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withdrawn through the perfusion cap 15 NH 4 + added to ~95%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infused slowly back in the soil Porewater withdrawn with gas tight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