1. CumulativeN2(µmolsN2h-1
)
0.00
0.10
0.20
0.30
CumulativeN2O(µmolsN2Oh-1
)
0.00
0.03
0.06
NecromassSterile Media
(a) (b)
Live Fungi NecromassSterile Media Live Fungi
Unveiling Fungal Contributions to Agricultural Soil Nitrogen Cycling Following
Application of Organic and Inorganic Fertilizers
Rebecca L. Phillips1,2*, Bongkeun Song3, Craig Tobias4, Andrew McMillan1,
Gwen Grelet1, Thilak Palmada1, Mikhail Forkin1, Bevan Weir1
1Landcare Research, NZ, 2Ecological Insights, 3Virginia Institute of Marine Science, 4University of Connecticut
Rebecca Phillips:
phillipsr@landcareresearch.co.nz
rebecca.phillips@ecologicalinsights.org
Relevance
Removal of excess nitrogen (N) can best be achieved through
denitrification processes that transform fixed N to di-nitrogen
(N2) gas in various ecosystems. The greenhouse gas nitrous
oxide (N2O) is considered an intermediate or end-product in
denitrification pathways. Some fungi reportedly produce N2 by
combining two N sources through codenitrification. The only
other known source of hybrid N2 is anammox , so formation of
hybrid N2 is reported as evidence of anammox or
codenitrification (Fig. 1). However, fungal codenitrification
reports are inconsistent. We used Bipolaris sorokiniana as a
model fungal species to examine fungal denitrification and
codenitrification using established techniques at nuetral pH. In
addition, we rigorously investigated abiotic N2 and N2O
production under not only anoxic, but also 20% oxygen
conditions.
Objectives
1. Examine effects of N (both organic and inorganic N) on N2
and N2O production and the expression of N metabolism
genes in Bipolaris sorokiniana, a common cereal pathogenic
fungus.
2. Determine if O2 (anoxic vs. 20% O2) affects production of N2
and N2O.
3. Examine how the absence of live fungi and presence of
necromass affects production of N2 and N2O.
4. Determine if N2O and N2 are produced abiotically in the
presence or absence of O2, give N source and sterile media
commonly used in fungal codenitrification experiments.
Results
Summary
1. Di-nitrogen was produced in the presence of live and dead fungi
and in sterile media only with no evidence of N2O consumption.
2. Isotope pairing experiments indicated N2 was produced abiotically
and all N2 was formed using a combination of glutamine N and
nitrite N.
3. Di-nitrogen was produced abiotically under anaerobic and fully
aerobic conditions.
4. Differential gene expression of N uptake in B. sorokiniana was
observed under different N conditions while the gene expression
in N dissimilation is still under investigation.
5. These results call into question the assumptions that (1) N2O is an
intermediate required for N2 formation, (2) production of N2 and
N2O requires anaerobiosis, and (3) hybrid N2 is evidence of
codenitrification and/or anammox.
Acknowledgements
This research is funded by the AFRI program of National Institute of Food
and Agriculture and Landcare Research Ltd, New Zealand. Special
thanks to Veronica Rollinson, Megan Peterson and Duckchul Park for
contributing their technical expertise needed for this project.
Table 1. Average (SD) of 29N2 and 30N2 recovered in the headspace following aerobic
and anaerobic incubation of sterile media at three levels of N addition, where added N
comprised 50% N from unlabelled C5H10N2O3 and 50% N from labelled 15N-NaNO2
(n=5). Values were adjusted according to helium or heliox blanks.
Figure 3. Abiotic and biotic production rates of (a) N2O and (b) N2 accumulated per
hour following N addition under both aerobic and anaerobic conditions for sterile
medium, necromass and live fungi. Boxes represent the 90th percentile data; error
bars, s.d.; n=4. Median values are the lines horizontally bisecting each box.
Figure 1. Schematic of codenitrification, anammox and known chemical
denitrification pathways, a modified adaptation from Butterbach-Bahl et al. (2013).
Selected processes potentially leading to N2O and N2 formation, involved N
compounds, their reaction pathways as well as their oxidation states are shown.
Closed circles are biotic and open circles are abiotic reactions. The last process,
abiotic hybrid N2 formation, was observed in this study.
Impact
1. Findings will encourage development of new approaches to
removal of excess nitrogen in aquatic and terrestrial
ecosystems.
2. Findings challenge researchers to re-assess abiotic
contributions to what is known as codenitrification.
3. Unveiling chemical, rather than fungal, formation of hybrid di-
nitrogen may help explain discrepancies in the N budget.
N
addition
(mmol N)
29
N2 (14
N,15
N)
(µmol)
aerobic
30
N2 (15
N,15
N)
(µmol)
aerobic
29
N2 (14
N,15
N)
(µmol)
anaerobic
30
N2 (15
N,15
N)
(µmol)
anaerobic
0
-0.012
(<0.001)
0.000
(0.000)
-0.005
(0.005)
<0.001
(<0.001)
0.5
16.274
(2.045)
0.001
(<0.001)
15.759
(0.995)
0.003
(0.002)
1.0
40.289
(2.318)
0.002
(<0.001)
31.025
(3.144)
0.007
(0.001)
1
Figure 2. Experimental set-up to test how live fungi, necromass and media only
incubated in an anoxic and 20% O2 atmosphere affects production of N2O and N2
following addition of both organic and inorganic forms of N to pure cultures under
aseptic conditions. Terms are defined as: Glut, glutamine; NaNO2, sodium nitrite; He,
helium; HeOx heliox.
Methods
log2FPKM
6
4
2
0
Glutamine Nitrite N depleted
Nitrate/nitrite transporter
Nitrate reductase
Nitrite reductase
Glutamine synthase A
Glutamine synthase B
Glutamine dehydrogenase
Glutamine dehydrogenase
(NADP+)
Glutamine synthase
Figure 4. Differential gene expression of the nitrogen uptake metabolism in B.
sorokiniana under anoxic conditions for three N treatments. RNA-seq was conducted
with triplicate samples of each incubation conditions using Illumina HiSeq.
Transcriptomes were analyzed with Topha2/Bowtie 2 and Cufflinks pipeline.
NH2-OH
Fungal Denitrifica4on
Codenitrifica4on with NO
ANAMMOX
Chemical decomposi4on NH2OH
Chemodenitrifica4on-abio4c
Surface decomposi4on NH4NO3
Abio4c hybrid N2 forma4on
-3 -1 +5 +3 +2 +1 0
Oxida4ve State
N-Org
R-NH2
NH4
+
NH3
NO3
- NO2
- N2O NO N2
?
NH2OH