1. Humic Substances and Electron Transport Reactions in Aquatic Ecosystems: the Role of Quinone Moieties Diane McKnight Collaborators: Robin Fulton, Derek Lovley, Pat Hatcher, Mark Williams Students: Lisa Klapper, Durelle Scott, Eran Hood

2. Humic substances: Ubiquitous, chemically functional, biomolecular junk Humic substances modulate ecosystem function by controlling light regime, soil fertility, electron transfer, etc. Biogeochemical reactions involving humic substances control elemental cycles for C, N, S, P, Fe, Mn and other trace metals Heterogeneous class with similar properties (color, hydrophobicity, acidic groups, etc)

3. Humic substances as electron acceptors All humics have some electron accepting capacity Anoxic microbial respiration Reduced humics can readily transfer electrons to ferric iron ESR studies indicates that quinones are involved.

4. Biological reduction of humics Microbial reduction by GS15 (Geobacter metallireducens) with acetate

5. The electron mediator role of humics Electrons are microbially transferred from acetate to HS in an anoxic environment. Electrons are transferred from HS to Fe(III), reducing the metal to Fe(II). Lovley, D. R.; Coates, J. D.; Blunt-Harris, E. L.; Phillips, E. J. P.; Woodward, J. C. Nature, 1996, 382, 445-447

6. Quinones

7. Redox state of humic substances in natural environments? Necessary to detect redox state in order to compare to other microbially mediated redox processes Measurement by reduction of iron not possible in natural systems containing iron ESR is not practical, requiring high concentrations

8. Typical excitation emission matrices (EEMs) of fulvic acids Suwannee River FA terrestrial source Lake Fryxell FA microbial source Senesi, N.; Miano, T. M.; Provenzano, M. R.; Brunetti, G, Soil Science, 1991, 152, 259-271

9. Fluorescence

10. Emission spectra ( excit = 370 nm) varies with source of fulvic acid Fluorescence index: intensity ratio between 450 and 500 nm McKnight, D. M.; Boyer, E. W.; Westerhoff, P K.; Doran, P T.; Kulbe, T.; Andersen, D. T. Limnology and Oceanography, 2001, 46, 38-48

11. Quinones mediate electron transfer Play an important role in electron transfer several biologically important enzymes Redox potential is tunable by attaching various substrates to the quinone

12. EEM’s of AQDS and AHDS

13. San Diego Bay Coronado Cayes Paleta Creek Shelter Island

14. Humics as electron acceptors in San Diego Bay sediments Paletta Creek –PAH contaminated –High rates of anaerobic degradation of PAH’s have been observed –Terrestrial source of humics Shelter Island –Commercial/recreational boat basin –Contaminants: copper and tin (from anti-fouling hull paints) –Contains humic substances from both terrestrial and microbial sources Coronado Cayes –“Pristine” site –Microbial source of humics

15. Fluorescence index and % aromaticity of SDB sediment FA’s * Fluorescence index of sediment FA’s are lower than aquatic FA’s; however, the same source trend is followed.

16. Humic substances isolated from San Diego Bay sediments Microbial reduction by GS15 (Geobacter metallireducens) with acetate as substrate Measured amount of ferrous iron produced after removal of cells (electron accepting capacity) Recorded EEM’s before and after reduction

17. Electron accepting capacity of isolated humic substances

18. Change in EEM of Paletta Creek FA with microbial reduction Isolated Paletta Creek FA Reduced Paletta Creek FA

19. Change in EEM of Shelter Island FA with microbial reduction Isolated Shelter Island FAReduced Shelter Island FA

20. Change in EEM of Coronado Cayes FA with microbial reduction Isolated Coronado Cayes FAReduced Coronado Cayes FA

21. Continuing experiments… Isolated humic substances from sediments stored at 4 ºC for 13 months Discovered that EEM’s of these humic samples were same as those of microbially reduced humics!!!

22. Aged sediment FA’s have similar EEM’s to that of reduced FA’s FA’s isolated after 13 months incubation at 4°C Aged Paletta Creek FAReduced Paletta Creek FA

23. Aged sediment FA’s have similar EEM’s to that of reduced FA’s Microbial processes did not shut down at 4 o C Limited supply of Fe(III)??? Aged Coronado Cayes FAReduced Coronado Cayes FA

24. Aged FA’s can be oxidized by Fe(III): Original Coronado Cayes FAReoxidized Coronado Cayes FA

25. Change in EEM’s for reduced humics from sediments Possible to detect in natural environment Consistent with quinone moieties as main electron accepting moiety Greater alteration of EEM’s for microbially- derived fulvic acids with lower eelctron accepting capacity, suggesting more of quinones act as electron acceptors

26. Lake Fryxell fluorescence index depth profile

27. Lake Fryxell: Whole water samples Samples filtered through 4 micron filters Samples sealed directly after acquisition 5 m16 m

28. Similar changes in EEM’s seen in laboratory experiments Photo-oxidation of FA’s lead to change in EEM 16 m wholewater 8 hours under solar simulator 10 m

29. Hypothesis for relationship of humic source to electron accepting capacity Quinone moieties incorporated into humics from quinone-containing “enzymes” involved in electron transfer reactions Quinone moieties incorporated into humics from degradation products of lignin Quinones in humics from “enzymes” are more effective as electron acceptors than lignin-derived quinones

30. Implications of relationship of humic source to electron accepting capacity From geologic perspective, humics from microbial biomass came “first”, potentially preceding O 2 atmosphere. Evolutionary advantage to the preservation of the electron transfer function in detrital organic material (humics) to release nutrients to sustain growth

31. Implications of relationship of humic source to electron accepting capacity Because of O 2 atmosphere, terrestrial ecosystems not limited by availability of electron acceptors for degradation of OM. The land surface is “green”-higher plants have a higher standing biomass, more structural material, compete for light, water and nutrients

32. Implications of relationship of humic source to electron accepting capacity In terrestrial ecosystems, organic material cycling is slower than in aquatic environments Degradation of lignin is slow and is not “optimized” to produce humic substances containing “highly functional” quinone moieties for electron transfer.

33. San Diego Bay Coronado Cayes Paleta Creek Shelter Island

34. Conclusions Humic substances are probably involved as an electron shuttles or terminal electron acceptors in many aquatic environments Significant for designing C sequestration!! Significant for fate of organic contaminants in context of land use change!!

35. Humics as electron acceptors in San Diego Bay sediments Microbial reduction by GS15 (Geobacter metallireducens) with acetate Fluorescence of humics potentially caused by quinone moieties Humics with microbial source have less electron accepting capacity Electron accepting capacity may be related the lower excitation/emission maximum Potential for EEM’s to be used to map distribution of reactivity with concentration of extractable fulvic acid