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Nine cores, three treatments: Control (no vegetation), Wind (constant wind applied during incubation), and No Wind Plants incubated at ambient temperature under 12 h light cycle Porewater withdrawn through the perfusion cap 15 NH 4 + added to ~95% labeling of exchangeable + porewater NH 4 + Labeled porewater infused slowly back in the soil Porewater withdrawn with gas tight syringe through mini peepers Analyzed on EA GC-MS for 29/30 N 2 15 NO 3 - analyzed by reduction to N 2 O by a denitrifying culture (1) Plant tissue was analyzed for 15 N incorporation Oxygen profiles taken with microelectrodes O 2 flux from roots measured by growing taro in nutrient agar with a FeS oxygen scavenging bed Oxygen microelectrode readings taken over a 24 h period O 2 measured as a function of distance from root tip or lateral side Additional time-after-light-on experiment conducted to establish response to photosynthesis Time series 29/30 N 2 corrected for time of day the measurement was taken Values were integrated for 12h-based N 2 accumulation Revised isotope pairing technique (IPT) rate calculations (3) were based on the 14 NO 3 - / 15 NO 3 - ratio Use of Novel Whole-Core Incubations to Measure the Fate of Fertilizer N in a Flooded Agricultural System Penton C.R. 1, Deenik J. 2, Popp B. 3, Engstrom P. 4, Bruland G.L. 5, Worden A. 1, Brown G. 2, Tiedje J. 1 1 Michigan State University, Center for Microbial Ecology, 2 University of Hawaii at Manoa, Dept. of Tropical Plant and Soil Sciences, 3 University of Hawaii at Manoa Dept. of Geology and Geophysics, 4 University of Gothenburg, Dept. of Chemistry, Sweden, 5 University of Hawaii at Manoa Dept. of Natural Resources and Environmental Management,, Research supported by USDA-NIFA NRI no. 2008-35107-04526 We thank Garvin Brown for assistance in the field and laboratory Elizabeth Gier for technical assistance E. Tottori, R. Haraguchi, W.Tanji, and D. Murashige for allowing sample collection R. Yamakawa (CES Kauai) for coordinating sampling BACKGROUND AND OBJECTIVES This study was based on the following: A prior fertilization experiment found a >80% of added nitrogen (N) could not be accounted for using classic N balance calculations Fertilized taro (Colocasia esculenta) fields have been implicated as a source of inorganic N to Hawaiian coral reefs Previous slurry based 15 N experimental results drastically overestimated denitrification compared to modeled fluxes from porewater profiles Oxidized Fe 3+ is present in bulk soil, associated with root channels indicating that subsurface rhizosphere coupling of nitrification and denitrification may be a significant N loss pathway Aerenchyma in taro plants may be an efflux pathway for subsurface N 2 Subsurface fertilizer injection has been proposed to reduce surface nitrification and thus, total fertilizer N losses at an increased cost to the farmer What is the relative importance of nitrification/denitrification in surface waters versus soil? Is subsurface rhizosphere oxygenation significant? What is the overall N balance for these flooded soils? In this study we utilize a novel whole core method for investigating coupled nitrification/denitrification in Hawaiian flooded taro field soils. Taro plants were grown for eight weeks in perforated cores in the field, allowing porewater exchange. Fertilizer application was mimicked with perfusion of 15 NH 4 + labeled porewater, enabling uniform distribution of 15 NH 4 + in subsurface and surface sediments. The fate of 15 NH 4 + was traced through the possible pathways, from plant incorporation to coupled nitrification/denitrification in the rhizosphere. Taro plants grown in 25.4 cm diameter PVC cores inserted~12 cm into loi soils for 8 weeks Holes along the sides of PVC core tubes allowed for porewater exchange Perfusion caps (Fig 1) were placed on the bottom upon retrieval Mini-peepers were inserted in holes placed in 1 cm increments down the core profile Figure 1. Perfusion cap used for removal and labeling of porewater. Diurnal Root O 2 Production An oxygen microelectrode was placed at a root tip for a 24 hr period Without wind there was no O 2 accumulation With wind applied there was a 7 h lag after the lights turned on until O 2 accumulated (Fig 2A) Once lights turned off there was no further O 2 accumulation O 2 Flux From Root O 2 fluxed ~0.6 mm from the root tips at a maximum [O 2 ] of 70 uM Lateral flux of O 2 varied widely: From 0.2 to 2 mm distance away from the lateral side surface From 23 to 66 uM O 2 concentration at the lateral surface Whole Core O 2 Profiles Vegetated cores showed deeper O 2 penetration than controls (Fig 2B) Wind treatment had significantly higher O 2 in the surface water Figure 2. (A) Diurnal O 2 production by a young taro root tip planted in agar and (B) whole core vertical O 2 profiles for each treatment (n=3) METHODS RESULTS Oxygen Profiles and Root Production N BALANCE Figure 4 (A-C). Accumulation of 29+30 N 2 during the light incubation series on day 6. (A) Aerenchyma, (B) Surface water, and (C) Subsurface. Note the 6 to 7 hr lag time for nitrification/denitrification to begin following light on. The light incubation series showed that time after light on significantly influenced the 29+30 N 2 signal (Fig 4) A lag time of 6-7 h was present until N 2 production ramped up Aerenchyma N 2 production was the same in wind and no wind treatments Surface water N 2 production occurred most rapidly in the wind treatment Control surface water did not show increased 29+30 N 2 production with time In the sub-surface the control treatment had higher dark 29+30 N 2 production The wind and no wind treatments increased dramatically after a 9 h lag The time of the day in which sampling occurred was as important as the elapsed time since label addition Light Incubation 29/30 N 2 Production 29/30 N 2 rates were corrected by means of a revised isotope pairing technique calculation using the 14/15 NO 3 - ratio (Fig. 6) 15 NO 3 - determined by denitrification of extracted porewater (4,5) Maximum rates determined from the first three days after label addition showed that subsurface nitrification/denitrification dominated over the surface Maintenance rates were significantly lower in the surface and in the control subsurface Revised IPT Calculations for Total N 2 Figure 7. Revised IPT-corrected total 28+29+30 N 2 production rates Surface Subsurface Total NH 4 + loss over the incubation period (Fig. 8A) showed that the loss in the control was significantly less NH 4 + loss than the vegetated cores Some surface loss was due to NH 4 + flux from the surface to the near-subsurface layers 5.4x more NH 4 + was lost in the vegetated sub-surface compared to the control, presumably due to coupling between nitrification and denitrification Plant uptake of 14+15 N over the entire incubation period showed substantial N accumulation (Fig 8B) Significantly more N was incorporated into the above ground biomass than the root and corm Figure 8. (A) Average total NH 4 + loss in the three treatments in the surface water and subsurface layers over the total incubation time (~10 days). (B) Plant uptake of 14+15 N in the above ground biomass (AGB), root, and taro corm. (A)(B) Core-Based N Balance The current N balance accounts for between 80-90% of NH 4 + lost according to porewater NH 4 + (Fig. 9) Wind treatment resulted in a greater proportion of N loss through denitrification (44%) compared to the no wind (35%) N uptake by plants was lower in the wind treatment (56%) compared to the no wind (65%) Likely due to increased NO 3 - diffusive distance with higher O 2 flux with more competition for NO 3 - by denitrifiers Control cores lost of 5.82 mmol porewater NH 4 + over the incubation period Excess subsurface NH 4 + also became bound to exchangeable matrix (1.1 mmol) due to lack of subsurface demand Net NH 4 + loss of 4.72 mmol over the incubation period N balance accounted for 78% of NH 4 + lost Taro plants had a significant impact on N losses in these flooded agricultural soils, with the majority of N lost through subsurface pathways Wind had a significant effect, increasing subsurface nitrification/denitrification coupling via O 2 flux to the subsurface Mass flow of O 2 down to the subsurface resulted in increased N 2 accumulation in the aerenchyma Denitrification accounted for between 35-44% of NH 4 + loss over the 10 day incubation period Results indicate that subsurface placement of fertilizer N may not mitigate N losses in these fields due to an extensive root system supporting subsurface coupled nitrification/denitrification and most N is lost through coupling between nitrification and denitrification Porewater NH 4 + loss=9.1 89.5% accounted for 0.58 CORM 0.79 ROOT 3.921.9 AGB 0.89 Surface Denit 1.93 Subsurf Denit 0.04 Aeren N 2 Accum NO WIND Porewater NH 4 + loss=10.1 82.7% accounted for 0.750.35 CORM 0.81 0.49 ROOT 3.091.11 AGB 0.76 Surface Denit 2.83 Subsurf Denit 0.11 Aeren N 2 Accum WIND Figure 9. N balance within the cores for the wind and no wind treatments. Values are in mmols per core volume over the 10 day incubation Q-PCR of N Functional Genes CONCLUSIONS Quantitative PCR was carried out for five N functional genes and the 16S rRNA gene. The five genes were: nosZ (nitrous oxide reductase), the nitrite reductases nirS and nirK, and the archaeal and bacterial amoA (ammonia monooxygenase) NirK genes were present at