ANAEROBIC DIGESTION LAB OVERVIEW Level: Grades 9-16 Estimated Time to Completion: Two 180 Minute Sessions Prior Knowledge: Background Provided In this lab investigation, students will become familiar with anaerobic digestion of a waste stream. Anaerobic digestion is a method similar to composting where the goal is to produce a stable product that can be utilized for many purposes such as fertilizer. Unlike composting, the gas produced during digestion contains a large portion of methane, which can be harnessed for energy needs. Characteristics of anaerobic digestion products include: 1. The gas component contains mostly methane (~60%) and carbon dioxide (~40%), with small amounts of hydrogen sulfide and water vapor. 2. About 2/3 of the nutrients from the source materials are maintained in a liquid component that can be used for fertilizer 3. If a solid separator is used, 1/3 of the nutrient value is removed with the solids and is a concentrated natural fertilizer Upon completion, students will be able to: • Test a variety of a waste streams (substrates) for biogas production capability • Perform titrations to determine the amount of CO2 contained within a biogas sample • Understand how pH affects the productivity of anaerobic bacteria • Understand how multiple bacteria can living symbiotically within a system MATERIALS REQUIRED Lab Balance pH Meter Temperature Bath (~35-38 °C) 2 50ml Burets 2-4 150 to 250ml Erlenmeyer Flasks 2-3 60ml Vial with Caps and Rubber Stoppers 60ml Syringe with Needle and Open/Close Valves Food Waste (sugar as control, spoiled fruit, etc…) Anaerobic Seed Stock (obtained from a local city digester or dairy farm digester) Nitrogen .5M NaOH .1M HCl Phenolphthalein Methyl Orange NOTES TO INSTRUCTOR • Instead of the digestion being performed in a sealed bottle, a high quality balloon is placed on the top and the gas produced is measured this way. • Instead of flushing the bottle for 2 minutes with nitrogen, you could bubble through the solution, or not flush at all. • The use of a gas chromatograph is optional depending on the resources available.
Name:
Date:
Class/Hour:
ANAEROBIC DIGESTION INTRODUCTION An anaerobic digester is a biogas recovery system. During anaerobic digestion, bacteria break down manure in an oxygen-free environment. One of the natural products of anaerobic digestion is biogas, which contains between 60 to 70 percent methane, 30 to 40 percent carbon dioxide, and trace amounts of other gases. This is different than composting because composting uses aerobic bacteria that need oxygen. The biogas released is then only carbon dioxide. The key to any anaerobic digester is the substrate, which is the feedstock of the digester. Generally speaking, the feedstock can be comprised of anything that can be converted into methane by anaerobic bacteria. Cities have been using anaerobic digesters to deal with wastewater since 1895, when England developed an anaerobic digester that used wastewater to power streetlamps. It was not until the 1970s when farms in the U.S. experimented with digesters. Farm digesters have seen a lot of growth since 2000, and a number of cities are converting their aerobic treatment systems into anaerobic digesters. One component of most substrates can be manure. However, co-substrates can be added to increase production of methane. There is a balance to this addition, though. Too much high strength substrate can cause an imbalance in the bacteria population equilibrium. In a digester system there are at least three different active bacteria populations: acetogens, hydrogenic bacteria, and methanobacteria. Acetogens produce acetate, through acetogenesis, from a variety of energy and carbon sources. They are anaerobic bacteria. Hydrogenic bacteria are characterized by the consumption of organic acids and production of molecular hydrogen. Methanobacteria reduce carbon dioxide and produce methane as a waste product. The acid forming bacteria are the first to start breaking down the substrates into smaller organic molecules which are typically carboxylic acids. If too much acid is formed, the pH drops below the optimal range for the other bacteria and they become dormant. If they become dormant, there will be no methane formed. In this experiment, you will prepare a range of concentration of substrate to seed stock and determine the optimal concentration for methane production. These samples will be maintained at a constant temperature for one to two weeks. Each week, you will determine the total amount of gas produced and the percentage of carbon dioxide by titration. The methane concentration will then be determined by the difference.
PROCEDURE PART 1: SETUP 1. Weigh out about 30-35g of a seed solution obtained from a nearby anaerobic digester. This is either from a wastewater treatment plant or a farm digester. 2. Place in the bottle that will be sealed for seven days. 3. Add varying amounts of the substrate (waste stream) that will be tested. Typically, this is about 1.0%, 1.5%, 2.0%, or 3.0% by mass of seed solution. 4. Flush nitrogen through the bottle for 2 minutes at a steady, gentle flow. 5. Seal the container and place in a constant temperature apparatus at 35째C or 38째C until the next week. 6. Determine the pH, total solids, volatile solids, and COD (chemical oxygen demand) of the seed solution and substrate if time allows. (Some of this information may be provided by the instructor.) PART 2: TOTAL GAS DETERMINATIONS After 1 week, determine the amount of biogas produced over the last week. Note: Extra caution is required, depending on what type of container the biogas is produced in. If it is produced in a solid container, the valve needs to be closed after the syringe is filled to only about 40% capacity. Otherwise, the syringe plunger will expand beyond the end of the syringe to reach room pressure. A 60mL syringe is used, along with a needle and needle valve in the closed position, when inserted into the sealed bottle. 1. Keep a hand on the plunger with pressure to keep it from blowing out the end. The syringe is only allowed to fill up partway so that the pressure is equilibrated to room pressure before it is removed. The syringe is equilibrated to room pressure after closing the valve by pulling the plunger further out and letting it pull back in slightly. 2. After the syringe pressure is equilibrated, remove it from the valve. This decreases the amount of gas leakage from the bottle.
ANAEROBIC DIGESTION
NOTES
PROCEDURE (cont.) 3. Inject this sample into the carbon dioxide trap prepared in the next steps. Only 50-60mL total of the biogas is injected into the carbon dioxide trap. 4. Continuously remove gas until there is no more. Follow the pressure equilibrating step each time. BIOGAS RATIO BACKGROUND Biogas quality is typically determined by the percent methane and amount of hydrogen sulfide that needs to be removed. The biogas sample is too small to determine the hydrogen sulfide content without the use of a gas chromatograph (GC). Typically, the methane content is determined by using a GC and a different column and detector than that used for hydrogen sulfide. If such a GC is available and is used the following titration step is bypassed. The remainder of the gas not determined to be methane in the GC is deduced to be carbon dioxide. The following titration is used to determine the amount of carbon dioxide. The amount of methane is determined from the difference. In this experiment, a two-step titration is performed on a gas trap solution. The first step in the titration is to neutralize the strong base. The second step is the titration of HCO3- to H2CO3. This titration determines how much CO2 from the gas sample is trapped by the sodium hydroxide. Trap NaOH + CO2 -> Na+ + HCO3Neutralization that happens above pH of 8 NaOH + HCl -> NaCl + H2O Neutralization that happens around pH of 5 HCO3- + HCl -> H2CO3 + Cl-
ANAEROBIC DIGESTION
NOTES
PROCEDURE (cont.) PART 3: TITRATING SAMPLES PREPARING THE CO2 TRAP 1. Pipette 5mL of 0.50M NaOH into a 60mL sample vial and close with a rubber stopper and aluminum cap using the crimper. 2. Take a 50 to 60mL gas sample from the test vial and inject into the sealed vial. Record the volume injected. 3. Swirl the solution for 2-4 minutes to ensure that all of the carbon dioxide reacts with the hydroxide solution. DETERMINATION OF CO2 BY TITRATION 1. Remove the aluminum cap and rubber stopper. 2. Add 2 to 3 drops of Phenolphthalein to the solution. 3. Titrate the solution with 0.10M HCl. (Note: pink color in solution may initially disappear after the first 5mL of titration, well before the equivalence point is reached. If this happens, add 2 more drops of indicator to solution and continue titration.) 4. Once the end point is reached, record the volume on the burette. 5. Add 2 to 3 drops of Methyl Orange indicator to the solution. 6. Continue titration with 0.10M HCl to the second end point. Note: The same titration should be performed using a blank sample of 0.50M NaOH solution to determine if there is any carbon dioxide dissolved in the stock solution. This titration is performed on all samples prepared in the previous week.
ANAEROBIC DIGESTION
NOTES
PROCEDURE (cont.) To determine the % of the gas sample that is carbon dioxide, determine the moles of carbon dioxide from the titration. mL HCl (for second end point) x ~0.10M HCl -> moles HCl = moles CO2 The formula 22.4L = 1 mole of a gas at standard conditions is used to determine the mL of gaseous CO2. To be more precise, the temperature and pressure of the room is used along with PV = nRT. The remaining volume of the biogas contains a very small amount of water vapor and other gases, such as hydrogen sulfide. For this laboratory, these other small volumes are ignored, and the remainder of the gas is assumed to be methane and the original nitrogen gas.
ANAEROBIC DIGESTION
NOTES
Name:
Date:
Class/Hour:
DATA AND CALCULATION Trial #:
Blank
1
2
Volume of Head Space of Vial: Total Volume of Gas Removed from Vial: Volume of Biogas added to the CO2 Trap: Initial Volume in Burent before CO2 Titration: Volumbe in Buret at First End Point of CO2 Titration: Volume in Buret at Second End Point of CO2 Titration:
Molarity of Acid Used:
Molarity of Base Used:
Pressure in the Lab:
Temperature in the Lab:
Refer to the Lab Manual for information and equations needed to complete the next page of Data and Calculation.
ANAEROBIC DIGESTION
3
Name:
Date:
Class/Hour:
DATA AND CALCULATION (cont.) Trial #:
Blank
1
CO2 Concentration in Gas Sample CO2 Volume in Gas Sample
Average CO2 Concentration in Gas Sample (all trials): Average CO2 Volume in Gas Sample (all trials): Show work below:
ANAEROBIC DIGESTION
2
3