Kox e al. AMB Exp (2018) 8:76
h ps://doi.o g/10.1186/s13568-018-0607-2
ORIGINAL ARTICLE
The in luence o oxygen andme hane
onni ogen ixa ion insuba c ic Sphagnum
mosses
Ma ine A. R. Kox1* , Sanni L. Aal o2,3, Timo Pen ilä4, Ka ha ina F. E wig1, Mike S. M. Je en1
and Maa je A. H. J. an Kessel1
Abs ac
Biological ni ogen ixa ion is an impo an sou ce o bioa ailable ni ogen in Sphagnum domina ed pea lands. Sphag-
num mosses ha bo a di e se mic obiome including ni ogen- ixing and me hane (CH4) oxidizing bac e ia. The inhibi-
o y e ec o oxygen on mic obial ni ogen ixa ion is documen ed o many bac e ia. Howe e , he ole o ni ogen-
ixing me hano ophs in ni ogen supply o Sphagnum pea mosses is no well explo ed. He e, we in es iga ed he
ole o bo h oxygen and me hane on ni ogen ixa ion in suba c ic Sphagnum pea mosses. Fi e species o Sphagnum
mosses we e sampled om wo meso ophic and h ee oligo ophic si es wi hin he Lakkasuo pea land in O i esi,
cen al Finland. Mosses we e incuba ed unde ei he ambien o low oxygen condi ions in he p esence o absence
o me hane. S able iso ope ac i i y assays e ealed conside able ni ogen- ixing and me hane-assimila ing a es a
all si es (1.4 ± 0.2 µmol 15N–N2 g−1 DW day−1 and 12.0 ± 1.1 µmol 13C–CH4 g−1 DW day−1, espec i ely). Addi ion o
me hane did no s imula e inco po a ion o 15N-ni ogen in o biomass, whe eas oxygen deple ion inc eased he ac i -
i y o he ni ogen- ixing communi y. Analysis o he 16S RNA genes a he bac e ial communi y le el showed a e y
di e se mic obiome ha was domina ed by Alphap o eobac e ia in all si es. Bona ide me hane-oxidizing axa we e
no e y abundan ( ela i e abundance less han 0.1%). Based on ou esul s we conclude ha me hano ophs did no
con ibu e signi ican ly o ni ogen ixa ion in he in es iga ed pea lands.
Keywo ds: Diazo ophy, Me hane oxida ion, Oxygen, Pea land, Sphagnum moss, 16S RNA amplicon sequencing
© The Au ho (s) 2018. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License
(h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium,
p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license,
and indica e i changes we e made.
In oduc ion
Biological ni ogen (N2) ixa ion is o g ea impo ance o
he Ea h’s biosphe e as i is he majo na u al p ocess o
eplenish biologically a ailable ni ogen. In nu ien -lim-
i ed ecosys ems, he be e compe i o s a e o en hose
ha ind al e na i e ways o gain hei equi ed nu i-
en s, i.e. by engaging in a symbiosis wi h o he o ganisms
( an de Heijden e  al. 2008). Pea lands a e nu ien -
limi ed ecosys ems ha ha e been s udied in ensi ely
due o hei signi ican ole in he global ca bon (C)
cycle. App oxima ely 1/3 o he global e es ial ca bon
is s o ed as dead o ganic ma e in pea lands (Go ham
1991). In Sphagnum-domina ed pea lands, Sphagnum
mosses a e he ecosys em enginee s. They ou compe e
ascula plan species in a ious ways (Malme e  al.
2003), bu mainly by c ea ing and main aining acidic (pH
3–5) and wa e logged condi ions. In addi ion, hei own
biomass is di icul o deg ade, which con ibu es o he
slow decomposi ion and consequen ial accumula ion o
dead o ganic ma e (Clymo 1963, 1964; an B eemen
1995). Pea ens ha a e meso ophic o oligo ophic
ecei e N om a mosphe ic deposi ion and g ound wa e
in low. Compa ed o meso ophic ens, oligo ophic ens
ecei e less nu ien s, leading o nu ien limi a ion and
lowe p oduc i i y (La mola e al. 2014). In bo h sys ems,
Sphagnum mosses minimize nu ien a ailabili y o as-
cula plan s by apid and e icien nu ien up ake (F i z
e al. 2014).
Open Access
*Co espondence: [email p o ec ed].nl
1 Depa men o Mic obiology, Radboud Uni e si y, Nijmegen, The
Ne he lands
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Page 2 o 9
Kox e al. AMB Exp (2018) 8:76
In hese nu ien -limi ed pea lands, Sphagnum ci cum-
en s N-limi a ion by engaging in a ela ionship wi h N2
ixing mic oo ganisms (diazo ophs). Diazo ophs con-
e a mosphe ic N2 o ammonia (NH3). This is a cos ly
p ocess (16 ATP pe N2 molecule) ca alyzed by an oxy-
gen (O2) sensi i e ni ogenase enzyme (Pos ga e 1982).
In na u e, diazo ophs a e abundan , di e se and exis as
ee-li ing s a e as well as in symbiosis wi h plan s. Many
moss species a e known o ha bo a di e se diazo ophic
communi y (Leppänen e al. 2013; Vile e al. 2014; Kno
e al. 2014; Kox e al. 2016; Wes on e al. 2015). The diazo-
ophic ac i i y associa ed wi h Sphagnum suppo s and
explains high concen a ions o N in Sphagnum biomass
(Vile e al. 2014). Al hough i is e iden ha he mosses
bene i om he N supply by diazo ophs, he bene i s
o he mic oo ganism a e less appa en . Especially, since
i was ecen ly pos ula ed ha op imal condi ions o
diazo ophic mic oo ganisms and he moss-hos a e e y
di e en ( an den Elzen e al. 2017).
Biological N2 ixa ion ac i i y is mos commonly de e -
mined using he ace ylene educ ion assay (Ha dy e al.
1968). Ini ial s udies on he Sphagnum associa ion wi h
N2 ixing pa ne s indica ed ha mainly cyanobac e-
ia con ibu ed o he inco po a ion o N in Sphagnum
biomass (Be g e al. 2012; Lindo e al. 2013). Al hough
he ace ylene educ ion assay is a sensi i e and easy
way o de e mine N2 ixing ac i i y, ace ylene i sel is an
i e e sible inhibi o o he me hane monooxygenase
enzyme in me hane (CH4) oxidizing bac e ia (me hano-
ophs). This p e en s me hano ophs om me aboliz-
ing ca bon, ul ima ely leading o cell dea h. Due o he
use o ace ylene o measu e N2 ixa ion a es, he ole
o diazo ophic me hano ophs may ha e been unde -
es ima ed (Leppänen e al. 2013; Vile e al. 2014). As an
al e na i e me hod o measu e N2 ixa ion, 15N–ni ogen
(15N–N2) s able iso ope inco po a ion can be used. Wi h
his me hod, Vile e al. (2014) showed a signi ican con-
ibu ion o CH4 dependen N2 ixa ion o he Sphagnum
N-pool (Vile e al. 2014). Se e al Sphagnum 16S RNA
gene-based mic obiome s udies indica ed ha Alphap o-
eobac e ia we e he mos abundan N2 ixing bac e ia
(B agina e al. 2012, 2014; Shche bako e al. 2013; Wa -
en e al. 2017). In addi ion, di e si y s udies based on
ni ogenase ni H gene also showed ha Alphap o eobac-
e ia we e highly ep esen ed (B agina e al. 2012; Vile
e al. 2014; Kox e al. 2016; Wa en e al. 2017). Some o
hese N2 ixing Alphap o eobac e ia we e ound o be
me hano ophs (i.e. ype II Me hylosinus spp., Me hylo-
cys is spp.) as well.
Me hano ophs associa ed wi h Sphagnum mosses
ha e been shown o suppo moss g ow h by p oducing
ca bon dioxide (CO2) which is subsequen ly aken up
by Sphagnum, especially unde CO2 limi ing condi ions
(Raghoeba sing e al. 2005; Kip e al. 2010). The me ha-
no ophs a e hypo hesized o bene i om O2 p oduced
by Sphagnum and by being p o ec ed om p eda o s
inside Sphagnum’s hyaline cells (Kos ka e  al. 2016).
Howe e , ac i i y measu emen s o me hano ophic
diazo ophs in en i onmen al Sphagnum moss samples
ha e yielded con as ing esul s (La mola e  al. 2014;
Leppänen e al. 2014). Due o he high ene gy demand o
N2 ixa ion, a me hano ophic diazo oph equi es high
me hano ophic ac i i y in o de o sus ain N2 ixa ion.
The e o e, pa ame e s con olling me hano ophy sup-
posedly con ibu e o he obse ed a iabili y in he CH4
dependen N2 ixa ion (Ho and Bodelie 2015). To his
poin , he composi ion o he ac i e N2 ixing communi y
associa ed wi h Sphagnum and ac o s ha a ec his
ac i i y ha e been in es iga ed in only a ew s udies (Ho
and Bodelie 2015). The mic oo ganisms in ol ed in bio-
logical N2 ixa ion ha e been iden i ied, bu hei ela i e
impo ance and ac o s con olling hei ac i i y emain
un esol ed (Ho and Bodelie 2015).
The aim o his s udy was o elucida e he e ec o O2
and CH4 on N2 ixa ion in Sphagnum mosses o oligo-
ophic and meso ophic suba c ic Sphagnum-domi-
na ed pea lands. Sphagnum mosses we e sampled om
wo meso ophic and h ee oligo ophic si es (Lakkasuo
pea land in O i esi, Finland). Sphagnum mosses we e
incuba ed unde ei he ambien o low oxygen condi-
ions in he p esence o absence o 13C-labeled CH4 and
15N-labeled N2. Highe N2 ixa ion ac i i y is expec ed o
se e e nu ien limi ed condi ions (oligo ophic) com-
pa ed o meso ophic condi ions. On he o he hand,
mo e bu e ed condi ions and highe pH p e ailing in
meso ophic pea lands ha e p e iously been shown o be
bene icial o N2 ixa ion ( an den Elzen e al. 2017). We
hypo hesize ha CH4 will s imula e N2 ixa ion ac i i y
and his ac i i y will be highes in low O2 condi ions.
Ma e ials andme hods
Si e desc ip ion andexpe imen al se ‑up
The s udy was pe o med in he suba c ic, nu ien lim-
i ed mi e complex Lakkasuo in cen al Finland (61°47′N
24°18′E; 150m.a.s.l.). Lakkasuo is a well-s udied bo eal
mi e complex, wi h an annual N inpu ia ainwa e o
0.40 g N m−2 yea −1 (Laine 2004). Sphagnum mosses
we e collec ed in Sep embe 2014 om oligo ophic
and meso ophic ens and a ainwa e - ed bog wi hin
he same pea land basin (Addi ional ile 1: Table S1).
The meso ophic en si e domina ed by Sphagnum sub-
secundum (si e A) was a we en sys em, whe eas meso-
ophic en si e B wi h Sphagnum ob usum was d ie .
The oligo ophic en si e was na u ally di ided in wo
pa ches whe e Sphagnum allax (si e C) and Sphagnum
papillosum (si e D), espec i ely, we e dominan . The
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Kox e al. AMB Exp (2018) 8:76
ainwa e - ed bog si e was domina ed by Sphagnum
majus (si e E). See Addi ional ile1: Table S2 o a ull
o e iew o he si e ege a ion index.
Incuba ions
To es whe he a ailabili y o O2 and/o CH4 would
a ec mic obial N2 ixa ion a es, Sphagnum mosses
we e incuba ed unde wo di e en O2 concen a ions
(ambien and deple ed) and wi h o wi hou 13C–CH4.
N2 ixa ion and CH4 oxida ion ac i i y we e es ima ed
by measu ing he inco po a ion o 15N–N2 and 13C–CH4
espec i ely. To co ec o he na u al p esence o 15N
and 13C in he moss, con ols we e incuba ed wi hou any
labelled gasses.
Pe ea men , moss samples (10 plan le s pe bo -
le, op 3cm) om each si e we e incuba ed in 180ml
plas ic Scholl lasks and closed wi h sep um con aining
sc ew-caps. Be o e incuba ion, he esh weigh (FW)
o he mosses was de e mined. All bo les ecei ed 5%
15N–N2 in he headspace, excep o he backg ound con-
ols. 13C–CH4 ea ed samples ecei ed addi ionally 5%
13C–CH4 in he headspace. The headspace o he ambi-
en O2 condi ion consis ed o ai . Low O2 condi ions we e
achie ed by eplacing ( ou cycles o acuum—helium
gassing) he headspace wi h an a i icial gas mix u e con-
sis ing o N2 gas (80%), CO2 (0.04%) and helium (20%).
Oxygen was no added, so ha he only sou ce o oxygen
was pho osyn hesis by he mosses. Samples we e incu-
ba ed o 48h ou side unde p e ailing ligh and empe -
a u e condi ions (Jy äskylä, Finland, Sep embe 2014).
S able iso ope inco po a ion
A e he incuba ion, moss samples we e s o ed a
− 20°C. Nex , samples we e eeze-d ied using an Alpha
1-4LD plus (Ma in Ch is GmbH, Os e ode am Ha z,
Ge many) and s o ed a − 20°C. D ied s ems om each
ea men we e pooled and dis up ed using a beadbea e
a 1500 pm o 3min (Mic odismemb a o U, B. B aun
Bio ech In ., Meisungen, Ge many). To de e mine he
ac ion o 15N and 13C inco po a ed in plan biomass,
app oxima ely 5 and 0.225mg, espec i ely, we e pu in o
5 × 8mm in cups in duplo. Nex , samples we e com-
bus ed by lash combus ion (1800°C) on a CNS analyze
(EA 1110 Ca lo E ba, The mo Fishe Scien i ic, Wal ham,
MA, USA) coupled o an Iso opic Ra io Mass Spec ome-
e (Finnigan Del aPlus, The mo Elec o GmbH, B emen
Ge many) ia an in e ace (Con lo III, The mo Elec o
GmbH, B emen, Ge many). 15N and 13C con en o all
mosses be o e incuba ion we e uni o m.
The 15N–N2 ixa ion a es we e calcula ed by co ec ing
he δN o he en iched plan ma e ial o he na u al 15N
con en , a e which he co ec ed 15N inc ease was con-
e ed o 15N–N2 ixa ion a es (µmolNg−1DWday−1).
13C–CH4 oxida ion a es we e calcula ed in a simila ash-
ion as he 15N–N2 ixa ion a es. Fi s , he backg ound
13C label was sub ac ed om he measu ed inc ease in
13C o he biomass. The co ec ed 13C inc ease was sub-
sequen ly con e ed in o 13C–CH4 oxida ion a es.
Po ewa e composi ion
A each si e pH was measu ed and po e wa e samples
we e aken using hizons (po e size 2µm; 5cm leng h)
a he su ace (0cm), 5, 10 and 15cm dep h. Elemen
concen a ions o Al, Ca, Fe, K, Mg, Mn, Na, P, S, Si and
Zn in po e wa e (10ml, acidi ied wi h 1ml HNO3) we e
analyzed by induc i ely coupled plasma op ical emission
spec oscopy (ICP-OES iCAP 6000, The mo Fishe Sci-
en i ic, Wal ham, MA, USA). Concen a ions o NH4+,
NO3−, PO43− we e analyzed colo ime ically wi h a 3
Au o Analyze sys em (B an and Luebbe GmbH, No de -
s ed , Ge many) using ammonium molybda e (Hen iksen
1965), hyd azine sul a e (Kamphake e al. 1967) o salicy-
la e (G assho and Johannsen 1972). Cl was de e mined
wi h a Technicon Flame Pho ome e IV Con ol (B an
and Luebbe, No de s ed , Ge many).
To de e mine he concen a ion o dissol ed me hane,
3ml exe aine s (Labco, Ce edigion, UK) we e p epa ed
wi h 1g o NaCl and closed wi h a sep um cap. Nex ,
1ml o he po ewa e collec ed ia hizons was added
o he closed exe aine s immedia ely a e sampling. The
p essu e in he ials was measu ed and he CH4 concen-
a ion in he headspace o he exe aine was measu ed
using he GC as desc ibed abo e.
Olsen‑P de e mina ion
Diges ion o d ied and g ound Sphagnum issue was pe -
o med o ob ain he Olsen P concen a ion as desc ibed
( an den Elzen e al. 2017). In b ie , samples we e hea ed
o 120 °C o 45 min in a mix u e o 500 µl HNO3
(65%w/w) wi h 200µl H2O2 (30%w/w). Nex , samples
we e dilu ed wi h demine alized wa e and measu ed by
induc i ely-coupled plasma emission spec ome y (IRIS
In epid II, The mo Elec on co po a ion, F anklin, MA,
USA).
DNA ex ac ion and16S RNA gene ampli ica ion
Sphagnum mosses o molecula analysis we e sam-
pled di ec ly om he ield and immedia ely pu in liq-
uid ni ogen. In he labo a o y, samples we e s o ed
a − 80°C. DNA ex ac ion was pe o med on 0.5g o
sample ( esh weigh ), using he Fas DNA SPIN ki o
soil (MP Biomedicals, San a Ana, CA, USA), ollow-
ing manu ac u e s p o ocol. Beadbea ing was inc eased
o 2 × 1.5 min a 50 Hz using a issue lyse (LT, Qia-
gen, Hilden, Ge many) DNA yield was assessed using
Qubi luo ome ic analysis (The mo Fishe Scien i ic,
Page 4 o 9
Kox e al. AMB Exp (2018) 8:76
Wal ham, MA, USA). Ba coded 16S RNA gene amplicon
lib a y was p epa ed wi h a 2 s ep PCR p o ocol (Be y
e al. 2011). Used p ime s a ge ed he V3–V4 egion o
he 16S RNA gene o mos bac e ia (341F-785R; Klind-
wo h e al. 2013). The 25μl PCR eac ions o he i s
PCR con ained 12.5μl Quan a pe ec a mix (Quan abio,
Be e ly, MA, USA), 1μl o each p ime (20μM), 1μl
DNA (0.5ng/μl). The PCR p og am consis ed o 25 cycles
o 95°C 1min, 60°C, 1min, 72°C o 2min, a e which
inal elonga ion 10min 72°C. The ob ained PCR p od-
uc s we e checked o pu i y and size on 1.5% aga ose
gel. PCR p oduc s o 7 pa allel eac ions we e pooled and
pu i ied using QIAquick pu i ica ion ki (Qiagen, Hilden,
Ge many) ollowing manu ac u e s p o ocol. The second
PCR was pe o med o ba code all samples (bcPCR). Fo
bcPCR he gene-speci ic The same PCR p ime s (341F
and 785R) as o he i s PCR eac ion we e agged wi h
adap e sequences, speci ic ba codes and key sequences
a he 5′ end, compa ible wi h Ion To en sequencing
echnology ( o al 60–62 nucleo ides pe p ime ). Fo
each sample six nes ed bcPCR eac ions we e conduc ed
in pa allel, which we e combined a e p oduc pu i y
and size con ol. Subsequen ly, nes ed PCR p oduc s
we e pu i ied using QIAquick pu i ica ion ki (Qiagen,
Hilden, Ge many) ollowing manu ac u e s p o ocol.
Amplicon sequencing
P io o Ion o en lib a y cons uc ion, concen a ion
and agmen leng h o samples was de e mined wi h a
Bioanalyze 2100 and he High Sensi i i y DNA ki (Agi-
len Technologies, San a Cla a, CA, USA). The lib a ies
we e dilu ed o a inal concen a ion o 26pM. Acco d-
ing o manu ac u e ’s p o ocol he lib a y agmen s
we e a ached o Ion Sphe e pa icles using he One
Touch ins umen and Ion PGM Templa e OT2 400 ki
(Li e Technologies, Ca lsbad, CA, USA). Subsequen ly,
Ion Sphe e pa icles we e loaded on an Ion 318 2 Chip
o he i s un and an Ion 314 2 chip o he second
un, a e which he amplicon lib a ies we e sequenced
acco ding o manu ac u e ’s p o ocol using he ION
PGM Sequencing 400 Ki , using 850 nucleo ide lows.
Run 1 (318 chip) esul ed in 35,790 eads wi h an a e age
ead leng h o 132 base pai s. Run 2 esul ed in 180,870
eads wi h an a e age ead leng h o 275 base pai s.
Sequence analysis
Sequences om bo h uns we e me ged esul ing in
a o al o 216,660 eads wi h an a e age leng h o 252
nucleo ides. The eads we e analysed using Mo hu
( 1.38; Schloss e  al. 2009) and he Mo hu 454 SOP
(Schloss e al. 2011). Fi s , eads we e quali y il e ed on
ead leng h (200–450 bp) allowing o homopolyme s
(maximum 8), a maximum o 2 di e ences wi h he
p ime sequence and minimum a e age quali y sco e o
20 o e a window o 50 base pai s. Imp o ed eads we e
aligned o he Sil a n da abase ( elease 123) (Quas
e al. 2013). Chime a’s we e emo ed using Uchime. Nex ,
eads we e classi ied a boo s ap alue o 80%, a e
which he unwan ed and non- a ge lineages A chaea,
Euka yo a, chlo oplas , mi ochond ia and unknown we e
emo ed. The inal da ase om which OTUs we e clus-
e ed consis ed o 49,975 sequences o which 16,493 we e
unique, wi h an a e age ead leng h o 221 nucleo ides
( o a de ailed o e iew o quali y il e ing see Addi ional
ile1: Table S3). OTU’s we e clus e ed using he a e age
clus e ing algo i hm a a cu o o 0.03, ollowed by sin-
gle on emo al and esul ed in 2001 OTUs. Sequences
we e deposi ed in NCBI SRA unde p ojec numbe
PRJNA432031.
S a is ical analysis
Da a was analyzed using R e sion 3.4.0 by he R De el-
opmen Co e Team (2017). No mali y o he esiduals
was es ed using Shapi o–Wilk’s no mali y es (s a s-
package). Homogenei y o a iance was es ed using
Le ene’s es (ca -package). In he analysis o N2 ixa ion,
CH4 oxida ion ac i i y and po e wa e composi ion, he
si es we e g ouped based on hei nu i ional ophic
s a e, esul ing in he le els: meso ophic and oligo-
ophic si es. G ouping was only allowed i he di e ence
be ween species was no signi ican (p > 0.05), which was
es ed i s using a one-way ANOVA. N2 ixa ion ac i -
i y da a was ans o med by squa e oo ans o ma ion
o pe mi pa ame ic es s. Nex , di e ences in N2 ixa-
ion ac i i y unde he di e en O2 condi ions, p esence
o 13C–CH4 and si es (oligo ophic o meso ophic), we e
es ed using a 3-way ANOVA (s a s-package; o sample
size (n) see Addi ional ile1: Table S5). 13C–CH4 oxida-
ion ac i i y da a was no mally dis ibu ed. Di e ences
in ac i i y be ween he wo oxygen ea men s and si es
we e es ed using a 2-way ANOVA (s a s-package). All
g aphs we e cons uc ed in R using ggplo 2.
Fo downs eam analysis o he communi y compo-
si ion he OTU able and axonomy iles gene a ed by
Mo hu , we e impo ed in o R and analyzed using phy-
loseq (McMu die and Holmes 2013).
Resul s
Po ewa e composi ion
The oligo ophic si es we e mo e acidic compa ed o
he meso ophic si es and con ained less dissol ed
CH4 (see Addi ional ile 1: Figu e S1). Concen a-
ions o NH4+, NO3− and PO4− we e lowes in he
ainwa e ed bog (no shown) and we e p esen in
sligh ly highe concen a ions in bo h oligo ophic
and meso ophic ens. Mo eo e , bo h oligo ophic
Page 5 o 9
Kox e al. AMB Exp (2018) 8:76
and meso ophic ens showed o e all highe elemen-
al concen a ions o S, Fe Mg, K and Ca, compa ed o
he bog. Olsen P was highe in mosses o igina ing om
meso ophic si es (0.65 ± 0.02 μmol P g−1 DW, see
Addi ional ile 1: Figu e S2) han in oligo ophic si es
(0.54 ± 0.01μmolPg−1DW).
N2 ixa ion and CH4 oxida ion ac i i y
N2 ixa ion a es as measu ed by 15N–N2 inco po a ion
we e simila in all si es (meso ophic 1.6 ± 0.4 and oli-
go ophic 1.5 ± 0.4 μmol 15N–N2g
−1 DW day−1). The
N2 ixa ion a es we e no a ec ed by 13C–CH4 addi ion
(p > 0.05), bu we e a ec ed by absence o O2 (F1,16 = 6.40
p = 0.022; see Fig.1). Incuba ion unde low O2 condi ions
yielded highe N2 ixa ion ac i i y (2.0 ± 0.5 μmol 15N–
N2g−1DWday−1) compa ed o incuba ions a ambien
O2 condi ions (0.8 ± 0.2 μmol 15N–N2g−1DWday−1).
CH4 oxida ion occu ed in all incuba ions, and 13C–
CH4 inco po a ion was simila (p > 0.05) in all incuba ions
(o e all a e 12.0 ± 1.1 µmol 13C–CH4g
−1 DW day−1)
(Fig.2).
Mic obial communi y analysis
The bac e ial communi y analysis showed ha he P o eo-
bac e ia we e he mos dominan phylum in all samples
(Fig.3). In he oligo ophic en samples Alphap o eobac-
e ia we e he mos abundan class p esen (> 50% wi hin
phylum P o eobac e ia; see Fig.3 and Addi ional ile1:
Figu e S4). The mos abundan amilies wi hin he Alp-
hap o eobac e ia a e: Ace obac e aceae and he Caulo-
bac e aceae. The o he phyla ha made up a leas 1% o
Meso ophic Oligo ophic
Ambien Low Ambien Low
0
1
2
3
4
5
O condi ion
15N-N2 inco po a ion
µmol g−1DW day−1
Labelling
15
15
2
N-N
N-N + 4
13
2C-CH
2
**
Fig. 1 Inco po a ion o 15N–N2 (μmol g−1 DW day−1) in he meso ophic ens (n = 2) and oligo ophic ens and bog (n = 3) Sphagnum mosses
incuba ed wi h ambien O2 condi ions (da k g ey ba s) o low O2 condi ions (ligh g ey ba s), supplemen ed wi h 5% 15N–N2 (da k g ey ba s) o 5%
15N–N2 + 5% 13C–CH4 (ligh g ey ba s)
0
5
10
15
20
25
Meso ophic Oligo ophic
Si e
13C-CH4inco po a ion
µmolg
−1DW day−1
O condi ion
Ambien
Low
2
Fig. 2 Inco po a ion o 13C–CH4 (μmol g−1 DW day−1) in Sphagnum
mosses om meso ophic ens and oligo ophic ens and bog si es,
incuba ed wi h ambien O2 condi ions (da k g ey ba s) o low O2
condi ions (ligh g ey ba s)

Page 6 o 9
Kox e al. AMB Exp (2018) 8:76
he mic obial communi y and ha appea ed o be p esen
in all si es we e he Acidobac e ia, Ve ucomic obia and
he Planc omyce es (Fig.3). The phylum o Acidobac e ia
had a compa able ela i e abundance ac oss all si es. In
con as , he Bac e oide es and Cyanobac e ia we e only
p esen in some si es. Genuine me hano ophic p o eo-
bac e ial 16S RNA sequences ( ype I and ype II) we e
less han 0.1% in ela i e abundance in all samples.
In he meso ophic si es, highe mic obial di e si y was
obse ed (see Addi ional ile1: Figu e S3) compa ed o
he oligo ophic si es. Ve ucomic obia we e ela i ely
less abundan in he meso ophic si es compa ed o he
oligo ophic si es, whe eas he Bac e oide es (4% o o al
eads) and Cyanobac e ia (3% o o al eads) we e mo e
abundan ly ound in he meso ophic si es.
Discussion
This s udy aimed o elucida e he e ec o CH4 and O2
a ailabili y on N2 ixa ion ac i i y in oligo ophic and
meso ophic Sphagnum-domina ed pea lands. Biologi-
cal N2 ixa ion ac i i y was expec ed o be s imula ed by
he p esence o CH4 and he absence o O2. Fo he si e
e ec (oligo ophic s. meso ophic), expec a ions we e
no so clea -cu , as di e en con ounding ac o s play a
ole. Highe a ailabili y o N in meso ophic si es may
dec ease he demand o biological N2 ixa ion, while
highe P con en may lead o ela i e N sca ci y. Also, he
be e bu e ing and highe pH o meso ophic si es may
be a o able o N2 ixa ion.
In he oligo ophic s udy si es, howe e , e y low con-
cen a ions o he N compounds ni a e and ammonium,
ypical o oligo ophic condi ions, we e no ound (Addi-
ional ile1: Figu e S1); hey we e compa able o concen-
a ions a he meso ophic si es. Also he N:P ela ion in
po ewa e was simila , so ha no bigge ela i e N sca -
ci y could be diagnosed o he oligo ophic si e. Only in
he biomass, sligh ly highe Olsen P e lec ed a gene ally
highe a ailabili y o P in he meso ophic si e. Taken
oge he , his may explain why N2 ixa ion a es as meas-
u ed by 15N–N2 inco po a ion did no di e be ween
nu i ional meso ophic and oligo ophic ophic s a es.
This is in con as wi h ecen obse a ions by a s udy o
Van den Elzen e al. (2017), whe e i was ound ha N2
ixa ion is s imula ed by mo e bu e ed condi ions and
highe phospho us a ailabili y. I is possible ha N2 ixa-
ion a es in ou esea ch we e a ec ed by o he ac o s
such as he ela i e d y pe iod in which samples we e
aken o lack o ce ain ace elemen s (Vi ousek e al.
2013; Wa en e al. 2017).
Me hane addi ion lead o signi ican up ake o 13C–
CH4 de i ed ca bon, bu did no a ec N2 ixa ion a es
compa ed o con ols, indica ing ha CH4 dependen N2
ixa ion is p obably no a majo con ibu o o N2 ixa-
ion in he s udied ecosys em. These esul s a e simila
o indings o bo h Leppänen e al. (2014) and Wa en
0.00
25.0
50.0
75.0
100
Si e A
Meso ophic en
S. subsecundum
Si e B
Meso ophic en
S. ob usum
Si e C
Oligo ophic en
S. allax
Si e D
Oligo ophic en
S. papillosum
Si e E
Oligo ophic bog
S. majus
Rela i e Abundance
(Phyla >1%)
Phylum
Acidobac e ia
Ac inobac e ia
Bac e oide es
Chlo o lexi
Cyanobac e ia
Gemma imonade e
s
Pa cubac e ia
Planc omyce es
P o eobac e ia
Ve ucomic obia
WD272
unclassi ied
Fig. 3 Taxonomic composi ion (16S RNA) o he mic obial communi y associa ed wi h Sphagnum moss om si e A–E. Ba cha s ep esen he ela-
i e abundance o he di e en phyla p esen in each si e. Only phyla wi h a RA > 1% a e shown
Page 7 o 9
Kox e al. AMB Exp (2018) 8:76
e al. (2017). In bo h s udies he addi ion o me hane o
ba ch incuba ions wi h Sphagnum did no a ec N2 ixa-
ion ac i i y ei he . Fu he mo e, bo h s udies showed
much lowe CH4 oxida ion ac i i y han obse ed in he
p esen s udy, ques ioning he possibili y o an N2- ixing
li e s yle o he me hano oph (Ho and Bodelie 2015).
Al hough me hano ophs comp ised less han 0.1% o he
16S RNA, his alone does no exclude hem om being
a majo con ibu o o N2 ixa ion. Recen ly Bae e al.
(2018) showed ha a chaea cons i u ing only 0.27% o
he mic obial communi y could accoun o 44% o he N
ixed in an Flo ida pea sys em. In addi ion, N2 ixa ion
a es may di e o e seasons (Le and Michelsen 2014)
and hus he mic obial guilds ac i ely pe o ming N2 ixa-
ion may a y wi h season. To u he con i m which N2
ixing mic oo ganisms a e ac i e, ansc ip omic s udies
combined wi h ac i i y assays o e di e en seasons a e
equi ed in u u e s udies.
In con as o CH4 addi ion, O2 deple ion did s imula e
N2 ixa ion. While he mosses in he incuba ion bo les
did p oduce O2 as hey pe o med oxygenic pho osyn-
hesis du ing he day, he ne e heless lowe O2 le el
esul ed in highe N2 ixa ion a es compa ed o he
ambien O2 le els, which is in line wi h ou hypo hesis.
The ni ogenase enzyme ha is esponsible o N2 ixa-
ion is i e e sibly inhibi ed by O2 in mos mic oo gan-
isms (Vi ousek e al. 2002). Ou inding indica es ha
ac i i y o diazo ophs migh be s ongly con olled by
he oxygen concen a ion, and he e o e by dep h, posi-
ion unde o abo e he wa e le el and he cu en a e
o pho osyn hesis o Sphagnum. N2 ixa ion migh be
highe in he da k han in he ligh , due o O2 elease by
Sphagnum du ing he ligh pe iod.
The CH4 oxida ion a es obse ed in his s udy a e
compa able o CH4 oxida ion a es measu ed in o he
Sphagnum mosses (Kip e al. 2010). The CH4 oxida ion
a es we e sligh ly highe in he meso ophic si es p ob-
ably due o highe pH. High CH4 oxida ion ac i i y is
essen ial o me hano ophs in o de o ul il he high
ene gy equi ed o N2 ixa ion (Ho and Bodelie 2015).
Wi h hese high CH4 oxida ion a es, ene gy seems no
he limi ing ac o o CH4 dependen N2 ixa ion. Taken
oge he i seems highly unlikely ha CH4 dependen N2
ixa ion is a majo N-supplie o he N-pool o he Sphag-
num mosses in es iga ed in his s udy.
Mic obial communi y composi ion
The obse ed mic obial communi y composi ion o he
Sphagnum associa ed mic obial communi y is compa a-
ble o he composi ion ound in o he s udies (B agina
e al. 2012, 2014; Kox e al. 2016) wi h he P o eobac e-
ia as dominan phylum (B agina e al. 2011, 2014; Pu -
kinen e al. 2014). The po en ial o pe o m N2 ixa ion
has been epo ed o many phylogene ic g oups (Zeh
e al. 2003; Dixon and Kahn 2004; Khadem e al. 2010),
o which he Alphap o eobac e ia, Cyanobac e ia, Bac e-
oide es and Ve ucomic obia we e p esen in ou s udy.
The Alphap o eobac e ia which we e p esen in all si es,
ha e p e iously been iden i ied as po en ially impo an
N2 ixing pa ne s o Sphagnum mosses (B agina e al.
2012; Vile e al. 2014). RT-qPCR and/o ansc ip omic
sequencing we e no pe o med in his s udy, he e o e
we can only specula e which N2 ixe s we e ac i e.
When we compa e he di e en si es, i is appa en ha
he oligo ophic ens only con ain a limi ed numbe o
cyanobac e ial species, whe eas he meso ophic ens do
con ain mo e Cyanobac e ia. Po en ially he Cyanobac e-
ia may ha e been a ec ed by he nu ien a ailabili y in
he pea land, wi h hem becoming mo e abundan wi h
highe nu ien and p oduc i i y le els. The Ve ucomi-
c obial OTU coun inc eased om meso ophic o he
oligo ophic si es, his sugges s ha species o Ve ucomi-
c obia migh h i e be e unde mo e oligo ophic con-
di ions (B agina e al. 2015). The high ela i e abundance
o Ve ucomic obia p esen in he pea mosses ha e been
ound in ea lie s udies as well (Pu kinen e al. 2014; B a-
gina e al. 2014). Fu u e isola ion o Ve ucomic obial spe-
cies and physiological s udies should e eal hei me abolic
po en ial wi h espec o N2 ixa ion and CH4 oxida ion.
Up o his poin , ac o s con olling N2 ixa ion in
Sphagnum mosses ha e yielded con as ing esul s. This
s udy has ocused on he e ec o O2 and CH4 on N2 ixa-
ion ac i i y in Sphagnum mosses in oligo ophic and
meso ophic suba c ic pea lands. Based upon he esul s
we conclude ha CH4 dependen N2 ixa ion was no a
majo sou ce o ni ogen o Sphagnum mosses a he
s udied si es. The mic obial communi y associa ed wi h
Sphagnum was domina ed by P o eobac e ia (mainly
Alphap o eobac e ia), which is compa able o o he s ud-
ies. Fu u e s udies should combine ield and mesocosm
s udies, wi h ac i i y assays, communi y analysis and
ansc ip omic da a o unco e con ols o biological
ni ogen ixa ion in Sphagnum mosses.
Au ho s’ con ibu ions
MARK, KFE, MSMJ and SLA designed he expe imen s; MARK, KFE, SLA, and TP
sampled he pea mosses and pe o med he expe imen s; MARK and MAHJ K
analysed he da a. The manusc ip was w i en by MARK, MAHJ K and MSMJ.
All co-au ho s i e a i ely e iewed he manusc ip p io o submission. All
au ho s ead and app o ed he inal manusc ip .
Au ho de ails
1 Depa men o Mic obiology, Radboud Uni e si y, Nijmegen, The Ne h-
e lands. 2 Depa men o Biological and En i onmen al Science, Uni e si y
Addi ional ile
Addi ional ile1. Supplemen a y ables and igu es.
Page 8 o 9
Kox e al. AMB Exp (2018) 8:76
o Jy äskylä, PO Box 35, 40014 Jy äskylä, Finland. 3 Depa men o En i on-
men al and Biological Sciences, Uni e si y o Eas e n Finland, PO Box 1627,
70211 Kuopio, Finland. 4 Na u al Resou ces Ins i u e Finland, PO Box 2,
00791 Helsinki, Finland.
Acknowledgemen s
Ma ja Tii ola is hanked o eedback on he expe imen s and manusc ip .
Je oen F ank is hanked o se ing up bioin o ma ics pipelines and o use ul
discussions on 16S RNA gene di e si y analysis.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
A ailabili y o da a and ma e ials
All sequencing da a has been deposi ed in he NCBI SRA da abase, P ojec
Numbe PRJNA432031.
Consen o publica ion
No applicable.
E hics app o al and consen o pa icipa e
No applicable.
Funding
M.A.R.K. was suppo ed by Eu opean Resea ch Council Ecomom 339880 o
M.S.M.J., who was u he suppo ed by he Ne he lands O ganisa ion o
Scien i ic Resea ch (SIAM G a i a ion G an 024 002 002 and Spinoza Awa d).
K.F.E. was suppo ed by he Ne he lands O ganisa ion o Scien i ic Resea ch
(Veni G an 863.13.007). M.A.H.J. .K. was suppo ed by Technology Founda-
ion STW (G an 13146). S.L.A. was suppo ed by Academy o Finland p ojec
260797 and he Eu opean Resea ch Council Consolida o p ojec Mic o-RIP
(615146).
Publishe ’s No e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub-
lished maps and ins i u ional a ilia ions.
Recei ed: 24 Ma ch 2018 Accep ed: 28 Ap il 2018
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