Lab - 4 - Biodegradation - Continuation

1. Lab - 4 - Biodegradation - Continuation • Objectives: • Learn the principles of biodegradation process through hands-on experience with aerobic biodegradation processes (Biochemical Oxygen Demand (BOD)) and anoxic biodegradation processes (Denitrification). Also quantify the microorganism biomass in activated sludge biodegradation: Mixed Liquor Volatile Suspended Solids (MLVSS). • Lab Overview: • A) Finish BOD • B) Finish MLSS, MLVSS • C) Denitrification kinetic studies

2. Lab - 4 - Biodegradation (A) Biological Oxygen Demand (BOD): measures the amount of biodegradable organic matter • Goal: measure BOD of lake water, treated wastewater and activated sludge. • Procedure • 1) Locate the following materials: • BOD bottles prepared last week • Dissolved Oxygen (DO) meter previously warmed up for 30 minutes • 2) Add enough water into the quiver (gray plastic housing) to damp the foam pad and insert the probe, check that the dissolved oxygen is 8-9 mg/L, if not press (std) twice and wait until measurement mode. • 3) Remove the probe from the quiver and unscrew the black protective cover, rinse the probe with DI water and insert in the top of each of the BOD bottle to measure, register the stable value of DO for each of the containers your group prepared. • 4) Calculate the corresponding BOD of the sample: • Group 1: (BOD1)7= [(DO1,I-DO1,7)-270/300ml(DO4,I-DO4,7)] *300ml/30ml • Group 2: (BOD2)7= [(DO2,I-DO2,7)-297/300ml(DO4,I-DO4,7)] *300ml/3ml • Group 3: (BOD3)7= [(DO,3,I-DO3,7)-30/300ml(BOD1)7 ] *300ml/270ml • 5) Share the results among the different groups (everybody should include the BOD results for all samples)

3. Lab - 4 - Biodegradation (B) Mixed Liquor Volatile Suspended Solids (MLVSS): is an indication of microorganism biomass present in the activated sludge • Goal: Measure MLVSS for the denitrification activated sludge • Procedure • 1) Locate the following materials: • Dried glass fiber filters in dessicator after last Week MLSS determination • Porcelain container for use in 550C oven • 2) Weigh the glass fiber filter containing last week’s MLSS - W1 (grams) (should be close to last week’s measurement) • 3) Place the glass fiber filter in the porcelain container and introduce into the oven at 550C CAUTION: use appropriate gloves (located below the oven) as temperatures are high enough to calcinate the skin upon contact, keep your body away from the oven. • 4) After 20 minutes, remove the porcelain container from the oven and place it in the dessicator for 30 minutes. • 5) Remove the sample from the dessicator and weigh the filter again - W2 (grams) • MLVSS = (W1-W2)/Vs (remember, Vs is the volume of the sample, 10 ml) • 6) Report the MLSS and MLVSS values in mg/L and share these results with the rest of the group (everybody should include these values in their notebook and find the average, standard deviation and error-95) • 7) Find the ratio of MLVSS/MLSS and compare with common values of 0.8

4. Lab - 4 - Biodegradation (C) Denitrification: Removal of nitrate (NO3-) ions by anoxic biodegradation of a carbon substrate • Goal: measure nitrate removal rates in bioreactors • Procedure • 1) Locate the following materials: • Denitrification reactor setup from last week’s experiments • Container with a solution of 2,000 mg/L NO3--N • 1,000 ul pipette and tips • Solution of 5% carbon source (methanol, ethanol, acetic acid) and dropper. • Reagents 1&2 for nitrate measurement • Nitrate spectrophotometric standards (0.0 , 0.1, 0.2, 0.4, 1, 2 mg/L NO3--N) • 10 ml HACH vials • 50 ml centrifuge sample vials. • Watch, pH meter and DO meter • 2) Uncap your denitrification reactor and add 3,000 ul (3 ml) of the 2,000mg/L nitrate solution (use the 1000 ul pipette), also add the required amount of carbon source you determined last week (use the dropper - you determined the # of droplets last week), cap the reactor and register the time you capped it. • 3) After 30 minutes uncap the reactor again and pour 10 ml of the sample in the centrifuge vials, cap the centrifuge vial, refill the reactor with additional sludge (ask instructor) and cap the reactor again.

5. Lab - 4 - Biodegradation Denitrification: Removal of nitrate (NO3-) ions by anoxic biodegradation of a carbon substrate • Procedure • 4) Centrifuge the 50 ml vial for 5 minutes, then remove from the centrifuge and take a 1000 ul (1ml) sample from the clear liquid and pour it into a HACH vial, then add DI water until reach the center of the HACH label (~10 ml) • 5) Add 10 droplets of Nitrate Reagent 1, screw in the lid and shake the vial for 30 seconds. • 6) Shake the Nitrate Reagent 2 for 30 seconds and add 10 droplets to the vial, screw in the lid shake it for 30 seconds and wait 5 minutes before taking the absorbance reading. • 7) Repeat steps 3 through 6 at 1 h and 2 h • 8) To measure the concentration of nitrate: • Turn on the spectrophotometer in absorbance mode at 540 nm of wave length - red color (ask instructor for details) • Take the blank standard (0.0 NO3--N) and place it in the sample housing, then set the blank absorbance (ask instructor for details) • Measure the absorbance of the standards provided and plot the concentration of the standard versus its absorbance - this is called the calibration curve • Read the absorbance of the samples collected at 30min, 1hr and 2 hr, and use the calibration curve to obtain the concentration of the diluted sample, the actual concentration is 10 times (dilution factor) this value

6. Lab - 4 - Biodegradation Denitrification: Removal of nitrate (NO3-) ions by anoxic biodegradation of a carbon substrate • Procedure • 9) Measure the pH and Dissolved Oxygen after 2 hrs. • 10) Obtain from the instructor the concentration of nitrate after 6hr, 12hr, 24hr, and 72 hr • 11) Measure the concentration of nitrate in treated wastewater, tap water and lake water, pour 10ml of the sample directly into the HACH vial, without further dilution • 12) Plot the concentration of time versus nitrate as described below: • Semilog plot of concentration (log scale) versus time (linear scale), apply an exponential regresion of the data (we’ll do in class) • Obtain the decay constant (Kd) of the reaction. • 13) Clean glassware using desinfectant and rinse twice with DI water. • CAUTION: the activated sludge has a substantial concentration of bacteria, therefore all disposable material is considered a biohazard and should be disposed in the appropriate containers, use gloves and goggles at all times. In case of contact, wash thoroughly with water and desinfectant. • CAUTION: Nitrate Reagent 1 is highly acidic and corrosive, in case of contact wash with sufficient water and add sodium bicarbonate to the affected area. • CAUTION: Nitrate Reagent 2 contain Cadmium metal, avoid direct contact or ingestion

7. Lab - 4 - Biodegradation Nitrate Measurement principles The most common nitrate test kit is one that uses cadmium metal (Reagent 2) to reduce the nitrate (NO3-) to nitrite (NO2-). The resulting nitrite can then be tested based on a version of the Griess Reaction. With this type of kit, you place a known volume of water sample in a clear test vial. Three to ten drops of an acidic sulfanilamide reagent is then added to the test vial(Reagent 1). This is followed by the addition of three to ten drops of N-1-naphthylethylenediamine dihydrochloride solution (NED for short) containing also the cadmium suspension (Reagent 2). After ~5 minutes, the reaction produce a red colored component whose intensity is a reflect of the concentration of the nitrate in solution. The intensity of the red color (540 nm) can be measured using the Beer- Lambert law: Absorbance= log(Intensity of incident light/Intensity of transmitted light) = a*b*C a = absortivity (a property of each solute) b = light path length C = concentration of the solute Sample - red color 540 nm Light Filter Light 540 nm Light detector - Photocell Light Source