Developing Inoculum to Increase Anaerobic Digestion Efficiency in Winter Months

Developing Inoculum to Increase Anaerobic Digestion Efficiency in Winter Months

Summary

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.

Wetlands 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 temperatures that could be achieved when anaerobic digestion systems are inoculated with cold-adapting microorganisms obtained from wetland soils and landfill leachates.

In 2012, a second set of specific methanogenic activity (SMA) test was conducted to determine the two best landfill and wetlands sites for this study. Two wetlands and two landfills were recognized to be the ideal inoculum sources for this study.

Wetland sediment from two different sites, landfill leachates from two different sites, and digestate from a mesophilic anaerobic digester treating dairy manure were collected and incubated at 15 degrees celcius, 25 degrees celcius, and 35 degrees celcius to increase the methanogenic population that are adapted to these temperatures within these inocula. On the third month and sixth month of incubation, samples were extracted from selected reactors and used as inoculum sources for 90-day biochemical methane potential (BMP) tests.

The first BMP test, conducted after three months of inocula incubation, demonstrated that adding the right type and quantity of inoculum can increase the amount of methane produced from the digestion of manure by approximately 540 percent, 2,566 percent, and 3,070 percent at 15 degrees celcius, 25 degrees celcius, and 35 degrees celcius, respectively.

Higher inoculum to substrate ratio (50 percent (w/w) compared to 35 percent, 20 percent, and 0 percent) also led to higher methane production at all the temperatures studied. At 15 degrees celcius and 35 degrees celcius, the highest methane production was observed in bottles where incubated digestate was used as the source of inoculum, while at 25 degrees celcius, highest methane production was observed when selected incubated landfill leachate was used as inoculum source.

The second BMP test is being performed to determine if varying the incubation time (3 versus 6 months) for the inocula could increase the methane produced when they are used for the digestion of dairy manure.

Objectives/Performance Targets

There are two main objectives for this project:
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 celcius, 15 degrees celcius, and 25 degrees celcius.

2.) Determine the optimal incubation period and inoculum to substrate ratio at 5 degrees celcius, 15 degrees celcius, and 25 degrees celcius. 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.) As noted in Annual Report 2011, 15 degrees celcius, 25 degrees celcius, and 35 degrees celcius were chosen as the new temperature ranges. Specific methanogenic activity (SMA) test was also introduced into the study to determine which two wetlands and landfills should be used.

2.) A second set of SMA tests was conducted and two wetlands and two landfills were chosen as the ideal candidate for this study.

3.) The preliminary results from the SMA tests indicated that there was a need to substantially increase the number of microorganisms before the inoculum sources were used in biochemical methane potential (BMP) testing. Hence, a higher number of reactors (15 instead of 6), which allowed regular feeding, were constructed.

4.) Samples were collected from two wetlands, two landfills, and a running mesophilic anaerobic digester and a six month incubation period with frequent feeding regimen to increase the methanogenic population was performed.

5.) On the third month, samples were extracted from three of the reactors in each of the temperature chamber and used as inocula in biochemical methane potential (BMP) tests conducted 15 degrees celcius, 25 degrees celcius, and 35 degrees celcius.

6.) Samples have also been extracted on the sixth month and a second set of BMP test is currently being performed.

7.) The need to incubate the methanogens before they are used for inoculation meant that the total number of BMP tests are reduced from four to two. The number of days for the BMP tests is also reduced from 120 days to 90 days since it was recognized that 90 days are sufficient to determine which inoculum is more suited for digestion at the different temperatures.

8.) The revised timeline can be found in Appendix A (Table 1). While there have been changes made to the project, it is currently on track. The field tour, which is part of the NE SUN Grant (NE10-404) awarded to the PI, has been postponed to Spring 2013 to allow researchers in the projects to collect additional data to be presented to the farmers.

• Appendix A

Accomplishments/Milestones

A second set of SMA tests was conducted to identify the ideal wetland and landfill sites for this project. The following modifications were made in the second run:
1.) Nutrient media was added to all bottles. As a result, the pH’s of all, except one bottle, were maintained at the optimum level for methane production (6.8-7.2) (Gerardi 2003).
2.) Bigger bottles (250 mL instead of 160mL) to allow more landfill leachates or wetland soils to be added to each bottle.
3.) A longer time period was used (up to four weeks) to give sufficient time for the production of quantifiable amount of methane.
4.) All bottles were shaken during the test period to increase substrate-microorganisms contact time.

The two wetlands chosen for this project were Sites 1 and 2 (with approximate coordinates of 38 degrees 46 minutes 51.36 seconds N, 76 degrees 42 minutes 26.61 seconds W and 38 degrees 46 minutes 49.63 seconds N, 76 degrees 42 minutes 45.75 seconds W, respectively) since they produced the highest amount of methane during the SMA test (Figure 1 in Appendix B).

Low quantity of biogas was measured during the second landfill leachate SMA test, but percent changes in methane within the bottles’ headspace indicated that methane was produced by some of these leachates. Charles County and Stafford County landfills had the greatest increase in headspace percent methane indicating that there were methanogens present in these leachates. These two sites were thus chosen to supply landfill leachates for this project (Figure 2 in Appendix B).

While the SMA tests illustrated that the above sites contain methanogens, the amount of methane produced were low. It was thus concluded that samples from these sites needed to be incubated and fed at regular intervals to increase the methanogenic population before they are used as sources of inoculum.

Reactors, with approximate volume of 4L, were designed and constructed using PVC pipes and ball valves. Test plugs and closet flanges were used to seal the reactors. Biogas was collected in multi-layer foil gas sampling bags (Figure 3 in Appendix B).

A total of 15 reactors were constructed and they incubated the different types of inocula at 3 different temperatures (15 degrees celcius, 25 degrees celcius, and 35 degrees celcius). There are five reactors in each temperature chamber incubating microorganisms obtained from the following sources: soils from the wetland sites 1 and 2, landfill leachates from the Stafford County and Charles County landfills, and digestate from a mesophilic digester treating separated dairy manure located in the United States Department of Agriculture (USDA) Beltsville Agricultural Research Center (BARC) in Maryland.

Each reactor was filled with equal amount (1.25L) of the inoculum source (wetland soil, landfill leachate, or the digestate) and nutrient media. The rectors were fed with autoclaved dairy manure obtained from the USDA BARC facility at approximately four-day intervals. Autoclaved manure was used to minimize the introduction of methanogens present in the raw manure. Biogas production and percent methane in the gas were measured twice a week.

On the third month of incubation, the reactors incubating the best-performing wetland soil and the best-performing landfill leachate (highest methane producers) at all three temperatures (with greater emphasis placed on the results at 15 degrees celcius), and the anaerobic digestate (Figures 4, 5, and 6 in Appendix B) were selected to supply the inocula for the BMP test. Samples were extracted from reactors incubating wetland site 1 soil, Stafford County Landfill leachate, and the digestate at each of the three temperatures. Different inoculum to substrate ratio (0 percent (w/w), 20 percent, 35 percent, and 50 percent) was also used during the BMP test to determine the effect of different inoculum to substrate ratio on methane production. The BMP bottles were placed at 15 degrees celcius, 25 degrees celcius, and 35 degrees celcius chambers (corresponding to the temperatures at which the inocula were previously incubated at).

Preliminary results from the third month BMP test demonstrate that in general, higher amount of inoculum leads to higher production of methane per gram of manure at the three different temperatures. At each of the temperature, the highest methane production was observed in bottles inoculated with 50 percent (w/w) inoculum to substrate ratio (Figures 7, 8, and 9 in Appendix B).

Compared to the manure only treatment, adding the right type and quantity of inoculum can lead to approximately 540 percent, 2,566 percent, and 3,070 percent increase in methane production at 15 degrees celcius, 25 degrees celcius, and 35 degrees celcius, respectively (Figures 7, 8, and 9 in Appendix B). This illustrates further the importance of adding inoculum and the impacts that different types and quantity of inoculum have on methane production in anaerobic digesters.

At 15 degrees celcius, highest amount of methane was produced when the incubated digestate was used as inoculum. The treatment produced 0.96 mL CH4/g manure, which was 129 percent more than the amount produced by the next best treatment (0.42mL CH4/g manure produced when the incubated Stafford County landfill leachate with a 35 percent inoculum to substrate ratio was used) (Figure 7 in Appendix B).

At 25 degrees celcius, the highest amount of methane was produced when the incubated Stafford County landfill leachate was used as the source of inoculum. This suggests that the Stafford County landfill leachate may contain methanogens more adapted to this temperature. The amount produced by this treatment was 25.86 mL CH4/g manure, which was 18 percent more than the amount of methane produced by the next best treatment (21.98 mL CH4/g manure was produced when the incubated digestate with a 50 percent inoculum to substrate ratio was used) (Figure 8 in Appendix B).

At 35 degrees celcius, highest methane production was observed when the incubated digestate was used as inoculum source (27.26 mL CH4/g manure). Incubated Stafford County landfill leachate produced lower but approximately the same amount of methane (26.32 mL CH4/g manure). The amounts produced by these two treatments were at least 23 percent higher than the next best treatment (21.48 mL CH4/g manure produced when the incubated wetland site 1 soil with a 50 percent inoculum to substrate ratio was used) (Figure 9 in Appendix B).

Statistical analyses, however, still need to be conducted to determine if there are any significant differences between the methane produced when different types and/or quantity of inoculum were used.

After the inocula had been incubated for 6 months, a second BMP test was started and is currently being monitored. Samples were extracted from the same reactors used in the first BMP. After analyzing the results from the first BMP test, the following changes were made in the second BMP test:
1.) The BMP test conducted at 25 degrees celcius and 35 degrees celcius do not include the 20 percent: 80 percent (inoculum: manure) ratio since these ratios produced low amount of methane during the first BMP test.
2.) At 15 degrees celcius, only the most promising inocula (wetland site 1 soil and incubated digestate) with 50 percent inoculum to substrate ratio are used.

Geradi, M.H., 2003. The Microbiology of Anaerobic Digesters. Hoboken, NJ: John Wiley & Sons, Inc.

• Appendix B

Impacts and Contributions/Outcomes

This project aims to increase the methane production capacity of anaerobic digesters at low temperatures by using different sources and quantity of inoculum during low-temperature digestion. 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.

So far, this project has further illustrated the importance of adding inoculum to anaerobic digesters operating at various temperatures. Higher inoculum to substrate ratio is also beneficial towards increasing the amount of methane produced at these temperatures. Furthermore, the addition of incubated landfill leachate as inoculum resulted in the highest methane production during the digestion of dairy manure at 25 degrees celcius, suggesting that, compared to conventional sources of inoculum (i.e. the anaerobic digestate), the leachate used in this study may contain methanogens that are more adapted to this temperature.

Collaborators: