1. Application of microbial fuel cells for the recovery of animal waste water William C Rice, Adam Holman, and Byron Neal

2. Acknowledgements CRIS Projects under National Programs NP213 and NP214 Ogallala Aquifer Funding Texas Cattle Feeders Association Funding Technical Support, Lana Castleberry and Will Willis Ken Casey, Texas AgriLife

3. Topics Evaluation of manure as a biofuel feedstock for the production of electricity sediment-Microbial Fuel Cells (s-MFC) single chamber-Microbial Fuel Cells (sc-MFC) Hydrogen production Evaluation of purple nonsulfur bacteria (PNSB) for the production of hydrogen gas

4. Hydrogen and electrical fuel cells (dual cells)

5. Assembled sediment- MFC (sMFC) for the selection of microbial consortia and strains capable of generating electricity. Milled Graphite electrodes (10 cm diameter)

6. s-MFC in operation; playa sediments and waster water are the fuel feedstock for s- MFC experiments

7. Alpha-proteobacteria associated with s-MFC exp1 Electrode Biofilm and sediments DDGE-PCR using Alpha-proteobacteria specific PCR primers; Note the presence and absence of bands specifically associated with Control and Load Electrodes (both cathode and anode)

8. Power density curves from s-MFC exp2 s-MFC harvested at three time intervals after initiation of experiment; sporadic power produced in Cell #5, sustained power produced in Cell #4 and #6 prior to harvest.

9. Alpha-proteobacteria associated with s-MFC exp2 Electrode Biofilm and sediments DDGE-PCR using Alpha-proteobacteria specific PCR primers; Note the presence and absence of bands specifically associated with Control and Load Electrodes (both cathode and anode)

10. sc-MFC experiments Employed specially designed single cell MFC made by a scientific glass blowing company Carbon cloth anode and Platinum-carbon black-carbon cloth electrode coated with Teflon Feedlot playa sediment wastewater slurry used a fuel feedstock inoculum +/- seeding of microbial consortia grown from fuel cell #6

11. Carbon cloth anode on the left and Pt-carbon cloth cathode on the right

12. Schematic depicting the material layers used in the construction of a sc-MFC

13. sc-MFC (volume ~125 mL)

14. Single chamber-Microbial Fuel Cell (sc-MFC): experiment 1 Power densities (W/m 2 )

15. Single chamber-Microbial Fuel Cell (sc-MFC): experiment 2 Power densities (W/m2)

16. Cell 4 Control R1 Cell 6 ATCC #1 Dark R2 Cell 8 Pooled Light R2 Cell 5 Control R2 Cell7 ATCC #5 Dark R2 Cell1 ATCC #5 Dark R1 Cell3 ATCC #1 Dark R1 Cell2 Pooled Light R1 Single chamber-Microbial Fuel Cell (sc-MFC): experiment 3 Power densities (W/m2)

17. MFCs for generation of electricity; general conclusions Much longer time to develop stable currents from sediment MFCs than single chamber MFCs Lower power densities observed with s-MFCs than with sc-MFCs sc-MFCs seeded with electricity producing microbial consortia tend to produce higher amounts of power than unseeded sc-MFCs Data suggests that the initial inoculation step of 7-10 days in the sc-MFCs is sufficient to obtain the final power densities observed

18. MFCs for generation of electricity; general conclusions cont’d Different electrode materials and geometries of electrodes affect power densities Length of time of initial exposure to inoculating materials import for ultimate power density Type of bacterial media (substrates) appears to affect power density observed

19. Evaluation of PNSB for the production of hydrogen gas Sample materials obtained from playa waste waters and sediments from USDA and commercial feedlots Bieble - Pfanning media under anaerobic conditions plus tungsten light (~2,000 lux) in Balch tubes Two to three weeks incubation at 30 ⁰C in Balch tubes followed by plate isolation on Bieble – Pfanning media under anaerobic conditions

20. Characteristics of PNSB isolates and 16S DNA sequence classification Isolate16S IDSourcePhenotype Strict Anaerobe 409Rubrivivax sp.sedimentredY 320 Rhodobacter sp. sedimentreddish-brownN 390 Rhodopseudomonas sp. sedimentredN 400 Rhodobacter sp. waterredN 385 Rhodobacter sp. waterredN 399 Rhodobacter sp. waterredN 388 Rhodopseudomonas sp. waterredN 326 Rhodobacter sp. waterreddish-brownN 382 Rhodobacter sp. sedimentredN 319 Rhodobacter sp. sedimentreddish-brownN 408 Rhodopseudomonas sp. waterreddish-brownN 339 Rhodobacter sp. sedimentbrownN 379 Rhodobacter sp. waterbrownN 396 Rhodobacter sp. waterreddish-brownN 369 Rhodobacter sp. waterbrownN 325 Rhodobacter sp. waterreddish-brownY 324 Rhodobacter sp. sedimentreddish-brownY 395 Rhodopseudomonas sp. sedimentredN 389 Rhodopseudomonas sp. waterredN 346 Rhodobacter sp. sedimentbrownN 373 Rhodocyclus sp. waterredY 318 Rhodobacter sp. sedimentreddish-brownN 394 Rhodopseudomonas sp. waterredN 358 Rhodobacter sp. waterbrownN 401 Rhodopseudomonas sp. waterredN 375 Rhodocyclus sp. waterreddish-brownN 334 Rhodobacter sp. sedimentbrownN 323 Rhodobacter sp. sedimentreddish-brownY 351Rubrivivax spwaterbrownN 403Rubrivivax spsedimentbrownN 406 Rhodobacter sp. sedimentreddish-brownN 411 Rhodopseudomonas sp. waterredN

21. Members of Rhodocyclus have been shown to possess a novel polyphosphate kinase, conferring the ability of these strains to perform enhanced biological phosphorus removal. Strains of R. capsulatus, R. sphaeroides, R. palustris, and Rubrivivax gelatinosus are all known to produce hydrogen gas. R. gelatinosus however is not a classical PNSB. It produces hydrogen via a hydrogenase while PNSB use a nitrogenase to generate hydrogen. Thus, R. gelatinosus can produce H gas from the CO oxidation- hydrogen pathway. Biomass, such as dried manure, may be burned to generate thermal energy and CO which can be bubbled through a bioreactor to generate H gas.

23. Isolate339326346323396319334325358324R. palustrisR. capsulatusR.sphaeroides H2H2 1.662.060.752.462.582.962.171.600.011.930.071.980.79 CO 2 0.350.070.190.020.250.600.080.290.090.530.060.560.03 H 2 /CO 2 4.728.33.9101.110.24.928.55.40.13.71.23.527.4 Gas production by isolates, measured in L of gas / L of media

24. PNSB general conclusions Five confirmed genera that are metabolically diverse were recovered from feedlot wastewater and sediments Five Rhodobacter isolates produced higher amounts of H gas than the Rhodobacter standards Three Rhodobacter isolates had a higher H 2 /CO 2 gas production ratio than the Rhodobacter standards Isolates that are very similar based on 16S DNA sequence data are very dissimilar based on metabolic profiles probably due to lateral gene transfer and diverse environmental selection pressures

25. Where do we go?? DNA pyrosequencing analysis to confirm the relationships of exoelectrogenic to one another and correlations with power density Use of large scale sc- and dc-MFC operated under continuous flow and batch conditions to determine reduction in BOD and COD Theoretical paper evaluating energy content of manure feedstocks using a combination of energy generating methods such as pyrolysis plus CO capture to generate H (Rubrvivax sp) vs. H via MFC vs. electricity production or combinations Enhanced recovery of P from manure wastes using Rhodocyclus sp