The goal of the project was to quantify the increase in methane production when alternative sources of inocula, specifically landfill leachate and wetland sediment, were used in manure anaerobic digesters operating at temperatures lower than the conventional mesophilic digesters, which operate at 35ºC. Anaerobic reactors were constructed to incubate these alternative inoculum sources as well as digestate from a mesophilic digester, a traditional inoculum source, at three different temperatures (15ºC, 25ºC, and 35ºC) to acclimate and grow the populations of methane-producing microorganisms at each temperature range.
Samples were extracted from the incubation reactors to supply inocula for two biochemical methane potential (BMP) tests that ran for approximately 90 days at each incubation temperature (15ºC, 25ºC, and 35ºC). BMP 1 was conducted after 3 months of incubation, and BMP 2 was conducted after 6 months of incubation to determine the effect of incubation time on methane production. In addition, the effect of inoculum to substrate ratios on methane production was determined using four ISR rates: (50% (w/w), 35%, 20%, and 0%).
The 6 month incubation time in BMP 2 increased maximum methane production by 3,500%, 15%, and 28% at 15ºC, 25ºC, and 35ºC, respectively, compared to BMP 1 which had only 3 months of inoculum incubation. In addition to incubation time, temperature had a strong effect on methane production, with the maximum methane produced at 15ºC in both BMP 50%-99% lower than quantities observed at 25ºC and 35ºC. At 25ºC, the treatment with mesophilic digestate (at 50% ISR) that was incubated for 6 months could produce up to 9% more methane than the highest methane producing treatment at 35ºC that used inocula incubated for 3 months, indicating that digestion can be carried out at 25ºC given the appropriate inoculum source and inoculum incubation time.
High ISR (50%) generally produced higher quantity of methane although longer incubation time (6 months) resulted in treatments with 35% ISR at 25ºC and 35ºC to produce only 10% less methane than the highest methane quantity observed at each temperature with 50% ISR. With increasing inoculum incubation time, less inoculum and more manure can be placed in a digester of fixed volume, which in turn, can yield at least 18% more methane.
Incubated landfill leachate generally performed as well as, if not better, than the incubated mesophilic digestate in the two BMP experiments. At 25ºC in BMP 1, the treatments with the incubated landfill leachate (at 50% ISR) produced significantly higher amount of methane than the other treatments. The amount produced at the end of the 90 day BMP test was equivalent to 95% of the maximum methane produced by the treatments that received incubated mesophilic digestate at 35ºC. This indicates the possibility of operating digesters at 25ºC without compromising methane production.
Incubated wetland sediment could be used as an inoculum source, but the amount of methane produced was not comparable to the other two (at 50% ISR), except at 35ºC and when the inoculum incubation time was extended. All inoculum sources produced drastically less methane when performed at 15ºC, illustrating the limits to psychrophilic digestion. Further studies in methanogenic community and numbers of these inocula are currently being conducted using q-PCR and T-RLFP techniques.
The purpose of this project is to improve the efficiency of dairy manure anaerobic digestion in Maryland during the winter months by using different local sources of inocula, such as wetlands and landfills, that produce methane and are more acclimated to colder temperatures than anaerobic digestion inoculum, which is maintained at 35ºC. The hypothesis is that these alternative inoculum sources would increase the biogas production in the winter and allow the digesters to be kept at lower operating temperatures, thereby reducing the need to utilize the biogas produced and allowing more of it to be available for on-farm use in the winter when biogas need is the greatest.
The 2007 census reported 663 dairy farms with 57,172 milking cows operated in Maryland (USNASS, 2009). Assuming that each dairy cow is 1,000 lbs and produces 86 lbs of manure per day (ASAE, 2003), a total of 4.92 million lbs of manure are produced daily in the State. Anaerobic digestion offers farmers a method to treat the waste produced, while reducing odors and greenhouse gas emissions, and creating renewable energy from this waste.
In the US, digestion research has focused on industrialized digesters, but with an average cost of $1 million, these systems are inaccessible to medium and small-scale farmers (AgSTAR, 2010). Due to these capital requirements, the AgSTAR (2006) recommends digester installation for farms with more than 500 cows, but 94% of dairies in both Maryland and the wider Chesapeake Bay have less than 200 dairy cows (USNASS, 2004).
Low-cost anaerobic digestion is a proven technology in developing countries with over 35 million low-cost digester in India, China, and Latin America (Abraham et al., 2007; Lansing et al., 2008). Transfer of this technology to temperate zones has been limited by the high cost of heating mechanisms needed to produce methane in the winter. This project seeks to fulfill this development gap by investigating the addition of environmentally derived microbial inocula to anaerobic digester, thus allowing higher methane production at lower temperatures due to the expected greater diversity of microorganisms in these alternative sources. This could lower the cost of anaerobic digestion systems in the Chesapeake Bay watershed, leading to higher adoption rates of this technology.
Anaerobic digestion is a process in which organic matter is degraded in the absence of oxygen. Initially, hydrolytic bacteria break down and convert organic polymers into volatile fatty acids, carbon dioxide, hydrogen, and alcohols. Acetogens then convert the volatile fatty acids into acetate, hydrogen, and carbon dioxide, which are then converted by methanogens into methane.
Anaerobic digestion can be used to treat manure, while producing methane, which could be used as a source of heat and electricity (Nelson and Lamb, 2002). However, the efficiency of anaerobic digesters to produce methane fluctuates with temperature variations. For instance, Massé et al. (2003) found that decreasing digestion temperatures decreases the amount of methane produced. In addition, lower- temperature digesters have a longer lag-phase before methane production commences. Dairy cow manure digested at less than 15ºC did not produce methane for 165 days, while manure digested at 30ºC and 25ºC experienced shorter lag-phases of 33 and 66 days, respectively (Zeeman et al., 1988).
Mesophilic, thermophilic, and psychrophilic digestions refer to digestion processes at temperature ranges of 25-35ºC, greater than 45ºC, and below 20ºC, respectively. Because of the inefficiency of psychrophilic digestion, most digesters are run in the mesophilic and thermophilic temperatures. Digesters in temperate climates are thus equipped with costly internal heating systems that use the biogas produced to heat the systems.
Considering that digestion is a microbial-based process, adding microorganisms, known as inoculum, can hasten and increase methane production. To improve digestion at low temperatures during seasonal fluctuations, it is ideal to introduce inoculum that has been exposed to similar fluctuations and can adapt to low temperatures. Potential sources of these methane-generating microorganisms include landfills and freshwater marshes.
Past research has shown stable digestion of various wastewaters at 20ºC when river sediment was used as a source of inoculum (Bardulet et al., 1990). Additionally, after incubation at 15ºC for 225 days, Xing et al. (2010) was able to obtain inoculum from lake sediment that resulted in higher methane production from glucose at 15ºC compared to an inoculum obtained from a mesophilic digester. It should be noted that it was not clearly stated why a 225 incubation period was chosen in this study.
While the above-mentioned studies indicate the usefulness of developing and introducing environmentally derived microorganisms to anaerobic digesters during the colder months, there were gaps in the research that needed to be addressed before the widespread use of inoculum on a field-scale can be conducted:
(1) There were not any previous studies conducted in the US using environmentally derived inoculum, such as landfill leachate and wetland sediments, for anaerobic digestion at lower temperatures;
(2) No studies were conducted to determine the optimal incubation period for environmentally derived inoculum;
(3) No studies were conducted to determine the optimal inoculum to substrate ratio;
In order to use inocula in the field, the substrate to inoculum ratios and the incubation periods were determined in this study in order to understand inoculum management conditions.
Abraham, E.R., S. Ramachandran, and V. Ramalingam. 2007. Biogas: Can it be an important source of energy? Environmental Science and Pollution Research 14 (1): 67-71.
AgSTAR. 2006. Market Opportunities for Biogas Recovery Systems: A Guide to Identifying Candidates for On- Farm and Centralized Systems. USEPA. EPA-430-8-06-004.
AgSTAR. 2010. Anaerobic Digestion Capital Costs for Dairy Farms. Retrieved 5/24/ 2011, from http://www.epa.gov/agstar/documents/digester_cost_fs.pdf
ASAE. 2003. Manure Production and Characteristics. St. Joseph, MI: ASAE. D384.1
Bardulet, M., J. Cairo, and J.M. Paris. 1990. Start-up of Low Temperature Anaerobic Reactors Using Freshwater Methanogenic Sediments. Environmental Technology 11: 619-624.
Lansing, S., J. Víquez, H. Martínez, R. Botero, and J. Martin. 2008. Quantifying electricity generation and waste transformations in a low-cost, plug-flow anaerobic digestion system. Ecological Engineering 34 (4): 332-348.
Massé, D.I., L. Masse, and F. Croteau. 2003. The effect of temperature fluctuations on psychrophilic anaerobic sequencing batch reactors treating swine manure. Bioresource Technology 89 (1): 57-62.
Nelson, C., and J. Lamb. 2002. Final Report: Haubenschild Farms Anaerobic Digester Updated. St. Paul, Minnesota: The Minnesota Project.
USNASS. 2004. 2002 Census of Agriculture, United States Summary and State Data, Volume 1, Geographic Area Series, Part 51. Washington D.C.: USDA. AC-02-A-51.
USNASS. 2009. 2007 Census of Agriculture, United States Summary and State Data, Volume 1, Geographic Area Series, Part 51. Washington D.C.: USDA. AC-07-A-51.
Xing, W., Y. Zhao, and J. Zuo. 2010. Microbial Activity and Community Structure in a Lake Sediment Used for Psychrophilic Anaerobic Wastewater Treatment. Journal of Applied Microbiology 109: 1829- 1837.
Zeeman, G., K. Sutter, T. Vens, M. Koster, and A. Wellinger. 1988. Psychrophilic Digestion of Dairy Cattle and Pig Manure: Start-up Procedures of Batch, Fed-batch, and CSTR-type digesters. Biological Wastes 26: 15-31.
Due to the lack of research on environmentally derived inoculum and the need for increasing biogas production in the winter using cost-effective digesters, the PI conducted laboratory experiments in which inoculum from local sources, specifically landfills and freshwater marshes, were used for digesting dairy manure obtained from the United States Department of Agriculture (USDA) Beltsville Agricultural Research Center (BARC), Maryland. Inoculum from a mesophilic anaerobic digester, which was readily available, from the Beltsville farm digester, was also compared to these environmentally derived inocula.
The main objective was divided into two objectives:
(1) Obtain suitable inoculum from two landfills, two freshwater marshes, and a mesophilic anaerobic digester and determine the quantity of methane produced from inoculum source when digested with dairy cow manure at 15ºC, 25ºC, and 35ºC.
Hypothesis: The inocula from landfills and freshwater marshes will produce more methane at 15ºC and 25ºC than the inoculum obtained from mesophilic anaerobic digester due to their exposure to seasonal fluctuations and low temperatures during the winter months resulting in higher diversity of methanogenic microorganisms from the environmentally derived inoculum sources.
(2) Determine the optimal incubation period and inoculum to substrate ratio at 15ºC, 25ºC, and 35ºC. This experiment will be conducted only with the ideal inoculum source at each of the temperature determined in Objective 1. These specific inoculum details (optimal incubation period and inoculum quantity needed) will allow for farmer recommendations to be made.
Hypothesis: Higher inoculum to substrate ratio will result in higher amount of methane generated per gram of dairy manure added due to the presence of a larger quantity of microorganisms. Longer incubation periods will also result in a higher production of methane due to the microbial adaptation to the temperature and the substrate.
The experiments for both objectives were completed with a few modifications, as detailed below. Originally, the proposed digestion temperatures were 5ºC, 15ºC, and 25ºC, but were changed to 15ºC, 25ºC, and 35ºC since an experiment conducted in 2011 indicated that the methane production during manure digestion at 3ºC was too low and the amount of methane produced deemed not practical for any use (Zeeman et al., 1988). Since many studies on anaerobic digestions are conducted at 35ºC, adding this temperature was considered useful for cross-referencing results from this study to others. A sequential approach was originally proposed (conducting Objective 1 before Objective 2), but both objectives were completed simultaneously due to the need to incubate the inoculum at the beginning before the ideal inoculum source could be selected.
Zeeman, G., K. Sutter, T. Vens, M. Koster, and A. Wellinger. 1988. Psychrophilic Digestion of Dairy Cattle and Pig Manure: Start-up Procedures of Batch, Fed-batch, and CSTR-type digesters. Biological Wastes 26: 15-31.
Four landfills and four wetland sites were sourced as potential sites for the project. Potential landfills around the Maryland area were selected by using data from the Landfill Methane Outreach Program (LMOP) (USEPA, 2011). The database, which contains information about the amount of landfill gas produced, was used as a resource to determine which landfill sites in the region produce the largest amount of methane. Additional information about gas production for landfills in Maryland was obtained from a list compiled by the Land Management Administration at Maryland Department of Environment (MDE, 2011).
The amount of landfill gas collected per ton of waste for each of the landfill was calculated using these databases to compare the different landfills. If this information was absent, the amount of landfill gas produced per ton of waste was used to determine the normalized methane production. Landfills with the highest amount of gas per ton of waste were chosen as candidates for this project.
Landfill superintendents were contacted and permissions to sample leachate were requested. It is important to note that while there may have been other higher methane producing landfills in the region, there were difficulties in obtaining permission to obtain leachate from some of these landfills, and thus, the highest producing sites where permission was obtained were selected.
A literature review was conducted in order to determine appropriate wetland sites for this study. Soils and wetland experts including Dr. Martin Rabenhorst, Dr. Andrew Baldwin, Dr. Megan Lang, Dr. Patrick Megonigal, Chris Swarth, and Dr. Scott Neubauer were consulted to determine which wetlands in the area could be used for this research.
The following points were used to search for appropriate wetland sites:
(1) Nahlik and Mitsch (2010) found that a natural wetland emitted greater amount of methane than a restored wetland.
(2) Freshwater wetlands produce higher amount of methane compared to brackish wetlands (DeLaune et al., 1983; Neubauer et al., 2005)
(3) Wetlands with higher water tables have been known to emit larger amount of methane (Koh et al., 2009). However, this occurs only up to a depth of 1 0cm where any changes in water table above that depth does not change the emission amount (Christensen et al., 2003).
(4) Tidal freshwater marshes located in the Jugbay Wetland Sanctuary have been reported to produce high amount of methane (Neubauer et al., 2005).
(5) Beaver and farm ponds have been reported to produce high amount of methane (Yavitt et al., 1990; Baker-Blocker et al., 1977).
While it was preferable to find natural, freshwater wetlands with water depths of 10 cm or more in the local area, they were difficult to find (personal communication, Dr. Megan Lang, 2011) due to the seasonality and the inter-annual variations of the water depths. Hence, a decision was made to find wetlands that were inundated with water most of the year.
Specific methanogenic activity (SMA) tests were performed to determine which 2 landfill sites and 2 wetland sites from at least 4 potential sites for wetlands and landfills would be used in the subsequent steps. The purpose of the SMA test is to minimize the risk of conducting 90-day biochemical methane potential (BMP) tests with sediments or leachate that do not harbor high numbers of methane-producing microorganisms (methanogens).
To conduct the SMA tests, duplicate sediment or landfill leachate samples collected from each location were placed in serum bottles and combined with acetate (treatment), which served as a substrate for the methanogens, or deionized water, which served as the control. Nutrient media was also added to each bottle. The bottles were purged with N2/CO2 gas (70:30), capped with butyl rubber stoppers and incubated on a shaker at 25ºC. Produced biogas was measured using a gas-tight wet-tipped glass syringe and the percent methane was monitored using an Agilent HP 7890A GC equipped with a thermal conductivity detector (TCD). In most SMA tests, biogas is measured every three hours for up to 72 hours. Due to low biogas production, however, biogas and percent methane were measured at least once a day for the first three days and every couple of days afterwards. The tests lasted for a period of approximately 25 days due to the lack of the methanogens and longer lag phases needed for methane production to commence and discernable differences in inoculum sources to emerge.
Due to the low biogas production during the SMA tests, incubation of the inoculum sources was conducted to increase the number of methanogens before using them as inocula in the BMP testing. The two wetland sediments and two landfill leachate, which had the highest rate of methane production, were selected and incubated in anaerobic reactors. Additionally, digestate from a mesophilic anaerobic digester treating dairy manure at USDA BARC facility was also incubated in anaerobic reactors to act as the conventional inoculum source reference.
Each incubation reactor was made of 4 inch PVC pipes with a length of 20 inch. The bottom of the reactor was sealed with total knockout closet flange, while the top was sealed with mechanical test plug. Biogas flowed through white high-density polyethylene HDPE tubing inserted in the mechanical test plug and then through Tygon® tubing into a 5L multi-foil bag. A three-way stopcock was fitted into the Tygon® tubing to allow gas sample to be collected directly from the tubing (Figure 1 in Appendix A). A total of 15 reactors were built for the 5 inoculum sources at the three different temperatures (15ºC, 25ºC, 35ºC).
Equal amount (1.25L) of inoculum and nutrient media were added into each reactor. For the wetland, the density of the sediment was calculated to determine the mass of sediment needed to obtain 1.25L.
Following the addition of the inoculum and the media, the liquid mixture and headspace of each reactor were purged with N2/CO2 (70:30) mixture for ten minutes each to remove oxygen from the reactor. The reactors were sealed and incubated at the three temperatures of interest: 15ºC, 25ºC, and 35ºC.
Biogas measurements and percent methane analysis were conducted at approximately 4-day interval. Biogas production in the multi-foil bag was measured using 140 and 60 mL gas tight syringes and percent methane was analyzed using an Agilent HP 7890A GC equipped with a thermal conductivity detector (TCD).
The reactors were fed at approximately 4-day intervals with unseparated manure. Fresh unseparated manure was collected approximately every two weeks from the USDA BARC facility. In order to minimize the introduction of methanogens present in fresh manure, the manure were autoclaved before being stored at 4ºC. During each of the feedings, the amount needed for each reactor was weighed out before being added to the reactor, as detailed below. The reactors were shaken by hand after each feeding session.
A step-wise loading of manure over time with methanol comprising up to 50% of the chemical oxygen demand (COD) content of the feedstock in the first few weeks was chosen because this method had been shown to allow anaerobic digesters reactors to achieve stable operation and high efficiencies of COD reduction (Bull et al., 1983; Bardulet et al., 1990). The percent methanol in feedstock was gradually reduced from 50% of total COD loaded to 25% and finally to 0% by Day 35 (Bull et al., 1983). An initial loading rate of 0.1kg COD/m^3/day and a final loading rate of 1.3kg COD /m^3/day was used (Bardulet et al., 1990). Table 1 (Appendix A) describes the feedstock additions over the 6 month period.
While it is ideal to use COD for calculating the amount needed to meet the loading rate requirements, the difficulty of obtaining accurate COD data from viscous unseparated manure made an extra step necessary. Volatile solids in the manure were measured for the feedstock instead of COD and a ratio of 1.088 g COD/g VS was used as the conversion factor (ASAE, 2003; Barth et al., 1999; NCSU, 1994 (cited in USEPA, 2002)).
Due to the nature of the feeding and the removal of inoculum for BMP testing at various stages, the reactors functioned as semi-batch reactors until about Day 160 when it began to function as semi continuous reactors where wasting of the digester content equal to the feedstock was performed before feedstock was added.
After three months of incubation, the reactors with the best-performing of the two types of wetland sediment and landfill leachate (highest methane production) across all three temperatures (with special emphasis given to 15ºC) were selected and along with the mesophilic anaerobic digester digestate, were used as the inocula for the modified biochemical methane potential (BMP) test. This BMP test (BMP 1) was performed to determine the ideal inoculum source for the digestion of dairy manure at 15ºC, 25ºC, and 35ºC. A detailed description of the BMP process using agricultural wastes can be found in Moody et al. (2009). Fresh manure was obtained from the USDA BARC facility and inocula from the selected reactors were added to 250 mL serum bottles at different inoculum to substrate (manure) ratio (ISR) (20% (w/w), 35%, and 50%). The bottles were purged with nitrogen and hydrogen gas and then topped with rubber septa, after which, they were placed in 15ºC, 25ºC, and 35ºC chambers (corresponding to the temperature at which the inocula was incubated).
The quantity of biogas and methane produced were monitored for approximately 90 days. Each treatment was conducted in triplicates, except for the control (manure only and inoculum only bottles for each inoculum source), which were conducted in duplicates. The amount of biogas and methane produced each inoculum source in the control at that temperature was subtracted from the total methane to determine the methane production attributed to the manure substrate. This was followed by normalization of methane production to grams of volatile solids from the manure added.
After six months of inocula incubation, the process of inocula extraction from the reactors and the BMP experiment (BMP 2) was repeated. In BMP 2, the 20% ISR treatments at 25ºC and 35ºC was not included due to the low amounts of methane during BMP 1. At 15ºC, only the most promising inocula (wetland sediment and incubated digestate) and 50% ISR were used, in addition to manure and inocula only controls. In addition, BMP 2 lasted approximately 100 days, 10 days longer than BMP 1.
Total solids (TS) and volatile solids (VS) of the substrates pre-digestion and the post digestion mixtures were measured using standard methods (APHA, 2005). The pH of pre and post-BMP mixtures were also analyzed.
Where applicable, two-way ANOVA were used to analyze the effects of inoculum and/or the ISR on methane production for the two BMP tests. Tukey-Kramer analysis was conducted to analyze pair-wise comparisons between the different experimental units. An alpha value of 0.05 was used in the statistical analyses. Statistical analysis was conducted using SAS® 9.2 (SAS Institute Inc., Cary, NC, USA).
APHA. 2005. Standard Methods for the Examination of Water and Wastewater, 21st edition.Edited by Eaton, A.D., L.S. Clesceri, E.W. Rice, A.E. Greenberg, and M.A.H. Franson. Washington D.C.: APHA.
ASAE. 2003. Manure Production and Characteristics. St. Joseph, MI: ASAE. D384.1
Baker-Blocker, A., T.M. Donahue, and K.H. Mancy. 1977. Methane flux from wetlands areas. Tellus 29 (3): 245-250.
Bardulet, M., J. Cairo, and J.M. Paris. 1990. Start-up of low temperature anaerobic reactors using freshwater methanogenic sediments. Environmental Technology 11: 619-624.
Barth, C., T. Powers, and J. Rickman. 1999. Agricultural Waste Characteristics. In: USDA and NRCS. 1999. Agricultural Waste Management Field Handbook. Washington D.C.: USDA and NRCS.
Bull, M.A., R.M. Sterritt, and J.N. Lester. 1983. An evaluation of four start-up regimes for anaerobic fluidized bed reactors. Biotechnology Letters 5 (5): 333-338.
Christensen, T.R., A. Ekberg, L. Ström, M. Mastepanov, N. Panikov, M. Öquist, B.H. Svensson, H. Nykänen, P.J. Martikainen, and H. Oskarsson. 2003. Factors controlling large scale variations in methane emissions from wetlands. Geophysical Research Letters 30 (7): 1414.
DeLaune, R.D., C.J. Smith, and W.H. Patrick, Jr. 1983. Methane release from gulf coast wetlands. Tellus B 35B (1): 8-15.
Koh, H.S., C.A. Ochs, and K. Yu. 2009. Hydrologic gradient and vegetation controls on CH4 and CO2 fluxes in a spring-fed forested wetland. Hydrobiologia 630 (1): 271-286.
MDE, 2011. Land Management Administration: LMOP Datasheets MD.xls. Obtained 8/23/2011, from Land Management Administration at MDE.
Moody, L., R. Burns, W. Wu-Haan, and R. Spajic. 2009. Use of biochemical methane potential (BMP) assays for predicting and enhancing anaerobic digester performance. Agricultural Engineering, 44th Croatian & 4th International Symposium on Agriculture: 930-934.
Nahlik, A.M., and W.J. Mitsch. 2010. Methane emissions from created riverine wetlands. Wetlands 30 (4): 783-793.
Neubauer, S.C., K. Givler, S.K. Valentine, and J.P. Megonigal. 2005. Seasonal patterns and plant-mediated controls of subsurface wetland biogeochemistry. Ecology 86 (12): 3334-3344.
USEPA. 2002. Development Document for the Final Revisions to the National Pollutant Discharge Elimination System Regulation and the Effluent Guidelines for Concentrated Animal Feeding Operations. Washington D.C.: USEPA. EPA-821-R-03-001.
USEPA. 2011. LMOP: LMOP Landfill and Project Database, Sorted by State, Project Status, and Landfill Name (XLS). Retrieved 10/4/2011, from http://www.epa.gov/lmop/projects-candidates/index.html.
Yavitt, J.B., G.E. Lang, and A.J. Sexstone. 1990. Methane fluxes in wetland and forest soils, beaver ponds, and low-order streams of a temperate forest ecosystem. Journal of Geophysical Research 95 (D13): 22463-22474.
The SMA was conducted using sediments from 4 wetlands and leachate from 5 landfills. The following list describes the 4 wetlands and 5 landfills chosen for the SMA test:
(1) Wetland Site 1(approximate coordinates: 38 degrees 46 minutes 51.36 seconds N, 76 degrees 42 minutes 26.61 seconds W): A tidal freshwater marsh located within the Jugbay Wetland Sanctuary adjacent to the Patuxent River, Anne Arundel County, Maryland, USA.
(2) Wetland Site 2 (approximate coordinates: 38 degrees 46 minutes 49.63 seconds N, 76 degrees 42 minutes 45.75 seconds W): A tidal freshwater marsh located within the Jugbay Wetland Sanctuary adjacent to the Patuxent River, Anne Arundel County, Maryland, USA.
(3) Wetland Site 3 (approximate coordinates: 38 degrees 48 minutes 34.99 seconds N, 76 degrees 42 minutes 33.48 seconds W): A beaver pond located within the Patuxent Wetland Park, in close proximity to the Jugbay Wetland Sanctuary, and adjacent to the Patuxent River, Anne Arundel County, Maryland, USA.
(4) Wetland Site 4 (approximate coordinates: 38 degrees 44 minutes 45.94 seconds N, 76 degrees 40 minutes 52.05 seconds W): Open water pond located in a housing community in Culvert County, Maryland, USA.
(5) Landfill Site 1 (approximate coordinates: 38 degrees 22 minutes 57.77 seconds N, 77 degrees 24 minutes 56.34 seconds W): The Stafford County Landfill located in Virginia. This landfill was opened in 1987 and contains 715 thousand tons of waste. The amount of landfill gas flowed to project per ton of waste is 1.61×10^-6 million standard cubic feet/day/ton of waste (USEPA, 2011).
(6) Landfill Site 2 (approximate coordinates: 38 degrees 34 minutes 12.53 seconds N, 76 degrees 52 minutes 54.91 seconds W): The Charles County Landfill in Maryland. This landfill was opened in 1994 and contains 877 thousand tons of waste. The amount of landfill gas collected per ton of waste is 1.03×10^-6 million standard cubic feet/day/ton of waste (MDE, 2011)
(7) Landfill Site 3 (approximate coordinates: 38 degrees 40 minutes 43.50 seconds N, 77 degrees 46 minutes 39.05 seconds W): Corral Farm Landfill located in Fauquier County, Virginia. This landfill was opened in 1996 and contains 1.24 million tons of waste. The amount of landfill gas flowed to project per ton of waste is 5.22×10^-7 million standard cubic feet/day/ton of waste (USEPA, 2011).
(8) Landfill Site 4 (approximate coordinates: 39 degrees 23 minutes 55.70 seconds N, 76 degrees 23 minutes 57.39 seconds W): Eastern Sanitary Landfill located in Baltimore County, Maryland. This landfill was opened in 1982 and contains 5.21 million tons of waste. The amount of landfill gas collected per ton of waste is 2.49×10^-7 million standard cubic feet/day/ton of waste (MDE, 2011).
(9) Landfill Site 5 (approximate coordinates: 39 degrees 38 minutes 05.96 seconds N, 76 degrees 40 minutes 21.78 seconds W): Parkton Landfill located in Baltimore County, Maryland. This landfill was operational between 1976 and 1983, was approximately 200 acres, and received 100% shredded trash (Lippy, 2006 cited in (Beemt, 2006)).
The SMA tests indicated that sediments from Wetland Sites 1 and 2, and leachate from Landfill Sites 1 and 2 produced the highest amount of methane (Figures 1 and 2 in Appendix B). It should be noted, however, that the percent methane changes in the bottles’ headspace were used as the means of comparison for the different landfill sites due to low biogas production. These four sites were chosen as ideal candidates for the next step of the project. Incubation of wetland sediments and landfill leachate from these four selected locations, in addition to digestate obtained from a mesophilic digester operating at BARC, were conducted using the anaerobic reactors.
After three months of incubation, samples were extracted from the highest methane producing reactors of each inoculum type to supply inocula for the BMP test. Comparison was conducted across all three temperatures to decide which wetland site and landfill site would be chosen for the next phase. Methane production from the incubation of reactors can be found in Figures 3-5 (Appendix B).
Wetland Site 1 was chosen for the subsequent step because its sediment produced 875% and 29% higher amount of methane than Wetland Site 2 sediment at 15ºC and 25ºC, respectively. At 35ºC, Wetland Site 2 sediment produced higher amount of methane than Wetland Site 1 sediment, but only by 2%.
Landfill Site 2 leachate produced 21% more methane than Landfill Site 1 at 25ºC, but the latter produced 33% and 10% more methane than the former at 15ºC and 35ºC, respectively. Therefore, Landfill Site 1 was chosen for the subsequent step.
At all the three temperatures, BARC mesophilic digestate produced the highest amount of methane with the lowest production at 15ºC (190 mL/g VS) and the highest at 35ºC (402 mL/g VS). Reactors incubating this digestate were selected since there was only one type of mesophilic digestate.
Compared to the manure only treatment, the addition of inocula resulted in up to 2,700% and 3,300% increase in methane production at 25ºC and 35ºC, respectively (Figures 6-9 in Appendix B). These results illustrate the importance of adding inoculum to anaerobic digesters to increase methane production.
At 25ºC, the treatment with Landfill Site 1 leachate and 50% ISR produced 194±7 mL CH4/g VS, which was significantly higher than the other treatments (p‹0.05) (Figures 6 and 7 in Appendix B). This suggests that the landfill leachate may contain methane-producing microorganisms that are better adapted to 25ºC.
At 35ºC, the Landfill Site 1 leachate and BARC mesophilic digestate at 50% ISR produced significantly higher methane than the other treatments (205±4 mL CH4/g VS for incubated BARC mesophilic digestate and 198±15 mL CH4/g VS for Landfill Site 1 leachate) (p‹0.05) (Figures 8 and 9 in Appendix B). Compared to the manure only treatment, the use of either of these two inocula equally increased methane production by at least 3,200%.
The treatment with Landfill Site 1 leachate at 25ºC produced 78% of the highest methane yield observed (by the BARC mesophilic digestate) at 35ºC on Day 30, but 95% of the BARC mesophilic digestate on Day 90. Therefore, given enough retention time, this particular inoculum could allow farmers to operate their digesters at 25ºC instead of 35ºC. This would reduce the heating, fuel, and costs requirements for operating the digesters.
At 15ºC, both the treatments with the BARC mesophilic digestate at 50% ISR and Landfill Site 1 leachate at 35% ISR produced the highest amount of methane (approximately 3 mL CH4/g VS) (Figure 10 in Appendix B). While this value was 200% higher than the amount produced by the manure only treatment (1 mL CH4/g VS), it was more than 98% lower than the maximum amount of methane produced at 25ºC or 35ºC. This means that the inocula tested were not able to substantially increase methane production at 15ºC. It should also be noted that some of the cumulative methane curves as shown in Figure 10 (in Appendix B) had a drop in the amount of methane produced due to the higher rate of methane production by the inocula controls compared to manure + inoculum treatments as the experiment proceeded, likely due to inhibition.
At 15ºC, the pH of all the treatments fell by 0.24 to 1.14 units during the digestion process, except for the incubated wetland sediment control, which increased by 0.14 units (Figure 11 in Appendix B). Most of the treatments started out within the acceptable pH range (the pH range of 6.5-8 is stated as the acceptable range for mesophilic digestion, but slight variation may exist at different temperature ranges (Seadi et al., 2008)) for anaerobic digestion, except for two of the treatments that were the Landfill Site 1 control and Landfill Site 1 50% ISR, but by the end of the experiment, only two of the treatments (the BARC mesophilic digestate and Wetland Site 1 sediment) maintained their pH values within this acceptable range. The ability for these two inocula to maintain within this pH range could explain their continuous methane production while the methane production rate for manure + inoculum treatments associated with these two inocula fell after some time. The low starting pH value for the incubated landfill site 1 leachate control could also explain its lower rate of methane production than the other treatments with the same inoculum source, allowing the cumulative methane curve to continuously increase instead of dropping.
The pH of the 25ºC and 35ºC BMP tests were within the acceptable range for digestion (6.5-8 (Seadi et al., 2008)) (Figures 12 and 13 in Appendix B). At both temperatures, the manure only treatment had the lowest pH of approximately 6.7. At 25ºC, the pH of most treatments increased by 0.03-0.43 units during the digestion process and all treatments had final pH values within the acceptable range. At 35ºC, however, two of the treatments’ pH values fell below 6 (the manure only treatment and incubated landfill site 1 leachate at 20% ISR). The inability of these two treatments to maintain the pH explains why these two treatments produced the lowest amount of methane. This likely means that the rate of methaneogenesis (methane-formation) was lower than the rate of acetogenesis (acetic acid formation) due to a low quantity of methanogens resulting in a build up of acid within the reactors.
At 15ºC, the percent of TS destroyed for the manure in the manure + inoculum treatments ranged from 4% to 19%, while the percent VS destroyed for the manure ranged from 3% to 20% (Figure 14 in Appendix B). These ranges were lower than those observed for 25ºC and 35ºC and partly explains the lower methane production observed at 15ºC. The percent TS and VS destroyed for manure in the manure + inoculum treatments was higher at 35ºC (ranging from 14% to 44% for TS, and 16% to 50% for VS) than at 25ºC (ranging from 10% to 39% for TS, and 11% to 44% for VS) (Figures 15 and 16 in Appendix B). At 25ºC and 35ºC, there were clear general trends between percent of manure VS destroyed and the methane (mL/g manure VS) produced. This was expected since methane is produced from the destruction of volatile solids, which is a proxy for organic matter (Figures 15 and 16 in Appendix B). At 15ºC, however, this trend is less clear, likely due to the low overall methane production and the higher rate of methane production by the some controls compared to the associated inoculum + manure treatments (Figures 14 in Appendix B).
Compared to the manure only treatment, the addition of inocula incubated for 6 months resulted in up to 5,600%, 3,700%, and 770% increase in methane production at 15ºC, 25ºC, and 35ºC, respectively (Figures 17-21 in Appendix B). Once again, these results indicate the importance of adding inoculum to anaerobic digesters to increase methane production.
At 15ºC, the incubated BARC mesophilic digestate at 50% ISR produced the significantly higher amount of methane (114±1 mL CH4/g VS) than the other treatments (p‹0.05) (Figures 17 and 18 in Appendix B). This amount, however, was still at least 50% lower than the amount produced by the highest producers at 25ºC and 35ºC.
At 25ºC, both the treatments with BARC mesophilic digestate and Landfill Site 1 leachate at 50% ISR produced the highest quantity of methane (229±1 mL CH4/g VS and 219±6 mL CH4/g VS, respectively) (p‹0.05) (Figures 19 and 20 in Appendix B). No significant difference in methane production was observed between the two treatments. The treatments with BARC mesophilic digestate and Landfill Site 1 leachate at 35% ISR produced 10% less methane (208±2 and 206±7 mL CH4/g VS, respectively) than the highest methane producing treatment that had incubated BARC digestate at 50% ISR. This means that less inoculum (35% ISR instead of 50% ISR) and more manure could be placed in the digester, allowing an 18% increase in the total volume of methane produce from the additional manure placed in the same sized reactor. If the reactor space were not limiting, then larger ISR (50%) would increase methane production by 10%.
At 35ºC, treatments with 50% ISR (250±6 mL CH4/g VS) produced significantly higher amount of methane compared to treatments with 35% ISR (228±6 mL CH4/g manure VS), followed by the manure only treatment (30.6±0 mL CH4/g manure VS) (p‹0.05) (Figures 21 and 22 in Appendix B). Regardless of the inoculum source, using 50% ISR increased methane production by approximately 10%, as compared to 35% ISR Again, this indicates the possibility of using 35% ISR, instead of 50% ISR, allowing more manure content in a fixed volume digester and allowing at least 18% increase in methane yield.
At 35ºC, BARC mesophilic digestate produced the highest amount of methane (258±6 mL CH4/g VS), although this was not significantly greater than the treatments with Landfill Site 1 leachate (238±7 mL CH4/gVS) (Figures 21 and 23 in Appendix B). This incubated landfill leachate could thus be used as an alternative to the BARC mesophilic digestate as a source of inoculum. The relative ease of obtaining one inoculum over the other, however, needs to be considered. The treatments with the Wetland Site 1 sediment did not produce significantly different amount of methane (222±4 mL CH4/g manure VS) than the treatments with incubated landfill site 1 leachate. The manure only treatment produced significantly lower amount of methane than the other treatments (31±0 mL CH4/g manure VS) (p‹0.05).
Inoculum incubation at 25ºC resulted in a treatment which produced 81% of the highest methane yield at 35ºC on Day 30 and 85% by Day 90. Should the amount of extra energy needed to operate the digesters at 35ºC instead of 25ºC exceeds the energy needed to incubate the inoculum and the difference between the methane energy yield at these two temperatures, then the digester operator should operate the digester at 25ºC.
At 15ºC, before the BMP started, the pH of all the treatments were within the acceptable range of anaerobic digestion (6.5-8 (Seadi et al., 2008)) (Figure 24 in Appendix B). While the pH dropped between 0.03-1.43 units, most of the treatments were still within the acceptable pH for digestion, except for two treatments. Both the manure only treatment and the treatment with Wetland Site 1 sediment at 50% ISR had pH’s of approximately 6.18, a value likely too low for digestion to occur. The inability for both of these treatments to maintain their pH within the acceptable range explains the low amount of methane produced by these two treatments.
At both 25ºC and 35ºC, all the treatments started out with pH values within the acceptable range for digestion to occur (6.5-8 (Seadi et al., 2008)) (Figures 25 and 26 in Appendix B). While all the treatments at 35ºC remained within this range after the BMP process, the pH values of two treatment within the 25ºC BMP experiment fell outside this range. The pH of the manure only treatment fell to a value of 6.18, while the treatment with Wetland Site 1 sediment at 35% ISR increased to a value of 8.23.
Percent of the TS and VS destroyed for the manure in the manure + inoculum treatments was highest at 35ºC (ranging from 9% to 47% for TS, and 10% to 54% for VS), followed by 25ºC (ranging from 7% to 39% for TS, and 8% to 45% for VS), and lowest at 15ºC (ranging from 3% to 14% for TS, and 3% to 16% for VS). In all three scenarios, methane produced (mL/g VS) generally increased as percent VS destruction increased (Figures 27-29 in Appendix B).
The treatments that produced the highest amount of methane at each temperature range from BMP 1 and BMP 2 were compared to each other. Methane production on Day 90, or a day closest to it, was chosen as the point of comparison for BMP 1 and 2, which corresponded the final day of BMP 1 and 10 days before BMP 2 experiment ceased.
Up to 3,500%, 15%, and 28% increase in methane production could be achieved when the inocula used were incubated for a longer period of time (6 months instead of 3 months) at 15ºC, 25ºC, and 35ºC, respectively (Figure 30 in Appendix B). Using inocula with longer incubation time thus allowed greater methane yield during digestion.
The treatment with the highest methane production at 25ºC in BMP 2, which was the BARC mesophilic digestate at 50% ISR, (224±1 mL CH4/g VS) produced 9% more methane compared to the highest methane producer at 35ºC in BMP 1, which had the same inoculum source and ISR (205±4 mL CH4/g VS) (Figure 30 in Appendix B). This suggests that using the inocula that had been incubated for 6 months, instead of 3 months, allows a lower temperature to be used to generate higher methane yield.
At 25ºC, the treatment with BARC mesophilic digestate, 35% ISR and 6 months of incubation time (202±2 mL CH4/g VS) produced 4% higher amount of methane than the highest methane producing treatment that used Landfill Site 1 Leachate at 50% ISR after 3 months of inoculum incubation in BMP 1 (194±7 mL CH4/g VS). Similarly, at 35ºC, the treatment with BARC mesophilic digestate and 35% ISR in BMP 2 (244±7 mL CH4/g VS) produced 19% more methane than the highest methane producing treatment that had BARC mesophilic digestate and 50% ISR in BMP 1 (205±4.26 mL CH4/g VS) (Figure 31 in Appendix B). Less inoculum is thus needed if the microorganisms are incubated for a longer period of time. This allows more manure to be used in digester, which in turn, can produce more methane.
Caution, however, should be taken when comparing the use of inocula that had been incubated for 6 months versus ones that had been incubated for 3 months due to different circumstances that existed during the two BMP experiments. Firstly, the inoculum during the first BMP was extracted from the reactors and stored for one week at 4ºC before being used and this was not the case during BMP 2 where samples were extracted and used immediately. Secondly, the manure obtained for the two BMPs were obtained at two different times of the year (September and December, 2012) and hence could have different properties. For instance, the pH of the manure for BMP 1 was 6.76±0.04, while that for BMP2 was 7.58±0.03. This could affect the methane production capacity of the different treatments.
Beemt, P.v.d. 2006. MTBE found in well at Parkton Landfill, Association invites experts to speak June 12. North County News. Retrieved 7/31/13, from http://archives.explorebaltimorecounty.com/news/6048886/mtbe-found-well-parkton-landfill/
MDE, 2011. Land Management Administration: LMOP Datasheets MD.xls. Obtained 8/23/2011, from Land Management Administration at MDE.
Seadi, T.A., D. Rutz, H. Prassl, M. Köttner, T. Finsterwalder, S. Volk, R. Janssen. 2008. Biogas Handbook. Edited by Seadi, T.A. Esbjerg, Denmark: University of Southern Denmark. Retrieved 7/29/13, from http://lemvibiogas.com
USEPA. 2011. LMOP: LMOP Landfill and Project Database, Sorted by State, Project Status, and Landfill Name (XLS). Retrieved 10/4/2011, from http://www.epa.gov/lmop/projects-candidates/index.html
This project aims to increase the methane production capacity of anaerobic digesters at low temperatures by creating a better understanding of the impacts of the type and quantity of inoculum used during low-temperature digestion. One of the main barriers towards the installation of anaerobic digesters is its high capital cost (AgSTAR, 2010). By increasing the amount of methane generated from anaerobic digesters during the colder months and reducing the quantity of biogas used to heat the digesters, farmers will have higher net return from the biogas produced. This will reduce the risk of installing anaerobic digestion and increase incentives for farmers to install this technology.
This project has illustrated the importance of inoculum to anaerobic digesters operating at various temperatures. In BMP 1 (using inocula that had been incubated for 3 months), higher ISR was observed to be beneficial towards increasing methane yield at 25ºC and 35ºC. Using the incubated landfill leachate developed in this project, digesters operating at 25ºC could produce 78% to 95% of the methane produced at 35ºC, depending on the retention time used. This indicates the possibility of operating the digester at 25ºC, instead of 35ºC, when the incubated landfill leachate is used as inoculum. This technique could lead to reduced heating and fuel requirements for operating digesters.
In BMP 2 (using inocula that had been incubated for 6 months), the use of appropriate inocula at 25ºC and 35ºC could allow the use of less inocula (35% ISR instead of 50% ISR) per digester. More manure can be placed in the digestion system, which in turn, can lead to 18% higher methane yield. Compared to the inocula in BMP 1, the inocula in BMP 2 increased the maximum methane yield at all the studied temperatures. At 25ºC and 35ºC, some of the treatments with 35% ISR in BMP 2 produced higher methane yield than the highest methane producers observed in BMP 1 that had 50% ISR. Again, this suggests that using appropriate inocula that had been incubated for a longer period of time allows less inoculum to be used and could increase methane yield by 4% at 25ºC and 19% at 35ºC. Caution, however, should be taken when comparing the results between BMP 1 and BMP 2, as stated in the results section.
The incubated BARC mesophilic digestate generally did well over all the scenarios studied when incubated at the desired temperatures, except at 25ºC in BMP 1 (using inocula that was incubated for 3 months) where the incubated landfill site 1 leachate produced the highest amount of methane. Incubated landfill site 1 leachate performed as well, if not better, as the incubated BARC mesophilic digestate at 25ºC and 35ºC in both BMP experiments, indicating the possibility of using the incubated landfill leachate as an alternative inoculum source. This project also illustrates the possibility of using incubated wetland sediment as a source of inocula, although it did not perform as well as the other two inocula (at 50% ISR).
Education & Outreach Activities and Participation Summary
On November 1st 2011, the graduate student assisted in an anaerobic digestion workshop that is part of the NE Sun Grant (NE10-404) awarded to the PI, Dr. Stephanie Lansing, and the Sustainable Agriculture Research Education (SARE) fellowship awarded to graduate student Andrew Moss. Andrew Moss and Dr. Gary Felton conducted the workshop at the USDA BARC site. Approximately 40 people from Maryland, Pennsylvania, and Washington DC attended the event. Presentations on the process, operation, and economics of anaerobic digestion were given. In addition, fact sheets about anaerobic digestion were also handed out to the audience. During the workshop, this funded NESARE research project was introduced to the farmers. From this workshop, the graduate student was able to gain experience on how to organize successful workshops in the future.
On June 6th, 2013, a second workshop was held at the USDA BARC site as part of the NE Sun Grant (NE10-404) awarded to the PI, Dr. Stephanie Lansing. Dr. Gary Felton and Dr. Lansing organized the workshop and approximately 30 participants from dairy farms, government agencies, extension agencies, and business entrepreneurs attended the event. The range of topics discussed and presented includes the background, economics, process, and operation of anaerobic digestion, and co-digestion. Fact sheets about anaerobic digestion were distributed to the attendees. The graduate student presented a topic titled “Digesters Worldwide and Research to Overcome Limitations in a Temperate Climate,” in which he discussed the different types of digesters both in the US and in other countries, the problem with digesters in temperate climates such as the US, and research that Dr. Stephanie Lansing’s lab team are undertaking to solve this problem. The research and results from this NESARE project were presented and lively discussion ensued after the presentation surrounding topics such as the importance of using inocula, the quantity of inocula needed, how this SARE-funded project can be applied in the field, the possibility of digesting at lower temperatures, and how digestion at lower temperatures affect pathogen kill.
Results from this project were also presented at three conferences where more than 100 people, many whom are graduate and undergraduate students, faculty, and part of the digester research community, were exposed to the abstract and/or results from this project.
The following lists the full details of the presentation:
(1) Witarsa, F., S. Lansing, V. Zhiteneva, H. Bowen, and C. Kenny. 2013. Alternative Sources of Inoculum to Increase Methane Production in Psychrophilic Anaerobic Digesters Treating Dairy Manure. Graduate Research Interaction Day, University of Maryland, College Park, Maryland (Oral).
(2) Witarsa, F., S. Lansing, V. Zhiteneva, H. Bowen, and C. Kenny. 2013. Alternative Sources of Inoculum to Increase Energy Production in Anaerobic Digesters. Energy and Water Nexus, University of Maryland, College Park, Maryland (Oral).
(3) Witarsa, F. and S. Lansing. Digesters Worldwide and Research to Overcome Limitations in a Temperate Climate. 2013. Small-Scale Anaerobic Digester for Dairy – Field Workshop, United States Department of Agriculture Beltsville Agricultural Research Center, Beltsville, Maryland (Oral).
(4) Witarsa, F., S. Lansing, V. Zhiteneva, H. Bowen, and C. Kenny. 2013. Alternative Sources of Inoculum to Increase Methane Production in Psychrophilic Anaerobic Digesters Treating Dairy Manure. Annual American Ecological Engineering Society (AEES) Meeting, Michigan State University, East Lansing, Michigan (Oral).
Manuscripts (in preparation):
(1) Witarsa, F., S. Lansing, V. Zhiteneva, H. Bowen, and C. Kenny. 2013. Alternative Sources of Inoculum to Increase Methane Production in Psychrophilic Anaerobic Digesters Treating Dairy Manure. Ecological Engineering.
(2) Witarsa, F., S. Lansing, S. Yarwood, M. Mateu, V. Zhiteneva, H. Bowen, and C. Kenny. 2013. Methanogenic Quantity and Community in Alternative Sources of Inocula and Their Relationship to Methane Production in Dairy Manure Digesters. Bioresource Technology
No economic analysis was conducted during this study.
This project aims to increase the amount of methane produced in temperate climate digesters by using alternative sources of inocula. Farmers that have heard about this project have recognized the necessity of this project. One dairy farmer from Rising Sun, Maryland, for instance, expressed his well wishes to the graduate student in his search to find new types of microorganisms for low-temperature digestion and asked him to share his final results with him.
While this project have shown the importance of the use, types, quantity, and time of incubation of inocula for anaerobic digestion, a number of studies still need to be conducted before the results can be applied in the field scale. Nevertheless, this study can be used to show farmers the importance of adding inocula to digesters as opposed to adding manure and letting the microbial population grow. Depending on the source (types and age) of the inocula, the farmers can also use less inoculum during the start up of digesters or operate digesters at 25ºC instead of 35ºC, allowing them to save fuel and money. In the future, results from this research can also be used to create digesters that are operated at low-temperatures to incubate inoculum, which could be introduced to low-temperature digesters during the winter months to boost methane production.
Areas needing additional study
A number of studies have been identified to further this idea of using inocula in low-temperature digesters to increase their methane production. In this project, incubated landfill leachate could produce at least the same if not more methane than incubated mesophilic digestate at 25ºC and 35ºC. However, the safety of using landfill leachate as the basis for inoculum should be studied before it is considered for use in the field scale. This is especially so since many farmers use digested material on their fields to fertilize their crops.
In addition, at 25ºC, incubated inocula could produce 85% to 95% of the amount of methane achievable at 35ºC. The benefits and costs of incubation, however, should be analyzed in order to determine if this process will bring more savings, in terms of energy and money, to the farmers than if they use un-incubated inoculum at 25ºC or if they heat their digesters to 35ºC.
In addition, the graduate student is currently conducting further studies to determine if the microorganisms in these different inocula sources were different and how these differences played a role in determining the amount of methane produced during the BMP experiments. Molecular techniques, including quantitative polymerase chain reaction (q-PCR) and terminal restriction fragment length polymorphism (T-RFLP) are currently being used to study the quantity and community of methanogens present within samples extracted from the reactors. This study is on going and was outside the scope of this SARE-funded research proposal. It is expected that this microbial research will be completed by October 2013.