The scope of our research is to use respirometry to examine the biokinetics of anaerobic microbiota, co-digesting organic waste streams such as wastewater sludge and food scrap. We attempted to answer the question of how different substrate concentrations influence the rate at which these anaerobic microbiota produce methane. While several studies have measured methane production from co-digestion of waste activated sludge, coupled with other organic substrates, such as a biowaste, fats, oils, grease, and agricultural waste, this research presents a respirometer protocol using a mix of dilute wastewater, waste activated sludge, and food scrap waste as substrates.
Similar studies use batch assays in serum bottles where methane production is measured at discrete points throughout the experiment, while others use flow meters connected directly to bench or pilot scale rioreactors. Our protocol uses a respirometer that provides continuous and precise methane measurements from samples run in different experimental conditions. To begin, collect 1.5 liters of primary effluent, one liter of waste activated sludge.
Obtain two liters of anaerobic sludge immediately before experimentation. Next, store the food waste at four degrees Celsius. To prepare mineral base solution one, mix 800 milliliters of deionized water with the given salts.
Make up the volume to one liter with deionized water. To prepare the mineral base two solution, dissolve 27.7 grams of calcium chloride and 101 grams of magnesium chloride in 800 milliliters of deionized water. Dilute the solution to one liter.
To prepare a nutrient base, mix 38.2 grams of ammonium chloride and 15 grams of sodium sulfate in 800 milliliters of water. Use 3.64 normal sodium hydroxide to adjust the pH of the solution to seven. Then dilute the solution to a final volume of one liter.
For sample preparation, combine the food waste in a blender to create a mixture. Add deionized water to the mixture to help blend it better. Transfer the working waste into a one liter plastic bottle.
Store it at four degrees Celsius until further use. Next, place four labeled two liter beakers on a stir plate. Place a large stir bar on the plate.
To each beaker, add 12 milliliters of both mineral base solutions and the nutrient base solution. Combine the food waste, waste activated sludge and dilutions as shown in the table. Add anaerobic cultures to the beaker.
Then add 2.4 grams of sodium bicarbonate to each beaker. Next, place a magnetic stir bar in the eight labeled respirometry bottles. Immediately transfer 500 milliliters of a mixture from the beaker into a labeled respirometer bottle.
Flush the mixture with nitrogen gas before capping. Next, set the respirometer to a low anaerobic setting. Push the reset button and the power on button simultaneously.
Set the chiller to 35.5 degrees Celsius. Fill a carbon dioxide and moisture scrubber with a 50/50 mix of calcium sulfate and potassium hydroxide pellets at the center. Surround the pellets with glass wool on each side.
Connect the tubes and needles from the sample bottles to the scrubber, and then from the scrubber to the gas inlet. Launch the respirometer program RSA 8 version 2 on the computer. Select all the bottles in the program.
Then choose Edit, followed by Data Labels. Name all the bottles then press the start button to begin gas production measurement. Set the program to measure data at the end of every half hour.
Select the rate chart or volume chart to monitor the gas production. After the experiment has concluded, stop the run, turn off the chiller and the RSPF module. Save the data as a CSV file.
The blended food scrap waste was 44%carbohydrates, 36%proteins, 16%fats, and 4%other materials. The initial volatile suspended solids concentrations increased from the control to the largest FM ratio. The concentration decreased towards the end of the experiment.
The initial oxygen demand concentrations increased from the control to the largest FM ratio. The BOD concentrations decreased between initial and final concentrations with the exception of the control. The rate of methane production and length of production increased as the FM increased.
