Problem Statement:
The ETP plant of ASK chemicals stops getting disturbed at intervals of time. This disturbance is caused because the factors facilitating the growth of microbial culture in the ETP plant are disturbed. This results in not dropping of COD (Chemical Oxygen Demand) of the effluent as per norms.
Objectives:
- Finding out and rectifying the factors causing disturbance of ETP
- Finding out the Nutrient Requirement and its level for effective functioning of microbial culture in ETP
- To drop the COD level of ETP as per norms
- Setting up SOP for culture maintenance in the ETP plant
Actionable Item:

Discussion on understanding the Project- 26 Feb 2022:
To understand the problem statement of the ETP project and the work required to be done a meeting was held with Dr Arun Dixt Sir, Dr Anuradha Jape Ma’am, & Sonal Shinde Ma’am. Following are the bullet points that were discussed/decided in the meeting:
- The biomass left after the elute treatment is taken to the dumping yard without its retreatment and reuse. To treat biomass and find its further applications, its biological and chemical analysis is needed to be done.
- By chemical analysis we can find out N, P, K, Ca, Mg, Fe, Zn, Cu, Mn, Ni, Co, Mo, etc. content.
- Also, Total Biomass dry Weight has to be determined by doing its centrifugation & taking filtrate.
- Identification of bacteria and fungi contributing to the effective functioning of the ETP plant needs to be done. For this identification step, we can approach a nearby lab.
- Doing BOD and COD analysis to know the efficiency of the ETP plant.
- Design of Experiment (DOE) has to be done to standardize the media Composition and chemical composition for running the ETP plant effectively.
Discussion on Design of Experiment (DOE)- 17 March 2022:
To resolve the problem of disturbance in ETP functioning, DOE was decided in a meeting with Dr Arun Dixit Sir and Dr Anuradha Jape Ma’am. Following are the bullet points that were discussed and decided in the meeting:
- Batch setup can be done in a bucket or small IBC tank.
- On evaporation of water from culture can be made up by adding water free from any salts or additional nutrients.
- Levels of C, N, and P will not be changed.
- The ratio of C: N:P: K in ETP is 100:10:1:1 respectively.
- There is no heavy requirement for Phosphorus, a trace amount of 1m mg/L is sufficient.
- Mn, Mg, and S will not be added as it is likely to get precipitated.
- Ca not be added as it is already available in the ETP.
- In 1st trial, only standard trace elements should be added to the reactor viz. are Fe, Cu, Mo, Zn, Co.
- Following are the nutrients and their concentration level to be added:
Iron | 0.3 mg/L |
Copper | 0.03 mg/L |
Molybdenum | o.5 mg/L |
Zinc | 0.8 mg/L |
Cobalt | 3 mg/L |
- BOD & COD analysis should be done on the 1st day, 3rd day, and day when the COD reduction stops.
- Find out the COD, BOD ratio to know the biodegradability.
Listing of requirements required for the Experiment Setup (29 March 2022):
Following is the list of chemicals, glassware, and other materials required for the experiment:
Sr. No. | Chemical / Glassware | Quantity |
1 | Potassium dichromate (K2Cr2O7). | 500 gm |
2 | Mercuric sulphate (HgSO4) | 100 gm |
3 | Sulfuric acid (H2SO4) | 5 Liter |
4 | Ferrous ammonium sulphate (FAS) | 500 gm |
5 | Ferron indicator | 100 ml |
6 | Potassium hydrogen phosphate (KHP) | 100 gm |
7 | Sodium hydroxide | 500 gm |
8 | Sodium sulfite | 500 gm |
9 | Acetic acid | 500 ml |
10 | Potassium iodide | 500 gm |
11 | Starch indicator | 250 ml |
12 | Potassium dihydrogen phosphate (KH2PO4) | 500 gm |
13 | Dipotassium Hydrogen Phosphate (K2HPO4) | 500 gm |
14 | Disodium hydrogen phosphate ( Na4HPO4.7H2O) | 500 gm |
15 | Ammonium chloride (NH4Cl) | 500 gm |
16 | Sodium iodide | 250 gm |
17 | Calcium Carbonate | 500 gm |
18 | Magnesium Sulphate | 500 gm |
19 | Ferric Chloride | 500 gm |
20 | Manganous sulphate (MnSO4.H2O) | 500 gm |
21 | Sodium thiosulphate | 500 gm |
22 | BOD Bottles | 10 |
23 | Erlenmeyer Flask (1000 ml) | 2 |
24 | Erlenmeyer Flask (500 ml) | 2 |
25 | Erlenmeyer Flask (250 ml) | 6 |
26 | Burette | 1 |
27 | Reflux condenser | 6 |
28 | COD tubes | 6 |
29 | Glass Measuring Cylinder (100 ml) | 1 |
30 | Hand Gloves | 1 Box |
31 | Buckets | 5 |
32 | Motor | 5 |
33 | Air Bubbles | 5 |
34 | Ferrous sulphate | 100 gm |
35 | Cupric Sulphate | 100 gm |
36 | Zinc suphate | 100 gm |
37 | Molybdenum Trioxide | 100 gm |
38 | Cobalt Sulphate | 100 gm |
39 | NH4NO3 | 100 gm |
Experiment Setup 1 at ASK Chemicals (1 April 2022):
As per discussions on Project in a meeting, we arranged a setup of the experiment at ASK Chemicals. Following is a detailed procedure of the Experiment to know the nutrient requirement of the Microbial culture.
A) Multiplication of Microbial Culture (30 March 2022) :
- 5 Liters of tap water were taken and boiled for 30 min and allowed to cool again.
- On the cooling of water, add 50 g Jaggery and 0,5 g Urea to it.
- Inoculate 250 ml microbial mother culture to it. This culture is used for experiments later.
B) Experiment Setup at ASK Chemicals (1 April 2022):
- 5 Buckets were taken.
- Addition in 5 buckets is done as follows:
Bucket 1 | Bucket 2 | Bucket 3 | Bucket 4 | Bucket 5 | |
ETP Effluent | 5 L | 5 L | 1 L | 1 L | 1 L |
Tap Water | 10 L | 10 L | 14 L | 14 L | 14 L |
Fe | 0.3 mg/L | – | 0.3 mg/L | – | – |
Cu | 0.06 mg/L | – | 0.06 mg/L | – | – |
Zn | 0.8 mg/L | – | 0.8 mg/L | – | – |
Mix Nutrients | – | 14 g | – | 14 g | – |
Soil | – | – | – | – | 1 kg |
Microbial culture | 1 L | 1 L | 1 L | 1 L | 1 L |
Note:
- In Bucket No. 1 & Bucket No. 2; 5 Lit of effluent and 10 L of tap water were added. This dilution was done to get the effluent COD 5000 mg/L.
- In Bucket No. 3, Bucket No,4 & Bucket No. 5; 5 Lit of effluent and 10 L of tap water was added. This dilution was done to get the effluent COD 1000 mg/L.
- As a source of Fe, Cu, and Zn; Ferrous sulphate, Cupric Sulphate, and Zinc suphate salts were taken respectively.
- By calculation: Ferrous sulphate- 0.045 gm, Cupric Sulphate- 0.005 gm, and Zinc suphate- 0.15 gm was weighed (to get 0.3mg/L of Fe, 0.03 mg/L of Cu, and 0.8 mg/L of Zn) and taken in a beaker. Made up volume up to 100ml using distilled water. 50 ml of this solution was added to Bucket No.1 and another 50ml was added to Bucket No. 3.
- After the addition of all components given in the above table, aeration was provided in the bucket using aero tubes.
- COD (Chemical Oxygen Demand) analysis was done after every 24 hours.
COD Readings:
COD analysis was done after every 24 hours. Following are the readings of COD of effluent in all buckets:
Bucket No. | 1/4/2022 | 2/4/2022 | 3/4/2022 | 4/4/2022 | 5/4/2022 | 6/4/2022 | |
1 | 5 lit Diluted to 15+ Micro Nutrients | 3828 | 3904 | 3864 | 3513 | 3006 | 3279 |
2 | 5 lit Diluted to 15+ Mix Nutrients | 3368 | 3864 | 3825 | 3201 | 2849 | 3006 |
3 | 1 lit Diluted to 15 + Micro Nutrients | 918 | 918 | 1140 | 1327 | 1132 | 1249 |
4 | 1 lit Diluted to 15 + Mix Nutrients | 918 | 1288 | 1167 | 1171 | 702 | 858 |
5 | 1 lit Diluted to 15 + Soil | 918 | 1249 | 1096 | 1015 | 624 | 741 |
% of COD Decrease From the previous day | ||||
1.02 | 9.08 | 6.66 | 8.33 | |
1.01 | 16.31 | 6.09 | 5.22 | |
14.09 | -16.40 | 5.88 | 9.37 | |
9.39 | -0.34 | 26.73 | 18.18 | |
12.25 | 7.39 | 27.00 | 15.79 | |
% of COD Decrease From 2 April | ||||
1.02 | 10.02 | 16.01 | 23.00 | |
1.01 | 17.16 | 22.20 | 26.27 | |
14.09 | 0.00 | 5.88 | 14.69 | |
9.39 | 9.08 | 33.39 | 45.50 | |
12.25 | 18.73 | 40.67 | 50.04 |
Observations:
- 16% COD reduction was observed in Bucket No. 1
- 22% COD reduction was observed in Bucket No. 2
- 6% COD reduction was observed in Bucket No. 3
- 33% COD reduction was observed in Bucket No. 4
- 40% COD reduction was observed in Bucket No. 5
Understanding and Learning outcome from Experiment Setup No.1:
- COD reduction was observed more in Bucket no. 5. This could be because of:- a) Microbes which we introduced through microbial culture have got nutrients from the soil that contributed to the growth of microbes that also fed on effluent in bucket resulting in a decrease in COD; b) Microbes from soil got introduced in the system from soil which fed on effluent in bucket resulting in a decrease in COD; c) The content in the effluent may have got adsorbed on soil resulting in COD reduction.
- COD in Bucket No. 1, 2, 3, and 4 had not reduced remarkably as compared to Bucket No. 5. This could be because we just gave micronutrients to the microbes and failed to provide an NPK source. Also in Bucket No. 1 & Bucket No. 3, micronutrients like Mo and Co were not added (Mo and Co were not added because it was not available in the lab during the experiment setup); because of this enzyme activity of microbes may not be seen at the optimum level.
- Hence, considering the above mistakes and learning outcomes we decided to set up a 2nd Experiment trial.
B) Experiment Setup 2 at ASK Chemicals (9 April 2022):
- 5 Buckets were taken.
- Addition in 5 buckets is done as follows:
Bucket 1 | Bucket 2 | Bucket 3 | Bucket 4 | Bucket 5 | |
ETP Effluent | 5 L | 5 L | 1 L | 1 L | 1 L |
Tap Water | 10 L | 10 L | 14 L | 14 L | 14 L |
Fe | 0.3 mg/L | – | 0.3 mg/L | – | – |
Cu | 0.06 mg/L | – | 0.06 mg/L | – | – |
Zn | 0.8 mg/L | – | 0.8 mg/L | – | – |
Mix Nutrients | – | 14 g | – | 14 g | – |
KH2PO4 | 0.5 gm/L | 0.5 gm/L | 0.5 gm/L | 0.5 gm/L | – |
K2HPO4 | 0.5 gm/L | 0.5 gm/L | 0.5 gm/L | 0.5 gm/L | – |
NH4NO3 | 0.1 gm/L | 0.1 gm/L | 0.1 gm/L | 0.1 gm/L | |
Soil | – | – | – | – | 1 kg |
Microbial culture | 750 ml | 750 ml | 750 ml | 750 ml | 750 ml |
Note:
- In Bucket No. 1 & Bucket No. 2; 5 Lit of effluent and 10 L of tap water were added. This dilution was done to get the effluent COD 5000 mg/L.
- In Bucket No. 3, Bucket No,4 & Bucket No. 5; 5 Lit of effluent and 10 L of tap water was added. This dilution was done to get the effluent COD 1000 mg/L.
- As a source of Nitrogen, NH4NO3 was added at 0.1 gm/L (1.5 gm for 15 Liters Bucket) in Buckets No. 1, 2, 3, and 4.
- As a source of Potassium and Phosphorous, KH2PO4 and K2HPO4 were added at 0.5 gm/L each (7.5 gm for 15 Liters Bucket) in Buckets No. 1, 2, 3, and 4.
- As a source of Fe, Cu, and Zn; Ferrous sulphate, Cupric Sulphate, and Zinc suphate salts were taken respectively.
- By calculation: Ferrous sulphate- 0.045 gm, Cupric Sulphate- 0.005 gm, and Zinc suphate- 0.15 gm was weighed (to get 0.3mg/L of Fe, 0.03 mg/L of Cu, and 0.8 mg/L of Zn) and taken in a beaker. Made up volume up to 100ml using distilled water. 50 ml of this solution was added to Bucket No.1 and another 50ml was added to Bucket No. 3.
- 0.75 g Urea was added as a Nitrogen source to Buckets 1, 2, 3 and 4.
- After the addition of all components given in the above table, aeration was provided in the bucket using aero tubes.
- COD (Chemical Oxygen Demand) analysis was done after every 24 hours.
COD Readings:
COD analysis was done after every 24 hours. Following are the readings of COD of effluent in all buckets:
Bucket No. | 10/4/2022 | 11/4/2022 | 12/4/2022 | 13/4/2022 | 14/4/2022 | 15/4/2022 | 16/4/2022 | 19/4/2022 | 20/4/2022 | 21/4/2022 | 22/4/2022 | |
1 | 5 lit Diluted to 15+ Micro Nutrients + NPK | 6910 | 6026 | 5037 | 5544 | 4782 | 5684 | 5540 | 4664 | 4530 | 4132 | 3817 |
2 | 5 lit Diluted to 15+ Mix Nutrients + NPK | 7027 | 6531 | 6136 | 6136 | 5448 | 5292 | 5099 | 5213 | 5196 | 5077 | 4526 |
3 | 1 lit Diluted to 15 + Micro Nutrients + NPK | 3474 | 3382 | 3304 | 3252 | 2273 | 2077 | 2020 | 1646 | 1630 | 1259 | 1102 |
4 | 1 lit Diluted to 15 + Mix Nutrients + NPK | 4099 | 4043 | 4082 | 3948 | 3880 | 3880 | 3770 | 3449 | 3296 | 2873 | 2715 |
5 | 1 lit Diluted to 15 + Soil | 967 | 505 | 583 | 493 | 274 | 392 | 320 | 274 | 236 | 196 | 118 |
Per day COD Reduction Rate:
In 5 days of experiment setup 2 COD reduction was observed as follows:
Bucket No. | 12/4/2022 | 13/4/2022 | 14/4/2022 | 15/4/2022 | 16/4/2022 | 19/4/2022 | 20/4/2022 | 21/4/2022 | 22/4/2022 |
1 | 18.42 % | 19.77% | 30.8 % | 17.74 % | 19.83 % | 32.50 % | 34.44 % | 40.20 % | 44.76 % |
2 | 8.71 % | 12.68 % | 22.47 % | 24.69 % | 27.44 % | 25.81 % | 26.06 % | 27.75 % | 35.59 % |
3 | 4.89 % | 6.39 % | 34.57 % | 40.21 % | 41.85 % | 52.62 % | 53.08 % | 63.76 % | 68.28 % |
4 | 0.41 % | 3.68 % | 5.34 % | 5.34 % | 8.03 % | 15.86 % | 19.59 % | 29.91 % | 33.76 % |
5 | 4.27 % | 49.49 % | 71.93 % | 59.64 % | 67.21 % | 71.93 % | 75.82 % | 66.38 % | 79.76 % |
Understanding and Learning outcome from Experiment Setup No.2:
- Buckets No. 1 & 3 are showing a remarkable reduction in COD of ETP plant effluent. This bucket contained N, P, K, Cu, Fe, & Zn.
- Bucket No. 5 showed the highest rate of COD reduction. But, it can be due to- 1) Content in effluent got adsorbed on the soil, 2) Microorganisms from soil got introduced into the bucket that fed on the organic and inorganic matter in effluent resulting in the dropping of COD.
Note:
- Bucket No. 5 having soil showed a remarkable result in dropping COD of the effluent of the ETP plant; soil cannot be used as a solution to drop COD at the Industrial level as the amount of soil required is huge and getting the same quality of soil is not possible.
- But, we can isolate the microbes responsible for the reduction of ETP effluent COD using appropriate media and prepare an effective microbial consortium for ETP effluent treatment.
- In Bucket No. 1 & 3, micronutrients like Co and Mo were not added because of their unavailability. Therefore, we had made a setup of Bucket No. 2 & 4 where we used mixed nutrients. But now Co and Mo micronutrients are available. So, in Experiment 3 setup we can add all the micronutrients decided in the ‘Design of Experiment’ viz. were Cu, Co, Mo, Fe, & Zn along with N, P and K.
- When calculations were reviewed again for the addition of Cupric Sulphate, Iron Sulphate and Zinc Sulphate as a source of Cu, Fe and Zn it was understood that the calculation for Cu and Zn went wrong. According to the correction, Copper sulphate and Zinc sulphate had to be added at 0.0036 gm and 0.108 gm respectively for 30 litres.
- As all the micronutrients as per decided in DOE of the experiment are available, no need to set an experiment setup of mixed nutrients separately.
Discussion on Design of Experiment (DOE)- 22 April 2022:
Results of ETP experiment setup 2 were recorded, studied and shared with Jape maám. The aphonic meeting was scheduled with Jape Maám to discuss the results, understanding and learning outcomes, and the next steps to be taken in the ETP project. The points raised through the discussion and Experiment Setup 3 were designed and decided to be done at ASK Chemicals as follows:
- Macronutrients and Micronutrients Levels: Levels of NH4NO3, K2HPO4, KH2PO4, Cupric Sulphate, Zinc Sulphate, and Iron Sulphate as a source of N, P, K, Cu, Zn, Fe respectively should not be changed and should be added just like added in Experiment Setup no.2. Also, Micronutrients Molybdenum Trioxide and Cobalt sulphate should be added as a source of Mo and Co in next Experiment Setup No.3 that was left to be added in previous experiment setup no.2; it should be added such that we should get Mo 0.5 mg/L and Co 3 mg/L. Hence, the addition of Macronutrients and Micronutrients should be as follows:
Sr.No. | Nutrients | Composition (for 15 Liters ) |
1. | NH4NO3 | 1.5 gm (0.1 gm/L) |
2. | KH2PO4 | 7.5 gm (0.5 gm /L) |
3. | K2HPO4 | 7.5 gm (0.5 gm /L) |
4. | Cupric Sulphate | 0.0025 gm |
5. | Zinc sulphate | 0.075 gm |
6. | Ferrous Sulphate | 0.0225 gm |
7. | Molybdenum Trioxide | 0.1136 gm |
8. | Cobalt Sulphate | 0.215 gm |
- The soil that was used in Bucket No. 5 should be taken and reused for effluent treatment. From this, we can understand whether the COD of effluent in Bucket No.5 was dropped due to adsorption or not.
- The treated effluent of Bucket No.5 can be used as an inoculum and study its effect on the COD of effluent. From this, we can understand if there is any effect of Microbes that were introduced from the soil used in Experiment Setup 2 in dropping the COD of effluent.
- BOD analysis should be done immediately on the day of Experiment Setup 3 arranged and on the day when COD drop is stabilized. ( Procedure followed for BOD: http://vadic.vigyanashram.blog/2022/04/13/experiment-setup-of-biological-oxygen-demand-bod/)
C) Experiment Set 3 at ASK Chemicals (03/05/2022):
As per understanding from the experiments setup 1 & 2 done at ASK Chemicals, and discussions held with Dr Arun Dxit Sir and Dr Jape Maám; Experiment Setup 3 was designed as follows:
Bucket No. 1 | Bucket No.2 | Bucket No.3 | Bucket No. 4 | Bucket No. 5 | Bucket No.6 | |
Nutrients+ NPK+ Culture (5000 COD) | Nutrients+ NPK+ Culture (1000 COD) | Mix Nutrients+ NPK+ Culture (1000 COD) | Nutrients+ NPK+ Effluent from Bucket No.5, Exp No.2 (1000 COD) | Nutrients+ NPK+ Culture + Soil from Bucket No.5, Exp No.2 (1000 COD) | NPK+ Culture + New Soil (1000 COD) | |
ETP Effluent | 5 L | 1 L | 1 L | 1 L | 1 L | 1 L |
Tap Water | 10 L | 14 L | 14 L | 14 L | 14 L | 14 L |
KH2PO4 | 7.5 g | 7.5 g | 7.5 g | 7.5 g | 7.5 g | 7.5 g |
K2HPO4 | 7.5 g | 7.5 g | 7.5 g | 7.5 g | 7.5 g | 7.5 g |
Urea | 0.75 g | 0.75 g | 0.75 g | 0.75 g | 0.75 g | 0.75 g |
Ferrous Sulphate | 0.0225 g | 0.0225 g | – | 0.0225 g | 0.0225 g | – |
Cupric Sulphate | 0.0025 g | 0.0025 g | – | 0.0025 g | 0.0025 g | – |
Zinc Sulphate | 0.075 g | 0.075 g | – | 0.075 g | 0.075 g | – |
Molybdenum Trioxide | 0.1136 g | 0.1136 g | – | 0.1136 g | 0.1136 g | – |
Cobalt Sulphate | 0.215 g | 0.215 g | – | 0.215 g | 0.215 g | – |
Mix micronutrients | – | – | 14 g | – | – | – |
Culture | 750 ml | 750 ml | 750 ml | – | 750 ml | 750 ml |
The effluent of Bucket No.5 in Exp Setup No.2 (as inoculum) | – | – | – | 750 ml | – | – |
The soil from Bucket No.5 in Exp Setup No.2 | – | – | – | – | Complete Soil | – |
Soil | – | – | – | – | – | 1 Kg |
COD Readings:
Following are the COD readings day wise:
Bucket No. | | 6/5/2022 | 7/5/2022 | 8/5/2022 | 9/5/2022 |
1 | Nutrients+ NPK+ Culture (5000 COD) | 3825 | 3708 | 3708 | 3669 |
2 | Nutrients+ NPK+ Culture (1000 COD) | 741 | 585 | 507 | 429 |
3 | Mix Nutrients+ NPK+ Culture (1000 COD) | 1991 | 1756 | 1639 | 1561 |
4 | Nutrients+ NPK+ Effluent from Bucket 5, Exp No. 2 (1000 COD) | 468 | 312 | 273 | 195 |
5 | Nutrients+ NPK+ Culture+ Soil from Bucket 5, Exp No. 2 (1000 COD) | 1522 | 1249 | 1210 | 1054 |
6 | NPK+ Culture+ New Soil (1000 COD) | 741 | 546 | 507 | 390 |