BOD (Biochemical oxygen demand) is the pollution index of any water sample. One of the main factors influencing the estimation of BOD is the nature of microorganisms used as seeding material. In order to meet the variation in wastewater characteristics, one has to be specific in choosing the biological component that is the seeding material. The present study deals with the estimation of BOD of dairy wastewater using a specific microbial consortium and compares of the results with seeding material (BODSEED). Bacterial strains were isolated from 5 different sources and were screened by the conventional BOD method. The selected microbial seed comprises of
Dairy industry is found all over the world, but their manufacturing process varies tremendously [
Due to the presence of high organic load, dairy effluents degrade rapidly and deplete the DO (dissolve oxygen) level of the receiving streams and become the propagation place for mosquitoes and flies carrying malaria and other perilous disease like dengue fever, yellow fever, and chicken guinea [
Strict guidelines have been established by government agencies to prevent water contamination [
Among the wastewater parameters, BOD is widely used as a primary indicator to gauge water pollution. BOD provides information about the amount of biodegradable substance present in wastewater. As this is a bioassay test, the results depend not only on the kinetics exerted during the incubation period, but also on the microorganisms used; thus, the test exhibits poor repeatability. Some of the industrial wastewaters have sufficient microbial population to perform the BOD5 test without providing an acclimated microbial seed. In comparison, there are other types of wastes, namely, untreated industrial wastes, disinfected wastes, and wastes that have been treated to a high temperature, that contain negligible bacterial population to perform the test. Thus, these samples need to be seeded with a population of microorganisms to exert an oxygen demand. Seeding is a process in which the microorganisms that oxidize the organic matter present in a wastewater are added to the BOD bottle. Pierce etal. [
The BOD5 values of dairy wastewaters are often misleading since the normal seeding materials used for BOD5 estimation are nonspecific bacteria that cannot biodegrade some of the nitrogenous compounds present in the effluent. Pepper et al. [
The aforementioned problems can be overcome by formulating a uniform microbial seed comprising selected bacterial isolates thatare acclimatized to the dairy industrial wastewater. Further, these bacterial isolates must be specific for the biodegradation of the organic compounds present in dairy effluent. Reproducible and reliable results may be obtained if a specifically designed formulated microbial consortium comprising selected bacterial strains is used as seed for the BOD5 analysis.
The objective of this study is to isolate autochthonous bacteria from the industrial premises in order to develop a microbial consortium specifically formulated for use as seeding material for the BOD5 analysis of dairy industrial wastewater which will incorporate the utilization of nitrogen present in the dairy effluent. Screening is done by conventional method and compared with already available seeding material. Statistical
D-glucose and D-glutamic acid were obtained from Sigma, Germany. Charged nylon membrane (SIGMA) with a pore size of 0.45
5 samples specially sludge samples and sample from equalization tank were collected from different sources. Different sources are Equalization tank (MET), aeration tank (sludge) (MAT), sludge (MS), inlet sludge (MIS), clarifier (MC). Samples were stored at 4°C and analyzed within 24 hr.
Different extracts were prepared for different media mentioned in (Table
Soil extract preparation.
Sample | Distill water |
---|---|
500 mL (MET/M1) | 500 mL |
500 mL (MAT/M2) | 500 mL |
1 litre (MS/M3) | — |
1 litre (MIS/M4) | — |
1 litre (MC/M5) | — |
The collected samples were enriched for the autochthonous bacterial population present theirin, by adding 5 gm of the sample in medium containing milk, tryptone, lactose, and soil extract. Extract used is different for different samples. This suspension was incubated at 37°C for 2 days under gentle shaking (150 rpm). Different media were designed (Table
Different media used.
Serial number | MET (M1) | MAT (M2) | MS (M3) | MIS (M4) | MC (M5) |
---|---|---|---|---|---|
1 | M1A | M2A | M3A | M4A | M5A |
2 | M1B | M2B | M3B | M4B | M5B |
3 | M1C | M2C | M3C | M4C | M5C |
4 | M1D | M2D | M3D | M4D | M6D |
Medium laboratory | Medium composition |
---|---|
M1A | 75% Soil extract + 25% Milk + Tryptone + Lactose |
M1B | Soil extract + 2% Agar |
M1C | 75% Soil extract + 25% Milk + 2% Agar |
M1D | Tryptone + Yeast extract + Glucose + Dipotassium hydrogen phosphate + 2% Agar |
Note: Soil extract is replaced depending upon the sample used.
The chemical oxygen demand (COD) and Biochemical oxygen demand 5-day (BOD) tests of effluent sample were carried out according to the method described in standard methods for examination of water and wastewaters. COD: A sample is refluxed in strongly acid solution with a known excess of potassium dichromate. After digestion, the remaining unreduced dichromate is titrated with ferrous ammonium sulfate to determine the amount of potassium dichromate consumed and the oxidizable matter is calculated in terms of oxygen equivalent. BOD: The method consists of filling with sample. To overflowing, an airtight bottle of the specified size and incubating it at the specified temperature for 5 days. Dissolved oxygen is measured initially and after incubation, and the BOD is computed from the difference between initial and final DO [
The bacterial strains selected as stated above were individually inoculated in 25 mL of TYG (tryptone yeast glucose). All the cultures were incubated at 37°C for 16–20 hrs at 150 rpm. Cells were harvested by centrifugation at 7000 rpm for 15–20 min. The pellet thus obtained was washed twice with 50 mM phosphate buffer, pH 6.8. The cell pellet of individual bacterial isolates thus obtained was resuspended in 2 mL of same buffer and used as seeding material for the BOD analysis of dairy wastewater.
The inoculums was prepared by inoculating one loopful of all the individual bacterial isolates separately in 25 mL sterilized nutrient broth. The inoculated broths were incubated in an orbital shaker at 35°C for 16–24 hours so as to obtain actively growing mother cultures. After achieving the desired growth (1.2 optical density), the cultures were centrifuged at 7000 rpm for 15 min at 4°C. The cell pellet of individual bacterial isolates thus obtained was resuspended in 2 mL of same buffer and mixed at the time of performing BOD analysis of dairy wastewater. Twenty consortia were designed from 13 selected isolates. Out of 20 microbial consortia prepared for BOD analysis (APHA 1998). Three microbial consortia were selected, which exhibited the best values for dairy wastewater.
Formulated microbial consortium was tested for reproducibility by testing the wastewater collected at different periods of time with the best identified consortium.
The ultimate BOD test is an extension of the 5-d dilution test. Formulated microbial consortium was compared with BODSEED by performing ultimate BOD for 90 days [
To statistically analyze the data
The selected organisms of the consortium were identified by Microbial Type Culture Collection at IMTECH, Chandigarh, India.
Alexandra in 1994 defined biodegradation as the biologically catalyzed reduction in complexity of chemical compound [
Industrial wastes are probably the greatest single water pollution problem as they contain large fraction of organic matter which acts as substrate for microorganisms when released in to water course.
Dairy wastewater is of great concern due to the presence of high nitrogenous load. The conventional estimation of biological oxygen demand estimates the load in 5 days, and as per rule, we will get the results in the form of carbonaceous demand and nitrogenous demand which requires 90 days for the measurement. So in order to avoid that, the consortia was designed which will contain the bacteria which is able to give you the nitrogenous demand in 5 days.
After isolation of 25 bacterial isolates were chosen randomly from all 45 bacterial isolate on the basis of their growth rate. Selected individual bacterial isolate were then used as seeding material for estimating BOD of inlet dairy industrial wastewater. The BOD in all cases was assessed and the results are presented (Table
Comparison of BOD values (mg/L) of dairy industrial wastewater sample using individual bacterial isolates (as seeding material) and using GGA as a reference standard.
Serial number | Seeding Source (Laboratory names of Individual bacterial isolates) | BOD mg/L |
---|---|---|
1 | BODSEED | 1999 |
2 | Isolate 1 | 100 |
3 | Isolate 2 | 893 |
4 | Isolate 3 | 260 |
5 | Isolate 4 | 580 |
6 | Isolate 5 | 1526 |
7 | Isolate 6 | 1886 |
8 | Isolate 7 | 1273 |
9 | Isolate 8 | 426 |
10 | Isolate 9 | 1000 |
11 | Isolate 10 | 1530 |
12 | Isolate 11 | 1875 |
13 | Isolate 12 | 1840 |
14 | Isolate 13 | 2185 |
15 | Isolate 14 | 1605 |
16 | Isolate 15 | 1230 |
17 | Isolate 16 | 1245 |
18 | Isolate 17 | 1175 |
19 | Isolate 18 | 235 |
20 | Isolate 19 | 560 |
21 | Isolate 20 | 104 |
22 | Isolate 21 | 369 |
23 | Isolate 22 | 489 |
24 | Isolate 23 | 1287 |
25 | Isolate 24 | 1111 |
26 | Isolate 25 | 1210 |
The pollutional strength of wastewater can be estimated by measuring oxygen demand. Primary parameters for monitoring wastewater quality are COD and BOD. COD gives the total load either in the form of organic or inorganic. It cannot differentiate between the two loads, or we can say COD tells us the total pollutional load of wastewater. The BOD test has been widely measured the organic load of wastewater in terms of carbonaceous matter. So we can say it can give a far more reliable estimation of the possible oxygen demand that a waste will have on a river than a COD test. So we can define BOD as a measure of oxygen required for the biochemical oxidation of the organic matter. Although the BOD test is not specific to any pollutant, yet it continues to be one of the important general indicators of the potential of a substance for environmental pollution of surface waters. For screening the single isolates and consortia BOD was performed. Those individual bacterial isolates, which exhibited BOD values higher to or comparable to BODSEED, were chosen. Out of the above screened isolates, 14 bacterial isolates (2, 5, 6, 7, 9, 10, 11, 12, 13, 14, 19, 23, 24, and 25) were selected for the formulation of different microbial consortia. In the subsequent experiment, seeding was carried out at 0.1% as in the case being done with BODSEED. The results of the BOD analysis performed using the microbial seeds (20 consortia) are illustrated in figure (Figure
Comparison of BOD values (mg/L) of dairy wastewater sample using different formulated bacterial consortia. BOD limit for selection of consortia depicted blue color (1405 mg/L) as calculated by using BODSEED. Selected consortia above the BOD limit are shown in green.
On the basis of the results obtained in the above experiment, further selections were carried out according to the ability of the screened consortia to biodegrade the constituents of dairy industrial wastewater. The selected bacterial consortia were again tested for the BOD analysis of a fresh lot of dairy industrial effluent. Out of 20 bacterial consortia selected for the BOD analysis, 3 consortia, which exhibited the best values for dairy effluent, were selected (Figure
Comparison of BOD : COD ratios of dairy industrial wastewater sample (
BOD : COD ratios exhibited by the above 3 consortia showed that the ratio can be increased with the help of selected and screened bacteria.
It was evident from the results that consortium 7 was performing the best in all the experiments conducted during the course of the study. The BOD : COD ratios increase remarkably to 0.75–0.8 as against 0.58–0.62 obtained with the conventional seeding material. Therefore, this consortium was selected for use as seeding material, specifically for BOD analysis of dairy wastewater sampled at various time intervals over a six-month period. The results of this study are presented in (Figure
BOD analysis of dairy industrial wastewater sample using the selected bacterial consortium 7 (
As mentioned in standards methods APHA 1998 biochemical oxygen demand estimation is divided into two groups carbonaceous oxygen demand requires 3–5 days for estimation and ultimate oxygen demand (carbonaceous + nitrogenous demand) requires 90 days for estimation and known as ultimate BOD.
Ultimate BOD was performed using consortium 7 and results were compared with BODSEED. The results revealed that the consortium will able to give 2005 mg/L of BOD after 90 days of incubation and BOD reaches to 1635 mg/L with BODSEED (Table
Comparison of ultimate BOD values (mg/L) of dairy industrial wastewater sample using consortium 7 (as seeding material) and BODSEED (as conventional seeding material).
COD = 2195 mg/L | ||||
---|---|---|---|---|
Time (in days) | BOD mg/L | Ratio BOD/COD |
||
Ratio BOD mg/L/COD mg/L | ||||
With BODSEED | With Consortia | BODSEED/COD | Consortia/COD | |
1 | 1049 | 1210 | 0.48 | 0.55 |
3 | 1262 | 1412 | 0.57 | 0.64 |
5 | 1276 | 1435 | 0.58 | 0.65 |
7 | 1322 | 1601 | 0.60 | 0.73 |
10 | 1345 | 1633 | 0.61 | 0.74 |
15 | 1399 | 1674 | 0.64 | 0.76 |
18 | 1425 | 1712 | 0.65 | 0.78 |
20 | 1463 | 1768 | 0.67 | 0.81 |
25 | 1498 | 1795 | 0.68 | 0.82 |
30 | 1515 | 1856 | 0.69 | 0.85 |
40 | 1549 | 1912 | 0.71 | 0.87 |
50 | 1578 | 1935 | 0.72 | 0.88 |
60 | 1610 | 1968 | 0.73 | 0.90 |
70 | 1629 | 1985 | 0.74 | 0.90 |
90 | 1635 | 2005 | 0.74 | 0.91 |
While comparing the ratios in case of consortia and BODSEED the BOD : COD ratio increased to 0.91 as against 0.74 obtained with the conventional seeding material (Figure
Comparison of ultimate BOD of dairy industrial wastewater sample using 3 selected bacterial consortia and conventional seeding material (BODSEED).
It was found that both the techniques are significantly different at
The bacterial strains comprised in this consortium were identified as
BOD : COD ratio determines the biodegradability of waste water. From the above studies, it is clear that specific bacteria can be identified for degrading particular compounds present in wastewater. Moreover, the ratio of BOD : COD showed considerable increase to 0.91 as against 0.74 obtained with the conventional seeding material after 90 days of incubation at 27°C, thereby changing the degree of biodegradability of industrial waste water.
The authors acknowledge the financial help provided by the Department of Biotechnology, Government of India.