UNIVERSITY OF WISCONSIN SYSTEM SOLID WASTE RESEARCH PROGRAM Student Project Report. Optimizing Biogas Output by Biological Means.

Transcription

1 UNIVERSITY OF WISCONSIN SYSTEM SOLID WASTE RESEARCH PROGRAM Student Project Report Optimizing Biogas Output by Biological Means May 2014 Student Investigators: Stephanie Hands and Ryan Bartell Advisor: Greg Kleinheinz University of Wisconsin-Oshkosh

2 INTRODUCTION Biogas is a gaseous fuel produced by the fermentation of organic matter. Biogas is typically comprised of 58% methane and 38% carbon dioxide (16). Pound for pound, methane is a potent green house gas that is 20 times more effective at trapping heat than CO 2 over a 100-year timeframe (14). The use of methane as a clean energy source provides a versatile carrier of renewable energy, as well as the opportunity to mitigate greenhouse gas emissions and simultaneously increase available energy supply. The production of biogas through anaerobic digestion has been evaluated as one of the most energy-efficient and environmentally beneficial technologies for bioenergy production (15). Anaerobic digesters mitigate waste through anaerobic composting. As oxygen is used up, anaerobic microbial communities thrive and metabolize carbon dioxide and/or acetic acid to produce methane (5). In turn, the methane produced from these microbial communities can be harnessed to produce energy. FIGURE 1. Composition of Biogas With the establishment of the dry anaerobic digester at UW-Oshkosh, campus researchers have the unique opportunity to explore biogas production (energy potential), by conducting extensive testing on the process of methanogenesis. Methanogenesis is the formation of the energy-yielding product methane produced by microbes known as methanogens. Organisms capable of producing methane are only found in the diverse group Archaea (1). Classically, the health of an anaerobic digester has been characterized by chemical parameters such as volatile fatty acids, organic dry matter, and ammonia

3 content. However, there have been few studies done on the succession of the methanogen microbial community present within a digester, the key component in the organic composting process. FIGURE 2. The dry anaerobic digester at University of Wisconsin-Oshkosh. The digester (left) receives solid organic waste (upper right) from various sources for use in large bays (bottom right) which create an anaerobic environment for digestion. To optimize biogas output from organic material, our study will attempt to define the normal community succession of methanogenic archaea. By defining the normal succession of microbes present in the anaerobic digestion system, we can make beneficial reuse of organic waste by optimizing the organic waste to its full potential. This will in turn reduce the amount of organic waste sent to a landfill, provide nutrient-rich composted soil at the end of the digestion process for reuse, and reduce our environmental footprint by using biogas as an energy source, rather than emitting it as a greenhouse gas. Using biological means to measure the efficiency of a dry anaerobic digester could allow plant managers to better determine which types of feedstocks are appropriate for their system. Where the microbiological community of one digester might be more suited to feedstocks such as food waste, another may be more suited to other organic material such as paper waste. With information from the microbiological feedback proposed in this study, managers will be able to make informed decisions concerning: 1) which feedstocks are appropriate for their digester; and 2) how to shift the microbial community within the digester to optimize it for other feedstocks. OBJECTIVES The primary objective of this study was to optimize the output of methane from a dry anaerobic digester by measuring the microbiological community within the system. Biogas output will be measured in order to determine which substrate is best suited for the microbiological community present within the system.

4 METHODS AND MATERIALS 1. Characterization of digestate used for the study Total solids (TS), volatile solid (VS), ph, and biogenic methane potential (BMP) content were measured in the original digestate material. a) Total Solids: This test determines what percent of the digestate is composed of water and how much of the material is solid. Digestate will be placed in an incubator set to 105C for a period of at least 24 hours. After 24 hours, the digestate will be removed from the incubator, weighed, and returned to the incubator. The digestate will then be weighed again 2 hours later to determine whether or not there was a change in mass. If the mass does not change, then the mass of the sample will be recorded (7). b) Volatile Solids: This test determines what percent of the digestate is composed of organic material. Dried digestate from the total solids method will be placed in a muffle oven at a temperature of 550 C for at least 4 hours. After cooling in a desiccator, the samples will be weighed. The resulting residue is composed of inorganic material (8). c) ph: ph was measure using a standard calibrated ion-selective electrode (11). d) Biogenic Methane Potential: Digestate will be placed in eudiometers (fermentation vessels) in a water bath maintained at a temperature of 37 C. Gas composition (methane, carbon dioxide, oxygen, and hydrogen sulfide)and volume will be measured from each vessel for a period of 28 days (12). 2. Determine the abundance of two groups of methanogenic microbes within the digestate This took place over a 28 day period and attempted to measure the abundance of two different groups of methanogens at different time points from a fermentation vessel containing digestate and food waste. Quantitative polymerase chain reaction (qpcr) was used to target each of the individual groups and determine their relative abundance. a) Primers/probe sets were used for two orders of methanogens (Methanobacteriales, Methanomicrobiales). b) Bench scale digesters i. Fermentation vessels were sampled once per week without replacement for 28 days. c) DNA was extracted from samples using a mobio soil extraction kit. d) qpcr was conducted with the primer/probes from 2 a) above in order to determine level of presence for each organism at different time points. The primer/probe-specific details of this method have been used before in previous studies (13). 3. Summarize the succession pattern of each methanogenic group and determine any relationship to methane output.

5 a) qpcr data was assimilated into figures and tables to better show the overall succession pattern of the chosen methanogenic groups. b) Compared abundance of each methanogen versus the output of methane at given time points. RESULTS Biogas Volume Eudiometers loaded with digestate and microcrystalline cellulose (MC) produced an average of 28.5L of biogas (n=4). Eudiometers loaded with only digestate (negative controls) produced significantly less biogas over the 28 day fermentation period (Figure 3). Negative control eudiometers only produced an average of 9.2L of biogas, significantly less than those loaded with microcrystalline cellulose. FIGURE 3. Total Volume of Biogas Produced. Biogas produced by eudiometers loaded with only digestate is represented by squares, and biogas produced by digestate with microcrystalline cellulose is represented by diamonds. Gas Quality Measurements Biogas from MC eudiometers had averages of 43.5, 51.0, 57.9, and 62.1% methane at 7, 14, 21, and 28-day time points, respectively. Biogas from negative control eudiometers averaged 51.5, 59.9, 61.3, and 60.5% methane at 7, 14, 21, and 28-day time points, respectively (Figure 4).

6 FIGURE 4. Methane production. Digestate loaded with MC produced biogas had an initially lower percent methane than that produced from the negative control digestate. Biogas from MC eudiometers had averages of 56.0, 48.5, 42.8, 37.0% carbon dioxide at 7, 14, 21, and 28-day time points, respectively. Biogas from negative control eudiometers averaged 35.3, 36.6, 37.7, 38.2% carbon dioxide at 7, 14, 21, and 28-day time points, respectively (Figure 5). FIGURE 5. Carbon Dioxide Production. Digestate loaded with MC had higher initial carbon dioxide production than the negative control digestate. In MC eudiometers, the biogas started with a lower percent methane content and trended upward, whereas the percent carbon dioxide started out high and declined over time. Biogas from negative control experiments started out with low methane and carbon dioxide content (at or below 20%) and increased every day until levelling off around day 14 (Figures 6 and 7).

7 FIGURE 6. Methane and Carbon Dioxide Production from Digestate Loaded with MC. FIGURE 7. Methane and Carbon Dioxide Production from Digestate Only. Dry Matter, Organic Dry Matter, and ph Measurements The dry matter of digestate from biogas experiments was measured at 4-day intervals. Dry matter remained consistent around 30%, while organic dry matter content varied slightly over the 28-day period. Organic dry matter began at approximately 52%, and declined slowly but consistently until reaching 45%. The ph of the digestate began very basic, at about 8.5, and increased to 8.9 by day 28 of the experiment. Real-time PCR Methanogen Analyses Two different orders of methanogens were quantified using real-time polymerase chain reaction. 16s ribosomal RNA (rrna) gene copies were quantified for Methanomicrobiale (MMB) and Methanosarcinales (MSL) over a 28-day period.

8 In MC eudiometers, MSL was present in consistently higher densities than MMB. MSL concentrations began at just above 10 7 gene copies/µl, dropped to 10 5 after 7 days, and then increased in density until reaching 10 7 gene copies/µl at day 28. MMB began the experiment at copies/µl and decreased at day 7 to MMB concentrations increased at day 14, decreased at day 21, and then increased again at day 28. FIGURE 8. MMB and MSL 16s rrna Gene Copy Concentrations in MC Eudiometers. MSL was consistently more present over the tested period. In biogas experiments where there was no substrate mixed into the digestate, MSL gene copies stayed consistent at about 10 7 gene copies/µl throughout the entire experiment. MMB copies began the 28-day experiment at copies/µl and fluctuated around 10 5 and 10 6 copies at each time point measured (Figure 9). FIGURE 9. MMB and MSL Concentrations in Digestate with No Substrate.

9 DISCUSSION Biogas Volume Biogas volume differences between the MC digestate and the negative control digestate were notable. Digestate which was not inoculated with microcrystalline cellulose had much lower gas production than MC digestate. This is expected because microbes are able to use the microcrystalline cellulose (when present) as a source of nutrients. When there is no substrate present, the microbes are forced to use whatever nutrients remain in the digestate, which typically has little or no long or shortchain fatty acids present. In anaerobic digestion, fatty acids are broken down by bacteria and used by archaea to make methane. Biogas Quality (CH 4 and CO 2 ) Biogas from MC digestate had lower initial methane percentages than biogas produced from negative control digestate. MC digestate had higher initial carbon dioxide content. In the first 7 days, the gas composition of MC digestate biogas may have been higher in carbon dioxide because bacteria may be using the microcrystalline cellulose as an energy source and producing carbon dioxide as a byproduct. The limited initial methane production may be due to the proliferation of bacteria, limiting nutrients for methanogens which need either carbon dioxide or acetic acid as an energy source to make methane. Also, in the initial days of the experiment, oxygen is present in trace amounts, which prevents strict anaerobes (such as methanogens) from growing. Although methane was a main constituent in biogas from negative control digestate, the digestate did not produce a high volume of biogas. In summation, digestate with no added substrate produced only a small amount of gas high in methane content. Methanogen Abundance and Relationship to Methane Production Methanosarcinales (MSL) was more abundant than Methanomicrobiales (MMB) in both the MC digestate and the negative control digestate. In MC digestate, MSL gene copies started at about 10 7 and then decreased before increasing as the experiment progressed. This increase in abundance parallels the increase in methane content and total biogas production. There are three families within the order of MSL, and methanogens within the three families are capable of using both acetic acid and carbon dioxide to make methane. Families within the order MMB are only capable of using carbon dioxide as an electron acceptor to produce methane. The wider range of molecules that MSL can use may account for its increase ability to survive and proliferate within digestate. While real-time PCR is a useful tool for measuring the amount of DNA in a sample, it cannot determine the number of live cells within a sample nor how the cells present are contributing to biogas

10 production. Future studies may be able to better elucidate the relationship between certain groups of methanogens and biogas production by employing techniques such as fluorescent in situ hybridization (FISH). Techniques such as FISH would help to show which archaea are producing methane along with which substrates are being used to produce the methane. By identifying how each group of methanogens contributes to biogas production, anaerobic digester plant managers can better determine how efficient their digester is operating by measuring the abundance of each type of methanogen. Literature Review 1. Ferry, J. G. (1992). Biochemistry of Methanogenesis (p. 1). CRC Press, Inc. 2. Municipal Solid Waste (MSW) in the United States: Facts and Figures. USEPA. Retrieved May 6, 2013, from 3. What is Organic Waste? In Organic Disposal LLC. Retrieved May 6, 2013, 4. Governor s Task Force on Waste Materials Recovery and Disposal. Final report, Retrieved May 6, Alliant Energy. Anaerobic digesters and methane production in the agricultural sector of states served by Alliant Energy. Technical report, Alliant Energy, Retrieved May 6, Governor s Task Force on Reducing Global Warming. Wisconsin s Strategy for Reducing Global Warming. Final report, Retrieved May 6, Total Solids Method: DIN EN : Volatile Solids Method: DIN EN : Total Phosphorus Method: Standard Methods, 20th Ed., 4500 P B5, 4500 P E. 10. Volatile Fatty Acids Method: Based on: Determination of Volatile Fatty Acids with House Method at Schmack Laboratories in Schwandorf, Germany and method development at the UW- Oshkosh. 11. ph Method: DIN EN : Biogenic Methane Potential Method: DIN and VDI Youngseob Yu, Changsoo Lee, Jaai Kim, and Seokhwan Hwang Group-Specific Primer and Probe Sets to Detect Methanogenic Communities Using Quantitative Real-Time Polymerase Chain Reaction. Wiley InterScience DOI: /bit US Environmental Protection Agency (2012) US government's global methane initiative accomplishments report December 2012: annual report. US Environmental Protection Agency, Washington, DC e872-4e91-aa09-7a5091dfc761%40sessionmgr114&vid=2&hid= Rasi, S Biogas composition and upgrading to biomethane. Dissertation. University of Jyväskylä.