Determination of Biochemical Oxygen Demand

What is Biochemical oxygen demand (BOD) ?

Biochemical oxygen demand is used as a measure of the quantity of oxygen required for oxidation of Biodegradable organic matter present in water samples by aerobic biological action. The oxygen demand of wastewater is exerted by three classes of materials.

1. Carboneous organic materials are used as a source of food by aerobic organisms (carbonaceous biochemical oxygen demand).

2. Oxidizable nitrogen derived from nitrite, ammonia, and organic nitrogen compounds which serve as food for specific bacteria (e.g. Nitrosomonas and Nitrobacter).

3. Chemically reducing compounds eg: Ferrous ion (Fe2+) sulfites (S03) and sulfide (S2‘), which are oxidized by dissolved oxygen.

So in the biological degradation of waste material which is brought about by diverse groups of living organisms or bacteria is converted into full acids mainly acetic and CO2 etc. In the presence of excess oxygen which is an electron acceptor is reduced while the organic matter is being oxidized to CO2 and H20.

To avoid the development of nuisance inside the BOD bottle it is a must keep:

  • Sufficient oxygen by aerating the waste water sample.
  • Addition of Buffer to maintain the neutral pH throughout the incubation period. In biological term the biodegradable matter are utilized by microorganisms for food. The oxidation process is predominant than the reduction, when oxygen is available. The end products obtained after the decomposition:

Substrate + Bacteria + 02 + growth factors ——————> CO2 + H2O + increased bacterial cell + Energy

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BOD BOTTLE METHOD (BOD Experiment Lab Report)

BOD bottle is a batch operation in which the following operations takes place:

  • A portion of the organic (and inorganic) material in the system is oxidized in chemical reactions: Organic carbon to carbon dioxide, organic hydrogen to water; Sulfide and organic sulfur to sulfates, organic nitrogen and ammonia to nitrites or nitrates, referred to as nitrification and other reactions.

    The purpose of the oxidation reaction is to provide energy, and the oxygen consumed is referred to as energy oxygen. Energy oxygen is defined as the amount of oxygen required in an energy reaction that supports the synthesis of organic materials into new biological cells or biological stable organic materials energy oxygen will be a fraction of that required for complete biological stabilization.
  • Approximately 60-70% energy is utilized in coupled reactions which synthesize the remaining organic material into new microorganisms or convert it into stable compounds.
  • As the synthesized microorganisms age, lysis of the cell wall occurs and the cell contents are released.

Thus the organic materials in the BOD bottle are eventually altered to the point where the materials remaining are chiefly the stable end products of the cell wall.

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So BOD represents oxygen utilized in energy reactions which support the synthesis of organic material into new cell material, with time, the older cells die, and organic materials associated with the cell are utilized in energy reactions and further synthesis.

The cyclic lysis-synthesis-lysis reactions continue until the number of viable cells is greatly reduced and only relatively stable organic materials remain.


Sample for biochemical oxygen demand analysis may degrade significantly during storage between collection and analysis, resulting in low BOD values. To minimize this quick analysis is required or stored at a low temperature near freezing.

If the analysis is begun within 2 hr. of collection cold storage is not required, but otherwise started at 4°C and report length of time and temperature of storage with the result.

Composite sample
Keep samples at or below 4°C during compositing. The limit of the compositing period is 24 hr. Before doing analysis shake the sample very well then conduct the experiment.

BOD TEST Apparatus

  1. Incubation bottles 300 ml capacity.
  2. BOD incubators
  3. Aspirator bottles


Following are the reagents used in a bod test:



Place the desired volume of water in a suitable bottle and add 1 ml each of phosphate buffer, MgSO4, CaCl2, and Fegi solutions/lit of water. If required added seed also 1.5 ml/L. Before use keeps the water at 20″C and aerate it through a pump or mechanically.


For oxidizing biodegradable organic matter in the sample, it is necessary to have a microbial population in the system. Municipal sewage and also biologically treated effluents are supposed to have these popula­tions. which may be absent in many industrial wastewaters.

Even wastewater generated at elevated tempe­rature or containing toxic substances also has a lack of microbial populations. Such types of wastewaters require the addition of appropriate microbial populations as seeds for ensuring bio-degradation of organic matter during the incubation.

Worth Reading: Wastewater Treatment Basics

Some samples may contain materials not degraded at normal rates by the microorganisms example. Some untreated industrial wastes, disinfected wastes, high-temperature wastes, or wastes with extreme pH values). In such a case acclimatized seed is required for treatment.

Acclimatized seed

During the initial period of incubation, in the BOD test (biochemical oxygen demand), selection among the microbial populations added and an increase in the size of the selected populations may occur.

Because of this, one may observe an initial lag in oxygen demand Fig. pattern, and the cumulative demand may not follow first-order kinetics. Due to this reason, negative errors may be made in the measurement of BOD and in the estimation of ultimate BOD for the sample. This initial lag in the oxygen demand can be eliminated through the use of an acclimated seed that has appropriate microbial populations in significant sizes.

To prepare acclimatized seed, a sample of settled domestic sewage contains a large number of microorganisms added into a small amount of industrial effluent with a little amount of carbon source 0.1 g/L. Aerate the sample 24 hr. and then gradually add industrial effluent in co the system in 24 hr. interval and removing also. Continue this process for 3-4 days. This culture does not present a lag period thus becoming a typical BOD curve.

lag period in bod test
the lag period in bod test


Oxygen is sparingly soluble in water and solubility is dependent on attitude, temperature, and salinity, At the SEA level, water dissolves 9.2 mg/L, of oxygen at 20″c. Diluted wastewater sample is aerated, its DO nearer to the saturated DO level before the incubation such aeration may increase the DO level to above 8 mg/L levels. Hence. the diluted sample’s initial DO may be in the region of 8 mg/L.

Oxidation of organic matter is assumed to follow first-order kinetics and the rate of oxidation is dependent on the concentration of biodegradable organic matter. Hence, with the use of the BOD kinetics model, oxidation must not be allowed to take place at the DO level less than 0.5 mg/L.

The DO concentration of the incubated sample should not fall below 1.0 mg/L throughout the incubation period. So from the above statement about 7 mg/L of DO may be available for utilization in the oxidation of the organic matter present in the sample (soluble biochemical oxygen demand).

Hence the samples should be diluted to the extent that their organic matter concentration is reduced to that level, the sample’s cumulative oxygen demand during the incubation period is < 7 mg/L.

In case, you don’t have any idea about the expected range of biochemical oxygen demand at 20°C. One can perform the COD test as it is 2 hr. test, and make the idea of COD. As always COD is higher than BOD except for nitrogenous compounds. The COD value is divided by 2 COD/2= X rough idea. Now, this x is the expected BOD in the sample.

BOD Graph
BOD Graph


For proper incubation of a sample, it is must to maintain favorable pH, constant temperature, and addition of acclimatized seed (if required).

For Biological oxidation of organic matter by micro-organisms, it is necessary to maintain pH within a specific range between 7.0-8.0.

In BOD5 pH 7.2 is considered optimum. This pH can be changed due to the production of carbon dioxide and its dissolution in the BOD Bottle. For adjusting the pH value and maintaining it throughout the experimentation. the sample is added with phosphate buffer, which ensures maintaining pH at 7.2.

Biological oxidation of organic matter involves the synthesis of new microbial biomass. This synthesis. requires nitrogen, phosphorus, and other inorganic materials. The absence of these nutrients or insufficient quantities causes nutrient-deficient conditions. To avoid such type of nutrient limiting conditions, a sufficient quantity of nutrients is added to the dilution water.

Phosphate buffer is a solution of potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4) disodium hydrogen phosphate (Na2HPO4. 7E120), and Ammonium chloride (NH4CI). These chemicals act as a good source of phosphorus and nitrogen source (as nutrients) and also jointly as a buffer to maintain the pH throughout the experimentation.

Download: Environmental Engineering Lab Notes

To ensure first-order kinetics, maintenance of osmotic conditions for the microorganisms is also very important. Without which micro-organisms involved in the oxidation process are badly affected. For ensuring the necessary osmotic conditions, salts like Ferric chloride, calcium chloride, and magnesium sulfate are added to the sample in dilution water.

These chemicals act as nutrients also in the bio-oxidation of organic waste in biochemical oxygen demand estimation.

BOD reaction is very sensitive to temperature. To study the BOD kinetics sample should be incubated at a constant temperature. Incubation of the sample for 5 days at 20°C has one advantage, nitrogenous BOD exertion will not interfere with the measurement of the carbonaceous BOD.

Similarly, this temperature degrade in the range of 60 to 70% of the ultimate BOD. The central pollution control Board (CPCB) has suggested. incubation of the sample at 27°C for 3 days for degradation of 60-70% of the ultimate BOD. In order to ensure that the sample will be incubated at a constant temperature.

PROCEDURE (BOD measurement method)

  1. First prepare dilution water, by (Tap water or Distilled water) adding 1m1/L of all four nutrients namely phosphate buffer, magnesium sulfate, calcium chloride, and ferric chloride in the same serial.
  2. According to the requirement decide whether the seed is required or not if yes use acclimatized seed (1-5 ml/L).
  3. Prepare dilution water according to the requirement of 5 lit or 10 lit. and keep it to 20°C. Thoroughly aerate the dilution water either by diffused aeration or by mixing to increase its DO concentration as near as possible to the saturation concentration. Collect the representative sample for .which BODE has to be measured, find out dilution factor through the procedure described in dilution factor.
  4. Transfer the water into BOD bottles through siphoning from the aspirator bottle. (Outlet of the aspirator bottle may be provided with a rubber tubing). Another end of the rubber tubing, be inserted to the bottom of the BOD bottle, which is placed in a one-liter capacity beaker, and the sample may be allowed to overflow the BOD bottle. After that remove the tubing from the bottle and stopper it.
  5. Fill at least five bottles for one sample three bottles keep at 20°C after numbering them for incubation for 5 days. Rest two measure the DO by Winkler’s method described in DO estimation (dissolved oxygen and biochemical oxygen demand).
  6. The bottle which incubated at 20°C, ensure that each bottle has the water in the funnel portion for acting as a water seal and cover the bottle mouth and neck with an aluminium foil in order to avoid drying out of the water seal during incubation. After the desired period of incubation (5 day) at 20°C and 3 day at 27°C) test the bottle for DO concentration consider this DO as the final DO.

For the samples that require the addition of seed, maintain a blank parallel with the diluted sample. Prepare the blank in a manner similar to that of the diluted sample preparation, but without adding the sample. Aerate the sample and fill it into the BOD bottles and 2 bottles titrate and 3 incubate at 20°C for five days.



Dilution factor =

Incubation period = 5 day and 3 days

Incubation Temperature = 20°C and 27°C


DObf = final DO of the blank sample
DF = Dilution factor


  1. It is the principal test applied to domestic and industrial wastes to determine strength in terms of oxygen required for stabilization. It is the only test applied that gives a measure of the amount of biologically oxidizable organic matter present that can be used to determine the rates at which oxidation will occur.
  2. The major criterion used in stream pollution control where organic loading must be restricted to maintain desired dissolved oxygen level.
  3. It is an important consideration in the design of treatment facilities.


  • The ratio of BOD to COD is important for determining the acceptability of waste for biological treatment.
  • Because phosphorus levels are nearly zero in biosolids and the BOD/COD ratio is greater than 0.8, wastewater is highly polluted and treatable with biological methods.
  • Assessing the potency of wastes, which often include poisons and organically resistant organic chemicals, is beneficial.
  • The COD value is always lower than the BOD value. The COD value is usually around 2.5 times the BOD value in domestic and certain industrial wastewater.
  • There is no such thing as a COD to BOD ratio that applies to all sorts of wastewater.
  • The quality of wastewater differs from one source to another, as well as from one area to the next.
  • However, in France, for example, it is common to think that the COD content of domestic wastewater varies depending on the type of wastewater.

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