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: Sample containing 500 mg/L of sucrose (C12H22O11) Sample containing 500 mg/L of ethanol (C2H6O) Sample containing 500 mg/L of oxalic acid (C2H2O4) 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. b. c. d.
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 ThOD of the sample =
500 mg / l X 364 grams = 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 ThOD of the sample =
500 mg / l X 96 grams =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 ThOD of the sample =
500 mg / L X 32 grams = 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 ThOD of the sample =
3000 mg / L X 7840 grams = 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 ThOD of the sample =
2.2
125 mg / L X 7456 grams =195 mg / L 4790 grams
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 (biooxidized) 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.
mg/L
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). 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
Bi oo
th yn -s
Nb soluble organic matter
o Bi
Nb. suspended organic matter Soluble organic matter
xi da
Hydrolysis
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-chloromethyl) 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.
