured on EMB to obtain pure cultures and they were thereafter streaked on nutrient agar slant and incubated at 37oC for 24h and stored as stock cultures. Measurement of Dissolved Oxygen of the Covenant University Oxidation Pond The dissolved oxygen of the samples collected at the four points was measured using the MW600 Dissolved Oxygen Meter. The device was calibrated according to manufacturer’s specification. The probe was verified to be polarized and probe meter calibrated. The tip of the probe was immersed in the samples (A, B, C, D) respectively. For accurate Dissolved Oxygen (DO) measurements a minimal water movement of 0.3m/sec was required and each sample was dispensed into a sterile beaker and placed upon a stirrer. To check if the water speed was sufficient, a waiting period was observed for the reading to stabilize and move the DO probe. Measurement of Biochemical Oxygen Demand of the Covenant University Oxidation Pond The Biochemical Oxygen Demand of the samples was carried out according to the methods described in UGA extension (2013) whereby a DO meter was used to measure the initial dissolved oxygen concentration in the sample bottle collected from point D of the oxidation pond and the bottle was placed in a dark incubator at 20oC for five days. After five days, the DO meter was used to measure a final dissolved oxygen concentration. The Final DO reading is then subtracted from the initial DO reading and the result is the BOD concentration. [BOD5] = [DO] Final – [DO] Initial  Mathematical Determination of CBOD This was determined according to the method described by Penn et al. 13 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016   (2009) whereby the equation for the determination of CBOD is:      C6H12O6 + 6O2 = 6CO2 + 6H2O     (1)                   2H3+ + O2- = 2H2O + 2H+              (2)               [BOD5] = [DO] Final – [DO] Initial    (3)         d [DO]/dt = d [CBOD]/dt = -K[CBOD]    (4)  The BOD exerted (Oxygen Demand) increases with time, therefore,        [CBOD] = [CBOD] o x e-kt            (5)  Where   K = First-order reaction rate constant  T = Time in days  [CBOD]o = initial CBOD concentration  Ultimate CBOD using the approximation of the BOD5 which is based on using the exponential (first-order) nature of oxygen demand is therefore,  Ultimate-CBOD = BOD5 x (1-ekt)-1   (6)  Ultimate – [CBOD] = [BOD5] x (1–ekt)-1 Where (BOD5) = the Biochemical Oxygen Demand exerted over the five day period  Results   Escherichia coli strains obtained from the Covenant University Sewage pond All the samples from the sewage oxidation pond investigated revealed the presence of E.coli as shown by the Most Probable Number (MPN) test whereby all the samples showed gas production (Table. 1). The appearance of greenish metallic sheen colonies on Eosin Methylene Blue agar further confirms the presence of E.coli (Table. 2). The biochemical characteristics of the E. coli isolates (Table. 3 ) revealed that the E.coli were Indole positive, Methyl red positive, Catalase positive and Voges Proskauer negative, Starch hydrolysis negative, Urease negative and Citrate negative. They appeared as Gram-negative rods under the microscope.  Determination of Dissolved Oxygen at points of collection  The Dissolved oxygen measurement for samples taken at four random points along the oxidation pond decreased from a value of 10.1 mg/l to 7.9 mg/l from point A to point D respectively (Fig. 1).  Determination of Biochemical Oxygen Demand The BOD values obtained is as follows: [BOD5] = [DO] Final – [DO] Initial [BOD5] = 39.5mg/l – 7.9mg/l [BOD5] = 31.6 mg/l  Mathematical Determination of Ultimate Carbonaceous Biochemical Oxygen Demand This was determined using the following: DO final   =39.5 mg/l DO Initial   =7.9 mg/l Time in Days = Five Days; 5 x 24=120h K = ranging from 0.3 to 0.7  Ultimate [CBOD] = [BOD5] x (1–e-kt)-1 Ultimate CBOD = 31.6mg/1 for K= 0.3 and K= 0.7   14 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 + +  Keys       +   indicates a positive test - Indicates a negative test  Table 3: Biochemical Characterization of Escherichia coli strains                 isolated from Covenant University Oxidation Pond  Sample Oxidase Test Catalase Test Urease test Citrate Test Voges Proskauer Methyl Red  Indole Test Starch  Hydrolysis test Gram Staining Reaction A - + - - - + + - Negative Rods B - + - - - + + - Negative Rods C - + - - - + + - Negative Rods D - + - - - + + - Negative Rods  Key       +     indicates a positive result - indicates a negative result   15  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  Discussion The results of this investigation revealed that the Biochemical Oygen Demand was employed as a parameter to define the strenght and examine the efficiency of the Covenant University Oxidation Pond. The goal of wastewater treatment is to protect and maintain healthy rivers and oceans which is the aim of evaluating the BOD5 and COD of waste water (Abdalla and Hamman, 2014). If pollutants in wastewater are not removed, they flow directly into our waterways and this can threaten public health, fisheries, wildlife habitat, recreation opportunities and ultimately, our quality of life (Metro Vancouver, 2013). Two of the important components of wastewater addressed through treatment are: Total Suspended Solids (TSS) and the Biochemical Oxygen Demand. The amount of total suspended solids and biochemical oxygen demand removed from wastewater is used to gauge the effectiveness of wastewater treatment plants (Penn et al., 2009).   The Dissolved oxygen values of the water samples obtained from the Covenant University oxidation pond decreased in value from the first point of collection from 10.1mg/l to 7.9mg/l respectively for four random collection points. This shows that the water is in a healthy condition and is fit for aquatic life. It also reveals that the Covenant University treatment plant is effective. Nester et al. (2001) reported that when there is excessive BOD, there will be deficiency of DO and water will be in anaerobic condition resulting in mortality of living aquatic organisms; release of 16 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 4 No 1 June, 2016   ammonia, methane, CO2 in the absence of oxygen, anaerobic bacteria becomes active. CBOD is a method defined test which is measured by the depletion of dissolved oxygen by biological organisms in a body of H2O in which the contribution from nitrogenous bacteria has been suppressed (Penn et al., 2009). It is used as an indicator of the pollutant removal from wastewater.   The results of this investigation revealed the presence of Escherichia coli in the four sewage samples obtained. The strains of E.coli isolated were found to be urease positive with the exception of the strains isolated from the last collection point (D). Also, the strains were found to be indole positive, Methyl red positive, Catalase positive fermenting Glucose, Lactose, Maltose and Sucrose, with exception of the strain from the third collection point(C).  E.coli has been used as an indicator for water pollution since it is entirely foreign to water (Akande et al., 2011; Health Canada, 2012). E.coli is a facultative anaerobe, mixed acid fermenter, able to convert formic acid to hydrogen and carbondioxide, lactose fermenter and unable to utilize citrate as the 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 from being an indicator of faecal pollution of water, E.coli has been implicated in diseases, although most strains are harmless (Nataro et al., 1998). The Ultimate CBOD has the same value as BOD5 for K = 0.3 and K = 0.7 when calculated mathematically with the time for five days recorded in hours but an undefined result was obtained for the time calculated in seconds. This is probably due to the exponential function. The importance of the oxygen demand of wastewater for a healthy living condition cannot be overemphasized for two major related purposes which are: To provide an indirect measure of the total amount of organic matter in the wastewater and to provide a basis for assessing the effects of the natural water receiving it. CBOD is sometimes advantageous when compared with BOD because it measures just the oxygen demand exerted by organic (carbonaceous) compounds, excluding the oxygen demand exerted by the nitrogenous compounds.  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Biochemical Oxygen Demand and Carbonaceous

Oxygen Demand of the Covenant University Sewage

Oxidation Pond

http://eprints.covenantuniversity.edu.ng/7488/1/BOD%20PAPER%20PUBLISHED.pdf

Biochemical Oxygen Demand and Carbonaceous Oxygen Demand of the Covenant University Sewage Oxidation Pond

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