Developing Inoculum to Increase Anaerobic Digestion Efficiency in Winter Months

2011 Annual Report for GNE11-030

Project Type: Graduate Student
Funds awarded in 2011: $14,974.00
Projected End Date: 12/31/2013
Grant Recipient: University of Maryland
Region: Northeast
State: Maryland
Graduate Student:
Faculty Advisor:
Stephanie Lansing
University of Maryland

Developing Inoculum to Increase Anaerobic Digestion Efficiency in Winter Months


Anaerobic digestion of dairy manure produces renewable energy in the form of methane-enriched biogas in addition to improving water quality, reducing odors, greenhouse gases, and pathogens. The production of biogas, however, decreases at low temperatures. To counter this problem, digesters in temperate climates are designed with internal heating systems that use the biogas produced as the source of heat. This is not always a cost-effective method, especially in the winter when there is a greater need for the energy produced. In addition, many small to medium-scale farms do not have access to waste heat from a generator to supplement digester heating as co-generation systems are often too expensive.

Wetland sediments and landfills emit methane and are exposed to temperature fluctuations. The goal of this project is to collect and quantify the increase in biogas production at lower temperature that could be achieved when anaerobic digestion systems are inoculated with cold-adapting microorganisms obtained from wetland sediments and landfill leachates.

In 2011, at least four wetlands and four landfills were selected for this study. Preliminary specific methanogenic activity (SMA) tests were conducted to determine the two highest performing sites with limited success. Modifications are being made for subsequent SMA tests to improve the test outcomes by adding buffer and adjusting the dilution ratios. Chemical and physical characterizations of samples from a number of these sites were also conducted.

Objectives/Performance Targets

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 5 degrees C, 15 degrees C, and 25 degrees C.

2.) Determine the optimal incubation period and inoculum to substrate ratio at 5 degrees C, 15 degrees C, and 25 degrees C. This experiment will be conducted only with the ideal inoculum source at each temperature range determined in Objective 1. These specific inoculum details (optimal incubation period and inoculum quantity needed) will allow recommendations to be made to farmers who are interested in installing anaerobic digesters in temperate regions of the US.

Project Updates

1.) Funds for this project were not released until October 24th 2011, and as a result of this, a revised timeline was created (Appendix A, Table 1). The revised timeline also takes into account the time needed to search for more landfill and wetlands sites, and for the introduction of specific methanogenic activity (SMA) tests (explained below).

2.) A preliminary study conducted in 2011 found that methane production at 3 degrees C was minimal (Appendix A, Figure 1) compared to the amount produced at higher temperatures. Methane production at 5 degrees C was hence predicted to be low and the amount deemed not practical for any use (Zeeman et al. 1988). Instead, temperature ranges of 15, 25, and 35 degrees C were chosen as the new temperature ranges. Since many studies on methanogenesis are conducted at 35 degrees C, adding this temperature will be useful for cross-referencing results from this study to others.

3.) A decision was made to search for at least four landfill and four wetland sites, from which two sites (of landfills and wetlands) will be selected for the project. This was done in order to minimize the risk of conducting a four month long biochemical methane potential tests (BMP) with soils or leachates that do not harbor any methane-producing microorganisms (methanogens). Specific methanogenic activity (SMA) test was introduced into the study to determine which two wetlands and landfills should be used. A brief procedure can be found under the “Accomplishment/Milestones” section. Specific methanogenic activity tests have been used in anaerobic sludge studies to determine sludges’ potential to produce methane (Raposo et al. 2006; Sorensen and Ahring 1993; Kettunen and Rintala 1998). The advantages of SMA are the short amount of time needed (less than three days) and its ability to indicate if sludges contain methanogens.

4.) A correction is made for the date of the field tour. Instead of the original date stated (2/1/2013), it is supposed to be conducted on 9/1/2012.


Based on the timeline (Appendix A, Table 1), the project is currently on track. At least four sites for landfills and four sites wetlands have been located. Preliminary SMAs for two wetlands and five landfills have been performed with limited success. Total and volatile solids, percent organic matter, and pH analyses of these wetlands and landfills have been conducted.

Conventionally, SMA has always been used for sludges obtained from anaerobic digesters where there are already large numbers of microorganisms. The SMA method is currently under revision to obtain better results for this project’s alternative sources of inoculum.


Kettunen, R.H., and J.A. Rintala. 1998. Performance of an on-site UASB reactor treating leachate at low temperature. Water Research 32 (3):537-546.

Raposo, F., CJ Banks, I. Siegert, S. Heaven, and R. Borja. 2006. Influence of inoculum to substrate ratio on the biochemical methane potential of maize in batch tests. Process Biochemistry 41 (6):1444-1450.

Sorensen, A.H., and B.K. Ahring. 1993. Measurements of the specific methanogenic activity of anaerobic digestor biomass. Applied microbiology and biotechnology 40 (2):427-431.

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 (1):15-31.


Studies Conducted for Wetlands

Literature review was conducted in order to determine which wetland sites should be chosen 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. Six sites were chosen as potential sites for the study (shown below).

SMAs for two of the wetland sites were conducted with limited success. Triplicate soil samples were collected from wetland Sites 5 and 6 (description of each site can be found below). They were placed in serum bottles and combined with acetate, a substrate for the methanogens or deionized water, the control. The bottles are purged with N2/CO2 gas (70:30), capped with butyl rubber stoppers and incubated at 25 degrees C. Biogas produced was measured using a glass syringe and the percent methane was monitored using a gas chromatograph. Biogas production was monitored every three hours for a period of up to 72 hours.

The initial linear slope of the graph of cumulative methane versus time was used to calculate the rate of methane production per unit time, indicating the potential for the sediments to produce methane. The numbers are normalized for the unit volume of sediments used (mL of methane/ mL of sediment/day).

Percent organic matter and pH analyses for these two wetlands were also conducted.

Studies Conducted for Landfills

Literature review was conducted to determine which types of landfills produce larger amounts of methane gas.

A database published by the Landfill Methane Outreach Program (LMOP) (USEPA, accessed 2011) containing information about the amount of landfill gas flowed to projects was used as a resource to determine which landfill sites in the region are producing large amount of methane. Additional information about gas production for landfills in Maryland was also 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 was used to compare the different landfills. If this information is absent, the amount of landfill gas flared per ton of waste or the amount of landfill gas flowed to project per ton of waste were used. 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 leachates were requested. Four sites were chosen as the candidates for our study. 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 leachates from some of these landfills.

SMA was conducted for the landfill leachates with limited success. The leachates were diluted to 20%, experiment performed in duplicates and conducted using a similar procedure for the SMA tests for wetland sediments.

Total and volatile solids, and pH analyses were also conducted for these leachates.

Results for Wetlands

The following points were identified and used to search for potential 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, Ochs, and Yu 2009). However, this occurs only up to a depth of 10cm 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, Lang, and Sexstone 1990; BAKER?BLOCKER, Donahue, and Mancy 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 (Dr. Megan Lang, personal communication, 2011) due to the seasonality and the inter-annual variations of the water depths. Hence, a decision was made to find wetlands that are inundated with water most of the year.

These sites are chosen for this study:

Site 1: A tidal freshwater marsh located within the Jugbay Wetland Sanctuary adjacent to the Patuxent River, Maryland, USA (38 degrees 46 minutes 51.96 seconds N, 76 degrees 42 minutes 30.00 seconds W). Previous study conducted by Neubauer et al. (2005) indicated high amount of methane production by soils in this marsh.

Site 2: A tidal freshwater marsh located within the Jugbay Wetland Sanctuary adjacent to the Patuxent River, Maryland, USA (38 degrees 46 minutes 51.30 seconds N, 76 degrees 42 minutes 43.56 seconds W). High methane percentage has been observed in gas produced by soils in this site (Dr. Patrick Megonigal, personal communication, 2011).

Site 3: A beaver pond located within the Patuxent Wetland Park, in close proximity to the Jugbay Wetland Sanctuary, and adjacent to the Patuxent River, Maryland, USA (38 degrees 48 minutes 34.99 seconds N, 76 degrees 42 minutes 33.48 seconds W).

Site 4: Open water pond located in housing community in Culvert County, Maryland, USA (38 degrees 44 minutes 45.94& seconds N, 76degrees 40 minutes 52.05 seconds W). The pond has a fast growing population of aquatic vegetation. Wetlands with high net primary productivity correspond to high emission of methane (Whiting and Chanton 1993).

Site 5: Wetland located along Paint Branch Trail along the Paint Branch Trail, which is a part of the Anacostia tributary trail system (39 degrees 0 minutes 19.54 seconds N, 76 degrees 56 minutes 17.49 seconds W). This site is continuously flooded throughout the year (personal observation).

Site 6: A tidal freshwater forested swamp located within the Patuxent Wetland Park, in close proximity to the Jugbay Wetland Sanctuary, and adjacent to the Patuxent River, Maryland, USA (38 degrees 48 minutes 42.74 seconds N, 76 degrees 42 minutes 26.88 seconds W). This site is recognized to contain hollows, which are wetland areas with high water table (Dr. Andrew Baldwin, personal communication, 2011).

SMA was performed for two of the wetlands (Sites 5 and 6) with limited success. The graph (Appendix B, Figure 2) should be positively linear for most of the test period since the substrate, acetate, was added in a non-limiting amount. In this test, however, there was a spike for the first reading and the amount of methane produced subsequently was 0. The experiment was stopped at 24 hours instead of the full 72 hours.

Several observations were made during the first run. The pH values of the soils were between 4.72-5.59 (Appendix B, Table 2) and these are too low for high rates of methanogenesis to occur. The ideal pH for methanogenesis to occur is 6.8-7.2 (Gerardi 2003). In addition, during the experiment, it was noticed that the wetland soils had moderate amount of clay and this prevented the soils from mixing thoroughly with the acetate solution.

In subsequent SMA tests, the following modifications will be made:

1.) Soils will be mixed with nutrient media to form slurries to allow soil particles to have sufficient contact with the acetate solution.

2.) Nutrient media will be used to make the slurry to provide macro- and micro-nutrients to ensure both soils have all the nutrients necessary for methanogenesis to occur. Furthermore, nutrient media will provide buffering capacity so that the pH can remain in the optimum range.

The percent soil organic matter for Site 5 was very low (2.80%). It is classified as mineral soil instead of organic soil. This site was thus taken off from the list of potential sites.

Results for Landfills

Higher abundance of methanogens are expected to be found in newer landfills than older ones due to the presence of lower amount of organic matter in the latter (Calli, Durmaz, and Mertoglu 2006; Mori et al. 2003). Hence, only landfills that are still opened are used as potential sites for the project.

The following landfill sites are chosen for this study:

Site 1: 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, accessed 2011).

Site 2: 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)

Site 3: 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, accessed 2011).

Site 4: 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).

Site 5: Parkton Landfill located in Baltimore County, Maryland. While this landfill was not scouted, it is located in the same region as the Eastern Sanitary Landfill and the personnel was kind enough to provide leachate samples from this landfill to be tested.

The landfills from these leachates had pH values close to the optimal pH range for methanogenesis (6.85-7.97). Charles County, Stafford County, and Corral Farm Landfill leachates have higher concentrations of volatile solids and are thus expected to produce greater amount of methane than Parkton and White Marsh Landfills (Appendix C, Table 3).

Preliminary SMA tests for landfill leachates had limited success. The amount of methane produced was very low and all the control treatment (bottles with no acetate solution added) performed better than those with acetate (Appendix C, Figure 3). The bottles were allowed to sit for a couple of weeks in order to determine if gas will be produced should sufficient time be given. The methane concentration in most of the bottles increased and as shown in Appendix C, Figure 4, Stafford County, Charles County, and White Marsh Landfills with acetate added produced more methane compared to the other treatments.

While the final pH of all the bottles were not optimal (5.97-6.60), the bottles with the highest methane production had higher pH than those with lower methane production (>6.27) (Appendix C, Table 4). Charles and Stafford County landfills did perform better as expected from the volatile solids results. Corral Farm landfill, however, did not perform as expected and this may be due to the pH effect.

The following modifications will be implemented in the subsequent SMA tests:
1.) The landfill leachates will be diluted to 50% concentration instead of 20% concentration using nutrient media to maximize the volume of leachates within each bottle.

2.) Nutrient media will be used to provide nutrients for the microorganisms and to provide leachates with buffering capacity.


BAKER?BLOCKER, A., T.M. Donahue, and K.H. Mancy. 1977. Methane flux from wetlands areas. Tellus 29 (3):245-250.

Calli, B., S. Durmaz, and B. Mertoglu. 2006. Identification of prevalent microbial communities in a municipal solid waste landfill. Water science and technology 53 (8):139-147.

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, JR Patrick, and H. WILLIAM. 1983. Methane release from Gulf coast wetlands. Tellus B 35 (1):8-15.

Gerardi, M.H. 2003. The microbiology of anaerobic digesters. Hoboken, New Jersey: John Wiley & Sons, Inc.

Koh, H.S., C.A. Ochs, and K. Yu. 2009. Hydrologic gradient and vegetation controls on CH 4 and CO 2 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.

Mori, K., R. Sparling, M. Hatsu, and K. Takamizawa. 2003. Quantification and diversity of the archaeal community in a landfill site. Canadian journal of microbiology 49 (1):28-36.

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, 2011. LMOP: LMOP landfill and project database, sorted by state, project status, and landfill name (XLS). Retrieved 10/4/2011, from

Whiting, GJ, and JP Chanton. 1993. Primary production control of methane emission from wetlands. Nature 364: 794-795.

Yavitt, JB, GE Lang, and AJ 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):22463-22,474.

Impacts and Contributions/Outcomes

Progress is being made for the development of methods to find sources of inoculum which can improve the methane production capacity of anaerobic digesters at low temperatures. One of the main barriers towards the installation of anaerobic digesters is its high capital cost. By increasing the amount of methane generated from anaerobic digesters during the colder months, 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.

Anaerobic Digestion Workshop

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 and Education (SARE) fellowship awarded to graduate student Andrew Moss. Andrew Moss and Dr. Gary Felton conducted the workshop on November 1st 2011 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.


Stanley Fultz

[email protected]
Frederick County Dairy Science Agent
University of Maryland Extension
Frederick County 5370 Public Safety Place
Frederick, MD 21704
Office Phone: 3016003578
Dr. Stephanie Lansing

[email protected]
Assistant Professor
University of Maryland
1445 Animal Sciences/Agricultural Engineering
Building 142
College Park , MD 20742
Office Phone: 3014051197
Dr. Gary Felton

[email protected]
Associate Professor
1433 Animal Sciences/Agricultural Engineering
Building 142
College Park, MD 20742
Office Phone: 3014058039