UNIVERSITY OF WISCONSIN SYSTEM SOLID WASTE RESEARCH PROGRAM Student Project Report Microbial Community Analysis of the University of Wisconsin Oshkosh Dry Biodigester May 2015 Student Investigator: Jessi Zimmerman Advisor: Dr. Eric Matson University of Wisconsin-Oshkosh 1 Abstract In natural environments, heterotrophic bacteria and methanogenic Archaea assist in the degradation of organic waste, which results in the production of a mixture of carbon dioxide and methane gas, together known as biogas. In the UW-Oshkosh dry anaerobic biodigester, these same naturally occurring microbial processes are utilized to benefit the University and surrounding community. Organic waste produced by local farms, grocery stores, and residential households are collected and delivered to the biodigester facility rather than being added to landfills. These waste products serve as the feedstock for biogas production. The combustible biogas is captured and used as fuel for a combined heat and power generator, which meets about 10% of the campus electricity demand and helps to heat the digester and other nearby campus facilities. The process reduces the University’s operating costs while diverting some forms of municipal waste away from landfills and provides a cleaner alternative to fossil fuels. The composition of the incoming feedstock is closely monitored as are the physicochemical conditions such as temperature, pH and hydration. These are measured at the beginning and throughout the cycle, because they can influence methane output. What is not measured (and not currently known) is how the growth of microbial species changes throughout the course of the digestion cycle. In particular, changes that may occur in methanogenic species richness and relative abundance are likely to influence biogas quality and output. Therefore, the aims of this study were to 1) extract and purify DNA samples suitable for genomic and metagenomic analysis from the microbial community at time points that span the duration of a digestion cycle and 2) measure community dynamics in some of these samples using high-throughput next- generation sequencing (NGS). Biodigester samples collected and sequenced from two digester bays showed that methanogenic Archaea populations change markedly over time and differ between separate digestion cycles. Through understanding how these populations are changing, this research may help to identify conditions that could stimulate rapid growth and 2 prolonged maintenance of methanogen populations and, thus, improve the efficiency of methane production in the biodigester. Statement of Objectives The primary objective of this project was to obtain high-quality microbial community DNA samples from two fermentation chambers of the UW-Oshkosh biodigester over the course of a 28-day cycle. These samples will allow researchers to examine several aspects of the Archaeal communities - now and in years to come. The sample collection will be used for species-based assessments of community structure, such as in the current study and functional gene-based studies in future research. The secondary objective was to use universal PCR primers to amplify Archaeal SSU rRNA genes from the microbial community DNA samples and to sequence the resulting amplicons. The SSU rRNA sequencing data provides species identity data as well as frequency of occurrence throughout the biodigester cycle. Analyses that reveal structural changes to the community over the course of a cycle and between digester cycles will be compared to data on physicochemical conditions in the biodigester bays. Together, these comparisons will show how the microbial community may be responding to conditions within the digester throughout the cycle. Introduction The biodigester on the UW-Oshkosh campus (Fig. 1) is the only commercial dry biodigester in the Western hemisphere and has been in operation since the fall of 2011 (Innovations in Sustainability and Renewable Energy, 2014). Because it is a “dry” biodigester, the input material used has a moisture content of 75% or less (BIOFerm Energy Systems, 2011). The majority of the input consists of organic materials such as food, agriculture and yard waste from 3 campus, local community sources and additional area partners (Innovations in Sustainability and Renewable Energy, 2014). Figure 1. UW-Oshkosh biodigester facility located at 755 Dempsey Trail, Oshkosh, WI. The organic waste is loaded into individual fermentation chambers (Fig. 2) and liquid percolate, which is rich in microorganisms such as methanogenic Archaea, is sprayed over the feedstock to assist in decomposition and methane production (BIOFerm Energy Systems, 2011). The organic waste is broken down throughout a series of steps, collectively known as anaerobic digestion (AD), which results in the production of carbon dioxide and methane, or biogas. The biogas is captured and combusted to produce heat and electricity. This environmentally beneficial process provides clean, renewable energy while reducing and reusing organic wastes. The amount of solid waste generated is reduced by 40% at the end of the 28-day cycle. 4 Figure 2. The UW-Oshkosh anaerobic digester has four separate bays that operate continuously on staggered 28 day cycles. Arrows indicate the location of floor drains for fermenter bays 1 (left) and 2 (right). The left over solid waste, known as digestate, is low in odor and rich in nutrients and serves two purposes: approximately half of the digestate is reserved and mixed in with the incoming fresh feedstock as a source of inoculum, while the remaining half is further composted off-site and can be later used as fertilizer or a soil supplement. This increases the lifespan of landfills and capacity of compost sites, while decreasing the energy consumption and costs involved with moving waste (Innovation in Sustainability and Renewable Energy, 2014). Each week, one of the four digester bays starts a new 28-day cycle, such that the facility on the whole is maintained in a state of continuous operation. Once the mixing and loading of feedstock is complete, the airtight fermenter doors close and the chamber air is evacuated. Aerobic bacteria consume residual oxygen within the chamber, creating an anaerobic environment and thus starting the AD process. Anaerobic digestion is a series of biological processes in which microorganisms decompose organic material in the absence of oxygen. The process involves four steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis, and is carried out by communities of hydrolyzing, acid-producing and acetate-producing bacteria and methane-producing Archaea (Fig. 3). During hydrolysis, hydrolytic bacteria convert complex insoluble polymers into simpler, soluble monomers, making them available for other bacteria. For instance, carbohydrates are 5 converted to simple sugars, lipids to fatty acids and proteins to amino acids. In the next step, acidogenesis, fermentative bacteria convert the soluble organic monomers into volatile fatty acids, ketones, ethanol, hydrogen and carbon dioxide. Acetic acid, carbon dioxide and hydrogen created at this step skip acetogenesis (Serna, 2009), as they can be used directly by hydrogen-consuming (hydrogenotrophic) methanogens to produce methane. The majority of methanogens are hydrogenotrophs (Sarmiento, et al., 2011), which use hydrogen, and sometimes formate and carbon monoxide, as the electron donors to drive the reduction of carbon dioxide (Ferry, 2010). During acetogenesis, hydrogen-producing acetogenic bacteria convert volatile fatty acids and ethanol into acetic acid, carbon dioxide and hydrogen. The majority of the methane (approximately 72%) is produced by hydrogen-producing aceticlastic methanogens, which produce methane and carbon dioxide via the decarboxylation of acetate. Most of the balance is produced by hydrogenotrophic methanogens (Kanhal, 2009) though small amounts of methane are produced by the conversion of methylotrophic substrates, such as methanol, methylamines and methyl sulfides as well (Ferry, 2010). 6 1) Hydrolysis (fermentative bacteria) 2) Acidogenesis (fermentative bacteria) 3) Acetogenesis (acetogenic bacteria) 4) Methanogenesis Hydrogenotrophic Acetoclastic (methanogenic Archaea) Figure 3. Anaerobic digestion process. Microorganisms decompose organic material through a series of biological processes in the absence of oxygen, resulting in the production of biogas. Biogas typically consists of 60-70% methane, 30-40% carbon dioxide (EPA, 2014), and trace amounts of other gases, such as hydrogen, nitrogen, carbon monoxide, oxygen and hydrogen sulfide. This gas is collected, treated to remove impurities, such as hydrogen sulfide, and used to generate energy, providing a cleaner alternative to traditional fossil fuels (BIOferm, 2011). 7 Materials and Methods Sample collection. Being that the biodigester is a closed system, one cannot enter to collect samples. However, the percolate that flows through the biomass is accessible via floor drains in front of the individual chambers. The floor drains allow access to microorganisms that are washed out with the percolate from each fermenter and should provide an estimate of the microorganisms within the biomass piles. These floor drains, immediately adjacent to the fermenter bays, were used to individually sample bay 1 and bay 2 (F1 and F2) over the duration of a 28-day cycle (Fig. 2 and Fig. 4a). Samples of percolate were also collected from the percolate holding tank, which is a large, 120,000 gallon reservoir where the percolate