The Effect of Earthworms on Soil CH 4 Flux and Potential Mechanisms in a Temperate Pasture

1. The Effect of Earthworms on Soil CH4 Flux and Potential Mechanisms in a Temperate Pasture Edward J. Avizinis

2. Earthworms have an effect on soil conditions •Burrows •Feeding activity These conditions have an effect on the microbial community within the soil •Moist burrow lining create more anaerobic areas •Alter bulk soil density •Provides microorganisms with organic carbon source and nitrogen from mucus •This may effect the microbial community responsible for methane production and consumption in soil

3. Methane is produced and consumed by microorganisms in the soil •Methanotrophs consume methane in aerobic conditions -Bacteria ex. Methylomonas methanica CH4 + O2CO2 + H2O •Methanogens produce methane in anaerobic conditions -Archaea Hydrogenotrophic Acetoclastic CO2 + H2  CH4 CH3COO- CH4 ex. Methanosarcina barkeri ex. Methanosaetae concilii *Anaerobic microsites can occur in aerated soil so these processes can occur simultaneously

4. What will happen? •I believe that earthworm activity will increase methane flux by- -Increasing methanogenesis •increase anaerobic microsites •increase soil moisture •decrease oxygen diffusion •and by- -Decreasing methane oxidation •introducing more nitrogen (ammonia is a methane-oxidation inhibitor) •decrease oxygen diffusion

5. My research will answer these questions… •Do earthworms have an effect on methane flux? •If so, what is the magnitude and direction? •What are the potential mechanisms that cause this?

6. Why is this important? •Methane is an important greenhouse gas which can contribute to global warming -30 times the thermal absorption potential as CO2 /mole •Agriculture is shifting toward more sustainable methods that support natural food web interactions -These practices promote earthworm activity

7. Experiment design •Multiple plots set up at two similarly managed pasture sites •Different treatments -Some plots with earthworms and some without •Gas samples taken from plots and analyzed for methane •Data collected on potential mechanisms -soil moisture -diffusion in the soil -soil properties •

8. Earthworm extraction •Three main different methods -Hot mustard solution -Electroshocking -Hand sorting •Hot Mustard may have an effect on the processes being assessed •Electroshocking not reliable -Extracted about 50% of population in general •Hand sorting soil for earthworms most effective

9. Earthworm extraction • 1.) weigh 53 grams of hot mustard powder into a glass jar or plastic container. Add about a half of a cup of water stirring until a paste forms. The hot mustard paste must stand for at least 2 hours so that the "hotness" can develop. 2.) At the field site, select a suitable sampling area and place a wooden frame 0.5 m x 0.5 m square on the ground surface. 3.) Add about 3 L (2.5 qts) of water to a household plastic bucket. Add the mustard paste to the water in the bucket. Stir vigorously to remove any lumps. Add water to the bucket up to the 7 L (6 qt) mark. Stir vigorously.

10. Earthworm extraction • 4.) Gradually pour the water-mustard mixture to the soil surface. Worms should appear within 1 to 2 minutes and all activity will end in about 20 minutes. Wait until each worm completely emerges or is nearly finished emerging before picking it up with the tweezers. If picked up too soon, the worm will pull back into its burrow. 5.) If the worms are to be returned to the field, the worms should be rinsed in clean tap water before releasing.

11.  In field methane flux •Experimental plots were set up at two sites -W. Alton Jones campus -Peckham Farm, URI •Each site has 24 plots with 3 treatments each -8 Disturbed (+) Earthworms removed, weighed, counted, and returned -8 Disturbed (-) Earthworms removed -8 Unaltered To determine whether or not there is an effect from disturbance

12. Site at W. Alton Jones •Both temperate pasture sites •Managed in the same way -mowed twice per year Site at Peckham Farm

13. Plot set up •Plots were dug 60 cm wide x 60 cm long x 30 cm deep •Soil was removed and sorted for earthworms then replaced •Fiberglass mesh was fenced around the plot 30 cm below ground and 15 cm above •Earthworms from the disturbed (+) plots were returned to their plots within six hours

14. Gas sampling from plots •Each plot is fitted with a gas sampling device -Cylinder anchored into the ground with an airtight screw-on lid with a septum built into it •Gas sampled at t=0, 15, and 30 minutes, once per week from April-October 2010 •Flux calculated by methane concentrations plotted vs. time to determine net methane production or consumption for a given time and volume

15. Gas sample analysis •In the lab gas samples are run through the gas chromatograph to analyze methane concentration •The gas chromatograph separates the gases and gives concentrations

16. Earthworm counts •Once in August and once at the end of the experiment •Destructive sampling of plots •Earthworm count, biomass, and community composition

17. Potential mechanisms •Soil moisture •Diffusivity •Soil properties •Earthworm community composition •Microbial processes

18. Soil moisture •Measurements taken at one plot per treatment per site every 2 hours •Soil moisture- Decagon EC-5 soil moisture meter and EM5b data logger •Water in the soil is responsible for limiting oxygen diffusion and creating anaerobic areas where methane can by produced

19. Soil Properties •NH4 and NO3 concentrations -can inhibit oxidation •Bulk density -can limit diffusion increasing methanogenesis and limiting oxidation •Water filled porosity -responsible for creating anaerobic microsites where methanogenesis can take place All measurements made on burrow lining, casts, and bulk soil

20. Microbial processes •Acetoclastic methanogenesis CH3COO- CH4 •Hydrogenotrophic methanogenesis CO2 + H2  CH4 •Methane oxidation CH4 + O2CO2 + H2O Perform a lab assay to determine rates of each in burrow, cast, and bulk soil. -These can be used to hypothesize what might be driving field findings Production Consumption

21. Acetoclastic methanogenesis • 10g soil with 20mM solution of acetate in water •Sealed in a glass vial with rubber stopper septum •Headspace gas replaced with high purity N2 •Gas sampled over time

22. Hydrogenotrophic methanogenesis • 1g soil with 5ml water •Sealed in a glass vial with rubber stopper septum •Headspace gas replaced with 20:80 -CO2:H2 •Gas sampled over time

23. Methane oxidation •10g soil with adjusted water content to maximize oxidation •Sealed in a glass vial with rubber stopper septum •Headspace replaced with atmospheric levels of methane (~2.0 ppm) or high levels (~50.0 ppm) •Gas sampled over time

24. Soil fauna

25. Particle size in soil Diameter(mm) Particle class >2 Coarsefragments 2 – 0.05 Sand 0.05 – 0.002 Silt < 0.002 Clay

26. Particle size in soil Soil texturing- http://www.youtube.com/watch?v=GWZwbVJCNec

27. Particle size in soil

28. Soil components • Solids: Mineral Organic matter Always mingled together- Ratio is variable • Voids (pores): Water Air Ratio is variable

29. Soil fauna http://www.youtube.com/watch?v=VuHznslr8aI

30. Nitrogen in soil

31. Nitrogen in soil

32. Acknowledgments •Dr. Jose Amador- Advisor •Carl Sawyer- Agronomy •Soil Ecology and Microbiology Lab -Janet Atoyan -Joe Fetter -Andrew Guigere •Volunteer hole diggers •United States Department of Agriculture- funding