G eenhouse gas me abolizing
p oka yo es in pea lands
Disse a ion
zu E langung des akademischen G ades
eines Dok o s de Na u wissenscha en
D . e . na .
de Fakul ¨a ¨u Biologie, Chemie und Geowissenscha en
de Uni e si ¨a Bay eu h
o geleg on
Ka ha ina Palme
Bay eu h, den 09.05.2012
Die o liegende A bei wu de on Sep embe 2008 bis Mai 2012 am Leh s uhl
¨u ¨
Okologische Mik obiologie de Uni e si ¨a Bay eu h un e de Lei ung on PD
D . Ma cus A. Ho n ange e ig . Teile de p ak ischen A bei wu den au den
Fo schungss a ionen Ke o (Uni e si y o Tu ku, Finland) und Oulanka (Uni e si y
o Oulu, Finland) du chge ¨uh .
E m¨oglich wu de die A bei du ch inanzielle Un e s ¨u zung de Deu schen Fo -
schungsgemeinscha (DFG HO 4020/2-2), aus dem LAPBIAT P ojek (Teil das
6. EU Rahmenp og amms ”In as uc u es”), des Deu schen Akademischen Aus-
auschdiens es (DAAD), de Suomen Aka emia (Academy o Finland) und de Uni-
e si ¨a Bay eu h.
P omo ionsgesuch einge eich am: 09.05.2012
Tag des wissenscha lichen Kolloquiums: 29.10.2012
P ¨u ungsausschuss:
PD D . Ma cus A. Ho n (E s gu ach e )
P o . D . O win Meye (Zwei gu ach e )
P o . D . Ge ha d Gebaue
P o . D . S e an Pei e
P o . D . Ge ha d Rambold (Vo si zende )
IV
We know wha we a e, bu no wha we may be.
William Shakespea e - Hamle
I shall be elling his wi h a sigh
Somewhe e ages and ages hence:
Two oads di e ged in a wood, and I -
I ook he one less a eled by,
And ha has made all he di e ence.
Robe F os - The oad no aken
A uly happy pe son is one who can enjoy he scene y while on a de ou .
Au ho Unknown
V
Con en s
Con en s
Con en s IX
Lis o Tables X
Lis o Figu es XIII
Lis o Abb e ia ions XIV
Summa y 1
Zusammen assung 6
Acknowledgemen s 12
1 Gene al in oduc ion 15
1.1 G eenhouse gases and hei impac on ea h’s clima e . . . . . . . . . 15
1.2 Pea land ecosys ems as impo an sou ces o g eenhouse gases . . . . 18
1.2.1 Pea land o ma ion and pea land ypes . . . . . . . . . . . . . 18
1.2.2 Pe ma os pea lands . . . . . . . . . . . . . . . . . . . . . . . 18
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land
soils .................................... 21
1.3.1 Fe men a ion ........................... 23
1.3.2 Me hanogenesis.......................... 25
1.3.2.1 Hyd ogeno ophic me hanogenesis . . . . . . . . . . 25
1.3.2.2 Ace oclas ic me hanogenesis . . . . . . . . . . . . . . 27
1.3.2.3 Me hylo ophic me hanogenesis . . . . . . . . . . . . 28
1.3.2.4 Fac o s in luencing me hanogenesis in soils . . . . . . 28
VI
Con en s
1.3.3 Deni i ica ion........................... 30
1.3.3.1 Deni i ying mic oo ganisms . . . . . . . . . . . . . . 30
1.3.3.2 Enzymes in ol ed in deni i ica ion . . . . . . . . . . 31
1.3.3.3 Fac o s in luencing deni i ica ion in soils . . . . . . . 36
1.3.4 Dissimila o y ni a e educ ion . . . . . . . . . . . . . . . . . 37
2 Lis o publica ions and manusc ip s included in he disse a ion 38
2.1 Published a icles in pee - e iewed jou nals . . . . . . . . . . . . . . . 38
2.2 Manusc ip s in p epa a ion . . . . . . . . . . . . . . . . . . . . . . . . 38
2.3 P e ious pee - e iewed publica ions no included in he disse a ion . 39
2.4 Published abs ac s a na ional and in e na ional con e ences . . . . . 39
2.5 Addi ional p esen a ions o pa s o he wo k a in e na ional mee ings 41
3 G eenhouse gas p oduc ion in p is ine pea lands 42
Con ibu ions.................................. 42
3.1 Hypo heses es ed in his wo k . . . . . . . . . . . . . . . . . . . . . . 44
3.2 P ocesses leading o o ma ion o CH4................. 46
3.2.1 Fe men a i e and me hanogenic p ocesses in a pH-neu al en 46
3.2.2 Di e si y o p oka yo es pu a i ely associa ed wi h e men a-
ions and me hanogenesis in a pH-neu al en . . . . . . . . . 49
3.2.3 Conclusions: Fe men a ion and me hanogenesis in pH-neu al
ensoil............................... 53
3.3 P ocesses in ol ed in u no e o N2O.................. 55
3.3.1 N2O p oduc ion and consump ion in p is ine pea lands . . . . 55
3.3.2 Di e si y o pea land deni i ie s . . . . . . . . . . . . . . . . . 61
3.3.3 En i onmen al ac o s shaping deni i ie communi ies in pea -
lands................................ 72
3.3.4 Conclusions: Deni i ica ion in pea land soils . . . . . . . . . . 76
VII
Con en s
3.4 E ec o wa e able manipula ions on anae obic p ocesses in a model
pea land.................................. 77
3.4.1 E ec o wa e able manipula ions on e men a ion po en ials
inacidic ensoil.......................... 77
3.4.2 E ec o wa e able manipula ions on me hanogenesis in an
acidic ensoil ........................... 79
3.4.3 E ec o wa e able manipula ions on deni i ica ion in an
acidic en ............................. 85
3.4.4 Conclusions: wa e able manipula ions . . . . . . . . . . . . . 89
3.5 Gene alconclusions............................ 91
Re e ences 95
4 Manusc ip s 125
4.1 Published a icles in pee - e iewed jou nals . . . . . . . . . . . . . . . 125
Genome-De i ed C i e ia o Assigning En i onmen al na G and nosZ Se-
quences o Ope a ional Taxonomic Uni s o Ni a e Reduce s . . . . . 131
Con as ing deni i ie communi ies ela e o con as ing N2O emission pa -
e ns om acidic pea soils in a c ic und a . . . . . . . . . . . . . . . 181
Pu a i e Ac inobac e ial ni a e educe s and P o eobac e ial deni i ie s
a e abundan in pe ma os a ec ed N2O me abolizing acidic palsa
pea soil..................................193
4.2 Manusc ip s in p epa a ion . . . . . . . . . . . . . . . . . . . . . . . . 193
Deni i ica ion ac i i y o a new and di e se deni i ie communi y in a pH
neu al en soil in Finnish Lappland is ni a e limi ed . . . . . . . . . 227
S abili y o me hanogenic di e si y in an acidic en unde he in luence o
expe imen ald ough ...........................260
VIII
Con en s
Deni i ie communi ies in an acidic en a e s able du ing expe imen al
d ough ..................................283
4.3 P e ious pee - e iewed publica ions no included in he disse a ion . 283
Associa ion o No el and Highly Di e se Acid-Tole an Deni i ie s wi h
N2O Fluxes o an Acidic Fen . . . . . . . . . . . . . . . . . . . . . . . 294
Cu iculum i ae 294
IX
high di e si y o bac e ial amilies was de ec ed in en soil by ba coded ampli-
con py osequencing o bac e ial 16S RNA genes, including Fi micu es,Alpha- and
Del ap o eobac e ia as well as many no el amilies. Wi hin he de ec ed amilies,
gene a known o syn ophic in e ac ions wi h me hanogens (e.g., Clos idium,Syn-
ophobac e ) we e ound, indica ing ha hose gene a migh be impo an p o ide s
o me hanogenic subs a es. Di e si y o me hanogens was lowe han bac e ial di-
e si y, as only Me hanomic obiales and Me hanocellales (me hanogens o bo h o -
de s a e hyd ogeno ophs) we e de ec ed by analysis o he s uc u al gene ma ke
mc A (encoding he me hyl-coenzyme M educ ase), likewise indica ing ha hy-
d ogeno ophic me hanogens a e domina ing in pH-neu al en soil. Bo h p ocess
da a and molecula da a sugges ha (i) hyd ogeno ophic me hanogenesis is he
main p ocess o CH4 o ma ion in pH-neu al en soil, and (ii) a high di e si y o
bac e ial amilies occu in pH-neu al en soil ha a e likely in ol ed in di e se
e men a ions, hus p o iding subs a es o en me hanogens.
Deni i ica ion is conside ed o be he main sou ce o N2O in wa e logged soils
such as pea land soils, as high wa e sa u a ion p omo es anoxia in he soil. As N2O
is an in e media e in he deni i ica ion p ocess, deni i ie s can be p oduce s as well
as consume s o N2O. Despi e hei impo an ole in he global N2O budge , ac o s
con olling deni i ica ion in p is ine pea lands and he associa ed di e si y o he
deni i ie communi y a e i ually unknown. Thus, deni i ica ion and N2O con-
sump ion po en ials as well as deni i ie communi y composi ion we e assessed in all
i e no he n pea lands. In si u N2O emissions ange om <0.01 mg N2O·m−2·d−1
in pH-neu al en soil o app oxima ely 10 mg N2O·m−2·d−1in c yo u ba ed pea
ci cle soil, hus c yo u ba ed pea soil is an impo an N2O sou ce, emi ing in he
same ange as opical o ag icul u al soils. In si u N2O emissions we e posi i ely
and nega i ely co ela ed wi h soil ni a e and ammonia con en s, espec i ely. All
2
soils p oduced and consumed N2O in anoxic mic ocosms wi hou appa en delay.
N2O p oduc ion capaci ies and appa en a ini ies ( max/KM) o ni a e we e like-
wise posi i ely co ela ed wi h soil ni a e con en . N2O p oduc ion capaci ies we e
especially high in acidic, pe ma os -a ec ed c yo u ba ed pea soil, and co-occu ed
wi h high in si u N2O emissions and high ni a e con en s. On he o he hand, N2O
consump ion capaci ies we e highes in pH-neu al en soil, and co-occu ed wi h
low in si u emissions and low ni a e con en . Phylogene ic analyses o he ni a e
educ ion- and deni i ica ion-associa ed genes na G,ni K/ni S, and nosZ (encod-
ing ni a e, ni i e, ni i e, and N2O educ ases, espec i ely) indica ed ha he
di e si y o he deni i ie communi y was highes in pH-neu al en soil, and ha
di e si y was posi i ely co ela ed wi h pH. De ec ed ni a e educ ase genes a ili-
a ed mainly wi h Be ap o eobac e ial and Ac inobac e ial na G.Be ap o eobac e ial
na G domina ed in pH-neu al en soil, while Ac inobac e ial na G we e p edomi-
nan in all mo e acidic soils, indica ing ha Ac inobac e ial ni a e educe s migh
ha e a highe ole ance o acidi y. The numbe and he iden i y o obse ed ope a-
ional axonomic uni s (OTUs) o ni K,ni S, and nosZ in pH-neu al en soil was
clea ly dis inc om hose o he mo e acidic soils, and indica ed Alpha-,Be a-, and
Gammap o eobac e ial deni i ie s in all pea lands. These esul s we e con i med by
canonical co espondence analysis (CCA) o ela i e OTU abundances in amplicon
lib a ies, sugges ing ha he deni i ie communi y o he pH neu al en was unique
among he analyzed pea lands. Pe ma os -a ec ed soils mainly clus e ed oge he
in he CCA plo s based on all analyzed genes. pH was he mos impo an ac o
de e mining he communi y composi ion o ni a e educe s (as indica ed by na G)
and deni i ie s (as indica ed by all analyzed genes). Signi ican in luences o soil
ca bon con en (on na G,ni K), p ecipi a ion (on na G), o empe a u e (on nosZ)
we e also de ec ed. The a io o na G/nosZ copy numbe s was posi i ely co ela ed
3
wi h N2O emissions, as was he occu ence o ce ain OTUs o ni K and ni S. The
collec i e da a indica e ha (i) deni i ica ion is an ongoing p ocesses in di e en
ypes o p is ine pea land soils, (ii) sou ce and sink unc ion o pea land deni i-
ie s o N2O a e in luenced by soil ni a e con en as well as deni i ie communi y
composi ion, and (iii) ni a e educe and deni i ie communi y composi ion a e
a ec ed by pH, empe a u e, p ecipi a ion, and soil ca bon con en .
Global wa ming is p edic ed o inc ease he equency o ex eme wea he e en s,
causing pe iods o p olonged d ough o excess ain all. Those e en s a ec he wa e
able le el in pea lands and migh hus a ec mic obial communi ies in ol ed in he
p oduc ion o CH4and N2O. Thus, he in luence o sho - e m wa e able manip-
ula ions including applica ion o a i icial d ough condi ions o excessi e looding
was assessed in he acidic en as a model sys em. Fe men a i e, me hanogenic and
deni i ying po en ials ha we e assessed in anoxic mic ocosm s udies wi h en soil
aken a di e en imepoin s o he wa e able manipula ion e ealed ha he po-
en ial ac i i y o me hanogens and deni i ie s was a ec ed by changing wa e ables
(i.e., me hanogenic ac i i y based on ins an aneous CH4p oduc ion po en ials was
lowe ed by d ough and inc eased by looding, deni i ying ac i i y was inc eased
by looding), whe eas he po en ial ac i i y o e men e s was la gely una ec ed.
Changes in he copy numbe s o mc A,na G, and nosZ de ec ed by quan i a i e
PCR we e a he small when compa ed o he obse ed changes in po en ial ac i -
i y, indica ing ha he communi y size o me hanogens, ni a e educe s, and N2O
educe s, espec i ely, is a he una ec ed by sho - e m wa e able manipula ions.
Communi y composi ion o me hanogens, ni a e educe s and deni i ie s (as as-
sessed by TRFLP- inge p in ing o mc A,na G, and nosZ, espec i ely) was simila
a all sampled imepoin s o he manipula ion expe imen s, indica ing ha he mi-
c obial communi y composi ion is no a ec ed by enhanced wa e able luc ua ions.
4
The collec i e da a indica e a s able mic obial communi y in en soil ha is able o
adap i s ac i i y o he changing condi ions qui e apidly.
The collec i e obse a ions unde line he impo ance o pea land ecosys ems o
g eenhouse gas luxes. CH4is p oduced in a model pea land, and he s udied pea -
lands can be sou ces as well as sinks o N2O, and hus migh con ibu e signi -
ican ly o he global N2O budge . Changing wa e ables a ec ed he po en ial
ac i i y o me hanogens and deni i ie s, e en hough he communi y composi ion
o me hanogens and deni i ie s we e qui e s able. Thus, g eenhouse gas me aboliz-
ing mic oo ganisms in pea land ecosys ems a e p one o eac sensi i ely o global
change, which migh in u n a ec he sou ce and sink s eng hs o pea land ecosys-
ems o CH4and N2O and as a consequence change hei con ibu ion o he global
budge o hose g eenhouse gases.
5
Zusammen assung
Moo gebie e de n¨o dlichen Hemisph¨a e speiche n be ¨ach liche Mengen Kohlen-
s o und S icks o , und es wi d o he gesag , dass sie emp indlich au die globale
E w¨a mung eagie en. Die meis en Moo gebie e sind Quellen des T eibhausgases
Me han (CH4), welches on Me hanogenen im Moo boden in ophische In e ak-
ion mi G¨a e n gebilde wi d. Das T eibhausgas Dis icks o monoxid (N2O) kann
im Moo boden on Deni i ikan en p oduzie und e b auch we den. Quellen-
und Senken unk ionen ¨u CH4und N2O a iie en s a k zwischen e schiedenen
Moo ypen. Das Haup ziel de A bei wa en dahe die Un e suchung de mik o-
biellen P ozesse, welche an T eibhausgas l¨ussen aus Moo gebie en be eilig sind,
und zwa G¨a ungen und Me hanogenese als wich ige mi wi kende P ozesse an CH4-
Fl¨ussen und Deni i ika ion als mi wi kende P ozess an N2O-Fl¨ussen. F¨un Beispiel-
Moo e mi un e schiedlichen Eigenscha en (zwei Niede moo e (pH 7 und pH 5),
Hochland und a (pH 4), ein Palsamoo (pH 4,5) und c yo u bie e To k eise (pH
4). Diese un e schiedlichen Moo gebie e di e ie en in ih en in si u CH4- und N2O-
Emissionen sowie z.B. im Ni a gehal , de Jah esdu chschni s empe a u und/ode
im pH.
P ozesse, die an de CH4-Bildung be eilig sind, wu den am Beispiel des pH-
neu alen Niede moo es un e such . Das Niede moo p oduzie e CH4in si u sowie
in unsupplemen ie en anoxischen Mik okosmeninkuba ionen mi Niede moo bo-
den. Supplemen ie ung mi N-Ace ylglucosamin (NAG) s imulie e die Bildung on
G¨a ungsp oduk en sowie CH4und esul ie e in de e s ¨a k en Akkumula ion on
G¨a ungsp oduk en (haup s¨achlich Ace a , H2/CO2, sowie ge inge Mengen E hanol,
Fo mia und P opiona ) als die Me hanogenese du ch B omoe hansul ona (BES)
inhibie wu de, was da au hindeu e , dass Me hanogene G¨a ungsp oduk e als Sub-
s a e e wenden und somi ophisch mi den G¨a e n e kn¨up sind. Supplemen-
6
ie ung mi H2/CO2und Fo mia s imulie e die Me hanogenese s a k, w¨ah end
die S imulie ung de Me hanogenese mi Ace a und Me hanol deu lich ge inge
wa . Dies deu e da au hin, dass hyd ogeno ophe Me hanogenese ein wich ige
CH4-bildende P ozess im pH-neu alen Niede moo boden is . Mi els kodie e
Amplicon-Py osequenzie ung on bak e iellen 16S RNA Genen wu de eine g oße
Di e si ¨a bak e ielle Familien im Niede moo boden de ek ie , dazu geh¨o en Fam-
ilien de Fi micu es,Alpha- und Del ap o eobac e ia sowie iele neue Familien. In-
ne halb de de ek ie en Familien wu den Ga ungen, welche ¨u syn ophe In e ak-
ionen mi hyd ogeno ophen Me hanogenen bekann sind (z.B. Clos idium,Syn o-
phobac e ), ge unden. Dies weis da au hin, dass diese Ga ungen wich ige Lie e an-
en ¨u me hanogene Subs a e da s ellen k¨onnen. Die Di e si ¨a de Me hanogenen
wa nied ige als die de Bak e ien, da nu Me hanomic obiales und Me hanocellales
(Me hanogene beide O dnungen sind Hyd ogeno ophe) du ch Analyse des s uk-
u ellen Genma ke s mc A (kodie end ¨u die Me hyl-Coenzym M Reduk ase) de ek-
ie wu den. Auch dies deu e au eine Dominanz de hyd ogeno ophen Me hano-
genen im pH-neu alen Niede moo hin. Sowohl P ozess- als auch molekula e Da en
deu en da au hin, dass (i) hyd ogeno ophe Me hanogenese de Haup p ozess de
CH4-Bildung im pH-neu alen Niede moo is , und (ii) eine hohe Di e si ¨a bak-
e ielle Familien im pH-neu alen Niede moo o komm , welche wah scheinlich an
di e sen G¨a ungsp ozessen be eilig sind und somi Subs a e ¨u die Me hanogenen
be ei s ellen.
Deni i ika ion wi d als Haup quelle on N2O in s aunassen B¨oden wie z.B. Moo -
b¨oden angesehen, da hohe Wasse s¨a igung Anoxia im Boden ¨o de . Da N2O
ein In e media im Deni i ika ionsp ozess is , k¨onnen Deni i ikan en sowohl N2O-
P oduzen en als auch -Konsumen en sein. Obwohl sie eine wich ige Rolle ¨u den
globalen N2O-Haushal da s ellen, sind Fak o en, welche die Deni i ika ion in un-
7
be ¨uh en Moo gebie en kon ollie en, sowie die Di e si ¨a de assoziie en Deni-
i ikan engemeinscha wei gehend unbekann . Dahe wu den Deni i ika ions- und
N2O-Ve b auchspo en iale sowie die Zusammmense zung de Deni i ikan engemein-
scha in allen ¨un n¨o dlichen Moo gebie en un e such . In si u N2O-Emissionen
bewegen sich im Be eich on <0,01 mg N2O·m−2·d−1in pH-neu alem Niede -
moo boden bis zu unge ¨ah 10 mg N2O·m−2·d−1in c yo u bie em To k eisboden,
dahe s ell c yo u bie e Moo boden eine wich ige N2O-Quelle da und emi ie
in de selben G ¨oßeno dnung wie opische B¨oden ode Acke b¨oden. In si u N2O-
Emissionen wa en posi i bzw. nega i mi dem Ni a - bzw. Ammoniumgehal
des Bodens ko elie . Alle B¨oden p oduzie en und e b auch en N2O in anoxis-
chen Mik okosmen-Inkuba ionen ohne e kennba e Ve z¨oge ung. N2O-P oduk ions-
kapazi ¨a en und appa en e A ini ¨a en ( max/KM) ¨u Ni a wa en eben alls posi i
mi dem Ni a gehal des Bodens ko elie . N2O-P oduk ionskapazi ¨a en wa en in
sau en, Pe ma os -beein luss en c yo u bie en To k eisb¨oden besonde s hoch und
a en gemeinsam mi hohen in si u N2O-Emissionen und hohen Ni a gehal en au .
N2O-Ve b auchskapazi ¨a en wa en dagegen am h¨ochs en in pH-neu alem Niede -
moo boden und a en zusammen mi nied igen in si u Emissionen und nied igem
Ni a gehal au . Phylogene ische Analysen de Ni a eduk ions- und Deni i i-
ka ions-assoziie en Genma ke na G,ni K/ni S und nosZ (kodie end ¨u Ni a -,
Ni i -, Ni i - und N2O-Reduk asen) deu e en da au hin, dass die Di e si ¨a de
Deni i ikan engemeinscha in pH-neu alem Niede moo boden am h¨ochs en wa
und dass die Di e si ¨a posi i mi dem pH ko elie wa . De ek ie e Ni a -
Reduk asegene wa en haups ¨achlich mi na G de Be ap o eobac e ia und Ac i-
nobac e ia e wand . Be ap o eobac e ia- e wand e na G dominie en in pH-neu-
alem Niede moo boden, w¨ah en Ac inobac e ia- e wand e na G in allen sau e en
B¨oden dominie en, was da au hindeu e , dass Ac inobac e ia- e wand e Ni a e-
8
duzie e eine h¨ohe e S¨au e ole anz au weisen k¨onn en. Die Zahl und Iden i ¨a de
beobach e en ope a i en axonomischen Einhei en (ope a ional axonomic uni s,
OTUs) o ni K,ni S und nosZ un e schieden sich kla on denen de sau e en
B¨oden, und deu e en au Alpha-,Be a- und Gammap o eobac e ia- e wand e Den-
i i ikan en in allen Moo gebie en hin. Diese E gebnisse wu den du ch kanonis-
che Ko espondenzanalysen (canonical co espondence analysis, CCA) de ela i en
OTU-H¨au igkei en in den Amplicon-Biblio heken bes ¨a ig , was da au hindeu e ,
dass die Deni i ikan engemeinscha des pH-neu alen Niede moo es un e den un-
e such en Moo gebie en einziga ig wa . Pe ma os -beein luss e B¨oden ielen in
den CCA-O dina ionen alle analysie en Genma ke meis zusammen. pH wa
de wich igs e die Gemeinscha szusammmense zung de Ni a eduzie e (abgelei-
e on na G) und Deni i ikan en (abgelei e on den ande en Genma ke n) bes im-
mende Fak o . Signi ikan e Ein l¨usse des Kohlens o gehal es des Bodens (au na G,
ni K) de Niede schlagsmenge (au na G) ode de Tempe a u (au nosZ ) wu den
eben alls de ek ie . Das Ve h¨al nis de na G/nosZ Kopienzahlen und das Au e en
gewisse OTUs on ni K und ni S wa en posi i mi den N2O-Emissionen ko elie .
Die gesammel en Da en weisen da au hin, dass (i) Deni i ika ion in e schiede-
nen A en unbe ¨uh e Moo gebie e abl¨au , (ii) N2O Quellen- und Senken unk io-
nen de Moo deni i ikan en on dem Ni a gehal des Bodens sowie de Gemein-
scha zusammense zung de Deni i ikan en beein luss we den und (iii) die Gemein-
scha szusammense zung de Ni a eduzie e und Deni i ikan en on pH, Tempe -
a u , Niede schlagsmenge und Kohlens o gehal des Bodens beein luss wi d.
Au g und de globalen E w¨a mung we den e h¨oh e H¨au igkei en on Ex emwe e -
E eignissen e wa e , welche l¨ange e D¨u epe ioden ode e s ¨a k e Niede schl¨age
bewi ken. Diese E eignisse wi ken sich au den Wasse s and in Moo gebie en aus
und k¨onn en dahe die mik obiellen Gemeinscha en, welche an de Bildung on CH4
9
und N2O be eilig sind, beein lussen. Dahe wu de de Ein luss on ku zzei igen Ma-
nipula ionen des Wasse s ands, welche k¨uns liche D¨u e und e s ¨a k e ¨
Ube lu ung
beinhal e en, am Beispiel des sau en Niede moo es un e such . G¨a ungs-, CH4-
Bildungs- und Deni i ika ionspo en iale, welche in anoxischen Mik okosmens u-
dien mi zu e schiedenen Zei punk en de Manipula ionsexpe imen e genommenen
Niede moo bodenp oben un e such wu den, zeig en, dass die po en ielle Ak i i ¨a
de Me hanogenen und Deni i ikan en on du ch die ¨
Ande ung des Wasse s ands
beein luss wu den (d.h. me hanogene Ak i i ¨a basie end au spon anen CH4-
Bildungspo en ialen wu de du ch die D¨u e e nied ig und du ch die ¨
Ube lu ung
e h¨oh , deni i izie ende Ak i i ¨a wu de du ch die ¨
Ube lu ung e h¨oh ), wohinge-
gen die po en ielle Ak i i ¨a de G¨a e kaum beein luss wu de. ¨
Ande ungen in
de Kopienzahl on mc A,na G und nosZ, welche mi els quan i a i e PCR bes-
imm wu den, wa en ehe ge ing im Ve gleich zu den beobach e en ¨
Ande ungen de
po en iellen Ak i i ¨a en, was da au hindeu e , dass die G ¨oße de Me hanogenen-
, Ni a eduzie e - und N2O-Reduzie e -Gemeinscha kaum du ch die ku zzei i-
gen Manipula ionen des Wasse s ands beein luss wi d. Die Gemeinscha szusam-
mense zung de Me hanogenen, Ni a - und N2O-Reduzie e (un e such mi els
TRFLP-Finge p in ing on mc A,na G bzw. nosZ) wa zu allen bep ob en Zei -
punk en de Manipula ionsexpe imen e ¨ahnlich, was da au hindeu e , dass die
mik obielle Gemeinscha zusammense zung nich du ch e s ¨a k e Wasse s andss-
chwankungen beein luss wi d. Die gesammel en Da en weisen au eine s abile
mik obielle Gemeinscha im Niede moo boden hin, welche ih e Ak i i ¨a ela i
schnell an sich ¨ande nde Bedingungen anpassen kann.
Die gesammel en Beobach ungen un e s eichen die Bedeu ung on Moo -¨
Okosys-
emen ¨u T eibhausgas l¨usse. CH4wi d in einem Modell-Moo gebie gebilde , und
die un e such en Moo gebie e k¨onnen Quellen sowie Senken ¨u N2O sein und somi
10
signi ikan zum globalen N2O-Haushal bei agen. Sich ¨ande nde Wasse s ¨ande bee-
in luss en die po en ielle Ak i i ¨a de Me hanogenen und Deni i ikan en, obwohl
die Gemeinscha szusammense zung de Me hanogenen und Deni i ikan en ela-
i s abil wa . T eibhausgas-me abolisie ende Mik oo ganismen sind dahe geneig
emp indlich au globale ¨
Ande ungen zu eagie en, was wiede um die CH4und N2O
Quellen- und Senkens ¨a ken de Moo -¨
Okosys eme beein lussen k¨onn e und als Kon-
sequenz da aus ih en Bei ag zum globalen T eibhausgas-Haushal .
11
1 Gene al in oduc ion
1.2 Pea land ecosys ems as impo an sou ces o g eenhouse
gases
1.2.1 Pea land o ma ion and pea land ypes
No he n we lands s o e subs an ial amoun s o ca bon and ni ogen and a e hus
impo an playe s in he ca bon and ni ogen cycles [133, 167]. We land soils a e
ei he empo a ily o pe manen ly wa e logged which p o ides anoxic condi ions in
mos pa s o he soil [23, 181]. Pea lands a e ca ac e ized by he accumula ion o
pea which o ms when assimila ion exceeds decomposi ion unde oxygen limi ed
condi ions [22, 153]. Pea lands de elop when o ganic ma e ial accumula es in a lake
o pond (Figu e 2). The ini ial s a e o a pea land is a en pea which is ich in
ino ganic nu ien s (mine o ophic) as i ecei es nu ien om g oundwa e , su ace
uno and ainwa e . Fens a e gene ally pH-neu al o sligh ly acidic habi a s and
a e ege a ed by g asses and mosses [173]. As pea accumula ion con inues, he pea
su aces is aised abo e he g oundwa e able and a bog pea is o med (Figu e 2).
Bogs ecei e all nu ien s om ainwa e and a e hus usually nu ien limi ed. The
main ege a ion a e Sphagnum mosses and he pH is gene ally mo e acidic han in
en soils [176, 181].
1.2.2 Pe ma os pea lands
Pe ma os -a ec ed soils in he no he n hemisphe e co e abou 16% o he global
soil su ace, and s o e subs an ial amoun s o ca bon and ni ogen [133, 167]. Pe ma-
os -a ec ed pea lands in he con inuous and discon inuous pe ma os zone o he
no he n hemisphe e include palsa pea s and c yo u ba ed pea ci cles [138, 150,
179]. Palsas a e ele a ions o pea soil abo e he g ound le el due o upli ing o
pea laye s by a ozen ice lense and a e widely dis ibu ed in he ci cuma c ic
egions (i.e., Canada, USA, Finland, Sweden, Iceland, Russia) [150, 194]. Palsa
18
1.2 Pea land ecosys ems as impo an sou ces o g eenhouse gases
Figu e 2: Pea land de elopmen . Accumula ion o o ganic ma e in a pond (A, B),
en (C), aised bog pea (D); based on [176]. B own=mine al subs a um,
blue=wa e , da k g een= en pea , ligh b own=bog pea , ligh g een= ees.
Vege a ion co e changes wi h ime due o changes in g ound p ope ies.
de elopmen is a ec ed by a ious en i onmen al ac o s including dep h o snow
co e , g ound wa e able dep h, ege a ion co e and deg ee o wind e osion [150].
Palsa de elopmen includes many eeze- haw cycles leading o accumula ion o ice
in he ac i e laye and subsequen uphea al o he pea soil [150]. Howe e , as
palsas ma u e he ice lenses will e en ually mel and he palsa collapses, o ming a
he moka s lake [150]. On he o he hand, c yo u ba ed pea ci cles a e ba e su ace
pea a eas in a c ic und a in which ege a ion has been emo ed due o mixing by
os ac ion [138, 177]. Vege a ion co e is absen om app oxima ely 12% o he
a c ic soil su aces, including c yo u ba ed pea ci cles [177]. C yo u ba ion occu s
mainly in soils wi h poo soil d ainage and equen eeze- haw cycles, leading
o o ma ion o pa e ned g ound [11, 128, 179]. C yo u ba ed soils con ain high
amoun s o incomple ely deg aded soil o ganic ma e [85]. O ganic ca bon s o ed
in pe ma os soil is edis ibu ed o he ac i e laye (i.e., he laye ha haws in
19
1 Gene al in oduc ion
summe mon hs) by c yo u ba ion, and c yo u ba ion can inc ease he amoun o
o ganic ca bon s o ed in pe ma os soil [11, 63].
We lands a e he mos impo an single CH4sou ce, as hei emissions accoun o
25% o he obse ed global CH4emissions [182]. In no he n pea lands, abou 20%
o he assimila ed CO2a e eleased in o he a mosphe e as CH4[139]. Howe e ,
o al CH4p oduc ion in we land soils is e en highe , as abou 20 o 40% o he
p oduced CH4is oxidized on oo su aces o in mo e oxic su ace laye s [182]. N2O
emission om no he n pea lands ha e been s udied o a much lesse ex en han
CH4emissions, and mos s udies ha e ocused on N2O emissions om managed
pea lands [93, 98, 99]. P is ine no he n pea lands can be ne sou ces o N2O, e en
hough hei emission a es a e gene ally low [93, 161]. On he o he hand, no he n
pea lands can ac as pe manen o empo a y sinks o N2O [16, 43, 117, 100, 161].
Pe ma os -a ec ed pea lands like ce ain palsa pea s and especially c yo u ba ed
pea ci cles a e la ge poin sou ces o N2O wi h emission a es compa able o hose
o opical and ag icul u al soils [100, 138]. N2O emissions om palsa pea soils a e
highly a iable [100]. The abili y o pe ma os -a ec ed pea lands o emi N2O is
inc eased by a low deg ee o plan co e ( esul ing in educed compe i ion o N-
sou ces), high ni a e con en , low C:N a ios and high g oss mine aliza ion ac i i ies
[20, 100, 138].
Pea land ecosys ems in he no he n hemisphe e and especially pe ma os -a ec ed
und a ecosys ems a e p edic ed o be se e ely a ec ed by global wa ming, as wa m-
ing occu s a as e han a e age a es in hose sys ems [4, 38, 150, 174]. Inc easing
empe a u es a e likely o lowe he wa e able in no he n pea lands and hus in-
c ease he amoun o CO2, CH4and N2O eleased om pea land soils [4, 99, 100].
Mo eo e , pe ma os hawing may cause high emissions o CO2, CH4and N2O om
s o ed ca bon and ni ogen [32, 147].
20
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land soils
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic
pea land soils
Pea land soils a e oxygen limi ed due o he high deg ee o wa e -sa u a ion, and
o ganic ma e is hus deg aded mainly anae obically o CO2and CH4(Figu e 3, Fig-
u e 4). Complex s uc u al polyme s like cellulose, chi in o lignin a e i s deg aded
o monome s (e.g., glucose, xylose, N-ace yl-gucosamine, o a oma ic compounds)
which a e u he deg aded by e men a ion (p oducing alcohols, sho chain a y
acids, CO2, and H2) [30, 82, 192]. Monome s as well as e men a ion p oduc s a e
elec on dono s in espi a o y p ocesses ha use al e na i e elec on accep o s like
ni a e, manganese, i on, sul a e o CO2(Table 2) [30, 192]. P ima y e men a-
ion p oduc s such as alcohols and sho chain a y acids a e u ilized by syn ophic
seconda y e men e s which p oduce ace a e, CO2and H2[30, 82]. P ima y and sec-
onda y e men a ion p oduc s a e inally con e ed o CH4by me hanogens [30, 82].
Figu e 3: Schema ic o e iew o he main p ocesses in he biological ca bon
(A) and ni ogen (B) cylcles. DNRA=dissimila o y ni a e educ ion o
ammonium, Anammox=anae obic ammonium oxida ion (based on [96, 59]).
Me hanogenesis compe es wi h o he anae obic p ocesses unde anoxic condi ions
[30]. The educ ion o al e na i e elec on accep o s like ni a e o i on is he mody-
namically mo e a o able han me hanogenesis, and me hanogens a e hus o en ou -
21
1 Gene al in oduc ion
compe ed (Table 2) [92, 94]. Howe e , concen a ions o al e na i e elec on accep-
o s in we lands a e spacially and empo ally a iable and o en low [1, 13, 125, 149],
and me hanogenesis can al hough occu simul aneously wi h hose p ocesses, gi en
a su icen ly high concen a ion o H2[23].
Figu e 4: Schema ic o e iew o anae obic p ocesses in we land soils ha
lead o he deg ada ion o o ganic ma e ial and he p oduc ion o
CH4. C1=one-ca bon o ganic compounds (excluding CH4) such as o ma e,
me hanol, and me hylamine. Based on [30].
Table 2: Redoxpo en ials o di e en hal -cell eac ions
unde s anda d condi ions (E0’), based on [89,
96, 168].
Hal -cell eac ion ne−
1E0’ (V) 2P ocess
O2/H2O 2 0.82 Ae obic espi a ion
NO−
3/0.5 N25 0.74 Deni i ica ion
NO−
3/NO−
22 0.43 Ni a e educ ion
Mn+
4/Mn+
22 0.39 Mn(IV) educ ion
Fe3+/Fe2+ 1 0.20 Fe(III) educ ion
SO2−
4/S2−8 -0.22 Sul a e educ ion
CO2/CH48 -0.24 Me hanogenesis
CO2/Ace a e 8 -0.29 Ace ogenesis
1Numbe o ans e ed elec ons
2Redoxpo en ials unde s anda d condi ions (Tempe a u e: 25 ◦C,
P essu e: 101.3 kPA, pH 7.0).
22
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land soils
1.3.1 Fe men a ion
In e men a i e p ocesses, elec ons ob ained by oxida ion o a subs a e a e ans-
e ed o a pa ly oxidized in e media e [168]. Subs a es include ca bohyd a es,
amino acids, alcohols, and ca bonic acids [168]. Ene gy is conse ed in o m o ATP
ia subs a e-le el phospho yla ion by kinase- eac ions om ac i a ed compounds
such as ace yl-coenzyme A (Figu e 5). Those ac i a ed compounds a e o med in
p e ious s eps ia lyase- o dehyd ogenase- eac ions [168] (Figu e 5).
Figu e 5: Examples o ATP gene a ion by subs a e-le el phospho yla ion
(SLP). Ace a e kinase o ms ATP om ace yl-CoA a e an ini ial lyase- (A)
o dehyd ogenase- eac ion (B). Based on [168].
Table 3: Examples o p ima y e men a ions, based on [96, 168].
Fe men a ion ype Reac ion 1∆G0’ (kJ/mol) 2Example o ganisms
Alcoholic e men a ion Hexoses →2 E OH + 2 CO2-218 Saccha omyces sp.
Lac ic acid e men a ion Hexoses →2 Lac a + 2 H+(homo e men a i e) -198 Lac obacillus sp., En e ococcus sp.
Hexoses →Lac a e + E OH + 2 H++ CO2(he e o e men a i e) -208 Leuconos oc sp., Lac obacillus sp.
Bu y ic acid e men a ion Hexoses →Bu y a e + Ace a e + H2+ 2 CO2-247 Clos idium sp.
Mixed acid e men a ion Hexoses →E OH + 2,3-Bu anediol
+ Succina e + Lac a e + Ace a e + Fo ma e + H2+ CO2-200 o -260 Esche ichia sp., En e obac e sp.
1Equa ions a e no s oiciome ically balanced.
2Gibbs ee ene gy unde s anda d condi ions (Tempe a u e: 25 ◦C, P essu e: 101.3 kPA, pH 7.0; gi en o glucose u iliza ion).
P ima y e men a ion p oduc s include e hanol, ace a e, H2and CO2(Table 3).
Alcohols (e.g. e hanol, p opanol), o ganic acids (e.g. ace a e, succina e, bu y a e)
o a oma ic compounds (e.g. benzoa e) a e subjec ed o seconda y e men a ions
by syn ophic o ganisms (Table 4) [145]. As hose seconda y e men a ions a e
ende gonic unde s anda d condi ions, syn ophic pa ne s (e.g. me hanogens) a e
23
1 Gene al in oduc ion
equi ed o emo e p oduc s such as H2o CO2and hus d i e he eac ion [96,
145, 168]. The abili y o conse e ene gy ia e men ion is widesp ead in bac e ia,
A chaea, and Euka ya, and o ganisms can be acul a i e o obliga e e men e s [168].
Table 4: Examples o seconda y e men a ions (syn ophic p ocesses coupled o me hanogenesis), based
on [96, 145].
Subs a e Reac ion 1∆G0’ (kJ) 2Example o ganisms 3
Bu y a e 2 CH3CH2CH2COO−+ 2 H++ 2H2O→5 CH4+ 3 CO2-177 Syn ophomonas sp.
P opiona e 4 CH3CH2COO−+ 4 H++ 2 H2O→7 CH4+ 5 CO2-249 Syn ophobac e sp.
E hanol 2 CH3CH2OH + CO2→2 CH3COO−+ 2 H++ CH4-112 Desul o ib io ulga is
Ace a e CH3COO−+ H+→CH4+ CO2-36 Clos idium sp.
1Chemical equa ion o he combined syn ophic eac ion including seconda y e men a ion and me hanogenesis.
2Gibbs ee ene gy unde s anda d condi ions (Tempe a u e: 25 ◦C, P essu e: 101.3 kPA, pH 7.0).
3Examples o seconda y e men e s in ol ed in his ype o syn ophic eac ion.
24
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land soils
1.3.2 Me hanogenesis
Me hanogenesis is an anae obic espi a ion p ocess ca alyzed by s ic ly anae obic
obliga ely me hanogenic A chaea [6, 92]. Me hanogens belong o he Eu ya chaeo a
[184] and o m 6 o de s (Me hanobac e iales,Me hanocellales,Me hanococcales,Me-
hanomic obiales,Me hanopy ales,Me hanosa cinales) including 32 gene a [51, 144,
169].
Me hanogens ha e a limi ed subs a e ange and p oduce CH4hyd ogeno op-
ically, ace oclas ically o me hylo ophically [51, 92] (Figu e 6, Table 5). Hyd o-
geno ophic me hanogens educe CO2wi h H2 o o m CH4[6, 92]. Many hy-
d ogeno ophic me hanogens also u ilize o ma e, while some can u ilize seconda y
alcohols like 2-p opanol o 2-bu anol, e hanol, o CO [92]. Hyd ogeno ophic me hano-
genesis is widesp ead and occu s in all me hanogenic o de s [6]. Ace oclas ic me ha-
nogens belong o he o de Me hanosa cinales (gene a Me hanosa cina and Me hano-
sae a) and u ilize ace a e by oxidizing he ca boxyl-g oup o CO2and educing he
me hyl-g oup o CH4[92]. While Me hanosa cina p oduce CH4hyd ogeno ophi-
cally, me hylo ophically and ace oclas ically, Me hanosae a a e s ic ly ace oclas-
ic [51, 92]. Me hylo ophic me hanogens u ilize me hyla ed compounds such as
me hanol, me hylamines, and me hyla ed sul ides and occu only wi hin he Me hano-
sa cinales (wi h excep ion o Me hanosae a) and wi hin he genus Me hanosphae a
(Me hanobac e iales) [6, 92]. The me hyl-coenzyme M educ ases Mc and M
(ca aly ical subuni s encoded by mc A o m A) a e cen al enzymes in ol ed in
all ypes o me hanogenic pa hways (Figu e 6; [34]), and mc A is equen ly used
as a s uc u al genema ke o assess he communi y composi ion o me hanogens in
en i onmen al samples [47, 55, 101, 191].
1.3.2.1 Hyd ogeno ophic me hanogenesis
In hyd ogeno ophic me hanogenesis, CO2is educed wi h H2o o ma e as a p i-
25
1 Gene al in oduc ion
Figu e 6: Schema ic o e iew o he h ee me hanogenic pa hways. Me hyl-
Coenzyme M educ ase unc ions as a key enzyme in all h ee me hanogenic
pa hways. Fu he enzymes, co ac o s and compounds a e no shown. Dashed
lines indica e ha mo e han one s ep is needed o he con e sion. R=e.g., -SH,
-OH, o NH2. Mc =Me hyl coenzyme-M educ ase I; M =Me hyl coenzyme-
M educ ase II. Based on [34].
Table 5: Types o me hanogenesis.
Type Reac ion ∆G0’ (kJ) 1O ganisms 2
Hyd ogeno ophic 4 H2+ CO2→CH4+ 2 H2O -35 Mos me hanogens
Ace oclas ic CH3COOH →CH4+ CO2-33 Me hanosa cina,Me hanosae a
Me hylo ophic 4 CH3OH →3 CH4+ CO2+ H2O -105 Me hanosa cina and o he s
1Gibbs ee ene gy unde s anda d condi ions (Tempe a u e: 25 ◦C, P essu e: 101.3 kPA, pH 7.0).
2Examples o seconda y e men e s in ol ed in his ype o syn ophic eac ion. [92]
ma y elec on dono o CH4[92]. When o ma e is used as an elec on dono , 4
molecule o o ma e a e oxidized o CO2and he ob ained educ ion equi alen s a e
used o educed 1 molecule o CO2[92]. In a i s s ep, CO2binds o me hano u-
an (MF) and is educed by e edoxin o a o myl-g oup, he e odoxin in u n
is educed by H2(Figu e 7; [169]). The o myl-g oup is ans e ed o e ahy-
d ome hanop e in (H4MPT), dehyd a ed o a me henyl-g oup and subsequen ly e-
duced o me hylene-H4MPT and me hyl-H4MPT by educed F420 [92, 169]. The
me hyl-g oup is hen ans e ed o coenzyme-M (CoM) and educed o CH4in a
inal s ep by he me hy CoM educ ase [92, 169].
26
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land soils
Figu e 7: Reac ions in ol ed in hyd ogeno ophic me hanogene-
sis. F420=coenzyme F420; Fd= e edoxin; MF=me hano u an;
H4MPT= e ahyd ome hanop e in; HS-CoB=coenzyme B; HS-
CoM=coenzyme M; based on [92, 169]
1.3.2.2 Ace oclas ic me hanogenesis
In ace oclas ic me hanogenesis, ace a e is spli , he ca boxyl-g oup is oxidized o
CO2, while he me hyl-g oup is educed o CH4[92]. Ace a e is i s ac i a ed wi h
ATP and ans e ed o coenzyme A by ace a e kinase-phospo ansace ylase, o m-
ing ace yl-CoA. Ace yl-CoA is clea ed o me hyl-H4MPT and CO-CoA by he CO
27
1 Gene al in oduc ion
logues o he bac e ial NosZ. In con as o hei bac e ial con e pa s hese N2O
educ ases a e memb ane-bound and ecei e elec ons om menaquinol [83, 197].
Howe e , he e a e a chaeal deni i ie s capable o N2O educ ion whose genomes
do no ha bo nosZ homologues, hus he e is likely a second a chaeal ype o N2O
educ ase [197]. Some deni i ie s lack N2O educ ases, hus N2O is he endp oduc
o deni i ica ion in hose o ganisms [61, 197]. On he o he hand, he e a e o -
ganisms like Wolinella succinogenes ha educe N2O using a sligh ly modi ied N2O
educ ase wi hou being capable o comple e deni i ica ion [197]. W. succinogenes
educes ni a e o ni i e and N2O o N2, bu he in e media e s eps a e missing
[197].
In ungal deni i ica ion, he educ i e enzymes a e loca ed in he mi ochond ia
and a e coupled o he mi ochond ial elec on anspo chain o p oduce ATP
[74]. The memb ane-bound ungal ni a e educ ase (Na ) educes ni a e u ilizing
ubiquinol as elec on dono [107, 165]. P ope ies o ungal Na esemble hose o
he bac e ial con e pa . Howe e , no o hologues o he bac e ial na genes ha e
so a been de ec ed in ungal genomes, indica ing ha ungal Na is e olu iona y
dis inc om bac e ial Na [165]. In con as , he ungal ni i e educ ase gene ni K
de ec ed in he genomes o many deni i ying ungi is an o hologue o he bac e ial
ni K [68]. Fungal Ni K is a coppe -con aining ni i e educ ase loc aed in he mi-
ochond ion [75, 107]. Fungal NO educ ases a e clea ly dis inc om p oka yo ic
NO educ ases. Fungal No is a membe o he cy och ome P450 supe amily and
is hus called P450no [107, 165]. The educ ion o NO is e y di e en om he
oxida ion eac ions o o he P450 p o eins, as hose equi e u he elec on dona -
ing p o eins [165]. P450no is a soluble enzyme ha u ilizes NADH o NADPH o
educe NO o N2O [165]. N2O is he majo end p oduc in ungal deni i ica ion,
e en hough some ungi a e also capable o N2p oduc ion [107, 151, 195]. So a , no
34
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land soils
N2O educ ase has been isola ed om ungi o iden i ied in ungal genomes [107].
35
1 Gene al in oduc ion
1.3.3.3 Fac o s in luencing deni i ica ion in soils
Deni i ica ion a es in soils as well as he ela i e con ibu ion o N2O o he o al
emi ed N gases depend he composi ion o he deni i ie communi y as well as on
a a ie y o en i onmen al ac o s. Soil pH is one o he mos impo an pa ame e s
a ec ing deni i ica ion ac i i ies in soil [152]. In gene al, low pH impai s deni i i-
ca ion and inc eases he con ibu ion o N2O o o al N2gases [25, 152]. Howe e
deni i ica ion a es o deni i ie communi ies om acidic soils a e highe a in si u
pH han a mo e neu al pH [117, 123]. Acidic pH has a mo e p onounced e ec on
N2O educ ase han on he o he educ ases in ol ed in deni i ica ion [8, 91]. This
esul s in highe N2O:N2 a ios in acidic soils. The exac mechanism o inhibi ion is
no ye esol ed, bu i is likely due o pos - ansc ip ional e ec s du ing ansla ion
o p o ein assembly [8, 91].
In gene al, deni i ica ion ac i i ies inc ease wi h inc easing empe a u es, e.g.
pu e cul u es o Pseudomonas deni i icans ha e a empe a u e op imum o 38◦C
[178]. In soils, he empe a u e op imum o deni i ica ion is o en highe han
he obse ed in si u empe a u es [97, 117, 143]. E en hough o al deni i ica-
ion ac i i y is inc eased a highe empe a u es, N2O emissions can be lowe , as
mos ly comple e deni i ica ion o N2occu s a hose empe a u es [93]. N2O e-
duc ases a e mo e sensi i e o cold empe a u es han he o he educ ases in ol ed
in deni i ica ion, hus he N2O:N2 a ios a e highe a colde empe a u es, and
win e - ime deni i ica ion ac i i ies signi ican ly impac on he annual N2O emis-
sions [53, 93, 110]. Howe e , he e a e also di e ences be ween soil ypes, and some
soils do no show inc eased N2O:N2 a ios a low empe a u es, indica ing ha den-
i i ie s and N2O educ ases can be adap ed o cold empe a u es [31]. F eeze- haw
cycles lead o enhanced N2O emissions om soil, and N2O emission peaks a e o en
obse ed in he end o win e [46, 79, 78, 131].
36
1.3 P ocesses in ol ed in g eenhouse gas p oduc ion in anoxic pea land soils
Apa om soil pH and empe a u e, deni i ica ion in soils is also a ec ed by
soil wa e con en , ca bon and ni a e a ailabili y, and soil C:N a ios [23, 21].
Soil ni a e con en o en limi s deni i ica ion in soils, while ca bon sou ces a e
no limi ing [5, 50, 7, 175]. Due o he in luences o he men ioned en i onmen al
pa ame e s, deni i ica ion ac i i ies and N2O emissions a e highly a iable in soils,
and e en so-called ”ho spo s” o deni i ica ion exis [50, 138, 189].
1.3.4 Dissimila o y ni a e educ ion
Dissimila o y ni a e educ ion o ammonium (DNRA) educes ni a e ia ni i e
o ammonium [83]. DNRA compe es wi h deni i ica ion o ni a e and can be
he dominan p ocess in some soils [142]. High ca bon con en and low ni a e
a ailabili y a o DNRA o e deni i ica ion [83, 172]. The p ocess occu s in Bac e ia
as well as in ungi [83, 164]. In Bac e ia, he educ ion o ni a e o ni i e is
ca alyzed by he pe iplasmic ni a e educ ase NapAB (1.3.3.2) [83, 129, 195]. The
educ ion o ni i e o ammonium is ca alyzed by he pen aheme cy och ome cni i e
educ ase N A [83, 195]. In ungal DNRA (also called ammonia e men a ion),
ni a e educ ion o ammonium is coupled o he oxida ion o e hanol o ace a e
[163, 164, 165]. The NADH-dependen assimila o y ni a e and ni i e educ ases
encoded by niaD and niiA a e u ilized o he s epwise educ ion o ni a e o
ammonium [163, 165]. The elec ons o he educ ion o igina e om he oxida ion o
e hanol o ace a e [163, 165]. The oxida ion is coupled o ATP-p oduc ion by ace a e
kinase [163, 165]. E en hough assimila o y enzymes a e used o he educ ion o
ni a e and ni i e, he e is no indica ion ha he p oduced ammonium is ac ually
assimila ed by he ungus [165].
37
2 Lis o publica ions and manusc ip s included in he disse a ion
2 Lis o publica ions and manusc ip s included in he
disse a ion
2.1 Published a icles in pee - e iewed jou nals
1. Palme , K., D ake, H. L., Ho n, M. A. (2009). Genome-de i ed c i e ia o
assigning en i onmen al na G and nosZ sequences o ope a ional axonomic
uni s o ni a e educe s. Applied and En i onmen al Mic obiology 75: 5170-
5174. Re e ence numbe [116]. Own con ibu ion: Concep (50%, oge he
wi h M. Ho n), expe imen al wo k (100%), pa o he w i ing (50%).
2. Palme , K., Biasi, C., Ho n, M. A. (2012). Con as ing deni i ie communi-
ies ela e o con as ing N2O emission pa e ns om acidic pea soils in a c ic
und a. ISME Jou nal 6: 1058-1077. Re e ence numbe [115]. Own con ibu-
ion: Expe imen al design (60%, oge he wi h M. Ho n) and p ac ical wo k
(100%), da a analysis (100%), pa o he w i ing (60%).
3. Palme , K., Ho n, M. A. (2012). Pu a i e Ac inobac e ial ni a e educe s and
P o eobac e ial deni i ie s a e abundan in pe ma os a ec ed N2O me ab-
olizing acidic palsa pea soil. Submi ed o Applied and En i onmen al Mi-
c obiology. Re e ence numbe [119]. Own con ibu ion: Expe imen al design
(70%, oge he wi h M. Ho n), p ac ical wo k (100%), da a analysis (100%),
pa o he w i ing (60%). This manusc ip was unde e ision when he hesis
was handed in.
2.2 Manusc ip s in p epa a ion
1. Palme , K., Ho n, M. A. (2012). Deni i ica ion ac i i y o a new and di e se
deni i ie communi y in a pH neu al en soil in Finnish Lapland is ni a e lim-
38
2.3 P e ious pee - e iewed publica ions no included in he disse a ion
i ed. Re e ence numbe [120]. Own con ibu ion: Expe imen al design (75%,
oge he wi h M. Ho n) and p ac ical wo k (100%), da a analysis (100%), pa
o he w i ing (70%).
2. Palme , K., D ake, H. L., Ho n, M. A. (2012). Deni i ie communi ies in an
acidic en a e s able du ing expe imen al d ough . Re e ence numbe [118].
Own con ibu ion: P ac ical wo k (100%), da a analysis (100%), pa o he
w i ing (70%).
3. Palme , K., Schulz, K., Mundinge , A., Me el, R., Ho n, M. A., D ake, H.
L. (2012). S abili y o me hanogenic di e si y in an acidic en unde he in-
luence o expe imen al d ough . Re e ence numbe [121]. Own con ibu ion:
P ac ical wo k (40%, oge he wi h K. Schulz, A. Mundinge , R. Me el), da a
analysis (90%), pa o he w i ing (70%).
2.3 P e ious pee - e iewed publica ions no included in he
disse a ion
1. Palme , K., D ake, H. L., Ho n, M. A. (2010). Associa ion o no el and highly
di e se acid- ole an deni i ie s wi h N2O luxes o an acidic en. Applied and
En i onmen al Mic obiology 76: 1125-1134. Re e ence numbe [117].
2.4 Published abs ac s a na ional and in e na ional
con e ences
1. Palme , Ho n, M.A. 2012. Unknown deni i ie di e si y in a pH neu al en
soil in Finnish Lapland. Annual Mee ing Ve einigung ¨u Allgemeine und
Angewand e Mik obiologie, BioSpec um. Abs ac SMV003, p. 210.
39
2 Lis o publica ions and manusc ip s included in he disse a ion
2. Palme , K., Ho n, M.A. 2012. Palsa pea s ep esen hi he o unde app ecia ed
ese oi s o new deni i ie di e si y associa ed wi h N2O luxes. In e na ional
Pola Yea 2012 Con e ence. Abs ac online.
3. Palme , K., Biasi, C., D ake, H.L., Ho n, M.A. 2011. C yo u ba ion a ec s
deni i ie communi ies in N2O-emi ing a c ic pe ma os pea soil. Annual
Mee ing Ve einigung ¨u Allgemeine und Angewand e Mik obiologie, BioSpec-
um. Abs ac EMP104, p. 116.
4. Palme , K., Biasi, C., D ake, H.L., Ho n, M.A. 2011. Impac o c yo u ba-
ion on deni i ie communi y s uc u e and ac i i y in N2O-emi ing a c ic
pe ma os pea soil. Ecology o Soil Mic oo ganisms. Abs ac 99.
5. Palme , K., Schulz, K., Ho n, M.A., D ake, H.L. 2010. S abili y o he
me hanogenic communi y in an acidic en o expe imen al d ough . Annual
Mee ing Ve einigung ¨u Allgemeine und Angewand e Mik obiologie, BioSpec-
um. Abs ac ECV02, p. 81.
6. Palme , K., Schulz, K., Ho n, M.A., D ake, H.L. 2010. E ec s o enhanced
d ough on he di e si y o me hanogens in an acidic en. Bay eu h Cen e o
Ecology and En i onmen al Resea ch (BayCEER) Wo kshop 2010, Abs ac
O 1.5.
7. Palme , K., Schulz, K., Ho n, M.A., D ake, H.L. 2010. Impac o a i icial
d ough on di e si y, abundance, and gene exp ession o me hanogens in an
acidic en. 13 h In e na ional Symposium on Mic obial Ecology (ISME-13),
Abs ac on disk.
40
2.5 Addi ional p esen a ions o pa s o he wo k a in e na ional mee ings
2.5 Addi ional p esen a ions o pa s o he wo k a
in e na ional mee ings
1. Palme , K., Biasi, C., Ho n, M.A. 2011. Deni i ie communi iy composi ion
impac s N2O emission pa e ns in acidic und a pe ma os soils. No dic Ne -
wo k o S able Iso ope Resea ch (No dSIR) Mee ing.
2. Palme , K., D ake, H.L., Ho n, M.A. 2011. E ec o high la i ude on deni i ica ion-
dependen N2O- luxes and deni i ie communi y s uc u e in pea lands. Go -
don Resea ch Con e ence on Applied and En i onmen al Mic obiology.
41
3 G eenhouse gas p oduc ion in p is ine pea lands
3 G eenhouse gas p oduc ion in p is ine pea lands
Con ibu ions o he synopsis
Da a p esen ed in his sec ion a e pa ly ep esen ed in he manusc ip s (see e -
e ences in bold ace). Addi ional da a no ep esen ed in he manusc ip s a e
de i ed om he bachelo hesis o Sonja Pe as (pa o he wo k on e men a ion
and me hanogenesis in pH-neu al Puukkosuo en soil) o om own esea ch (pa
o he wo k on e men a ion and me hanogenesis in pH-neu al Puukkosuo en soil,
wo k on he compa ison o deni i ie communi ies, manipula ion expe imen s).
42
We lands including p is ine pea lands a e sou ces and sinks o he g eenhouse
gases CH4and N2O [18, 37, 76, 100, 161]. Howe e , g eenhouse gas luxes om
pea lands a y be ween pea lands and a e dependen on many en i onmen al ac-
o s such as empe a u e and pH [19, 23, 161]. N2O emissions om p is ine pea lands
ha e been less in ensi ely s udied han CH4emissions, and en i onmen al pa am-
e e s a ec ing sou ce o sink s engh o pea lands o N2O a e la gely un esol ed.
Thus, i e pea lands belonging o di e en pea land ypes we e selec ed as model
sys ems o assess me hanogenesis as well as deni i ica ion-associa ed N2O u no e
and he possible in luence o en i onmen al pa ame e s (Table 6). The i e pea -
lands di e ed in bo h CH4and N2O emissions as well as in en i onmen al pa ame e s
such as ni a e, ammonium o wa e con en , pH, mean annual empe a u e o mean
annual p ecipi a ion (Table 6, Figu e 9; mo e de ailed in o ma ion abou sampling
si es is gi en in [115, 117, 120, 119]). Some pea lands emi ed la ge amoun s o
CH4( en soils), some pea lands emi ed la ge amoun s o N2O (Schl¨oppne b unnen
en, c yo u ba ed pea ci cles), and some pea lands showed only low emissions o
ei he gas (Pea pla eau pe ma os und a, Skallu aa a palsa pea ; Figu e 9).
Table 6: Pea lands used in he s udies.
Si e name Abb e ia ion Pea land ype pH
1wa e ni a e 1ammonium 1MAT 2MAP 3
con en (%) con en (µM) con en (µM) (◦C) (mm)
Puukkosuo PS Fen 6.8 90 50-150 77 −0.4 772
Schl¨oppne b unnen SB Fen 5.0 85 0 −500 10 5.3 1162
Pe ma os und a PT Pea pla eau pe ma os und a 4.0 80 <1 300 −5.6 505
Skallu aa a SV Palsa pea 4.5 73 10 125 −1.6 415
Pea ci cles PC C yo u ba ed pe ma os pea 4.0 70 1500 77 −5.6 505
1De e mined in wa e y ex ac s.
2Mean annual empe a u e.
3Mean annual p ecipi a ion.
43
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 12: E ec o supplemen al subs a es on me hanogenic po en ials in
anoxic mic ocosms wi h Puukkosuo en soil. Mic ocosms we e p ein-
cuba ed o 120 days be o e subs a e supplemen a ion. A gon was used as
headspace gas. 1 mM o ace a e, o ma e, o me hanol o 8% H2/2% CO2
( / ) in he a mosphe e. Mean alues o h ee eplica e mic ocosms and
s anda d e o s a e displayed.
i y il e ed (i.e., py osequencing and PCR-based e o s we e emo ed by denoising
wi h Py oNoise and SeqNoise algo i hms; me hod desc ibed in [119]) and clus e ed
a 87% ( amily le el) and 84% (species le el) simila i y o 16S RNA genes and
mc A, espec i ely, using he Needleman Wunsch algo i hm (me hod desc ibed in
[119]). Phylogene ic ees we e gene a ed o assign de ec ed sequences o phyla. In
o al, 159 and 352 amily-le el bac e ial 16S RNA OTUs we e de ec ed in ampli-
con lib a ies om o wa d and e e se eads, espec i ely. OTUs mainly a ilia ed
o Fi micu es and P o eobac e ia (app oxima ely 60% o all sequences, Figu e 13).
Among he P o eobac e ia,Alpha- and Del a-P o eobac e ial sequences we e mos
50
3.2 P ocesses leading o o ma ion o CH4
abundan . Addi ionally, amilies a ilia ed o Ac inobac e ia,Acidobac e ia,Bac e-
iode es,Chlo o lexi,Cyanobac e ia, and Ni ospi aceae we e de ec ed (Figu e 13).
15% o all sequences (i.e., 66 amilies) we e no closely ela ed o known bac e-
ial amilies and migh hus ep esen no el amilies. In acidic bog and en soil,
P o eobac e ia,Fi micu es,Acidobac e ia and Ac inobac e ia a e also equen ly
de ec ed [27, 48, 55, 62, 106, 122, 185]. Sequences and isola es o bac e ia in pH-
neu al pe ma os soil consis ed mainly o Ac inobac e ia,Fi micu es and Alpha-
and Del a-P o eobac e ia [157]. Some Del a-P o eobac e ia a e impo an sul a e
educe s, indica ing ha pH-neu al Puukkosuo en soil ha bo s he po en ial o
sul a e educ ion. Addi ionally, many gene a wi hin he Del a-P o eobac e ia and
Fi micu es such as Syn ophobac e spp. o Clos idium spp. a e capable o syn-
ophic in e ac ions wi h hyd ogeno ophic me hanogens and a e equen ly de ec ed
in we lands [30, 48, 145, 146, 156, 185], indica ing ha hose syn ophic o ganisms
migh p o ide subs a es o hyd ogeno ophic me hanogens in Puukkosuo en soil.
mc A sequences om Puukkosuo en we e assigned o 9 and 9 species-le el OTUs
based on o wa d and e e se eads, espec i ely. OTU dis ibu ions ob ained wi h
sequences om o wa d and e e se eads we e simila , hus only esul s om o wa d
eads a e desc ibed in mo e de ail. Th ee species-le el OTUs had a ela i e abun-
dance o >1% o all mc A sequences. OTU 1, 2, and 3 accoun ed o 48.3%, 48.2%,
and 1.1% o de ec ed mc A sequences and we e ela ed o uncul u ed mc A as well
as o mc A o Me hano egula spp., Me hanocella paludicola, and Me hano egula spp.,
espec i ely (Figu e 14), indica ing ha bo h Me hanomic obiales and Me hanocel-
lales a e impo an playe s in he CH4p oduc ion o Puukkosuo en. Me hanogens
o bo h g oups p oduce CH4hyd ogeno ophically [6, 92], and highe s imula ion
wi h subs a es useable by hyd ogeno ophic me hanogens was obse ed in mic o-
cosm expe imen s (Figu e 12). None o he de ec ed mc A a ilia ed wi h mc A o
51
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 13: Bac e ial amilies de ec ed in pH-neu al en soil by amplicon py-
osequencing o bac e ial 16S RNA genes. In o al, 2 375 quali y il-
e ed sequences we e de i ed om e e se eads, which we e assigned o 352
OTUs based on a amily-le el h eshold simila i y o 87 %. Resul s ob ained
wi h o wa d ead sequences we e simila and a e hus no shown. A ilia ion
o amilies o majo g oups and numbe o de ec ed amilies pe g oup (in
pa en heses) a e displayed.
he Me hanosa cinaceae, indica ing ha his g oup and hus ace oclas ic me hano-
genesis is o mino impo ance in Puukkosuo en soil. Indeed, he s imula o y e ec
o ace a e on me hanogenic po en ials in en soil mic ocosms was much lowe han o
H2/CO2(Figu e 12). 16S RNA gene sequences and mc A sequences a ilia ed wi h
hyd ogeno ophic me hanogenic g oups a e equen ly obse ed as he p edominan
g oups in pea land soils, e en hough sequences a ilia ed wi h Me hanosa cinales
a e also de ec ed [40, 54, 55, 191],[121].Me hanosa cinales seem o be mo e abun-
dan in we e han in d ie si es in an acidic meso ophic en in Lapland, and we e
iden i ied as CO2consume s in sligh ly acidic Schl¨oppne b unnen en soil [55, 191].
52
3.2 P ocesses leading o o ma ion o CH4
Figu e 14: Phylogene ic ee o ep esen a i e mc A sequences ( o wa d eads)
om Puukkosuo en soil. The ee is based on in silico ansla ed amino
acid sequences ob ained by amplicon-py osequencing. OTUs we e g ouped
a species-le el phylogene ic simila i y o 84% a e ampliconnoise quali y-
il e ing. Values in pa en hese ep esen ela i e abundances o he OTUs.
In o al, 1 785 quali y il e ed sequences we e used o OTU calcula ions.
G ay boxes indica e b anches whe e he majo i y o sequences g oup in o a
ce ain phylogene ic class. The pe cen age o eplica e ees in he boo s ap
analysis (10 000 eplica es), in which he associa ed axa clus e ed oge he ,
a e shown nex o he b anches ( alues below 50% ha e been omi ed). mc A
o Me hanococcus ma ipaludis S2 was used as ou g oup.
3.2.3 Conclusions: Fe men a ion and me hanogenesis in pH-neu al en soil
pH-neu al Puukkosuo en soil showed he po en ial o p oduce ace a e, o ma e,
e hanol, and H2/CO2 om NAG and hus p o ide p ecu so s o en me hanogen-
esis. Me hanogenic po en ials we e s imula ed mainly by H2/CO2and o ma e,
indica ing a p edominance o hyd ogeno ophic me hanogenesis in pH-neu al en
53
3 G eenhouse gas p oduc ion in p is ine pea lands
soil. This inding was suppo ed by analysis o mc A as a s uc u al gene ma ke
o me hanogens, as all de ec ed sequences a ilia ed wi h mc A o hyd ogeno ophic
axa. The bac e ial communi y in Puukkosuo en soil was domina ed by Fi micu es,
Alpha- and Del a-P o eobac e ia including syn ophic gene a as well as by no el
amilies, indica ing high bac e ial di e si y and no el y as well as di e se me abolic
capaci ies in pH-neu al en soil.
54
3.3 P ocesses in ol ed in u no e o N2O
3.3 P ocesses in ol ed in u no e o N2O
3.3.1 N2O p oduc ion and consump ion in p is ine pea lands
N2O-emissions om p is ine pea lands a e highly a iable (Figu e 9). Wa e -sa u a ed
en soils con aining mino amoun s o ni a e emi only small amoun s o N2O and
o en can display also sink- unc ions o N2O (Figu e 9) [43, 42, 93, 141, 161],[117,
120], while pe ma os -a ec ed und a soils like palsa pea s o c yo u ba ed pea
ci cles can emi high amoun s o N2O (Figu e 9) [100, 138],[119]. Especially in
palsa pea s, N2O emissions a e highly a iable depending e.g., on he hickness o
plan co e and ni a e a ailabili y, and ni a e-limi ed palsas also ac as N2O sinks
[100],[119].
S a is ical analyses we e conduc ed o assess he co ela ion be ween en i onmen-
al pa ame e s and in si u N2O emission o i e con as ing pea land soils (Table 6,
Figu e 9). Spea man ank co ela ion e ealed posi i e (R= 0.9, P= 0.08) and
nega i e (R=−0.8, P= 0.08) co ela ions be ween soil ni a e and ammonium con-
en and in si u N2O emission, espec i ely (Table 7), indica ing ha deni i ica ion
a he han ni i ica ion is he p ocess esponsible o obse ed N2O emission om
pea lands (Figu e 9). Indeed, pea land soil has a high deg ee o wa e -sa u a ion,
and deni i ica ion is he main sou ce o N2O in wa e -sa u a ed and hus mainly
anoxic soils [23, 130]. The amoun o o al ca bon in he s udied pea land soils
dec eased wi h inc easing wa e con en and inc easing mean annual empe a u e
(R=−0.9 and R=−0.8, espec i ely), indica ing ha we and wa m condi ions
acili a e he mine aliza ion o soil ca bon. In e es ingly, in si u N2O emissions did
no co ela e wi h soil pH (Table 7), indica ing ha ni a e a ailabili y a he han
soil pH is he main d i e o N2O emissions om pea soil.
N2O p oduc ion in unsupplemen ed anoxic mic ocosms wi h pea land soil anged
om <1 o 1200 nmol·g−1
DW (Figu e 15). N2O p oduc ion was highes in mic ocosms
55
3 G eenhouse gas p oduc ion in p is ine pea lands
Table 7: Co ela ion o soil pa ame e s and obse ed N2O emissions. Spea man ank co e-
la ions we e calcula ed based on mean alues o en i onmen al pa ame e s. Values o N2O
emissions we e based on Figu e 9, alues o en i onmen al pa ame e s we e aken om si e
desc ip ions [100, 119, 120, 185].
Pa ame e pH wa e con en MAT 1NO−
3NH+
4To al C 2To al N 3C:N
Co ela ion (R) 0.10 -0.20 0.21 0.87 -0.82 0.10 0.50 -0.60
Signi icane (P) 0.95 0.78 0.78 0.08 0.08 0.95 0.45 0.35
1Mean annual empe a u e.
2To al ca bon.
3To al ni ogen.
wi h c yo u ba ed pea soil and lowes in mic ocosms wi h en soil (Figu e 15). In
mic ocosms wi h palsa and c yo u ba ed pea soil, N2O ha was p oduced wi hin
he i s 50 o 100 hou s o incuba ion was subsequen ly consumed (Figu e 15),
demons a ing he capaci y o pea land soils o N2O educ ion. N2O p oduc ion
was always highe in mic ocosms ha we e amended wi h ace ylene o block N2O
educ ion [190] han in unamended mic ocosms (Figu e 15), indica ing ha N2O
was no he sole endp oduc o deni i ica ion in ei he o he sys ems, bu ha
comple e deni i ica ion o N2occu ed. In ace ylene-amended mic ocosms, highes
N2O p oduc ion (1800 nmol·g−1
DW ) was obse ed in mic ocosms wi h c yo u ba ed
pea soil (Figu e 15), e lec ing he high ni a e con en o he soil and he obse ed
high in si u emissions (Figu e 9) [100, 138],[115]. N2O p oduc ion in ace ylene-
amended mic ocosms wi h palsa pea soil and wi h pH-neu al en soil was abou
10 old lowe han in mic ocosms wi h c yo u ba ed pea soil (app oxima ely 200
nmol·g−1
DW ). Lowes N2O p oduc ion was obse ed in ace ylene-amended mic ocosms
wi h pe ma os und a soil (<14 nmol·g−1
DW ) (Figu e 15).
Spea man ank co ela ion o en i onmen al pa ame e s and obse ed N2O o
(N2O+N2) in anoxic mic ocosms wi h pea soil e ealed ha he con ibu ion o
N2O o o al N-gases dec eased wi h inc easing pH (R=−0.9, P= 0.08) and
56
3.3 P ocesses in ol ed in u no e o N2O
Figu e 15: P oduc ion o N2O in unsupplemen ed anoxic mic ocosms wi h
pea land soil om 0 o 20 cm dep h. Open symbols ep esen mi-
c ocosms wi hou addi ion o ace ylene, closed symbols ep esen mic ocosms
wi h addi ion o ace ylene o block N2O educ ase [190]. The inse s in (A),
(B), (C), and (D) ep esen enla gemen s o allow be e isualiza ion o el-
a i ely small amoun s o N2O p oduced in hose mic ocosms. A: Puukkosuo
en (pH 6.8), B: Schl¨oppne b unnen en (pH 5.0), C: Pe ma os und a (pH
4.0), D: Skallu aa a palsa pea (pH 4.5), E: C yo u ba ed pea ci cles (pH
4.0).
inc easing mean annual empe a u e (R=−1.0, P= 0.02), indica ing ha unde
wa me and pH-neu al condi ions N2is he a ou ed endp oduc o deni i ica ion.
Indeed, he ela i e con ibu ion o N2O o o al N-gases is highe unde mo e acidic
condi ions in soils and pu e cul u es as N2O educ ase is mo e se e ely a ec ed by
acidic pH han he o he N- educ ases in ol ed in deni i ica ion [8, 39, 91, 170, 171].
Mo eo e , N2O- educ ion is mo e s ongly a ec ed by low empe a u es han he
p eceeding educ i e s eps, hus N2O elease om soil is o en high also in win e
[31, 53, 110]. Howe e , deni i ie communi ies can be adap ed o low pH and low
empe a u es as well [31, 117].
Supplemen al ni a e (0 o 500 µM) s imula ed N2O p oduc ion in ni a e-deple ed
ace ylene-amended anoxic mic ocosms wi h pea land soil o di e en ex en s (Fig-
u e 16). Maximal ini ial N2O p oduc ion a es ( max) we e highes in mic ocosms
wi h Schl¨oppne b unnen en soil (pH 5), while max was lowes in mic ocosms wi h
57
3 G eenhouse gas p oduc ion in p is ine pea lands
Skallu aa a palsa pea soil (pH 4.5) (Figu e 16, Table 8). In mic ocosms wi h
pe ma os und a soil, N2O p oduc ion a es we e highes wi h 10 µM ni a e
and dec eased wi h inc easing ni a e concen a ions, indica ing ha deni i ica ion
in pe ma os und a soil is sa u a ed a low ni a e concen a ions. Supplemen-
al ni i e (0 o 500 µM) likewise s imula ed N2O p oduc ion in ni a e-deple ed
ace ylene-amended anoxic mic ocosms wi h pea land soil (Figu e 16). max we e
highes in mic ocosms wi h Puukkosuo en soil (52 nmol·g−1
DW ·h−1), in e media e
in mic ocosms wi h Schl¨oppne b unnen en and c yo u ba ed pea soil (32 −33
nmol·g−1
DW ·h−1), and lowes in pe ma os und a and Skallu aa a palsa pea soil (18
and 15 nmol·g−1
DW ·h−1, espec i ely) (Table 8). max alues we e in he same ange
in ni a e- and ni i e-supplemen ed mic ocosms wi h Schl¨oppne b unnen en soil,
while max alues we e 2- o 9- old highe in ni i e- han in ni a e-supplemen ed
mic ocosms wi h soil om he o he pea lands (Table 8).
max/KM(indica i e o a soils ni a e/ni i e a ini y) we e highes o ni a e-
and ni i e-dependen deni i ica ion in mic ocosms wi h c yo u ba ed pea soil (Ta-
ble 8). Ni a e-dependen max/KMwe e posi i ely co ela ed wi h he ni a e con-
en o he soil and in si u N2O emissions as well as nega i ely co ela ed wi h he
ammonium con en o he soil (Spea man ank co ela ion: R= 0.9, P= 0.08;
R≈1.0, P < 0.0001; R=−0.8, P= 0.08 o ni a e con en , in si u emissions,
and ammonium con en , espec i ely).
Table 8: Pa ame e s o appa en ni a e- and ni i e-dependen Michaelis-Men en kine ics in anoxic ace ylene-amended mic ocosms wi h pea land soil.
Ni a e amended Ni i e amended
Soil Puukkosuo Schl¨oppne b unnen Pe ma os Skallu aa a C yo u ba ed Puukkosuo Schl¨oppne b unnen Pe ma os Skallu aa a C yo u ba ed
en en und a palsa pea pea ci cles en en und a palsa pea pea ci cles
max (nmol·h−1·g−1
DW )118 25 2 3 17 52 33 18 15 32
KM(µM) 129 11 n.a. 217 3 62 7 21 39 6
max/KM(10−3·h−1)117 53 n.a. 211 466 22 105 41 25 407
1Kine ic pa ame e s calcula ed om Figu e 16.
2No applicable.
The a io o N2O o o al N-gases (i.e., he amoun o N2O p oduced in he ab-
sence o he amoun o N2O p oduced in he p esence o ace ylene) a di e en
58
3.3 P ocesses in ol ed in u no e o N2O
Figu e 16: Appa en Michaelis-Men en kine ics o ni a e- (A) and ni i e-
dependen (B) deni i ica ion in ace ylene-amended anoxic mic o-
cosms wi h pea land soil om 0 o 20 cm dep h. Blue squa es: Puukko-
suo en (pH 6.8), Red ci cles: Schl¨oppne b unnen en (pH 5.0), G een upwa d
iangles: Pe ma os und a (pH 4.0), Black diamonds: Skallu aa a palsa
pea (pH 4.5), O ange downwa d iangles: C yo u ba ed pea ci cles (pH
4.0). Indi idual alues o duplica e mic ocosms (c yo u ba ed pea ci cles)
o mean alues and s anda d e o s o h ee eplica e mic ocosms (all o he
soils) a e displayed. Michaelis-Men en cu es we e i ed o he da a whe e
applicable (solid lines).
concen a ions o supplemen ed ni a e and ni i e di e ed be ween he soils (Fig-
u e 17). In pH-neu al en soil, N2O/(N2O+N2) was below 10% when 10 µM ni a e
o ni i e we e supplied and did no exceed 30% a 100 µM supplied ni a e/ni i e.
In con as , N2O/(N2O+N2) was 100% a all supplied concen a ions o ni a e
o ni i e in mic ocosms wi h pe ma os und a soil. Indeed, N2O/(N2O+N2) was
nega i ely co ela ed wi h soil pH (Spea man ank co ela ion: R=−0.9, P= 0.08
o 100 µM supplied ni a e). Mo eo e , he e was a posi i e co ela ion be ween
N2O/(N2O+N2) when ni a e and when ni i e we e supplied (Spea man ank co e-
la ion: R≈1.0, P < 0.0001 o 10 µM supplied ni a e/ni i e). Ni a e concen a-
ions gene ally in luence he a ios o N2O/N2+N2O p oduced om deni i ica ion
59
3 G eenhouse gas p oduc ion in p is ine pea lands
and Me hylobac e ium sp., was de ec ed almos exclusi ely in pH-neu al en soil
om Puukkosuo, whe e i accoun ed o >60% o de ec ed ni K, indica ing ha
his OTU migh be adap ed o neu al pH and hus unable o ole a e highe le els
o acidi y (Figu e 18 B). OTU 4 was only de ec ed in sligh ly acidic en soil om
Schl¨oppne b unnen and was only dis an ly ela ed o known ni K (Figu e 18 B,
Figu e 20). Thus, he e migh be no el and specialized Ni K- ype deni i ie s in his
en, ha can ole a e sligh ly acidic condi ions bu a e mo e sensible o soil eezing.
De ec ed and es ima ed species-le el OTU numbe s we e highes in Puukkosuo and
Schl¨oppne b unnen en soils and lowes in pe ma os -a ec ed palsa pea and pea
ci cle soil (Table 9). Shannon di e si y and Species E enness indices we e simila
in all soils excep o c yo u ba ed pea ci cle soil, which showed e y low Shannon
di e si y and Species E enness (Table 9).
ni S sequences likewise g ouped in o 10 majo species-le el OTUs (i.e., OTUs
wi h a ela i e abundance o >3% in a leas one o he soils). Th ee di e en
ypes o ni S communi ies we e de ec ed: The i s ype was de ec ed in he pH-
neu al en Puukkosuo and consis ed mainly o OTUs 3 and 5 (app oxima ely 80%
o all sequences), which a ilia ed wi h uncul u ed soil and sedimen bac e ia in
he phylogene ic ee (Figu e 18 C, Figu e 21). Thus, his communi y ype is likely
domina ed by hi he o unknown Ni S- ype deni i ie s. The second ni S communi y
ype was de ec ed in Schl¨oppne b unnen en and c yo u ba ed pea ci cle soil. This
communi y ype was domina ed by OTU 1 (Figu e 18 C), which a ilia ed wi h ni S
o B ady hizobium sp., Rhodanobac e sp., and Magne ospi illum magne icum in he
phylogene ic ee (Figu e 21). The hi d ype was de ec ed in un u ba ed pe ma os
und a and Skallu aa a palsa pea soil and was domina ed by OTUs 2, 3, and 4
(Figu e 18 C). OTUs 2 and 3 we e ela ed o ni S o uncul u ed sedimen bac e ia,
while OTU 4 was ela ed o ni K o Azoa cus oluly icus (Figu e 21). Species-le el
66
3.3 P ocesses in ol ed in u no e o N2O
ni S di e si y as exp essed by numbe o obse ed and es ima ed OTUs as well as
by Shannon di e si y and Species E enness indices was high in Puukkosuo en soil
and low in c yo u ba ed pea ci cle soil, while i was a a simila le el in all o he
s udied soils (Table 9).
nosZ sequences g ouped in o 8 majo species-le el OTUs (i.e., OTUs wi h a ela-
i e abundance o >1% in a leas one o he soils). nosZ OTU dis ibu ion di e ed
be ween he 5 soils (Figu e 18 D). 7 majo OTUs we e de ec ed in Puukkosuo en
soil, OTUs 1, 4, 5, and 6 we e mos p ominen wi h ela i e abundances o 45%,
26%, 13%, and 10%, espec i ely (Figu e 18 D). OTU 1 was also de ec ed in he 4
o he soils, i domina ed de ec ed nosZ in Schl¨oppne b unnen en soil (87%), bu
had a e y low abundance in pe ma os und a and c yo u ba ed pea ci cle soil (2%
and 3% o de ec ed nosZ, espec i ely). OTU 2 was almos exclusi ely de ec ed in
pe ma os und a and c yo u ba ed pea ci cle soil, whe e i accoun ed o 97% and
94% o de ec ed nosZ, espec i ely (Figu e 18 D). OTU 3 was he p edominan OTU
in Skallu aa a palsa pea soil (52%) and was also abundan in Schl¨oppne b unnen
en soil (12%). OTUs 1, 2, 3, 4, 5, and 6 we e a ilia ed wi h nosZ o Azospi illum
lipo e um,Meso hizobium sp., B ady hizobium japonicum,Bosea sp., Azospi illum
la gimobile, and He baspi illum spp., espec i ely (Figu e 22), indica ing ha de-
ec ed pea land nosZ a e di e se and domina ed by Alpha- and Be a-P o eobac e ial
nosZ.Be a-P o eobac e ial nosZ we e almos exclusi ely de ec ed in Puukkosuo en
soil (Figu e 22), indica ing ha Be a-P o eobac e ia migh be mo e se e ely a ec ed
by he mo e acidic pH in he o he soil ypes, con i ming he e ec obse ed o na G
(Figu e 18 D, Figu e 19). Species-le el nosZ di e si y as gi en by he numbe o
obse ed and es ima ed OTUs and Shannon di e si y and Species E enness indices
was highes in Puukkosuo en soil (Table 9). This inding is in line wi h he esul s
ob ained wi h he o he gene ma ke s.
67
3 G eenhouse gas p oduc ion in p is ine pea lands
The collec i e da a indica e ha (i) Ac inobac e ial,Alpha- and Be a-P o eobac e ial
ni a e educe s and deni i ie s a e common in pea lands, (ii) some species occu in
all pea lands and migh hus con ibu e o a co e deni i ie communi y in pea lands,
(iii) ni a e educe and deni i ie communi y composi ions di e be ween di e en
pea land ypes, and (iii) highes di e si y is associa ed wi h pH-neu al en soil.
68
3.3 P ocesses in ol ed in u no e o N2O
Figu e 20: Phylogene ic ee o ep esen a i e ni K sequences om di e en
pea land soils. The ee is based on in silico ansla ed amino acid se-
quences. Sequences we e ob ained ia amplicon-py osequencing om pea -
land soils. OTUs we e g ouped a species-le el phylogene ic simila i y o
83% a e ampliconnoise quali y- il e ing. Values in pa en hese ep esen el-
a i e abundances o he OTUs in Puukkosuo en, Schl¨oppne b unnen en,
pe ma os und a, Skallu aa a palsa pea , and c yo u ba ed pea ci cles. In
o al, 19 292 quali y il e ed sequences we e used o OTU calcula ions. G ay
boxes indica e b anches whe e he majo i y o sequences g oup in o a ce ain
phylogene ic class. The pe cen age o eplica e ees in he boo s ap anal-
ysis (10 000 eplica es), in which he associa ed axa clus e ed oge he , a e
shown nex o he b anches ( alues below 50% ha e been omi ed). ni K o
Haloa cula ma ismo ui ATCC 43049 was used as ou g oup.
69
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 21: Phylogene ic ee o ep esen a i e ni S sequences ( o wa d eads)
om di e en pea land soils. The ee is based on in silico ansla ed
amino acid sequences. Sequences we e ob ained ia amplicon-py osequencing
om pea land soils. OTUs we e g ouped a species-le el phylogene ic simila -
i y o 82% a e ampliconnoise quali y- il e ing. Values in pa en hese ep esen
ela i e abundances o he OTUs in Puukkosuo en, Schl¨oppne b unnen en,
pe ma os und a, Skallu aa a palsa pea , and c yo u ba ed pea ci cles. In
o al, 5 218 quali y il e ed sequences we e used o OTU calcula ions. G ay
boxes indica e b anches whe e he majo i y o sequences g oup in o a ce ain
phylogene ic class. The pe cen age o eplica e ees in he boo s ap anal-
ysis (10 000 eplica es), in which he associa ed axa clus e ed oge he , a e
shown nex o he b anches ( alues below 50% ha e been omi ed). ni S o
Rhodo he mus ma inus DSM 4252 was used as ou g oup.
70
3.3 P ocesses in ol ed in u no e o N2O
Figu e 22: Phylogene ic ee o ep esen a i e nosZ sequences ( o wa d eads)
om di e en pea land soils. The ee is based on in silico ansla ed
amino acid sequences. Sequences we e ob ained ia amplicon-py osequencing
om pea land soils. OTUs we e g ouped a species-le el phylogene ic simila -
i y o 80% a e ampliconnoise quali y- il e ing. Values in pa en hese ep esen
ela i e abundances o he OTUs in Puukkosuo en, Schl¨oppne b unnen en,
pe ma os und a, Skallu aa a palsa pea , and c yo u ba ed pea ci cles. In
o al, 5 920 quali y il e ed sequences we e used o OTU calcula ions. G ay
boxes indica e b anches whe e he majo i y o sequences g oup in o a ce ain
phylogene ic class. The pe cen age o eplica e ees in he boo s ap anal-
ysis (10 000 eplica es), in which he associa ed axa clus e ed oge he , a e
shown nex o he b anches ( alues below 50% ha e been omi ed). nosZ o
Haloa cula ma ismo ui ATCC 43049 was used as ou g oup.
71
3 G eenhouse gas p oduc ion in p is ine pea lands
3.3.3 En i onmen al ac o s shaping deni i ie communi ies in pea lands
S a is ical analyses based on he communi y composi ion o sequences ecei ed om
py osequencing o na G,ni K,ni S, and nosZ om di e en pea land soils we e
conduc ed o assess he e ec o di e en en i onmen al pa ame e s on communi y
composi ion o ni a e educe s and deni i ie s. En i onmen al pa ame e s we e
classi ied as p ima y en i onmen al pa ame e s (i.e., pH, empe a u e, p ecipi a ion,
ni a e and ammonium con en , o al ca bon, o al ni ogen, C/N a io) o de i ed
pa ame e s (i.e., N2O emission, a io o N2O o o al N-gases, ela i e abundance
o na G o nosZ). Canonical co espondence analyses (CCA) we e conduc ed o
each gene ma ke on he basis o a i ied species-le el OTU ables o clus e he
communi ies acco ding o hei habi a and o elucida e he p ima y o de i ed
en i onmen al pa ame e s ha in luence communi y composi ion.
CCA o na G e ealed a clea sepa a ion o Puukkosuo and Schl¨oppne b unnen
en soil communi ies om each o he and om he pe ma os a ec ed communi ies
(Figu e 23 A), as was al eady obse ed o di ec compa ison o OTU ela i e abun-
dances (Figu e 18 A). Communi y composi ion was a ec ed by pH, o al soil ca bon
and mean annual p ecipi a ion (Figu e 23 A). The ela i e abundance o OTU 1 was
nega i ely co ela ed wi h pH and soil wa e con en (Spea man ank co ela ions
wi h R=−0.8, P= 0.08 and R≈ −1.0, P= 0.02, espec i ely), and posi i ely
co ela ed wi h o al soil ca bon (R= 0.9, P= 0.08), while OTU 2 was posi i ely
and nega i ely co ela ed wi h soil wa e con en and o al soil ca bon, espec i ely
(R≈1.0, P < 0.001 and R=−0.9, P= 0.08, espec i ely).
CCA o ni K showed a clea sepa a ion o all s udied pea land communi ies (Fig-
u e 23 B). The ni K communi y o Puukkosuo en was mos dissimila o he com-
muni ies in he o he pea land soil, as i was also indica ed ea lie (Figu e 18 B, Ta-
ble 9). pH and o al soil ca bon de e mined he communi y composi ion o de ec ed
72
3.3 P ocesses in ol ed in u no e o N2O
Figu e 23: Canonical co espondence analyses (CCA) based on ela i e
species-le el OTU abundances o na G (A), ni K (B), ni S (C),
and nosZ (D). Blue ci cles ep esen he di e en sampling si es (Table 6).
P ima y and de i ed en i onmen al pa ame e s a ec ing he communi y com-
posi ion o deni i ica ion-associa ed genes a e displayed in ed. OTU abun-
dances om a i ied da ase s(100 i e a ions a sampling dep hs o 500, 1000,
100, and 100 o na G,ni K,ni S, and nosZ, espec i ely) we e used o he
calcula ion o CCAs.
ni K (Figu e 23 B). Co ela ions be ween en i onmen al pa ame e s and indi id-
ual OTUs we e obse ed o OTU 1, which was p edominan in os -a ec ed soils
and was posi i ely, nega i ely and nega i ely co ela ed wi h o al soil ca bon, soil
wa e con en and soil pH, espec i ely (Spea man ank co ela ions wi h R= 0.9,
P= 0.08, R≈ −1.0, P= 0.02, and R=−0.8, P= 0.08, espec i ely). On he
o he hand, he ela i e abundance o OTU 5 which was abundan in Puukkosuo and
73
3 G eenhouse gas p oduc ion in p is ine pea lands
Schl¨oppne b unnen en soil was posi i ely and nega i ely co ela ed wi h soil wa e
con en and o al soil ca bon, espec i ely (R= 0.98, P= 0.02 and R=−0.87,
P= 0.08, espec i ely), indica ing ha o al soil ca bon does no only a ec he
ni K communi y composi ion in o al bu also mo e di ec ly he ela i e abundance
o indi idual OTUs. OTU 2 was he only OTU ha showed a co ela ion wi h in
si u N2O emission, he ela i e abundance o OTU 2 was nega i ely co ela ed wi h
N2O emission (R=−0.9, P= 0.08), indica ing ha deni i ie s ca ying his ype
o ni K migh be impo an o N2O educ ion in si u.
CCA based on ela i e abundances o de ec ed ni S e ealed 3 dis inc clus e s o
ni S communi ies, hus suppo ing he isual compa ison (Figu e 23 C, Figu e 18 C).
The ni S communi y om Puukkosuo en was clea ly dis inc om he communi y in
he o he pea land soils, and Skallu aa a palsa pea and pea land und a as well as
Schl¨oppne b unnen en and c yo u ba ed pea ci cle communi ies clus e ed oge he
(Figu e 23 C). Communi y composi ion o de ec ed ni S was in luenced by pH as
a p ima y en i onmen al ac o and by he ela i e abundance o na G and he
a io o N2O o o al N-gases (when 10 µM ni i e we e supplied) as de i ed ac o s
(Figu e 23 C). The ela i e abundance o se e al OTUs was co ela ed o obse ed
in si u N2O emissions: OTU 1 was posi i ely co ela ed wi h in si u N2O emissions
(Spea man ank co ela ion wi h R= 0.98, P= 0.02), while OTUs 2, 4 and 6 we e
nega i ely co ela ed wi h in si u N2O emissions (R=−0.98, P= 0.02, R=−0.87,
P= 0.08, and R=−0.98, P= 0.02, espec i ely), indica ing ha deni i ie s
ha bo ing hose ypes o ni S migh be in ol ed in p oduc ion and consump ion o
N2O in pea land soils, espec i ely.
CCA analysis based on ela i e abundances o nosZ in pea land soils likewise sup-
po ed he obse ed g ouping o pea land soils (Figu e 23 D, Figu e 18 D), as he
nosZ communi y o Puukkosuo en was clea ly sepa a ed om he o he soils, while
74
3.3 P ocesses in ol ed in u no e o N2O
nosZ communi ies o Schl¨oppne b unnen en and Skallu aa a palsa pea as well
as nosZ communi ies o pe ma os und a and c yo u ba ed pea ci cles g ouped
oge he (Figu e 23 D). Communi y composi ion o nosZ was in luenced by he
p ima y en i onmen al pa ame e s pH and empe a u e as well as by he ela i e
abundance o nosZ in pea land soils as a seconda y pa ame e (Figu e 23 D). Unlike
OTUs o ni K and ni S, OTUs o nosZ we e no co ela ed wi h obse ed in si u
N2O emissions. Howe e , OTUs 1 and 2 we e nega i ely and posi i ely co ela ed
wi h he a io o N2O o o al N-gases in unsupplemen ed anoxic mic ocosms, e-
spec i ely (Spea man ank co ela ions wi h R≈ −1.0, P= 0.02 and R= 0.9,
P= 0.08, espec i ely), indica ing ha deni i ie s ha bo ing nosZ o OTU 1 migh
be highly e icien in N2O consump ion and cause he obse ed di e ences in N2O
emissions.
The collec i e da a indica e ha (i) ni a e educe and deni i ie communi ies
in di e en pea land soils a e clea ly dis inc , (ii) he p ima y and de i ed en i on-
men al ac o s a ec ing communi y composi ion o deni i ica ion associa ed genes
di e be ween he genes, and (iii) se e al OTUs o ni K,ni S, and nosZ a e likely
de e mining N2O emissions om pea land soils.
75
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 26: E ec o 6 mon h looding on o ma e-dependen me hanogenesis
in anoxic mic ocosms wi h en soil om 0 o 20 cm (A) and 20 o
40 cm dep h (B). Mic ocosms we e supplemen ed wi h 0 o 1 mM o ma e
a e 7 days o anoxic p eincuba ion o educe al e na i e elec on accep o s.
max a e based on ini ial CH4p oduc ion a es in mic ocosms. Black ba s
ep esen mic ocosms wi h soil om con ol plo s, whi e ba s ep esen mic o-
cosms wi h soil om looded plo s. Mean alues o h ee eplica e mic ocosms
and s anda d e o s a e displayed. Pos - looding CH4p oduc ion a es in mi-
c ocosms wi h 0 o 20 cm soil om con ol plo s did no ollow appa en
Michaelis-Men en kine ics (*). The inse in (B) ep esen s a magni ica ion
o he y-axis o be e isualize he low obse ed max.
composi ion in 2008 and 2009 (Figu e 28 A and B, espec i ely), wi h Me hanocel-
laceae being o mos abundan g oup in 2008 and Me hanomic obiales/-bac e iales
being he mos abundan g oup in 2009. This indica es ha he composi ion o he
me hanogenic communi y is a he s able when subjec ed o sho - e m changes such
as expe imen al d ough o looding bu migh be in luenced by annual a ia ions.
82
3.4 E ec o wa e able manipula ions on anae obic p ocesses in a model pea land
Figu e 27: E ec o wa e able manipula ions on he abundance o mc A genes
and ansc ip s in an acidic en.mc A gene copy numbe s in ela ion
o a chaeal 16S RNA genes we e de e mined be o e and a e a 42 day long
expe imen al d ough in 2006 wi h soil om 0 o 40 cm dep h (A) as well as
be o e and a e 6 mon h o expe imen al looding in 2009 wi h soil om 0 o
40 cm dep h (B). mc A ansc ip numbe s we e de e mined be o e and a e
a 42 day long expe imen al d ough in 2006 wi h soil om 0 o 40 cm dep h
(C). Black ba s ep esen abundances in con ol plo s, whi e ba s ep esen
abundances in ea men plo s. Mean alues o h ee eplica e plo s ( ou
sampling dep hs pe plo ) and s anda d e o s a e displayed.
83
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 28: E ec o wa e able manipula ions on he communi y composi ion
o mc A genes and ansc ip s in an acidic en. Communi y compo-
si ion was de e mined by TRFLP analysis be o e and a e a 42 day long
expe imen al d ough in 2006 wi h soil om 0 o 40 cm dep h on gene (A)
and ansc ip (C) le el as well as on gene le el be o e and a e 6 mon h
o expe imen al looding in 2009 wi h soil om 0 o 40 cm dep h (B). All
TRFs ha could be assigned o a ce ain o de / amily o me hanogens we e
g ouped. Mean alues o h ee eplica e plo s ( ou sampling dep hs pe plo )
a e displayed.
84
3.4 E ec o wa e able manipula ions on anae obic p ocesses in a model pea land
3.4.3 E ec o wa e able manipula ions on deni i ica ion in an acidic en
The e ec o wa e able manipula ions on he abundance o ni a e educe s and
deni i ie s in acidic en soil was assessed by qPCR o na G and nosZ in con ol and
ea men plo s be o e and a e he expe imen al d ough in 2008 as well as be o e
and a e expe imen al looding in 2009 (qPCR assay desc ibed in [118]). na G and
nosZ copy numbe s anged om 2% o 14% and om 0.02% o 0.2% o bac e ial
16S RNA gene copy numbe s, espec i ely (Figu e 29). A e 42 days o expe i-
men al d ough , ela i e abundances o na G and nosZ in d ough plo s we e 2x
and 1.5 as high as be o e he d ough pe iod, espec i ely, while hey we e sligh ly
lowe in con ol plo s (0.75x and 0.6x as high as be o e he d ough pe iod, espec-
i ely) (Figu e 29), indica ing ha g ow h o ni a e educe s and deni i ie s was
s imula ed by he expe imen al d ough . Expe imen al d ough leads o inc eased
oxygen pene a ion in o he soil and migh hus p omo e he g ow h o acul a i ely
ae obic deni i ie s. The obse ed e ec o expe imen al looding on na G and nosZ
abundances was less p onounced han he e ec o expe imen al d ough . Rela i e
abundances o na G we e lowe pos - han p e- looding in looded and con ol plo s,
while ela i e abundances o nosZ we e simila pos - han p e- looding in bo h plo
ypes (Figu e 29), indica ing ha inc eased wa e sa u a ion does no in luence he
ela i e abundance o ni a e educe s o deni i ie s in en soil. Mo eo e , he ela-
i e abundance o ni a e educe s unde goes likely s onge seasonal a ia ions han
he ela i e abundance o deni i ie s, as he ela i e abundance o na G was lowe
in he end o he expe imen al pe iod in 2008 and 2009 (Augus and No embe ,
espec i ely), while he a ia ion in nosZ ela i e abundance was less p onounced
(Figu e 29).
The e ec o wa e able manipula ions on he communi y composi ion o ni a e
educe s and deni i ie s was assessed by TRFLP analysis o na G and nosZ in con-
85
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 29: E ec o wa e able manipula ions on he abundance o na G (A,B)
and nosZ (C,D) in an acidic en.na G and nosZ gene copy numbe s in
ela ion o bac e ial 16S RNA genes we e de e mined be o e and a e a 42
day long expe imen al d ough in 2006 wi h soil om 0 o 40 cm dep h (A,C)
as well as be o e and a e 6 mon h o expe imen al looding in 2009 wi h
soil om 0 o 40 cm dep h (B,D). Black ba s ep esen abundances in con ol
plo s, whi e ba s ep esen abundances in ea men plo s. Mean alues o
h ee eplica e plo s ( ou sampling dep hs pe plo ) and s anda d e o s a e
displayed.
ol and ea men plo s be o e and a e he expe imen al d ough in 2008 as well as
be o e and a e expe imen al looding in 2009 (expe imen al p ocedu e o TRFLP
desc ibed in [118]). Up o 8 and 9 TRFs we e obse ed o na G and nosZ, espec-
i ely (Figu e 30, Figu e 31). Communi y composi ion o na G and nosZ was simila
in con ol and ea men plo s a all sampling imepoin s (Figu e 30,Figu e 31), in-
dica ing ha he composi ion o he ni a e educe and deni i ie communi y is
likely una ec ed by sho - e m wa e able manipula ions o by seasonal a iabili y.
S abili y o deni i ie communi ies o wa e able luc ua ions is obse ed in many
we land sys ems. Deni i ie communi ies in a cons uc ed we land a y be ween
indi idual si es bu a e a he una ec ed by hyd ological pulsing (i.e., sho - e m
d ough and subsequen looding) [155]. Mo eo e , he communi y composi ion o
nosZ in en g assland soil does no change signi ican ly in esponse o a ia ions in
86
3.4 E ec o wa e able manipula ions on anae obic p ocesses in a model pea land
wa e con en [159].
Figu e 30: E ec o wa e able manipula ions on he communi y composi ion
o na G genes in an acidic en. Communi y composi ion was de e mined
by TRFLP analysis be o e and a e a 42 day long expe imen al d ough in
2006 wi h soil om 0 o 40 cm dep h (A) as well as be o e and a e 6 mon h
o expe imen al looding in 2009 wi h soil om 0 o 40 cm dep h (B). Mean
alues o h ee eplica e plo s ( ou sampling dep hs pe plo ) a e displayed.
The e ec o p olonged looding o he en soil on ni a e-dependen deni i i-
ca ion was assessed in anoxic mic ocosms wi h en soil aken be o e and a e a
6 mon hs looding pe iod in 2009 om 0 o 20 cm and 20 o 40 cm dep h om
con ol and ea men plo s. Appa en Michaelis-Men en kine ics we e de e mined
based on ini ial N2O p oduc ion a es in ace ylene-amended ni a e-supplemen ed
mic ocosms. max we e highe in 0 o 20 cm han in 20 o 40 cm dep h soil, hus
deni i ica ion po en ials like e men a i e and me hanogenic po en ials a e mainly
loca ed in he uppe soil laye s (Figu e 32). P olonged looding inc eased max in
87
3 G eenhouse gas p oduc ion in p is ine pea lands
Figu e 31: E ec o wa e able manipula ions on he communi y composi ion
o nosZ genes in an acidic en. Communi y composi ion was de e mined
by TRFLP analysis be o e and a e a 42 day long expe imen al d ough in
2006 wi h soil om 0 o 40 cm dep h (A) as well as be o e and a e 6 mon h
o expe imen al looding in 2009 wi h soil om 0 o 40 cm dep h (B). Mean
alues o h ee eplica e plo s ( ou sampling dep hs pe plo ) a e displayed.
bo h dep hs in ea men plo s, while max was simila in con ol plo s p e- and
pos - looding (Figu e 32 A, B). The a io o N2O o o al N-gases was lowe pos -
han p e- looding in ea men plo s, while i was simila in con ol plo s a bo h
ime poin s (Figu e 32 C, D). Thus, he highe wa e -sa u a ion o en soil as in-
duced by p olonged looding likely leads o inc eased deni i ica ion and highe N2O
consump ion po en ials. Highe in si u deni i ica ion a es a e de ec ed in a con-
s uc ed we land when soil is looded a e an ini ial d ough pe iod [155]. N2O
consump ion is s ongly a ec ed by he amoun o a ailable oxygen in he sys em,
and unde wa e -sa u a ed, mos ly anoxic condi ions he end-p oduc o deni i i-
88
3.4 E ec o wa e able manipula ions on anae obic p ocesses in a model pea land
ca ion is mainly N2[3, 26]. Wa e -sa u a ed pea soil can be a sink o N2O a he
han a sou ce, while pea soil wi h a lowe ed wa e able wa e able is a sou ce o
N2O [42, 93, 136, 137, 141]. Mo eo e , d ying o nu ien - ich pea soil inc eases he
amoun o emi ed N2O [99, 136, 137]. Thus, a aised wa e able due o inc eased
p ecipi a ion migh enhance he N2O sink s eng h in pea land ecosys ems.
Figu e 32: E ec o 6 mon h looding on ni a e-dependen N2O p oduc ion
and he a io o N2O o o al N-gases in anoxic mic ocosms wi h
en soil om 0 o 20 cm (A, C) and 20 o 40 cm dep h (B, D).
Mic ocosms we e supplemen ed wi h 0 o 100 µM ni a e and incuba ed wi h
o wi hou ace ylene (15% ol/ ol) in he gasphase. max a e based on ini ial
N2O p oduc ion a es in mic ocosms wi h ace ylene. The a io o N2O o
o al N-gases equals he a io o N2O p oduc ion in mic ocosms wi hou and
wi h ace ylene. Black ba s ep esen mic ocosms wi h soil om con ol plo s,
whi e ba s ep esen mic ocosms wi h soil om looded plo s. Mean alues o
h ee eplica e mic ocosms and s anda d e o s a e displayed.
3.4.4 Conclusions: wa e able manipula ions
The sho - e m wa e able manipula ions in he model en Schl¨oppne b unnen a -
ec ed me hanogenic and deni i ying po en ials in mic ocosms bu had no ob ious
e ec on e men a ion po en ials, hus u u e changes in wa e able heigh a e likely
o a ec he u no e and elease o g eenhouse gases om he en. Obse ed changes
89
3 G eenhouse gas p oduc ion in p is ine pea lands
in physiological po en ials we e concomi an wi h small bu de ec able changes in
ela i e abundances o he in ol ed mic obial g oups, hus highligh ing he capac-
i y o en mic oo ganism o espond o changing en i onmen al condi ions. The
communi y composi ion o me hanogens and deni i ie s was no a ec ed by he
manipula ion expe imen s, hus indica ing a a he s able mic obial communi y in
en soil. The collec i e da a hus indica e ha he acidic en ha bo s a a he s able
mic obial communi y ha is capable o adap ing i s ac i i y o changing hyd ological
condi ions qui e apidly.
90
3.5 Gene al conclusions
3.5 Gene al conclusions
CH4is p oduced om polyme s and suga s unde anoxic condi ions in we lands,
and di e en g oups o o ganisms a e in ol ed in he con e sion o suga s o CH4
(Figu e 4; [30]). In Puukkosuo en soil, he model suga NAG is i s con e ed
o H2/CO2, ace a e, and o ma e, which can hen be used di ec ly o indi ec ly by
me hanogens, lea ing CH4as he end p oduc (Figu e 33). Me hanogens in en soil
a e hus ophically linked o en soil e men e s, which suppo s he pos ula ed
Hypo hesis 1 (3.1).
Figu e 33: Schema ic model o p ocesses con ibu ing o me hanogenesis in
pH-neu al en soil. Suga s like NAG a e e men ed o a a ie y o e -
men a ion p oduc s, mos ly H2/CO2and ace a e, which in u n a e used by
me hanogens. The ela i e con ibu ion o each p ocess is indica ed by i s
a ow size. Do ed a ows indica e ha mo e han one s ep/o ganism migh
be in ol ed in he con e sion o he compound. Me hanol was no measu ed
in mic ocosm expe imen s, and possible sou ces o me hanol in en soil we e
no in es iga ed.
Deni i ica ion was in es iga ed in i e model pea lands, e ealing ni a e a ail-
abili y as he majo en i onmen al ac o de e mining di e ences in obse ed in si u
N2O emissions (Figu e 34), which is in ag eemen wi h he pos ula ed Hypo he-
91
Re e ences
B. H. Thawing sub-a c ic pe ma os : E ec s on ege a ion and me hane
emissions. Geophysical Resea ch Le e s 31, L04501 (2004).
[20] Ch is ensen, T. R., Michelsen, A., and Jonasson, S. Exchange o
CH4and N2O in a suba c ic hea h soil: e ec s o ino ganic N and P and
amino acid addi ion. Soil Biology & Biochemis y 31, 637–641 (1999).
[21] an Cleempu , O. Subsoils: Chemo- and biological deni i ica ion, N2O
and N2emissions. Nu ien Cycling in Ag oecosys ems 52, 187–194 (1998).
[22] Clymo, R. S. The ecology o pea lands. Science P og ess 71, 593–614 (1987).
[23] Con ad, R. Soil mic oo ganisms as con olle s o a mosphe ic ace gases
(H2, CO, CH4, OCS, N2O, and NO). Mic obiological Re iews 60, 609–640
(1996).
[24] Cos a, C., Dijkema, C., F ied ich, M., Ga cia-Encina, P.,
Fe nandez-Polanco, F., and S ams, A. J. M. Deni i ica ion wi h
me hane as elec on dono in oxygen-limi ed bio eac o s. Applied Mic obiol-
ogy and Bio echnology 53, 754–762 (2000).
[25] Cuhel, J., Simek, M., Laughlin, R. J., B u, D., Cheneby, D., Wa -
son, C. J., and Philippo , L. Insigh s in o he e ec o soil pH on N2O
and N2emissions and deni i ie communi y size and ac i i y. Applied and
En i onmen al Mic obiology 76, 1870–1878 (2010).
[26] Da idson, E. A., Kelle , M., E ickson, H. E., Ve cho , L. V., and
Veldkamp, E. Tes ing a concep ual model o soil emissions o ni ous and
ni ic oxides. Bioscience 50, 667–680 (2000).
[27] Dedysh, S., Pank a o , T., Belo a, S., Kuliche skaya, I., and Lie-
sack, W. Phylogene ic analysis and in si u iden i ica ion o bac e ia commu-
98
Re e ences
ni y composi ion in an acidic Sphagnum pea bog. Applied and En i onmen al
Mic obiology 72, 2110–2117 (2006).
[28] Denman, K. L., B asseu , G., Chid haisong, A., Ciais, P., Cox,
P. M., Dickinson, R. E., Hauglus aine, D., Heinze, C., Holland,
E., Jacob, D., Lohmann, U., Ramachand an, S., da Sil a Dias,
P. L., Wo sy, S. C., and Zhang, X. Couplings be ween changes in he
clima e sys em and biogeochemis y. In: Clima e Change 2007: The Physical
Science Basis. Con ibu ion o Wo king G oup I o he Fou h Assessmen Re-
po o he In e go e nmen al Panel on Clima e Change, edi ed by Solomon,
S., Qin, D., Manning, M., Chen, Z., Ma quis, M., A e y , K. B.,
Tigno , M., and Mille , H. L., pp. 499–588. Camb idge Uni e si y P ess
(2007).
[29] Dow ick, D. J., F eeman, C., Lock, M. A., and Reynolds, B. Sul-
pha e educ ion and he supp ession o pea land me hane emissions ollowing
summe d ough . Geode ma 132, 384–390 (2006).
[30] D ake, H. L., Ho n, M. A., and Wus , P. K. In e media y ecosys-
em me abolism as a main d i e o me hanogenesis in acidic we land soil.
En i onmen al Mic obiology Repo s 1, 307–318 (2009).
[31] D¨
o sch, P. and Bakken, L. R. Low- empe a u e esponse o deni i ica-
ion: Compa ison o soils. Eu asian Soil Science 37, S102–S106 (2004).
[32] Elbe ling, B., Ch is iansen, H. H., and Hansen, B. U. High ni ous
oxide p oduc ion om hawing pe ma os . Na u e Geoscience 3, 332–335
(2010).
[33] Ellis, M. J., G ossmann, J. G., Eady, R. R., and Hasnain, S. S.
99
Re e ences
Genomic analysis e eals widesp ead occu ence o new classes o coppe ni i e
educ ases. Jou nal o Biological Ino ganic Chemis y 12, 1119–1127 (2007).
[34] Fe y, J. G. How o make a li ing by exhaling me hane. Annual Re iew o
Mic obiology 64, 453–73 (2010).
[35] Fe ze , S., Bak, F., and Con ad, R. Sensi i i y o me hanogenic bac-
e ia om paddy soil o oxygen and desicca ion. FEMS Mic obiology Ecology
12, 107–115 (1993).
[36] Fo s e , P., Ramaswamy, V., A axo, P., Be n sen, T., Be s, R.,
Fahey, D., Haywood, J., Lean, J., Lowe, D., Myh e, G., Nganga,
J., P inn, R., Raga, G., Schulz, M., and VanDo land, R. Changes in
a mosphe ic cons i uen s and in adia i e o cing. In: Clima e Change 2007:
The Physical Science Basis. Con ibu ion o Wo king G oup I o he Fou h
Assessmen Repo o he In e go e nmen al Panel on Clima e Change, edi ed
by Solomon, S., Qin, D., Manning, M., Chen, Z., Ma quis, M.,
A e y , K. B., Tigno , M., and Mille , H., pp. 129–234. Camb idge
Uni e si y P ess (2007).
[37] Fowle , D., Pilegaa d, K., Su on, M. A., Ambus, P., Rai o-
nen, M., Duyze , J., Simpson, D., Fage li, H., Fuzzi, S., Schjo-
e ing, J. K., G anie , C., Ne el, A., Isaksen, I. S. A., Laj,
P., Maione, M., Monks, P. S., Bu kha d , J., Daemmgen, U.,
Nei ynck, J., Pe sonne, E., Wichink-K ui , R., Bu e bach-Bahl,
K., Flecha d, C., Tuo inen, J. P., Coyle, M., Ge osa, G., Lou-
be , B., Al imi , N., G uenhage, L., Ammann, C., Cieslik, S.,
Paole i, E., Mikkelsen, T. N., Ro-Poulsen, H., Cellie , P.,
Cape, J. N., Ho a h, L., Lo e o, F., Niineme s, U., Palme ,
100
Re e ences
P. I., Rinne, J., Misz al, P., Nemi z, E., Nilsson, D., P yo ,
S., Gallaghe , M. W., Vesala, T., Skiba, U., B ueggemann, N.,
Zechmeis e -Bol ens e n, S., Williams, J., O’Dowd, C., Facchini,
M. C., de Leeuw, G., Flossman, A., Chaume liac, N., and E isman,
J. W. A mosphe ic composi ion change: Ecosys ems-a mosphe e in e ac ions.
A mosphe ic En i onmen 43, 5193–5267 (2009).
[38] F onzek, S., Ca e , T. R., Raisanen, J., Ruokolainen, L., and
Luo o, M. Applying p obabilis ic p ojec ions o clima e change wi h impac
models: a case s udy o sub-a c ic palsa mi es in Fennoscandia. Clima ic
Change 99, 515–534 (2010).
[39] Fuji a, K. and Dooley, D. M. Insigh s in o he mechanism o N2O educ-
ion by educ i ely ac i a ed N2O educ ase om kine ics and spec oscopic
s udies o pH e ec s. Ino ganic Chemis y 46, 613–615 (2007).
[40] Galand, P. E., Saa nio, S., F i ze, H., and Y j¨
al¨
a, K. Dep h ela ed
di e si y o me hanogen a chaea in Finnish oligo ophic en. FEMS Mic obi-
ology Ecology 42, 441–449 (2002).
[41] Galand, P. E., Y j¨
al¨
a, K., and Con ad, R. S able ca bon iso ope ac-
iona ion du ing me hanogenesis in h ee bo eal pea land ecosys ems. Biogeo-
sciences 7, 3893–3900 (2010).
[42] Goldbe g, S. D., Kno , K. H., Blodau, C., Lischeid, G., and
Gebaue , G. Impac o al e ing he wa e able heigh o an acidic en
on N2O and NO luxes and soil concen a ions. Global Change Biology 16,
220–233 (2010).
[43] Goldbe g, S. D., Kno , K. H., and Gebaue , G. N2O concen a ion
101
Re e ences
and iso ope signa u e along p o iles p o ide deepe insigh in o he a e o N2O
in soils. Iso opes in En i onmen al and Heal h S udies 44, 377–391 (2008).
[44] Go ham, E. No he n pea lands - ole in he ca bon-cycle and p obable
esponses o clima ic wa ming. Ecological Applica ions 1, 182–195 (1991).
[45] Go ny, N., Wahl, G., B une, A., and Schink, B. A s ic ly anae -
obic ni a e- educing bac e ium g owing wi h eso cinol and o he a oma ic-
compounds. A chi es o Mic obiology 158, 48–53 (1992).
[46] G ogan, P., Michelsen, A., Ambus, P., and Jonasson, S. F eeze- haw
egime e ec s on ca bon and ni ogen dynamics in sub-a c ic hea h und a
mesocosms. Soil Biology & Biochemis y 36, 641–654 (2004).
[47] Hales, B. A., Edwa ds, C., Ri chie, D. A., Hall, G., Pickup, R. W.,
and Saunde s, J. R. Isola ion and iden i ica ion o me hanogen-speci ic
DNA om blanke bog ea by PCR ampli ica ion and sequence analysis. Ap-
plied and En i onmen al Mic obiology 62, 668–675 (1996).
[48] Hambe ge , A., Ho n, M. A., Dumon , M. G., Mu ell, J. C., and
D ake, H. L. Anae obic consume s o monosaccha ides in a mode a ely
acidic en. Applied and En i onmen al Mic obiology 74, 3112–3120 (2008).
[49] Ha o i, S. Syn ophic ace a e-oxidizing mic obes in me hanogenic en i on-
men s. Mic obes and En i onmen s 23, 118–127 (2008).
[50] Hayden, M. J. and Ross, D. S. Deni i ica ion as a ni ogen emo al
mechanism in a Ve mon pea land. Jou nal o En i onmen al Quali y 34,
2052–2061 (2005).
[51] Hedde ich, R. and Whi man, W. B. Physiology and biochemis y o
he me hane-p oducing a chaea. In: The P oka yo es, edi ed by Falkow,
102
Re e ences
S., Rosenbe g, E., and Dwo kin, M., ol. 2, pp. 1050–1079. Sp inge -
Ve lag, New Yo k (2006).
[52] Heylen, K., Ge e s, D., Vanpa ys, B., Wi ebolle, L., Gee s, J.,
Boon, N., and De Vos, P. The incidence o ni S and ni K and hei
gene ic he e ogenei y in cul i a ed deni i ie s. En i onmen al Mic obiology 8,
2012–2021 (2006).
[53] Hol an-Ha wig, L., D¨
o sch, P., and Bakken, L. R. Low empe a-
u e con ol o soil deni i ying communi ies: Kine ics o N2O p oduc ion and
educ ion. Soil Biology & Biochemis y 34, 1797–1806 (2002).
[54] Ho n, M., Ma hies, C., K¨
usel, K., Sch amm, A., and D ake, H.
Hyd ogeno ophic me hanogenesis by mode a ely acid- ole an me hanogens
o a me hane-emi ing acidic pea . Applied and En i onmen al Mic obiology
69, 74–83 (2003).
[55] Hunge , S., Schmid , O., Hilga h, M. A., M. Ho n, Kolb, S.,
Con ad, R., and D ake, H. L. Compe ing o ma e- and ca bon dioxide-
u ilizing p oka yo es in an anoxic me hane-emi ing en soil. Applied and
En i onmen al Mic obiology 77, 3773–3785 (2011).
[56] Jaa inen, K., Laiho, R., Vuo enmaa, A., del Cas illo, U., Minkki-
nen, K., Pennanen, T., Pen il¨
a, T., and F i ze, H. Responses o
ae obic mic obial communi ies and soil espi a ion o wa e -le el d awdown in
a no he n bo eal en. En i onmen al Mic obiology 10, 339–353 (2008).
[57] Jansen, E., O e peck, J., B i a, K. R., Duplessy, J.-C., Joos,
F., Masson-Delmo e, V., Olago, D., O o-Bliesne , B., Pel ie ,
W., Rahms o , S., Ramesh, R., Raynaud, D., Rind, D., Solomina,
103
Re e ences
O., Villalba, R., and D., Z. Palaeoclima e. In: Clima e Change 2007:
The Physical Science Basis. Con ibu ion o Wo king G oup I o he Fou h
Assessmen Repo o he In e go e nmen al Panel on Clima e Change, edi ed
by Solomon, S., Qin, D., Manning, M., Chen, Z., Ma quis, M.,
A e y , K., Tigno , M., and Mille , H. Camb idge Uni e si y P ess
(2007).
[58] Je man, V., Me je, M., Mandic-Mulec, I., and F enzel, P. We land
es o a ion and me hanogenesis: he ac i i y o mic obial popula ions and
compe i ion o subs a es a di e en empe a u es. Biogeosciences 6, 1127–
1138 (2009).
[59] Je en, M. S. M. The mic obial ni ogen cycle. En i onmen al Mic obiology
10, 2903–2909 (2008).
[60] Jones, C. M. and Hallin, S. Ecological and e olu iona y ac o s unde -
lying global and local assembly o deni i ie communi ies. ISME Jou nal 4,
633–641 (2010).
[61] Jones, C. M., S es, B., Rosenquis , M., and Hallin, S. Phyloge-
ne ic analysis o ni i e, ni ic oxide, and ni ous oxide espi a o y enzymes
e eal a complex e olu iona y his o y o deni i ica ion. Molecula Biology
and E olu ion 25, 1955–1966 (2008).
[62] Juo onen, H., Galand, P. E., Tui ila, E. S., Laine, J., F i ze,
H., and Y j¨
al¨
a, K. Me hanogen communi ies and bac e ia along an eco-
hyd ological g adien in a no he n aised bog complex. En i onmen al Mi-
c obiology 7, 1547–1557 (2005).
[63] Kaise , C., Meye , H., Biasi, C., Rusalimo a, O., Ba suko , P.,
104
Re e ences
and Rich e , A. Conse a ion o soil o ganic ma e h ough c yo u ba ion
in a c ic soils in sibe ia. Jou nal o Geophysical Resea ch - Biogeosciences 112,
G02017 (2007).
[64] Kelle , M. Con ols on soil-a mosphe e luxes o ni ous oxide and me hane
- e ec s o opical de o es a ion. In: Clima e-biosphe e In e ac ions: biogenic
emissions and en i onmen al e ec s o clima e change, edi ed by Zepp, R. G.,
pp. 121–138. John Wiley & Sons, Inc., New Yo k (1994).
[65] Kes e , R. A., Meije , M. E., Libochan , J. A., de Boe , W., and
Laanb oek, H. J. Con ibu ion o ni i ica ion and deni i ica ion o he
no and n2o emissions o an acid o es soil, a i e sedimen and a e ilized
g assland soil. Soil Biology & Biochemis y 29, 1655–1664 (1997).
[66] Kiehl, J. T. and T enbe h, K. E. Ea h’s annual global mean ene gy
budge . Bulle in o he Ame ican Me eo ological Socie y 78, 197–208 (1997).
[67] Kiene , A., O mejohnson, W. H., and Walsh, C. T. Re e sible con-
e sion o coenzyme F420 o he 8-OH-AMP and 8-OH-GMP es e s, F390-
A and F390-G, on oxygen exposu e and ees ablishmen o anae obiosis in
Me hanobac e ium he moau o ophicum.A chi es o Mic obiology 150, 249–
253 (1988).
[68] Kim, S. W., Fushinobu, S., Zhou, S. M., Wakagi, T., and Shoun, H.
Euka yo ic ni K genes encoding coppe -con aining ni i e educ ase: O igina -
ing om he p o omi ochond ion? Applied and En i onmen al Mic obiology
75, 2652–2658 (2009).
[69] Ki by, T. W., Lancas e , J. R., and F ido ich, I. Isola ion and cha -
105
Re e ences
ac e iza ion o he i on-con aining supe oxide-dismu ase o Me hanobac e ium
b yan ii.A chi es o Biochemis y and Biophysics 210, 140–148 (1981).
[70] Kno , K. H. and Blodau, C. Impac o expe imen al d ough and ewe -
ing on edox ans o ma ions and me hanogenesis in mesocosms o a no he n
en soil. Soil Biology & Biochemis y 41, 1187–1198 (2009).
[71] Kno , K. H., Glase , B., and Blodau, C. Fluxes and 13C iso opic
composi ion o dissol ed ca bon and pa hways o me hanogenesis in a en soil
exposed o expe imen al d ough . Biogeosciences 5, 1457–1473 (2008).
[72] Kno , K. H., Lischeid, G., and Blodau, C. Dynamics o edox p o-
cesses in a mine o ophic en exposed o a wa e able manipula ion. Geode ma
153, 379–392 (2009).
[73] Kno , K. H., Oos e woud, M. R., and Blodau, C. Expe imen al
d ough al e s a es o soil espi a ion and me hanogenesis bu no ca bon
exchange in soil o a empe a e en. Soil Biology & Biochemis y 40, 1781–
1791 (2008).
[74] Kobayashi, M., Ma suo, Y., Takimo o, A., Suzuki, S., Ma uo, F.,
and Shoun, H. Deni i ica ion, a no el ype o espi a o y me abolism in un-
gal mi ochond ion. Jou nal o Biological Chemis y 271, 16263–16267 (1996).
[75] Kobayashi, M. and Shoun, H. The coppe -con aining dissimila o y ni i e
educ ase in ol ed in he deni i ying sys em o he ungus Fusa ium oxyspo-
um.Jou nal o Biological Chemis y 270, 4146–4151 (1995).
[76] Kolb, S. and Ho n, M. A. Mic obial CH4and N2O consump ion in acidic
we lands. F on ie s in Mic obiology 3, 78 (2012).
106
Re e ences
[77] Kool, D. M., Dol ing, J., W age, N., and Van G oenigen, J. W.
Ni i ie deni i ica ion as a dis inc and signi ican sou ce o ni ous oxide
om soil. Soil Biology & Biochemis y 43, 174–178 (2011).
[78] Koponen, H. T., Jaakkola, T., Kein¨
anen-Toi ola, M. M.,
Kaipainen, S., Tuomainen, J., Se omaa, K., and Ma ikainen,
P. J. Mic obial communi ies, biomass, and ac i i ies in soils as a ec ed by
eeze haw cycles. Soil Biology & Biochemis y 38, 1861–1871 (2006).
[79] Koponen, H. T. and Ma ikainen, P. J. Soil wa e con en and eezing
empe a u e a ec eeze- haw ela ed N2O p oduc ion in o ganic soil. Nu i-
en Cycling in Ag oecosys ems 69, 213–219 (2004).
[80] Ko iaho, M., F i ze, H., Me il¨
a, P., Juo onen, H., Lepp¨
al¨
a, M.,
Laine, J., Laiho, R., Y j¨
al¨
a, K., and Tui ila, E. S. Me hanogen
ac i i y in ela ion o wa e able le el in wo bo eal ens. Biology and Fe ili y
o Soils 46, 567–575 (2010).
[81] Ko syu benko, O., Chin, K., Glagole , M., S ubne , S.,
Simanko a, M., Nozhe niko a, A., and Con ad, R. Ace oclas ic
and hyd ogeno ophic me hane p oduc ion and me hanogenic popula ions in
an acidic wes -sibe ian pea bog. En i onmen al Mic obiology 6, 1159–1173
(2004).
[82] Ko syu benko, O. R. T ophic in e ac ions in he me hanogenic mic obial
communi y o low- empe a u e e es ial ecosys ems. FEMS Mic obiology
Ecology 53, 3–13 (2005).
[83] K a , B., S ous, M., and Tege meye , H. E. Mic obial ni a e es-
107
Re e ences
zene, oluene, and m-xylene - kine ics, mass balance, and yields. Applied
Biochemis y and Bio echnology 119, 195–208 (2004).
[127] Pe e s, V. and Con ad, R. Me hanogenic and o he s ic ly anae obic
bac e ia in dese soil and o he oxic soils. Applied and En i onmen al Mic o-
biology 61, 1673–1676 (1995).
[128] Pe e son, R. A. and K an z, W. B. A mechanism o di e en ial os
hea e and i s implica ions o pa e ned-g ound o ma ion. Jou nal o Glaciol-
ogy 49, 69–80 (2003).
[129] Philippo , L. and Hojbe g, O. Dissimila o y ni a e educ ases in bac-
e ia. Biochimica e Biophysica Ac a - Gene S uc u e and Exp ession 1446,
1–23 (1999).
[130] Pihla ie, M., Sy ¨
asalo, E., Simojoki, A., Esala, M., and Regina,
K. Con ibu ion o ni i ica ion and deni i ica ion o N2O p oduc ion in pea ,
clay and loamy sand soils unde di e en soil mois u e condi ions. Nu ien
Cycling in Ag oecosys ems 70, 135–141 (2004).
[131] Pihla ie, M. K., Kiese, R., B uggemann, N., Bu e bach-Bahl, K.,
Kieloaho, A. J., Lau ila, T., Lohila, A., Mamma ella, I., Minkki-
nen, K., Pen ila, T., Schonbo n, J., and Vesala, T. G eenhouse
gas luxes in a d ained pea land o es du ing sp ing os - haw e en . Biogeo-
sciences 7, 1715–1727 (2010).
[132] Pomowski, A., Zum , W. G., K oneck, P. M. H., and Einsle, O.
N2O binding a a [4Cu:2S] coppe -sulphu clus e in ni ous oxide educ ase.
Na u e 477, 234–U143 (2011).
114
Re e ences
[133] Pos , W. M., Pas o , J., Zinke, P. J., and S angenbe ge , A. G.
Global pa e ns o soil-ni ogen s o age. Na u e 317, 613–616 (1985).
[134] Po h, M. and Foch , D. D. 15N kine ic-analysis o N2O p oduc ion by
Ni osomonas eu opaea - An examina ion o ni i ie deni i ica ion. Applied
and En i onmen al Mic obiology 49, 1134–1141 (1985).
[135] Ra ishanka a, A. R., Daniel, J. S., and Po mann, R. W. Ni ous
oxide (N2O): The dominan ozone-deple ing subs ance emi ed in he 21s
cen u y. Science 326, 123–125 (2009).
[136] Regina, K., Nyk¨
anen, H., Sil ola, J., and Ma ikainen, P. J. Fluxes
o ni ous oxide om bo eal pea lands as a ec ed by pea land ype, wa e able
le el and ni i ica ion capaci y. Biogeochemis y 35, 401–418 (1996).
[137] Regina, K., Sil ola, J., and Ma ikainen, P. J. Sho - e m e ec s
o changing wa e able on N2O luxes om pea monoli hs om na u al and
d ained bo eal pea lands. Global Change Biology 5, 183–189 (1999).
[138] Repo, M. E., Susiluo o, S., Lind, S. E., Jokinen, S., Elsako , V.,
Biasi, C., Vi anen, T., and Ma ikainen, P. J. La ge N2O emissions
om c yo u ba ed pea soil in und a. Na u e Geoscience 2, 189–192 (2009).
[139] Rinne, J., Riu a, T., Pihla ie, M., Au ela, M., Haapanala, S.,
Tuo inen, J. P., Tui ila, E. S., and Vesala, T. Annual cycle o
me hane emission om a bo eal en measu ed by he eddy co a iance ech-
nique. Tellus Se ies B - Chemical and Physical Me eo ology 59, 449–457
(2007).
[140] Risgaa d-Pe e sen, N., Langezaal, A. M., Ing a dsen, S., Schmid,
M. C., Je en, M. S. M., Op den Camp, H. J. M., De ksen, J.
115
Re e ences
W. M., Pina-Ochoa, E., E iksson, S. P., Nielsen, L. P., Re sbech,
N. P., Cedhagen, T., and an de Zwaan, G. J. E idence o comple e
deni i ica ion in a ben hic o amini e . Na u e 443, 93–96 (2006).
[141] Roob oeck, D., Bu e bach-Bahl, K., B ueggemann, N., and
Boeckx, P. Dini ogen and ni ous oxide exchanges om an und ained mono-
li h en: sho - e m esponses ollowing ni a e addi ion. Eu opean Jou nal o
Soil Science 61, 662–670 (2010).
[142] R¨
u ing, T., Boeckx, P., M¨
ulle , C., and Klemed sson, L. Assess-
men o he impo ance o dissimila o y ni a e educ ion o ammonium o
he e es ial ni ogen cycle. Biogeosciences 8, 1779–1791 (2011).
[143] Saad, O. A. L. O. and Con ad, R. Tempe a u e-dependence o ni i i-
ca ion, deni i ica ion, and u no e o ni ic-oxide in di e en soils. Biology
and Fe ili y o Soils 15, 21–27 (1993).
[144] Sakai, S., Imachi, H., Hanada, S., Ohashi, A., Ha ada, H., and Ka-
maga a, Y. Me hanocella paludicola gen. no ., sp no ., a me hane-p oducing
a chaeon, he i s isola e o he lineage ’Rice Clus e I’, and p oposal o he
new a chaeal o de Me hanocellales o d. no . In e na ional Jou nal o Sys-
ema ic and E olu iona y Mic obiology 58, 929–936 (2008).
[145] Schink, B. Ene ge ics o syn ophic coope a ion in me hanogenic deg ada-
ion. Mic obiology and Molecula Biology Re iews 61, 262–280 (1997).
[146] Schmid , O., D ake, H. L., and Ho n, M. A. Hi he o unknown [ e-
e]-hyd ogenase gene di e si y in anae obes and anoxic en ichmen s om a
mode a ely acidic en. Applied and En i onmen al Mic obiology 76, 2027–
2031 (2010).
116
Re e ences
[147] Schuu , E. A. G., Bockheim, J., Canadell, J. G., Euski chen,
E., Field, C. B., Go yachkin, S. V., Hagemann, S., Kuh y, P.,
La leu , P. M., Lee, H., Mazhi o a, G., Nelson, F. E., Rinke, A.,
Romano sky, V. E., Shiklomano , N., Ta nocai, C., Vene sky, S.,
Vogel, J. G., and Zimo , S. A. Vulne abili y o pe ma os ca bon o cli-
ma e change: Implica ions o he global ca bon cycle. Bioscience 58, 701–714
(2008).
[148] Sege s, R. Me hane p oduc ion and me hane consump ion: A e iew o p o-
cesses unde lying we land me hane luxes. Biogeochemis y 41, 23–51 (1998).
[149] Sege s, R. and Kengen, S. W. M. Me hane p oduc ion as a unc ion o
anae obic ca bon mine aliza ion: A p ocess model. Soil Biology & Biochem-
is y 30, 1107–1117 (1998).
[150] Sepp¨
al¨
a, M. Syn hesis o s udies o palsa o ma ion unde lining he impo -
ance o local en i onmen al and physical cha ac e is ics. Qua e na y Resea ch
75, 366–370 (2011).
[151] Shoun, H., Kim, D. H., Uchiyama, H., and Sugiyama, J. Deni i ica ion
by ungi. FEMS Mic obiology Le e s 94, 277–281 (1992).
[152] Simek, M. and Coope , J. E. The in luence o soil pH on deni i ica ion:
p og ess owa ds he unde s anding o his in e ac ion o e he las 50 yea s.
Eu opean Jou nal o Soil Science 53, 345–354 (2002).
[153] Si e, P., Weile , E., Kade ei , J., B esinsky, A., and K¨
o ne , C.
S asbu ge - Leh buch de Bo anik. Spek um Akademische Ve lag (2002).
[154] Smi h, K. A. The po en ial o eedback e ec s induced by global wa ming on
emissions o ni ous oxide by soils. Global Change Biology 3, 327–338 (1997).
117
Re e ences
[155] Song, K., Lee, S. H., Mi sch, W. J., and Kang, H. Di e en e-
sponses o deni i ica ion a es and deni i ying bac e ial communi ies o hy-
d ologic pulsing in c ea ed we lands. Soil Biology & Biochemis y 42, 1721–
1727 (2010).
[156] Sousa, D. Z., Smid , H., Al es, M. M., and S ams, A. J. M. Eco-
physiology o syn ophic communi ies ha deg ade sa u a ed and unsa u a ed
long-chain a y acids. FEMS Mic obiology Ecology 68, 257–272 (2009).
[157] S e en, B., B iggs, G., McKay, C. P., Polla d, W. H., G ee ,
C. W., and Why e, L. G. Cha ac e iza ion o he mic obial di e si y in
a pe ma os sample om he Canadian high A c ic using cul u e-dependen
and cul u e-independen me hods. FEMS Mic obiology Ecology 59, 513–523
(2007).
[158] S e en, B., Polla d, W. H., G ee , C. W., and Why e, L. G.
Mic obial di e si y and ac i i y h ough a pe ma os /g ound ice co e p o ile
om he Canadian high A c ic. En i onmen al Mic obiology 10, 3388–3403
(2008).
[159] S es, B., Dane cic, T., Pal, L., Fuka, M. M., Resman, L.,
Lesko ec, S., Hacin, J., S opa , D., Mahne, I., and Mandic-Mulec,
I. In luence o empe a u e and soil wa e con en on bac e ial, a chaeal and
deni i ying mic obial communi ies in d ained en g assland soil mic ocosms.
FEMS Mic obiology Ecology 66, 110–122 (2008).
[160] S ohm, T. O., G i in, B., Zum , W. G., and Schink, B. G ow h
yields in bac e ial deni i ica ion and ni a e ammoni ica ion. Applied and
En i onmen al Mic obiology 73, 1420–1424 (2007).
118
Re e ences
[161] Takakai, F., Desya kin, A. R., Lopez, C. M. L., Fedo o , A. N.,
Desya kin, R. V., and Ha ano, R. CH4and N2O emissions om a o es -
alas ecosys em in he pe ma os aiga o es egion, eas e n Sibe ia, Russia.
Jou nal o Geophysical Resea ch - Biogeosciences 113, G02002 (2008).
[162] Takao, M., Yasui, A., and Oikawa, A. Unique cha ac e is ics o
supe oxide-dismu ase o a s ic ly anae obic a chaebac e ium Me hanobac-
e ium he moau o ophicum.Jou nal o Biological Chemis y 266, 14151–
14154 (1991).
[163] Takasaki, K., Shoun, H., Yamaguchi, M., Takeo, K., Nakamu a,
A., Hoshino, T., and Takaya, N. Fungal ammonia e men a ion, a no el
me abolic mechanism ha couples he dissimila o y and assimila o y pa hways
o bo h ni a e and e hanol - ole o ace yl CoA syn he ase in anae obic ATP
syn hesis. Jou nal o Biological Chemis y 279, 12414–12420 (2004).
[164] Takaya, N. Dissimila o y ni a e educ ion me abolisms and hei con ol in
ungi. Jou nal o Bioscience and Bioenginee ing 94, 506–510 (2002).
[165] Takaya, N. Response o hypoxia, educ ion o elec on accep o s, and subse-
quen su i al by ilamen ous ungi. Bioscience Bio echnology And Biochem-
is y 73, 1–8 (2009).
[166] Takaya, N., Kuwazaki, S., Adachi, Y., Suzuki, S., Kikuchi, T.,
Nakamu a, H., Shi o, Y., and Shoun, H. Hyb id espi a ion in he
deni i ying mi ochond ia o Fusa ium oxyspo um.Jou nal o Biochemis y
133, 461–465 (2003).
[167] Ta nocai, C., Canadell, J. G., Schuu , E. A. G., Kuh y, P., Mazhi-
119
Re e ences
o a, G., and Zimo , S. Soil o ganic ca bon pools in he no he n ci cum-
pola pe ma os egion. Global Biogeochemical Cycles 23, GB2023 (2009).
[168] Thaue , R. K., Junge mann, K., and Decke , K. Ene gy-conse a ion
in chemo ophic anae obic bac e ia. Bac e iological Re iews 41, 100–180
(1977).
[169] Thaue , R. K., Kas e , A. K., Seedo , H., Buckel, W., and Hed-
de ich, R. Me hanogenic a chaea: ecologically ele an di e ences in ene gy
conse a ion. Na u e Re iews Mic obiology 6, 579–591 (2008).
[170] Thomas, K. L., Lloyd, D., and Boddy, L. E ec s o oxygen, pH and
ni a e concen a ion on deni i ica ion by Pseudomonas species. FEMS Mi-
c obiology Le e s 118, 181–186 (1994).
[171] Thomsen, J. K., Gees , T., and Cox, R. P. Mass-spec ome ic s udies
o he e ec o pH on he accumula ion o in e media es in deni i ica ion by
Pa acoccus deni i icans.Applied and En i onmen al Mic obiology 60, 536–541
(1994).
[172] Tiedje, J. M. Ecology o deni i ica ion and dissimila o y ni a e educ ion
o ammonium. In: Biology o Anae obic Mic oo ganisms, edi ed by Zehnde ,
A. J. B., pp. 179–244. Wiley, New Yo k (1988).
[173] T cko a, M., Ma lo a, L., Hudco a, H., Faldyna, M., Z aly, Z.,
D o ska, L., Be an, V., and Pa lik, I. Pea as a eed supplemen o
animals: a e iew. Ve e ina ni Medicina 50, 361–377 (2005).
[174] T enbe h, K., Jones, P., Ambenje, P., Boja iu, R., Eas e ling,
D., Klein Tank, A., Pa ke , D., Rahimzadeh, F., Renwick, J., Rus-
icucci, M., Soden, B., and Zhai, P. Obse a ions: Su ace and a mo-
120
Re e ences
sphe ic clima e change. In: Clima e Change 2007: The Physical Science Basis.
Con ibu ion o Wo king G oup I o he Fou h Assessmen Repo o he In-
e go e nmen al Panel on Clima e Change, edi ed by Solomon, S., Qin, D.,
Manning, M., Chen, Z., Ma quis, M., A e y , K. B., Tigno , M.,
and Mille , H., pp. 235–336. Camb idge Uni e si y P ess (2007).
[175] Ullah, S., B ei enbeck, G. A., and Faulkne , S. P. Deni i ica ion
and N2O emission om o es ed and cul i a ed allu ial clay soil. Biogeochem-
is y 73, 499–513 (2005).
[176] Vanb eemen, N. How Sphagnum bogs down o he plan s. T ends in Ecology
& E olu ion 10, 270–275 (1995).
[177] Walke , D. A., Raynolds, M. K., Daniels, F. J. A., Eina sson,
E., El ebakk, A., Gould, W. A., Ka enin, A. E., Kholod, S. S.,
Ma kon, C. J., Melniko , E. S., Moskalenko, N. G., Talbo , S. S.,
and Yu se , B. A. The ci cumpola a c ic ege a ion map. Jou nal o
Vege a ion Science 16, 267–282 (2005).
[178] Wang, J. H., Bal zis, B. C., and Lewandowski, G. A. Fundamen al
deni i ica ion kine ic-s udies wi h Pseudomonas deni i icans.Bio echnology
and Bioenginee ing 47, 26–41 (1995).
[179] Washbu n, A. L. Classi ica ion o pa e ned g ound and e iew o sugges ed
o igins. Geological Socie y o Ame ica Bulle in 67, 823–& (1956).
[180] We ne , C., Bu e bach-Bahl, K., Haas, E., Hickle , T., and
Kiese, R. A global in en o y o N2O emissions om opical ain o es
soils using a de ailed biogeochemical model. Global Biogeochemical Cycles
21, GB3010 (2007).
121
Re e ences
[181] Wes e mann, P. We land and swamp mic obiology. In: Aqua ic Mic obiol-
ogy, an Ecological App oach, edi ed by Fo d, T. E., pp. 215–238. Blackwell
Scien i ic Publica ions, Ox o d (1993).
[182] Whalen, S. C. Biogeochemis y o me hane exchange be ween na u al we -
lands and he a mosphe e. En i onmen al Enginee ing Science 22, 73–94
(2005).
[183] Williams, R. T. and C aw o d, R. L. Me hanogenic bac e ia, including
an acid- ole an s ain, om pea lands. Applied and En i onmen al Mic obi-
ology 50, 1542–1544 (1985).
[184] Woese, C. R., Kandle , O., and Wheelis, M. L. Towa ds a na u al
sys em o o ganisms - p oposal o he domains A chaea,Bac e ia, and Eu-
ca ya.P oceedings o he Na ional Academy o Sciences o he Uni ed S a es
o Ame ica 87, 4576–4579 (1990).
[185] W¨
us , P. K., Ho n, M. A., and D ake, H. L. T ophic links be ween
e men e s and me hanogens in a mode a ely acidic en soil. En i onmen al
Mic obiology 11, 1395–1409 (2009).
[186] Ya i , J. B., Fahey, T. J., and Simmons, J. A. Me hane and ca bon
dioxide dynamics in a no he n ha dwood ecosys em. Soil Science Socie y o
Ame ica Jou nal 59, 796–804 (1995).
[187] Yde, J. C., Fins e , K. W., Raiswell, R., S e ensen, J. P., Heine-
meie , J., Olsen, J., Gunnlaugsson, H. P., and Nielsen, O. B. Basal
ice mic obiology a he ma gin o he G eenland ice shee . Annals o Glaciology
51, 71–79 (2010).
122
Re e ences
[188] Ye geau, E., Bokho s , S., Kang, S., Zhou, J., G ee , C. W.,
Ae s, R., and Kowalchuk, G. A. Shi s in soil mic oo ganisms in e-
sponse o wa ming a e consis en ac oss a ange o an a c ic en i onmen s.
ISME Jou nal (2012).
[189] Yoshina i, T. Emissions o N2O om a ious en i onmen s - The use o s a-
ble iso ope composi ion o N2O as ace o he s udies o N2O biogeochemical
cycling. In: Deni i ica ion in Soil and Sedimen , edi ed by Re sbech, N. P.
and So ensen, J., pp. 129–150. FEMS Symposium No. 56 (1990).
[190] Yoshina i, T. and Knowles, R. Ace ylene inhibi ion o ni ous oxide e-
duc ion by deni i ying bac e ia. Biochemical and Biophysical Resea ch Com-
munica ions 69, 705–710 (1976).
[191] Y j¨
al¨
a, K., Tuomi i a, T., Juo onen, H., Pu kinen, A., Lappi,
K., Tui ila, E. S., Pen il¨
a, T., Minkkinen, K., Laine, J., Pel-
oniemi, K., and F i ze, H. CH4p oduc ion and oxida ion p ocesses in
a bo eal en ecosys em a e long- e m wa e able d awdown. Global Change
Biology 17, 1311–1320 (2011).
[192] Zehnde , A. J. B. and S umm, W. Geochemis y and biochemis y o
anae obic habi a s. In: Biology o anae obic mic oo ganisms, edi ed by Zehn-
de , A. J. B., pp. 1–38. John Wiley & Sons, Inc., New Yo k (1988).
[193] Zeno a, G. M., Manucha o a, N. A., and Z yagin se , D. G.
Ex emophilic and ex emo ole an ac inomyce es in di e en soil ypes.
Eu asian Soil Science 44, 417–436 (2011).
[194] Zol ai, S. C. Palsas and pea pla eaus in cen al Mani oba and
Saska chewan. Canadian Jou nal o Fo es Resea ch 2, 291–302 (1972).
123
nosZ-de i ed species-le el OTUs wi h a e y high p obabili y
o es ima ing co ec ly. The calcula ed h eshold simila i ies
a e in good ag eemen wi h hose calcula ed o he nosZ
agmen ampli ied by he p ime s nosZ661F/-1773R (9). Phy-
logene ic ees o nosZ and 16S RNA genes had simila clus-
e ing a he genus le el (Fig. 3). Mul iple copies o nosZ pe
o ganism we e no ound. The di e en h eshold simila i ies
o nosZ and na G sugges ha nosZ is mo e conse ed han
na G, indica ing ha species-le el OTU assignmen o nosZ
migh be mo e eliable han ha o na G.
Conclusions. The abo e conside a ions indica e ha na G o
nosZ analyses can be used o es ima e species-le el di e si y o
he associa ed bac e ia on he basis o sequence simila i ies.
Such analysis is conside ed o yield a minimum numbe o
species in a sample (i.e., he ue species-le el di e si y migh
be much highe ). Analyses o gene ma ke s o di e en unc-
ional g oups e ealed simila h eshold simila i ies indica ing
species-le el OTUs (e.g., o ds AB and amoA gene agmen s,
hey we e 80 o 90%) (11, 12). Since na G o nosZ sequences
om o ganisms o he same genus gene ally o m cohe en
clus e s in phylogene ic ees (Fig. 2 and 3), dis inc clus e s o
en i onmen al sequences could p o ide e idence o new ge-
nus-le el di e si y. Recen ho izon al gene ans e does no
appea o ha e occu ed o ei he na G (as indica ed by he
esul s in his s udy) o nosZ (as indica ed by he esul s in
e e ence 10). The adap a ion o genomic ea u es o an alien
gene o hose o a hos genome akes se e al hund ed million
yea s (15), and genes ha we e ho izon ally ans e ed 50
million yea s ago can be eliably de ec ed by sequence analyses
(26). Sequence di e ences migh also be caused by gene du-
plica ion and di e si ica ion (e.g., in species in which mul iple
gene copies a e p esen ) a he han by ho izon al gene ans-
e (1). Howe e , analysis o codon usage and oligonucleo ide
bias does no always e eal he ans e o genes om closely
ela ed o ganisms wi h simila genomic ea u es (27).
Suppo o his s udy was p o ided by he Deu sche Fo schungsge-
meinscha (DFG HO 4020/2-2 and DR 310/3-3) and he Uni e si y o
Bay eu h.
REFERENCES
1. Be g ho sson, U., D. I. Ande sson, and J. R. Ro h. 2007. Ohno’s dilemma:
e olu ion o new genes unde con inuous selec ion. P oc. Na l. Acad. Sci.
USA 104:17004–17009.
2. Che`neby, D., S. Halle , M. Mondon, F. Ma in-Lau en , J. C. Ge mon, and
L. Philippo . 2003. Gene ic cha ac e iza ion o he ni a e educing commu-
ni y based on na G nucleo ide sequence analysis. Mic ob. Ecol. 46:113–121.
3. Dandie, C. E., D. L. Bu on, B. J. Zeba h, J. T. T e o s, and C. Goye . 2007.
Analysis o deni i ica ion genes and compa ison o nosZ,cno B and 16S
DNA om cul u able deni i ying bac e ia in po a o c opping sys ems. Sys .
Appl. Mic obiol. 30:128–138.
4. Delo me, S., L. Philippo , V. Edel-He mann, C. Deul o , C. Mougel, and P.
Lemanceau. 2003. Compa a i e gene ic di e si y o he na G,nosZ, and 16S
RNA genes in luo escen pseudomonads. Appl. En i on. Mic obiol. 69:
1004–1012.
5. Felsens ein, J. 1985. Con idence-limi s on phylogenies—an app oach using
he boo s ap. E olu ion 39:783–791.
6. Ganesan, H., A. S. Raki ianskaia, C. F. Da enpo , B. Tummle , and O. N.
Re a. 2008. The SeqWo d genome b owse : an online ool o he iden i i-
ca ion and isualiza ion o a ypical egions o bac e ial genomes h ough
oligonucleo ide usage. BMC Bioin o ma ics 9:333.
7. G ego y, L. G., P. L. Bond, D. J. Richa dson, and S. Spi o. 2003. Cha ac-
e iza ion o a ni a e- espi ing bac e ial communi y using he ni a e educ-
ase gene (na G) as a unc ional ma ke . Mic obiology 149:229–237.
8. Hen y, S., D. B u, B. S es, S. Halle , and L. Philippo . 2006. Quan i a i e
de ec ion o he nosZ gene, encoding ni ous oxide educ ase, and compa -
ison o he abundances o 16S RNA, na G,ni K, and nosZ genes in soils.
Appl. En i on. Mic obiol. 72:5181–5189.
9. Ho n, M. A., H. L. D ake, and A. Sch amm. 2006. Ni ous oxide educ ase
genes (nosZ) o deni i ying mic obial popula ions in soil and he ea hwo m
gu a e phylogene ically simila . Appl. En i on. Mic obiol. 72:1019–1026.
10. Jones, C. M., B. S es, M. Rosenquis , and S. Hallin. 2008. Phylogene ic
analysis o ni i e, ni ic oxide, and ni ous oxide espi a o y enzymes e eal
a complex e olu iona y his o y o deni i ica ion. Mol. Biol. E ol. 25:1955–
1966.
11. Kjeldsen, K. U., A. Loy, T. F. Jakobsen, T. R. Thomsen, M. Wagne , and K.
Ing o sen. 2007. Di e si y o sul a e- educing bac e ia om an ex eme
hype saline sedimen , G ea Sal Lake (U ah). FEMS Mic obiol. Ecol. 60:
287–298.
12. Koops, H.-P., U. Pu khold, A. Pomme ening-Ro¨se , G. Timme mann, and
M. Wagne . 2003. The li hoau o ophic ammonia-oxidizing bac e ia, p. 778–
811. In M. Dwo kin, S. Falkow, E. Rosenbe g, K.-H. Schlei e , and E.
S ackeb and (ed.), The p oka yo es: an e ol ing elec onic esou ce o he
mic obiological communi y, 3 d ed. Sp inge -Ve lag, New Yo k, NY.
13. Kuma , S., M. Nei, J. Dudley, and K. Tamu a. 2008. MEGA: a biologis -
cen ic so wa e o e olu iona y analysis o DNA and p o ein sequences.
B ie . Bioin o m. 9:299–306.
14. Lane, D. J. 1991. 16S/23S RNA sequencing, p. 115–175. In E. S ackeb and
and M. Good ellow (ed.), Nucleic acid echniques in bac e ial sys ema ics,
1s ed. John Wiley and Sons, L d., Chiches e , NY.
15. Law ence, J. G., and H. Ochman. 1997. Amelio a ion o bac e ial genomes:
a es o change and exchange. J. Mol. E ol. 44:383–397.
16. Mounie , E., S. Halle , D. Cheneby, E. Beniz i, Y. G ue , C. Nguyen, S.
Piu i, C. Robin, S. Slezack-Deschaumes, F. Ma in-Lau en , J. C. Ge mon,
and L. Philippo . 2004. In luence o maize mucilage on he di e si y and
ac i i y o he deni i ying communi y. En i on. Mic obiol. 6:301–312.
17. Philippo , L. 2002. Deni i ying genes in bac e ial and a chaeal genomes.
Biochim. Biophys. Ac a 1577:355–376.
18. Philippo , L., S. Piu i, F. Ma in-Lau en , S. Halle , and J. C. Ge mon.
2002. Molecula analysis o he ni a e- educing communi y om unplan ed
and maize-plan ed soils. Appl. En i on. Mic obiol. 68:6121–6128.
19. Rich, J. J., R. S. Heichen, P. J. Bo omley, K. C omack, and D. D. My old.
2003. Communi y composi ion and unc ioning o deni i ying bac e ia om
adjacen meadow and o es soils. Appl. En i on. Mic obiol. 69:5974–5982.
20. Ro¨sch, C., A. Me gel, and H. Bo he. 2002. Biodi e si y o deni i ying and
dini ogen- ixing bac e ia in an acid o es soil. Appl. En i on. Mic obiol.
68:3818–3829.
21. Sai ou, N., and M. Nei. 1987. The neighbo -joining me hod—a new me hod
o econs uc ing phylogene ic ees. Mol. Biol. E ol. 4:406–425.
22. S ackeb and , E., and J. Ebe s. 2006. Taxonomic pa ame e s e isi ed: a -
nished gold s anda ds. Mic obiol. Today 33:152–155.
23. S ackeb and , E., and B. M. Goebel. 1994. A place o DNA-DNA easso-
cia ion and 16S ibosomal-RNA sequence-analysis in he p esen species
de ini ion in bac e iology. In . J. Sys . Bac e iol. 44:846–849.
24. S olz, J. F., and P. Basu. 2002. E olu ion o ni a e educ ase: molecula and
s uc u al a ia ions on a common unc ion. ChemBioChem 3:198–206.
25. Th oback, I. N., K. Enwall, A. Ja is, and S. Hallin. 2004. Reassessing PCR
p ime s a ge ing ni S,ni K and nosZ genes o communi y su eys o deni-
i ying bac e ia wi h DGGE. FEMS Mic obiol. Ecol. 49:401–417.
26. Ve nikos, G. S., N. R. Thomson, and J. Pa khill. 2007. Gene ic lux o e ime
in he Salmonella lineage. Genome Biol. 8:R100.
27. Waack, S., O. Kelle , R. Aspe , T. B odag, C. Damm, W. F. F icke, K.
Su o cik, P. Meinicke, and R. Me kl. 2006. Sco e-based p edic ion o
genomic islands in p oka yo ic genomes using hidden Ma ko models. BMC
Bioin o ma ics 7:142.
28. Wayne, L. G., D. J. B enne , R. R. Colwell, P. A. D. G imon , O. Kandle ,
M. I. K iche sky, L. H. Moo e, W. E. C. Moo e, R. G. E. Mu ay, E.
S ackeb and , M. P. S a , and H. G. T u¨pe . 1987. Repo o he ad hoc
commi ee on econcilia ion o app oaches o bac e ial sys ema ics. In . J.
Sys . Bac e iol. 37:463–464.
29. Zum , W. G. 1997. Cell biology and molecula basis o deni i ica ion.
Mic obiol. Mol. Biol. Re . 61:533–615.
30. Zum , W. G., and H. Ko¨ ne . 2007. Ni ous oxide educ ases, p. 67–81. In H.
Bo he, S. J. Fe guson, and W. E. New on (ed.), Biology o he ni ogen cycle,
1s ed. Else ie Academic P ess, Inc., Ams e dam, The Ne he lands.
5174 PALMER ET AL. APPL.ENVIRON.MICROBIOL.
4 Manusc ip s
130
Manusc ip in p epa a ion o ISME Jou nal Ve sion 2 04.10.11 1
2
3
Con as ing deni i ie communi ies ela e o con as ing N2O 4
emission pa e ns om acidic pea soils in a c ic und a 5
6
Ka ha ina Palme 1, Ch is ina Biasi2, and Ma cus A. Ho n1* 7
8
9 1 Depa men o Ecological Mic obiology, Uni e si y o Bay eu h, D .-Hans-F isch-S aße 1-3, 10
95440 Bay eu h, Ge many 11
2 Depa men o En i onmen al Science, Uni e si y o Kuopio, Yliopis on an a 1 E, FIN-70210 12
Kuopio, Finland 13
14
---------- 15
16
Key wo ds: Pe ma os a ec ed soil, global change, we land, ba coded amplicon 17
py osequencing, quan i a i e PCR 18
19
Running i le: Deni i ie s in c yo- and un u ba ed pea 20
21
---------- 22
23
* Co esponding au ho . Mailing add ess: Depa men o Ecological Mic obiology, 24
D .-Hans-F isch-S . 1-3, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many. Tel: (+49) 25
(0)921-555620. Fax: (+49) (0)921-555799. E-Mail: [email p o ec ed] 26
27
4.1 Published a icles in pee - e iewed jou nals
131
2
C yo u ba ed pea ci cles (i.e., ba e su ace soil mixed by os ac ion; pH 3-4) in he 1
Russian discon inuous pe ma os und a a e ni a e- ich ‘ho spo s’ o N2O-emissions in a c ic 2
ecosys ems, while adjacen un u ba ed pea a eas a e no . N2O was p oduced and 3
subsequen ly consumed a pH 4 in unsupplemen ed anoxic mic ocosms wi h c yo u ba ed 4
bu no in hose wi h un u ba ed pea soil. Ni a e, ni i e, and ace ylene s imula ed ne N2O 5
p oduc ion o bo h soils in anoxic mic ocosms, indica ing deni i ica ion as he sou ce o N2O. 6
Up o 500 and 10 µM ni a e s imula ed deni i ica ion in c yo u ba ed and un u ba ed pea 7
soil, espec i ely. Appa en maximal eac ion eloci ies o ni i e-dependen deni i ica ion 8
we e 28 and 18 nmol N2O gDW-1h-1, o c yo u ba ed and un u ba ed pea soil, espec i ely. 9
Ba coded amplicon py osequencing o na G, ni K/ni S, and nosZ (encoding ni a e, ni i e, 10
and N2O educ ases, espec i ely) yielded ≈49 000 quali y il e ed sequences wi h an 11
a e age sequence leng h o 444 bp. Up o 19 species-le el ope a ional axonomic uni s we e 12
de ec ed pe soil and gene, many o which we e dis an ly ela ed o cul u ed deni i ie s o 13
en i onmen al sequences. Deni i ica ion associa ed gene di e si y in c yo u ba ed and in 14
un u ba ed pea soil di e ed. Quan i a i e PCR (inhibi ion-co ec ed pe DNA ex ac ) 15
e ealed highe abundances o na G in c yo u ba ed han in un u ba ed pea soil. Copy 16
numbe s o ni S we e up o 1000x highe han hose o ni K in bo h soils, and ni S ni K-1 copy 17
numbe a ios in c yo- and un u ba ed pea soil di e ed. The collec i e da a indica e ha he 18
con as ing N2O emission pa e ns o c yo u ba ed and un u ba ed pea soils a e associa ed 19
wi h con as ing deni i ie communi ies. 20
21
4 Manusc ip s
132
3
In oduc ion 1
Ni ous oxide (N2O) is a majo ozone deple ing subs ance in he a mosphe e and he 2
hi d mos impo an g eenhouse gas on ea h (Fo s e e al., 2007; Ra ishanka a e al., 3
2009). The global wa ming po en ial o N2O is 300- old highe han ha o CO2 on a 100 yea 4
basis, and he a mosphe ic concen a ion o N2O inc eased om 270 ppb o 319 ppb om 5
1750 o 2005 (Fo s e e al., 2007). Ag icul u al and p is ine opical soils a e well ecognized 6
majo sou ces o N2O, while he impo ance o a c ic pea lands as sou ces o N2O is jus 7
eme ging (Denman e al., 2007; Repo e al., 2009; Ma ushchak e al., 2011). 8
A eas o ba e su ace soil mixed by os ac ion in acidic und a (pH 3 o 4) a e e med 9
c yo u ba ed pea ci cles, and emi N2O a a es documen ed o opical and ag icul u al soils 10
(We ne e al., 2007; Maljanen e al., 2007; Repo e al., 2009). The es ima ed global N2O 11
emission om c yo u ba ed pea ci cles is abou 0.1 Tg N2O y-1, which is equi alen o 4% o 12
he global wa ming po en ial o he a c ic me hane emissions and o 0.6% o he o al global 13
annual N2O emission (Ch is ensen, 1993; Denman e al., 2007; Repo e al., 2009). 14
Vege a ion is absen om abou 12% o he a ea in he a c ic, including c yo u ba ed pea 15
ci cles (Walke e al., 2005). Ni a e concen a ions app oxima e 2 mM in he po e wa e o 16
such un ege a ed c yo u ba ed pea soil, and a e app oxima ely 1000 x highe han in 17
adjacen ege a ed un u ba ed pea a eas whe e N2O emissions a e negligible (Repo e al., 18
2009). Repea ed eezing and hawing o he c yo u ba ed soil leads o b eakdown o soil 19
agg ega es, ende s decomposable o ganic ca bon mo e easily accessible o mic obes, and 20
may he eby ac i a e he mic obial communi y including N2O-p oduce s (Mø k ed e al., 21
2006; Sha ma e al., 2006). Thus, c yo u ba ed pea ci cles ep esen acidic ‘ho spo s’ o 22
mic obial N2O emission in und a (Repo e al., 2009; Ma ushchak e al., 2011). 23
The main sou ce o N2O in wa e -logged anoxic soils including pea lands is 24
deni i ica ion (Con ad, 1996; Pihla ie e al., 2004; Palme e al., 2010). Comple e 25
deni i ica ion is he sequen ial educ ion o ni a e o ni i e o dini ogen (N2) ia ni ic oxide 26
(NO) and N2O; ni i e is likewise an in e media e when ni a e is u ilized (Zum , 1997). The 27
oxido educ ases in ol ed in deni i ica ion include dissimila o y ni a e educ ases encoded 28
4.1 Published a icles in pee - e iewed jou nals
133
4
by na G o napA, coppe - and cy och ome cd1-con aining ni i e educ ases (encoded by ni K 1
and ni S, espec i ely), NO educ ases encoded by no BC, and N2O educ ases encoded by 2
nosZ (Zum , 1997). Ni a e educ ases likewise occu in dissimila o y ni a e educe s (S olz 3
& Basu, 2002). Ni K and Ni S a e s uc u ally di e en bu unc ionally equi alen (Jones e 4
al., 2008). O ganisms hos ing bo h ypes o ni i e educ ase a e unknown o da e (Heylen e 5
al., 2006). The genes coding o he abo e named oxido educ ases a e commonly used as 6
s uc u al gene ma ke s o he analysis o ni a e educe and deni i ie communi ies (e.g., 7
B u e al., 2011; Enwall e al., 2010; Jones & Hallin, 2010; Palme e al., 2010; Ho n e al., 8
2006; Rich e al., 2003; Philippo e al., 2002; P ieme e al., 2002; B ake e al., 2000). Main 9
p oduc s o deni i ica ion ha a e eleased in o he a mosphe e a e N2 o N2O. Deni i ie s 10
migh lack ni a e educ ases and/o N2O educ ases, and occupy di e se ecological niches 11
(Tiedje, 1988; Zum , 1997; Shapleigh, 2006). Deni i ica ion a es and he p oduc a io o 12
N2O o N2 a e egula ed by he deni i ying communi y and in si u condi ions (e.g., pH, 13
empe a u e, C- o-N- a io, as well as he a ailabili y o subs a es and elec on accep o s; 14
an Cleempu , 1998). Acidic pH < 5 impai s deni i ica ion and inc eases he p oduc a io o 15
N2O o N2 (Simek & Coope , 2002; Cuhel e al., 2010). The inc eased p oduc a io o N2O o 16
N2 is likely caused by pos - ansc ip ional e ec s o low pH on N2O educ ase o ma ion (Liu 17
e al., 2010). Howe e , in o ma ion on deni i ie communi ies ha h i e a pH < 5 in 18
pea lands is sca ce (Palme e al., 2010). 19
Deni i ie communi ies in pe ma os -a ec ed acidic und a soils a e un esol ed o 20
da e, despi e he ac ha such soils a e p one o eac sensi i ely o global wa ming, which 21
migh accele a e c yo u ba ion and in u n inc ease N2O emissions (Bockheim, 2007; Repo e 22
al., 2009). I is hypo hesized ha he obse ed con as ing N2O emission pa e ns o 23
c yo u ba ed and un u ba ed acidic pea soil a e associa ed wi h con as ing deni i ie 24
communi ies. The main objec i es o he p esen s udy we e (i) o compa e ecophysiological 25
ai s (i.e., capaci ies) o acid- ole an deni i ie communi ies in c yo u ba ed and un u ba ed 26
pea soil, (ii) o de elop py osequencing-based s a egies o he in-dep h analysis o 27
deni i ie communi ies by pa allel analysis o mul iple deni i ica ion associa ed genes, (iii) o 28
4 Manusc ip s
134
5
de e mine i con as ing and new deni i ie communi ies occu in c yo u ba ed and 1
un u ba ed pea soil by such py osequencing-based s a egies and quan i a i e PCR, and (i ) 2
hus o iden i y po en ial mic obial ca alys s o he excep ionally high N2O emissions om 3
c yo u ba ed pea soil. 4
5
4.1 Published a icles in pee - e iewed jou nals
135
6
Ma e ial and Me hods 1
Si e desc ip ion and soil sampling 2
The sampling a ea is loca ed in he Russian discon inuous pe ma os zone (62°57'E, 3
67°03'N) and was desc ibed p e iously (Repo e al., 2009; Supplemen al Ma e ial and 4
Me hods). Cumula i e N2O emissions in he ield om he c yo u ba ed soil a e 1.2 ± 0.3 g 5
N2O m-2, while hose o he un u ba ed soil a e negligible (<0.006 g N2O m-2). Topsoil was 6
iden i ied as he si e o highes N2O p oduc ion in he pea p o ile (da a no shown), and he 7
uppe 5 cm we e sampled om h ee c yo u ba ed pea ci cles and h ee adjacen , 8
un u ba ed a eas in Sep embe 2010. Roo s we e emo ed om un u ba ed soil, and soil o 9
mic ocosm s udies was s o ed a 4°C un il u he p ocessing. Soil o DNA ex ac ion was 10
suspended in RNAla e (Qiagen, Hilden, Ge many) immedia ely a e sampling o a oid 11
decomposi ion o nucleic acids, and s o ed a -20 °C upon a i al a he labo a o y. 12
Expe imen s we e conduc ed wi hin 2 mon hs a e sampling. Mois u e con en was 13
de e mined by weighing he soil be o e and a e d ying a 60°C o 3 days and was 71% and 14
81% in c yo u ba ed and un u ba ed pea soil, espec i ely. 15
16
Assessmen o deni i ica ion in soil mic ocosms 17
Soil o he h ee eplica e sampling si es was homogenized and pooled p io o 18
mic ocosm expe imen s. Soil slu ies a in si u pHH2O o app oxima ely 4 we e p epa ed wi h 19
4-5 g o soil and 3 olumes o deionized wa e in 125-ml in usion lasks, and sealed wi h gas-20
igh ubbe s oppe s. The gasphase was 100% a gon. Mic ocosms we e incuba ed a 20 °C 21
in he da k and pe o med in iplica e unless s a ed o he wise. 22
Ace ylene blocks ni ous oxide educ ases and hus he educ ion o N2O o N2
23
(Yoshina i & Knowles, 1976). Pa allel mic ocosms wi h and wi hou ace ylene (15% [ ol/ ol] 24
in headspace) we e used o di e en ia e be ween o al deni i ica ion and N2O-p oduc ion 25
po en ials. To al deni i ica ion s opped a e app oxima ely 4 days (90 h) in unsupplemen ed 26
mic ocosms wi h c yo u ba ed pea soil and ace ylene, indica ing ha in e nal ni a e and 27
ni i e we e deple ed (Figu e 1 A). 28
4 Manusc ip s
136
7
Fo appa en Michaelis-Men en kine ics, soil was p e-incuba ed o 7 days unde 1
anoxic condi ions o deple e in e nal ni a e and ni i e. Such soil was supplemen ed wi h 0 o 2
500 µM o NaNO3 o NaNO2. N2O did no accumula e in anoxic mic ocosms con aining 1 mM 3
ni i e in s e ile wa e a pH 4 wi hin 2 days (da a no shown). Appa en Michaelis-Men en 4
kine ics we e based on he p oduc ion o N2O in he p esence o ace ylene as desc ibed 5
(Segel, 1993; Palme e al., 2010; Supplemen al Ma e ial and Me hods). Soil ha was p e-6
incuba ed unde anoxic condi ions o 9 days was u ilized o s udy he e ec o he elec on 7
dono s ace a e, e hanol, o ma e, p opiona e, bu y a e, and lac a e on deni i ica ion in 8
mic ocosms supplemen ed wi h 1 mM ni i e and 0.5 mM o elec on dono s in he p esence 9
o ace ylene. A e ano he 47 days o anoxic incuba ion, 1 mM ni i e and 2 mM o elec on 10
dono s (0.5 mM o p opiona e only) we e esupplied. N2O p oduc ion, ni i e and elec on 11
dono s we e de e mined egula ly a e he ini ial supplemen a ion and a e 12
esupplemen a ion. N2O p oduc ion a es we e calcula ed om 3-4 da a poin s de e mined 13
wi hin 8-25 h a e addi ion o subs a es (ni i e and/o elec on dono s) when N2O p oduc ion 14
was linea . R2- alues o he linea eg essions we e always g ea e han 0.88. 15
Concen a ions o elec on dono s we e assessed by high-pe o mance liquid 16
ch oma og aphy, and ni a e as well as ni i e by ion ch oma og aphy (Palme e al., 2010; 17
Supplemen al Ma e ial and Me hods). 18
19
Ex ac ion o nucleic acids, and ampli ica ion o na G, ni K, ni S and nosZ 20
Nucleic acids we e ex ac ed om iplica e c yo u ba ed and un u ba ed pea soil 21
samples o accoun o la e al he e ogenei y in mic obial communi ies. A bead-bea ing 22
p o ocol ailo ed o he e icien emo al o PCR-inhibi ing humic acids by aluminum sul a e 23
p ecipi a ion p io o cell lysis was applied (Pe šoh e al., 2008; Supplemen al Ma e ial and 24
Me hods). 25
na G, ni K, ni S, and nosZ we e ampli ied using he p ime pai s na G1960 (TAY 26
GTS GGS CAR GAR AA)/na G2650 (TTY TCR TAC CAB GTB GC; Philippo e al., 2002), 27
F1aCu (ATC ATG GTS CTG CCG CG)/R3Cu (GCC TCG ATC AGR TTG TGG TT; Th obäck 28
4.1 Published a icles in pee - e iewed jou nals
137
8
e al., 2004), cd3aF (GTS AAC GTS AAG GAR ACS GG)/R3cd (GAS TTC GGR TGS GTC 1
TTG A; Th obäck e al., 2004), and nosZF (CGC TGT TCI TCG ACA GYC AG)/nosZR (ATG 2
TGC AKI GCR TGG CAG AA; Rich e al., 2003), espec i ely. Each p ime was p eceeded 3
by a 6 basepai -long ba code (AGCGTC o un u ba ed, and ATATAC o c yo u ba ed soil 4
samples) o sepa a e sequences a e py osequencing. Eigh eplica e 25 µl PCR eac ions 5
pe a ge gene we e pe o med a 8 di e en annealing empe a u es om 54.7 o 63.6 °C o 6
maximize he likelihood o de ec ing a high di e si y o a ge genes. All eplica e PCR 7
eac ions ha yielded p oduc s (i.e., amplicons) o he co ec size we e pooled p io o 8
subsequen analyses. Fo de ailed PCR p o ocols e e o Supplemen al Ma e ial and 9
Me hods. 10
11
Ba coded amplicon py osequencing o s uc u al genes 12
P e iously published amplicon py osequencing s a egies (Hube e al., 2007; Iwai e 13
al., 2010; Will e al., 2010) we e modi ied o maximize he likelyhood o speci ic ampli ica ion 14
o deni i ica ion associa ed s uc u al genes du ing amplicon gene a ion. Py osequencing 15
equi es amplicons used wi h sequencing adap o s. Published s a egies u ilize a ge gene 16
speci ic p ime s used wi h a ba code and an app oxima ely 30 bp long sequencing adap o , 17
esul ing in p ime s wi h mo e han 50% o he sequence being no complemen a y o he 18
a ge genes, and hus allowing o unspeci ic ampli ica ions. In his s udy, amplicons we e 19
gene a ed du ing PCR wi h a ge gene speci ic p ime s used wi h he ba code only (see 20
abo e) a he han u ilizing p ime s ha con ain ba code and sequencing adap o s. 21
Sequencing adap o s we e liga ed a e PCR o gel pu i ied amplicons. 22
Amplicons o simila leng hs o bo h soil ypes we e combined in equal amoun s (i.e., 23
na G and nosZ amplicons we e pooled, as well as ni K and ni S). Amplicon mix u es we e 24
ea ed wi h P eCR Repai Mix (New England Biolabs, F ank u am Main, Ge many) o 25
elimina e possible PCR-blocking DNA damage ha migh ha e occu ed du ing gel 26
pu i ica ion o s o age o amplicons, and pu i ied ia isop opanol p ecipi a ion. Sequencing 27
om 5’ ( o wa d) and 3’ ( e e se) ends o amplicons was pe o med a e liga ion o A (CGT 28
4 Manusc ip s
138
9
ATC GCC TCC CTC GCG CCA TCA G) and B (CTA TGC GCC TTG CCA GCC CGC TCA 1
G) sequencing adap o s a he Gö ingen Genomics Labo a o y employing he Roche GS-2
FLX 454 py osequence and GS FLX Ti anium se ies eagen s (Roche, Mannheim, 3
Ge many) acco ding o he manu ac u e ’s ins uc ions. 4
5
Sequence il e ing and analysis 6
Sequences wi h ambigui ies, and hose wi h inco ec p ime o ba code sequences 7
we e disca ded. na G as well as nosZ sequences sho e han 350 bp, and ni K as well as 8
ni S sequences sho e han 300 bp we e likewise excluded om u he analyses. Amplicon 9
sequences we e so ed acco ding o hei ba codes and p ime s, and combined subse s o 10
sequences o each s uc u al gene (i.e., con aining sequences om bo h c yo u ba ed and 11
un u ba ed pea soil) we e clus e ed [i.e., assigned o ope a ional axonomic uni s (OTUs)] a 12
species-le el h eshold dis ances o 33% [na G (Palme e al., 2009)], 17% [ni K, (Depka -13
Jakob e al., unpublished)], 18% (ni S, Depka -Jakob e al., unpublished), o 20% [nosZ 14
(Palme e al., 2009)] based on DNA sequences using he JAguc2 pipeline 15
(h p://wwwagak.in o ma ik.uni-kl.de/ esea ch/JAguc/; Nebel e al. unpublished 16
(wwwagak.in o ma ik.uni-kl.de/s a /nebel/www_jaguc/www_jaguc.pd ; Figu e S1). In b ie , 17
JAguc2 gene a es a pai wise sequence alignmen p io o calcula ion o a dis ance ma ix 18
and clus e ing wi h he a e age simila i y me hod. This app oach is mo e eliable [i.e., less 19
sensi i e o PCR- and py osequencing noise, and hus less sensi i e o an a i ical in la ion o 20
di e si y (Kunin e al., 2010)] han mul iple alignmen s and/o clus e ing wi h comple e 21
linkage algo i hms (Sun e al., 2009; Quince e al., 2009; Huse e al. 2010). Amplicon 22
sequences ob ained by py osequencing om de ined empla e mix u es we e essen ially a 23
mos 10% dissimila o empla e sequences due o PCR- and py osequencing noise (Quince 24
e al., 2011; Behnke e al., 2011). The h eshold-dis ances u ilized o call OTUs in his s udy 25
we e 17-33%, which is subs an ially g ea e han he abo e epo ed maximal PCR- and 26
py osequencing noise (Figu e S1). Thus, ou app oach was a he una ec ed by PCR- and 27
py osequencing noise, al hough we did no apply lowg am-based sequence co ec ion 28
4.1 Published a icles in pee - e iewed jou nals
139
3.4 E ec o expe imen al d ough on abundance o mc A genes
and ansc ip s in he acidic en
Be o e he expe imen al d ough , de ec ed mc A gene copy numbe s we e abou 1.7%
and 4.1% o de ec ed a chaeal 16S RNA gene copy numbe s in con ol and ea men
plo s, espec i ely (Figu e 5), indica ing a highe ela i e abundance o me hanogens
in d ough plo s be o e he onse o expe imen al d ough . A he end o he d ough
pe iod, he ela i e abundance o mc A genes was signi ican ly lowe in d ough plo s
han be o e he onse o d ough (1.5%), while i had emained a he simila in con ol
plo s (1.2%), sugges ing ha he ela i e abundance o me hanogens was lowe ed by
he expe imen al d ough . A e ewe ing, ela i e abundances o mc A emained in
he same ange in he con ol plo s (1.4%), while he ela i e abundance o mc A genes
in he d ough plo s inc eased o app oxima ely 3.1%, indica ing ha ewe ing led o
inc eased ela i e abundances o me hanogens in en soil.
Figu e 5: E ec o expe imen al d ough on he abundance o mc A genes and
ansc ip s. Black ba s ep esen con ol plo s, whi e ba s ep esen d ough plo s.
Mean alues o h ee plo s and ou soil laye s pe plo ype and s anda d e o s a e
shown. 0=be o e d ough (June 09 h 2008), 1=a e d ough (July 27 h 2008),
2=a e ewe ing (Augus 11 h 2008).
4 Manusc ip s
242
20 o 30 cm soil om bo h plo ypes yielded he highes ela i e abundances o mc A
(Figu e 5). Expe imen al d ough dec eased he ela i e abundance o mc A in 20 o
30 cm dep h-soil. A e ewe ing, ela i e abundance o mc A amoun ed o 10.4% in
he d ough plo s, in he con ol plo s hey we e abou 3.3%. In he o he soil laye s
ela i e mc A gene copy numbe s we e below 5%, and changes in he ela i e abundance
o mc A we e less p onounced han in he soil laye om 20 o 30 cm, indica ing ha
his soil laye was mo e sensi i e o expe imen al d ough han he o he soil laye s.
T ansc ip o gene a ios o mc A (i.e., a ios o mc A copies ob ained om cDNA o
mc A copies ob ained om DNA) we e highly a iable be ween plo s and imepoin s,
anging om 0.5% o 500%. The a e age ansc ip o gene a io o mc A (i.e., he
a e age o all 3 plo s and all 4 soil dep hs) was abou 50% in bo h con ol and d ough
plo s be o e he onse o d ying (Figu e 5). A he end o he d ough pe iod, he
ansc ip o gene a io o mc A was abou 100% in bo h plo ypes, indica ing inc eased
ac i i y o en me hanogens in bo h plo ypes. In he con ol plo s, he ansc ip o
gene a io o mc A s ayed a abou 100% a he ime a e ewe ing, while in he d ough
plo s he exp ession d opped again o 50%, indica ing a educed ac i i y o me hanogens
in d ough plo s. T ansc ip o gene a ios o mc A we e highes in he soil laye s om
0 o 20 cm in bo h plo ypes (14 o 260%), and lowes in 30 o 40 cm soil (3 o 7%)
(Figu e 5), indica ing a highe aci i i y o me hanogens in he uppe soil laye s.
3.5 E ec o expe imen al d ough on he communi y composi ion
o me hanogens
The e ec o he expe imen al d ough on he communi y composi ion o en me hanogens
was assessed by mc A TRFLP analysis om all plo s on gene and ansc ip le el. On
gene le el, 8 o 10 TRFs we e ound in each sample (Figu e 6). The same TRFs we e
ound on ansc ip le el wi h he excep ion o he 230 bp TRF, which was absen in
he ansc ip le el TRFLP p o iles.
The compa ison o he a e age gene-le el TRFLP p o iles (i.e., he a e age o all
sampled soil dep hs om all h ee plo s o a plo ype) in con ol and d ough plo s
e ealed sligh di e ences in he communi y composi ion o en me hanogens a he di -
e en imepoin s and be ween he wo plo ypes (Figu e 6 A). Di e ences in mc A
communi y composi ion be o e he onse o expe imen al d ough and a e he d ough
pe iod we e obse ed in he d ough plo s, while mc A communi y composi ion a hose
wo imepoin s was mo e simila in he con ol plo s. This indica ed a sligh e ec o
4.2 Manusc ip s in p epa a ion
243
Figu e 6: E ec o expe imen al d ough on he communi y composi ion o mc A
genes (A) and ansc ip s (B). Compa a i e TRFLP analysis o mc A ampli-
ied om DNA and cDNA o con ol and d ough plo s. PCR p oduc s we e di-
ges ed wi h Hin I. Combined ela i e abundances o he majo phylogene ic g oups
a e shown in colo ( igh ba s), ela i e abundances o indi idual TRFs a e in
black/whi e (le ba s). Mean alues o h ee plo s and ou soil laye s pe plo
ype a e shown. 0=be o e d ough (June 09 h 2008), 1=a e d ough (July 27 h
2008), 2=a e ewe ing (Augus 11 h 2008).
expe imen al d ough on he communi y s uc u e o en me hanogens. Se en majo
TRFs (i.e, TRFs wi h a ela i e abundance >1%) we e obse ed in bo h plo ypes be-
o e he expe imen al d ough . The communi y was domina ed by TRFs indica i e o
4 Manusc ip s
244
Me hanocellaceae, i.e., TRFs o 200 bp leng h (indica i e o OTU 1 and 6), 586 bp leng h
(indica i e o OTU 2), and 638 bp leng h (indica i e o OTU 2 and 7), wi h a combined
ela i e abundance o 57% and 47% in con ol and ea men plo s, espec i ely. A -
e he d ough pe iod, he combined ela i e abundance o hose TRFs was 53% and
37% in con ol and ea men plo s, espec i ely, indica ing a d ough induced shi
in communi y composi ion. Mo eo e , he ela i e abundance o TRFs indica i e o
Me hanomic obiales/Me hanobac e iales, i.e., he 305 bp and >760 bp TRFs (indica i e
o OTUs 4 and 5) inc eased in he ea men plo s, and 2 addi ional TRFs we e de ec ed
in he con ol plo s (64bp and 230 bp leng h, indica i e o OTU 3 and 4 and o OTU
8, espec i ely) and one in he d ough plo s (64bp leng h). A e ewe ing, TRFLP
p o iles o con ol and ea men plo s we e again simila o each o he , wi h combined
ela i e abundances o TRFs indica i e o Me hanocellaceae (i.e., TRFs o 200, 586
and 638 bp leng h) o 27% and 35% in con ol and ea men plo s, espec i ely, and
inc eased abundances o TRFs indica i e o Me hanomic obiales/Me hanobac e iales
(i.e., TRFs o 305 and >760 bp leng h) o 40% and 39%, espec i ely. TRFs indica-
i e o Me hanosa cinaceae (i.e., TRFs o 230 and 466 bp leng hs) had simila ela i e
abundances in con ol and d ough plo s a all imepoin s, equaling 14-15% be o e and
a e he d ough pe iod and 20-23% a e ewe ing. This indica es ha (i) a shi
in communi y composi ion occu ed in bo h con ol and d ough plo s h oughou he
expe imen al pe iod, (ii) d ough educed he ela i e abundance o Me hanocellaceae,
(iii) ewe ing migh equalize di e ences in communi y composi ion ha we e obse ed
a e he d ough pe iod, and (i ) Me hanosa cinaceae a e leas a ec ed by di e ences
be ween he wo plo ypes.
A he ansc ip le el, 8 majo TRFs (i.e, TRFs wi h a ela i e abundance >1%) we e
ound in he TRFLP p o iles o con ol and ea men plo s (Figu e 6 B). Communi y
composi ion o mc A ansc ip s di e ed be ween con ol and ea men plo s be o e he
onse o d ough . The combined ela i e abundances o TRFs indica i e o Me hanocel-
laceae (i.e, he 200 bp, 586 bp, and 638 bp leng h TRFs) amoun ed o 36% and 50%
in con ol and d ough plo s, espec i ely, and he ela i e abundance o TRFs indica-
i e o Me hanomic obiales/Me hanobac e iales (i.e., TRFs o 305 and >760 bp leng h)
was wice as high in con ol as in d ough plo s (36% and 18%, espec i ely). A e
he d ough pe iod, combined ela i e abundances o TRFs indica i e o Me hanocel-
laceae we e 39% and 49% in con ol and d ough plo s, espec i ely. The ela i e abun-
dance o TRFs indica i e o Me hanomic obiales/Me hanobac e iales was 21% and 34%
in con ol and d ough plo s, espec i ely, a e he expe imen al d ough . The ela i e
4.2 Manusc ip s in p epa a ion
245
abundance o TRFs indica i e o Me hanosa cinaceae inc eased in he con ol plo s om
12% o 30%, while i dec eased in he d ough plo s om 28% o 16%. This indica es
ha he exp ession o mc A (i) o Me hanocellaceae is a he s able unde expe imen-
al d ough condi ions, (ii) o Me hanomic obiales/Me hanobac e iales is up egula ed as
a esul o d ough condi ions, and (iii) o Me hanosa cinaceae is down egula ed as a
esul o d ough condi ions. A e ewe ing, Me hanosa cina- ela ed TRFs had he
highes ela i e abundance in bo h plo ypes (45% and 38% in con ol and d ough
plo s, espec i ely). The ela i e abundance o Me hanocellaceae- ela ed TRFs was low
in bo h plo ypes a e ewe ing (17% and 28% in con ol and d ough plo s, espec-
i ely), while he ela i e abundance o Me hanomic obiales/Me hanobac e iales- ela ed
TRFs was a ound 30% in bo h plo ypes. The da a indica es (i) ha ewe ing es o ed
simila exp ession pa e ns in bo h plo ypes and (ii) a shi in he ac i e me hanogenic
communi y om Me hanocellaceae in ea ly summe o Me hanosa cinaceae in la e sum-
me .
TRFLPs p o iles on ansc ip le el di e ed om TRFLP p o iles on gene le el (Fig-
u e 6). Me hanosa cina- ela ed TRFs gene ally had a highe ela i e abundance on an-
sc ip han on gene le el, while Me hanomic obiales/Me hanobac e iales- ela ed TRFs
we e gene ally mo e abundan on gene han on ansc ip le el. The a ios o ela i e
abundances o TRFs indica i e o ce ain phylogene ic g oups on gene and ansc ip
le el indica ed ha Me hanosa cinaceae we e inhibi ed by expe imen al d ough , while
Me hanomic obiales/Me hanobac e iales we e s imula ed.
4 Manusc ip s
246
4 Discussion
4.1 E ec o d ying and ewe ing on he me hanogenic communi y
in he acidic en
In he cou se o clima e change, a highe equency o mo e ex eme wea he e en s like
p olonged d ough pe iods o hea y p ecipi a ion e en s a e an icipa ed [61]. P olonged
d ough educes me hanogenic ac i i y in he en Schl¨oppne b unnen [33]. This is a -
ibu ed o egene a ion o elec on accep o s due o highe soil ae a ion [33]. Thus,
when condi ions become anoxic me hanogenesis compe es wi h o he anae obic edox
p ocesses o elec ons [8]. Indeed, a e expe imen al d ough me hanogenic po en ials
o 0 o 10 and 30 o 40 cm soil we e lowe and he ini ial lag phases we e longe in
d ough han in con ol plo s (Figu e 1). In an acidic en soil in Finland, educed CH4
emissions a e obse ed in d ained si es as compa ed o he we e si es along a d ainage
g adien nea a g oundwa e ex ac ion plan [67]. Reduced CH4emissions om d ained
we lands a e also caused by me hano ophy occu ing when O2pene a es in o deepe
soil laye s [67]. In mesocosm s udies wi h Schl¨oppne b unnen en soil, CH4emissions
dec ease due o me hano ophy in mo e ae a ed pa s abo e he wa e able when he
wa e able is expe imen ally lowe ed, while he me hanogenic pa hways a e mainly un-
a ec ed [32]. Me hano ophic ac i i y has also been de ec ed in mic ocosm s udies wi h
soil om Schl¨oppne b unnen en, and me hano ophic axa a e de ec ed in he en soil
[64]. A e ewe ing o pea land soil, eco e y o me hanogenesis is o e delayed as a
esul o inc eased a ailabili y o al e na i e elec on accep o s [7, 32, 33].
Me hanogenesis is con olled by in si u pa ame e es such as empe a u e and he le el
o he g ound wa e able [5, 31, 32, 57]. A wa e able d awdown like he one achi ed
du ing he expe imen al d ough esul s in highe soil ae a ion and a ise concomi an ise
in he edox po en ial [33]. Me hyl-coenzyme M educ ase is inac i a ed du ing pe iods
o aised O2concen a ions [28]. Dec eased abundances o me hanogens we e obse ed
in d ough plo s a e he d ough pe iod (Figu e 5), howe e , he composi ion o he
me hanogenic communi y was no signi ican ly a ec ed. De ec ed mc A we e mainly
a ilia ed wi h mc A o Me hanocella paludicola (Me hanocellaceae), ep esen a i e o
’Rice Clus e I’ [55], mo eo e mc A a ilia ed wi h mc A o he Me hanomic obiales,
Me hanobac e iaceae and Me hanosa cinaceae we e de ec ed (Figu e 3, Figu e 4). E en
hough small changes in communi y composi ion we e obse ed (Figu e 4, Figu e 6),
he ela i e abundances o he de ec ed g oups we e no signi ican ly a ec ed by expe -
imen al d ough . This is in good ag eemen wi h s udies om ice ield soils, whe e
4.2 Manusc ip s in p epa a ion
247
d ainage and O2exposu e educe he abundance o me hanogens while he communi y
composi ion o me hanogens is no a ec ed [38, 39, 68]. Thus, he obse ed changes in
he me hanogenic po en ial o en soil a e d ying and ewe ing migh be caused by
educed abundances o me hanogens.
T ansc ip o gene a ios o mc A anged om 50 o 110% in en soil (Figu e 5) and
we e hus o a simila magni ude as a ios o mc A in B i ish pea lands [13]. Howe e ,
his a io migh be unde es ima ed due o dead o inac i e me hanogens [13]. mc A
exp ession was simila in d ough and con ol plo s a e he d ough pe iod (Figu e 5).
Pea me hanogens main ain high abundances o mc A ansc ip s, e en when hei ac-
i i y is low, and simila gene and ansc ip abundances ha e been de ec ed in pea soil
wi h me hanogenic po en ial educed due o long- e m soil s o age in he lab [13]. Thus,
lowe me hanogenic po en ials a e p olonged expe imen al d ough a e likely caused
by pos - ansc ip ional e ec s on mc A, and migh also be a ec ed by o he pa ame e s
han O2. Mo eo e , me hanogens as well as copies o mc A we e also ound in he uppe
soil laye s in he acidic en Schl¨oppne b unnen, e en hough mo e oxic condi ions a e
ound he e oughou he yea [23]. Me hanogens a e also de ec ed in o he a he oxic
sys ems such as dese soils [50], and mino CH4p oduc ion and emission is known om
oxic upland soils [27, 66]. Reduced iabili y o me hanogens is a ibu ed o he e ec o
desicca ion a he han o he e ec o O2i sel [10]. The mechanism o he su i al o
e en g ow h unde oxic condi ions emains un esol ed, since he e is no e idence o he
exis ence o es ing s ages in me hanogens [10]. Howe e , enzyma ic p o ec ion agains
oxida i e s ess by he enzymes supe oxide dismu ase and ka alase has been epo ed in
me hanogenic species like Me hanobac e ium b yan ii and Me hanobac e ium he moau-
o ophicum [29, 60]. Some mc A sequences ob ained om he acidic en we e ela ed
o hose species, and i seems hus likely ha hey also ha bo he capaci ies o p oduce
p o ec i e enzymes. Addi ionally, we and anoxic mic oen i omen s in d ough plo s
migh p o ec me hanogens om O2du ing d ough pe iods [32]. Howe e , O2inhibi ed
me hanogenesis in en soil mic ocosms e en a e y low O2concen a ions (Figu e 2),
hus i is likely ha en soil me hanogens a e inac i e du ing pe iods o d ough s ess.
The composi ion o mc A ansc ip s was mo e a iable han ha o mc A genes
(Figu e 6). Howe e , di e ences we e mo e p onounced be ween he di e en sam-
pling imepoin s han be ween he wo plo ypes, e en hough ansc ip o gene
a ios o TRFs sugges inhibi o y and s imula o y e ec s o expe imen al d ough on
Me hanosa cinaceae and Me hanomic obiales/Me hanobac e iales, espec i ely. O2ex-
posu e al e s he composi ion o mc A ansc ip s in ice ield soil [69]. In Augus , a
4 Manusc ip s
248
g ea e abundance o Me hanosa cina- ela ed mc A ansc ip s han in June o July was
de ec ed, indica ing highe ac i i y o Me hanosa cina- ela ed me hanogens in la e sum-
me . Me hanogens o he o de s Me hanobac e iales,Me hanomic obiales, and Me hanocel-
lales use hyd ogeno ophic me hanogenesis o ene gy conse a ion [55, 63], while Me hanosa ci-
nales conse e ene gy ia ace oclas ic and hyd ogeno ophic me hanogenesis [43]. I is
hus easible ha he con ibu ion o ace oclas ic me hanogenesis is a iable oughou
he yea and is o g ea e in luence in he la e summe .
4.2 Conclusions and limi a ions
Fu u e clima e change will likely inc ease he equency o d ough e en s in pea lands
[61]. Expe imen al d ough in he acidic en Schl¨oppne b unnen dec eases me hane
emissions [33], and educed me hanogenic po en ials we e obse ed in mic ocosm s udies
wi h en soil. The abundance o me hanogens was educed in he d ough plo s while he
composi ion o he me hanogenic communi y was a he s able h oughou he d ough
expe imen based on analyses o mc A gene and ansc ip abundances and TRFLP
p o iles. Un o una ely, owing o he expe imen al design, he e ec o expe imen al
d ough on me hanogenic po en ials in mic ocosm incuba ions and on he abundance
and communi y s uc u e o en me hanogens we e s udied in wo di e en yea s. I is
he e o e no possible o di ec ly link he obse ed e ec s o expe imen al d ough on
p ocess le el o he obse ed changes in he me hanogenic communi y.
Wi hin hese limi a ions he da a collec ed in his s udy none heless indica e ha
expe imen al d ough (i) impac s on me hanogenic po en ials in he acidic en, and (ii)
mo eo e in luences he abundance bu no he communi y composi ion o en me hanogens.
Fu he in es iga ion would be needed o es ablish he link be ween obse ed e ec s on
me hanogenic po en ial and obse ed e ec s on communi y composi ion o en me hanogens.
5 Acknowledgmen s
Suppo o his s udy was p o ided by he Deu sche Fo schungsgemeinscha (DFG HO
4020/2-2). Wo k was also suppo ed by he DFG Resea ch g oup FOR 562.
4.2 Manusc ip s in p epa a ion
249
Re e ences
[1] Al schul, S. F., Gish, W., Mille , W., Mye s, E. W., and Lipman, D. J.
Basic local alignmen sea ch ool. Jou nal o Molecula Biology 215, 403–410 (1990).
[2] Bap es e, E., B ochie , C., and Bouche , Y. Highe -le el classi ica ion o
he A chaea: e olu ion o me hanogenesis and me hanogens. A chaea 1, 353–363
(2005).
[3] Blodau, C., Basiliko, N., and Moo e, T. Ca bon u no e in pea land
mesocosms exposed o di e en wa e able le els. Biogeochemis y 67, 331–351
(2004).
[4] Clymo, R. S. The ecology o pea lands. Science P og ess 71, 593–614 (1987).
[5] Con ad, R. Soil mic oo ganisms as con olle s o a mosphe ic ace gases (H2,
CO, CH4, OCS, N2O, and NO). Mic obiological Re iews 60, 609–640 (1996).
[6] Denman, K. L., B asseu , G., Chid haisong, A., Ciais, P., Cox, P. M.,
Dickinson, R. E., Hauglus aine, D., Heinze, C., Holland, E., Jacob, D.,
Lohmann, U., Ramachand an, S., da Sil a Dias, P. L., Wo sy, S. C., and
Zhang, X. Couplings be ween changes in he clima e sys em and biogeochemis y.
In: Clima e Change 2007: The Physical Science Basis. Con ibu ion o Wo king
G oup I o he Fou h Assessmen Repo o he In e go e nmen al Panel on Clima e
Change, edi ed by Solomon, S., Qin, D., Manning, M., Chen, Z., Ma quis,
M., A e y , K. B., Tigno , M., and Mille , H. L., pp. 499–588. Camb idge
Uni e si y P ess (2007).
[7] Dow ick, D. J., F eeman, C., Lock, M. A., and Reynolds, B. Sulpha e
educ ion and he supp ession o pea land me hane emissions ollowing summe
d ough . Geode ma 132, 384–390 (2006).
[8] D ake, H. L., Ho n, M. A., and Wus , P. K. In e media y ecosys em
me abolism as a main d i e o me hanogenesis in acidic we land soil. En i on-
men al Mic obiology Repo s 1, 307–318 (2009).
[9] Ege , M. and F ied ich, M. W. Fo ma ion o pseudo- e minal es ic ion
agmen s, a PCR- ela ed bias a ec ing e minal es ic ion agmen leng h poly-
mo phism analysis o mic obial communi y s uc u e. Applied and En i onmen al
Mic obiology 69, 2555–2562 (2003).
4 Manusc ip s
250
[10] Fe ze , S., Bak, F., and Con ad, R. Sensi i i y o me hanogenic bac e ia
om paddy soil o oxygen and desicca ion. FEMS Mic obiology Ecology 12, 107–115
(1993).
[11] Foken, T. Lu hygienische-Bioklima ische Kennzeichnung des obe en Ege ales,
Bay eu h (2003).
[12] Fo s e , P., Ramaswamy, V., A axo, P., Be n sen, T., Be s, R.,
Fahey, D., Haywood, J., Lean, J., Lowe, D., Myh e, G., Nganga, J.,
P inn, R., Raga, G., Schulz, M., and VanDo land, R. Changes in a -
mosphe ic cons i uen s and in adia i e o cing. In: Clima e Change 2007: The
Physical Science Basis. Con ibu ion o Wo king G oup I o he Fou h Assessmen
Repo o he In e go e nmen al Panel on Clima e Change, edi ed by Solomon, S.,
Qin, D., Manning, M., Chen, Z., Ma quis, M., A e y , K. B., Tigno ,
M., and Mille , H., pp. 129–234. Camb idge Uni e si y P ess (2007).
[13] F ei ag, T. E. and P osse , J. I. Co ela ion o me hane p oduc ion and
unc ional gene ansc ip ional ac i i y in a pea soil. Applied and En i onmen al
Mic obiology 75, 6679–6687 (2009).
[14] Galand, P. E., Juo onen, H., F i ze, H., and Y j¨
al¨
a, K. Me hanogen
communi ies in a d ained bog: E ec o ash e iliza ion. Mic obial Ecology 49,
209–217 (2005).
[15] Galand, P. E., Saa nio, S., F i ze, H., and Y j¨
al¨
a, K. Dep h ela ed
di e si y o me hanogen a chaea in Finnish oligo ophic en. FEMS Mic obiology
Ecology 42, 441–449 (2002).
[16] Galand, P. E., Y j¨
al¨
a, K., and Con ad, R. S able ca bon iso ope ac ion-
a ion du ing me hanogenesis in h ee bo eal pea land ecosys ems. Biogeosciences
7, 3893–3900 (2010).
[17] Go ham, E. No he n pea lands - ole in he ca bon-cycle and p obable esponses
o clima ic wa ming. Ecological Applica ions 1, 182–195 (1991).
[18] G i i hs, R. I., Whi eley, A. S., O’Donnell, A. G., and Bailey, M. J.
Rapid me hod o coex ac ion o DNA and RNA om na u al en i onmen s o
analysis o ibosomal DNA- and RNA-based mic obial communi y composi ion.
Applied and En i onmen al Mic obiology 66, 5488–5491 (2000).
4.2 Manusc ip s in p epa a ion
251
6 Supplemen al Figu es
Figu e S1: E ec o di luo ome hane on me hanogenesis and me hano ophy in
en soil mic ocosms. Mean alues and s anda d e o s o h ee eplica e mi-
c ocosms a e shown. Squa es ep esen anoxic mic ocosms, ci cles ep esen oxic
mic ocosms. Open symbols ep esen mic ocosms wi hou di luo ome hane, closed
symbols ep esen mic ocosms wi h di luo ome hane.
4 Manusc ip s
258
Figu e S2: Co ela ion o DNA sequence simila i ies o mc A e sus 16S RNA
gene simila i y. Do ed lines ep esen he simila i y alues, below which wo
sequences always had less han 97% 16S RNA gene sequence simila i y. The
dashed lines ep esen he 90% quan ile o pai wise sequence compa isons wi h a
16S RNA gene sequence simila i y o 97% (i.e., h eshold simila i y). The solid
lines ma k he 97% 16S RNA gene simila i ies.
4.2 Manusc ip s in p epa a ion
259
Deni i ie communi ies in an acidic
en a e s able du ing expe imen al
d ough
Manusc ip in p epa a ion
Ka ha ina Palme , Ha old L. D ake, Ma cus A. Ho n
Depa men o Ecological Mic obiology, Uni e si y o Bay eu h, 95440
Bay eu h, Ge many
4 Manusc ip s
260
We lands can ac as sou ces o sinks o he g eenhouse gas ni ous ox-
ide (N2O). The acidic en Schl¨oppne b unnen emi s deni i ica ion de i ed
N2O and is also capable o N2O consump ion. Global wa ming is p edic ed
o cause mo e ex eme wea he e en s in u u e yea s, including p olonged
d ough pe iods, which will in luence he deni i ie communi y in he acidic
en. Thus, he abundance and communi y composi ion o deni i ie s and
hei eac ion o enhanced d ough we e in es iga ed. Py osequencing o he
s uc u al gene ma ke s na G,ni K,ni S, and nosZ e ealed a high deni i-
ie di e si y and 6, 13, 18 and 6 species-le el ope a ional axonomic uni s
(OTUs). De ec ed species-le el OTUs we e mainly ela ed o Ac inobac e ia,
Alpha-,Be a-, and Gammap o eobac e ia. An expe imen o simula e p o-
longed d ough was conduc ed in summe 2008 by expe imen ally lowe ing
he wa e able in 3 d ough plo s o 8 weeks ollowed by subsequen ewe -
ing o he d ough plo s. 3 und ained plo s se ed as con ols. Samples
we e aken be o e and a e d ough and a e ewe ing. na G and nosZ
we e used o see assess changes in he ni a e educe and deni i ie com-
muni y o expe imen al d ough . Highe copy numbe s o na G and nosZ
(2x and 1.5x, espec i ely) we e de ec ed a e he expe imen al d ough
in d ough bu no in con ol plo s by quan i a i e PCR (qPCR), indica ing
ha ni a e educe s and deni i ie s a e posi i ely in luenced by he d ough
ea men . Rewe ing es o ed he copy numbe s o na G and nosZ o he
o iginal le el. Te minal es ic ion agmen leng h polymo phism (TRFLP)
pa e ns o na G and nosZ we e simila be o e and a e he d ough pe iod,
indica ing a high s abili y o ni a e educe s and deni i ie s in he en. The
collec i e da a indica e ha (i) a high deni i ie di e si y exis s in he acidic
en Schl¨oppne b unnen, (ii) expe imen al d ough inc eases deni i ie abun-
dances, and (iii) he communi y composi ion is una ec ed by expe imen al
d ough .
1 In oduc ion
Ex eme wea he e en s such as p olonged d ough pe iods o hea y ain alls a e p e-
dic ed o inc ease in equency as well as in in ensi y in u u e yea s due o global
wa ming [38]. This will a ec he wa e able in we land soils and hus many biological
p ocesses in hose soils, including p ocesses in ol ed in he u no e o ni ous oxide
4.2 Manusc ip s in p epa a ion
261
(N2O) [12, 33, 39]. N2O is a majo g eenhouse gas wi h a global wa ming po en ial
ha is 300 imes highe han ha o CO2and he majo ozone-deple ing subs ance
in he a mosphe e [9, 32]. The a mosphe ic concen a ion o N2O inc eased om 270
ppb o 319 ppb in he yea s 1750 o 2005 [9]. Soils a e majo sou ces o N2O, which
can be ni i ica ion- o deni i ica ion de i ed [6, 7, 30]. Mo eo e , such we land soils
can be sou ces o sinks ( ansien o pe manen ) o a mosphe ic N2O [11, 10, 27, 36].
In wa e -logged we land soils including ens deni i ica ion is he main sou ce o N2O
[6, 27, 30]. Deni i ica ion is he sequen ial educ ion o ni a e o ni i e o N2 ia
ni ic oxide (NO) and N2O [41]. The deni i ica ion p ocesses is ca alyzed by he en-
zymes ni a e educ ase Na o Nap (encoded by na GHJI o napEDABC, espec i ely),
coppe - o cy och ome-dependen ni i e educ ases Ni K o Ni S (encoded by ni K o
ni S, espec i ely), NO ecu ase No (encoded by no BC ), and N2O educ ase Nos (en-
coded by nosZ) [41]. Ni a e educ ases a e mo eo e ound in many non-deni i ying
ni a e- educing mic oo ganisms [41].
na G,ni K,ni S, and nosZ a e s uc u al gene ma ke s commonly used in he anal-
ysis o ni a e educe and deni i ie communi y composi ion [4, 15, 24, 29]. na G
and nosZ om he acidic en Schl¨oppne b unnen we e s udied ea lie by con en ional
cloning based sequencing, e ealing no el ni a e educe s and deni i ie s in he acidic
en [27]. Howe e , new sequencing me hods including amplicon py osequencing allow
mo e in-dep h analysis o ni a e educe s and deni i ie communi ies. Thus, one aim
o he p esen s udy was o eassess he di e si y o na G and nosZ om Schl¨oppne -
b unnen en soil along wi h he di e si y o he ni i e educ ase encoding ni K /ni S
using amplicon py osequencing.
In he con ex o global wa ming, mo e ex eme d ough pe iods a e expec ed o occu ,
which will e ec wa e ables in en soils [38]. Howe e , i is so a un esol ed how en
ni a e educe and deni i ie communi ies eac o changing wa e ables. Thus, a
u he aim o he s udy was o assess he e ec o simula ed d ough condi ions on
ni a e educe and deni i ie abundance and di e si y in acidic Schl¨oppne b unnen en
soil o gain a be e unde s anding o possible eedback e ec s o global wa ming.
4 Manusc ip s
262
2 Ma e ial and Me hods
2.1 S udy si e and expe imen al se up
The mine o ophic en Schl¨oppne b unnen is loca ed in he Lehs enbach ca chmen in
he Fich elgebi ge, Ba a ia, Ge many (50◦07’ 53” N, 11◦52’ 51” E), a app oxima ely
700 m abo e sea le el. The sampling si e is desc ibed in mo e de ail in [14, 25, 27]. In
2008, he wa e able was a i icially lowe ed in 3 ea men (i.e., d ough ) plo s (size
7.2 m x 5 m). Roo s we e e ec ed o shield he plo s om ain wa e , and addi ional
d ainage was accomplished by di ches. The oo s had la ge open side walls o minimize
empe a u e and wind speed e ec s. Addi ionally, 3 un ea ed plo s se ed as con ol
si es. Roo s and d ainage on he ea men plo s we e ins alled on June 10 h 2008 and
kep in place un il Augus 7 h. On a e age, expe imen al d ough lowe ed he wa e able
o 0.60 m below he su ace. A e he expe imen al d ough pe iod, oo s and d ainage
we e emo ed o allow ewe ing o he d ough plo s. Fo mo e de ailed desc ip ion o
he expe imen al se up e e o [25]. Soil was sampled o molecula analyses be o e he
s a o he d ough pe iod (June 09 h), a he end o he expe imen al d ough pe iod
(July 27 h), and 4 days a e ewe ing (Augus 11 h). Soil samples we e aken om
each plo om soil dep hs 0 o 40 cm a 10-cm in e als wi h a soil co e . Soil samples
we e quick- ozen in liquid ni ogen and s o ed a -80◦C un il u he p ocessing.
2.2 Ex ac ion o nucleic acids
Nucleic acids om all sampled imepoin s, plo s, and soil laye s we e ex ac ed using
a bead-bea ing p o ocol [13] ollowed by sepa a ion o DNA and RNA using he Qia-
gen RNA/DNA Mini Ki (QIAGEN GmbH, Hilden, Ge many) o he manu ac u e ’s
ins uc ions.
2.3 Ba coded amplicon py osequencing o na G, ni K, ni S, and
nosZ
na G,ni K,ni S, and nosZ we e ampli ied om pooled DNA ex ac s (i.e., om all
imepoin s, plo s, and soil laye s) using published p ime s agged wi h ba codes, pu-
i ied and py osequenced as p e iously desc ibed [26]. Quali y il e ing o sequences
included denoising (i.e., emo al o py osequencing and PCR-ampli ica ion e o s by
Py oNoise and SeqNoise algo i hms [31]) as desc ibed [28]. Denoised sequence da a se s
4.2 Manusc ip s in p epa a ion
263
we e g ouped in o species-le el ope a ional axonomic un is (OTUs) a h eshold simi-
la i ies o 67%, 83%, 82%, and 80% o na G,ni K,ni S, and nosZ, espec i ely. Fo
na G and nosZ only sequences ob ained om o wa d eads we e used, as mean sequence
lengh s o app oxima ely 400 bp yielded o li le o e lap o o wa d and e e se eads,
while o wa d and e e se eads o ni K and ni S we e combined [26]. OTU ep esen-
a i es we e aligned wi h sequences o cul u ed and uncul u ed e e ences (de e mined
by BLAST analysis [1]) using Clus alW and phylogene ic ees we e cons uc ed using
he neighbo joining algo i hm [34] based on pai wise sequence dis ances in MEGA 5.0
[22]. The s abili y o ee opologies was es ed using he boo s ap me hod wi h 10,000
eplica ions.
2.4 Quan i a i e PCR
Quan i a i e PCR (qPCR) o na G and nosZ was modi ied om [26]. Reac ions we e
se up in iplica e eac ions pe DNA ex ac . The o wa d p ime s we e luo esecen ly
labeled o allow he use o he p oduc s om qPCR in downs eam TRFLP analyses.
This app oach allows di ec coupling o quan i ica ion and analysis o communi y com-
posi ion o a speci ic gene ma ke . Inhibi ion o qPCR assays by con amina ing humic
subs ances was conduc ed as desc ibed in [26].
2.5 Te minal es ic ion agmen leng h polymo phism analysis
(TRFLP)
T iplica e qPCR eac ions o na G and nosZ we e pooled and gel pu i ied using he
Mon age Gel Ex ac ion Ki (Millipo e Co po a ion, Bed o d, MA, USA) p io o TR-
FLP analysis. The pu i ied PCR p oduc s we e diges ed wi h Mung Bean Nuclease
(New England Biolabs, F ank u am Main, Ge many) o emo e single s anded DNA
and educe he p obabili y o pseudo- e minal es ic ion agmen s [8]. The es ic ed
DNA was pu i ied using he Millipo e Mul isc een 96-well Fil a ion Sys em (Millipo e
Co po a ion, Bed o d, MA, USA). PCR p oduc s o na G we e diges ed wi h he e-
s ic ion enzyme C oI, nosZ PCR p oduc s we e diges ed wi h Fnu4HI. Polyac ylamide
gel elec opho esis was pe o med as desc ibed p e iously [27]. Te minal es ic ion ag-
men s (TRFs) we e assigned o na G and nosZ sequences ia in silico TRF analysis
wi h sequences ob ained om he en Schl¨oppne b unnen [27].
4 Manusc ip s
264
3 Resul s
3.1 Di e si y o deni i ica ion-associa ed genes in
Schl¨oppne b unnen en soil
Di e si y o Schl¨oppne b unnen en deni i ie s was assessed by ba coded amplicon py-
osequencing o deni i ica ion-associa ed genes. Amplicon py osequencing o na G o -
wa d eads yielded 1,325 quali y il e ed (i.e., denoised) sequences. Sequences we e
g ouped in o 6 species-le el OTUs, 3 OTUs had ela i e abundances g ea e han 1%.
O hose 3 OTUs, OTU 1 had a ela i e abundance o 71% and hus domina ed de ec ed
na G in Schl¨oppne b unnen en soil (Figu e 1). Rep esen a i e sequences o OTU 1
a ilia ed wi h he Ac inobac e ia and we e dis an ly ela ed o na G o Ac inosynnema
mi um and S ep omyces coelicolo (Figu e 1). OTU 2 accoun ed o 22% o na G, a -
ilia ed wi h he Gammap o eobac e ia, and was ela ed o na G o Pseudomonas spp.
(Figu e 1). OTU 3 accoun ed o 6% o na G and was dis an ly ela ed o he Deinococcal
gene a The mus,Oceani he mus, and Ma ini he mus (Figu e 1).
2,401 quali y il e ed sequences we e ob ained o ni K. Sequences g ouped in o 13
species-le el OTUs, and 7 OTUs had a ela i e sequence abundance g ea e han 1%
(Figu e 2). Those OTUs occu ed wi h ela i e abundances o 55%, 21%, 11%, 7%,
2%, 2%, and 1% (OTUs 1, 2, 3, 4, 5, 6, and 7, espec i ely; Figu e 2). Sequences
a ilia ed wi h P o eobac e ial ni K (Figu e 2). OTU ep esen a i es o OTUs 1 and 6
we e dis an ly ela ed o ni K o En e ococcus sp., OTUs 3 and 5 we e ela ed o ni K
o B ady hizobium sp., OTU 2 was ela ed o Rhizobial ni K, while OTUs 4 and 7 did
we e no clea ly ela ed o published ni K (Figu e 2).
334 quali y il e ed sequences we e ob ained o ni S which g ouped in o 16 species-
le el OTUs. 6 OTUs had ela i e sequence abundances g ea e han 1%. OTUs 1 and
2 domina ed ni S in Schl¨oppne b unnen en soil wi h ela i e abundances o 52% and
33%, espec i ely, while OTUs 3, 4, 5, and 6 we e de ec ed wi h ela i e abundances
o 4%, 3%, 2%, and 2%, espec i ely (Figu e 3). OTU ep esen a i es a ilia ed wi h
Alpha-,Be a-, and Gammap o eobac e ia. Rep esen a i e sequences o OTUs 1 and 2
we e ela ed o ni K o B ady hizobium sp., while ep esen a i e sequences o OTUs 3,
4, 5, and 6 we e ela ed o Pseudomonas spp., Rals onia sp., Thiobacillus deni i icans,
and T. deni i icans, espec i ely (Figu e 3).
384 quali y il e ed o wa d ead sequences we e ob ained o nosZ which g ouped
in o 7 species-le el OTUs, o which 3 had ela i e sequence abundances g ea e han
1%. OTU 1 domina ed nosZ in Schl¨oppne b unnen en soil wi h ela i e abundances
4.2 Manusc ip s in p epa a ion
265
Figu e 1: Phylogene ic ee o na G OTU ep esen a i es. Sequences we e e ie ed
om pooled DNA ex ac s. The ee is based on in silico anla ed amino acid
sequences. OTUs we e calcula ed a a species-le el h eshold simila i y o 67%
om a o al o 1,325 o wa d ead sequences. Only OTUs ha con ain ≥1%
o sequences a e displayed, ela i e abundances o OTUs a e gi en in pa en heses.
Codes p eceeding sequence names ep esen sequence accession numbe s o e e ence
sequences in public da abases. G ey boxes indica e b anches we e he majo i y o
he sequences belong o a gi en phylogene ic g oup (indica ed on he igh ). The
pe cen age o eplica e ees ha p oduced he same clus e ing in he boo s ap
analysis (10,000 eplica ions) a e shown nex o he b anches, boo s ap suppo
alues below 50% a e omi ed. The ee was oo ed using na G o Haloa cula
ma ismo ui ATCC 43049 as he ou g oup (NC 006396).
o 92%, while OTUs 2, and 3 we e de ec ed wi h ela i e abundances o 6%, and 1%,
espec i ely (Figu e 4). OTU ep esen a i es a ilia ed wi h Alphap o eobac e ial nosZ.
Rep esen a i e sequences o OTUs 1, 2, and 3 we e ela ed o nosZ o Ach omobac e
sp., Meso hizobium sp., and Azospi illum sp., espec i ely (Figu e 4).
4 Manusc ip s
266
Figu e 2: Phylogene ic ee o ni K OTU ep esen a i es. Sequences we e e ie ed
om pooled DNA ex ac s. The ee is based on in silico anla ed amino acid
sequences. OTUs we e calcula ed a a species-le el h eshold simila i y o 83%
om a o al o 2,401 sequences. Only OTUs ha con ain ≥1% o sequences a e
displayed, ela i e abundances o OTUs a e gi en in pa en heses. Codes p eceeding
sequence names ep esen sequence accession numbe s o e e ence sequences in
public da abases. G ey boxes indica e b anches we e he majo i y o he sequences
belong o a gi en phylogene ic g oup (indica ed on he igh ). The pe cen age o
eplica e ees ha p oduced he same clus e ing in he boo s ap analysis (10,000
eplica ions) a e shown nex o he b anches, boo s ap suppo alues below 50%
a e omi ed. The ee was oo ed using ni K o Alcaligenes sp.CJANPY1 as he
ou g oup (EF202175).
4.2 Manusc ip s in p epa a ion
267
4 Discussion
4.1 De ec ed di e si y o ni a e educe s and deni i ie s in acidic
Schl¨oppne b unnen en soil
Up o 16 species-le el OTUs o deni i ica ion-associa ed genes we e de ec ed in he
acidic en Schl¨oppne b unnen (Figu e 1, Figu e 2, Figu e 3, Figu e 4), indica ing a
high species-le el di e si y o en deni i ie s. OTUs o de ec ed na G a ilia ed wi h
Ac inobac e ia,Gammap o eobac e ia, and Deinococci (Figu e 1). Ea lie cloning-based
s udies o na G om Schl¨oppne b unnen en soil also indica e he p esence o he
men ioned g oups, howe e also Alpha- and Be ap o eobac e ial na G a e de ec ed by
cloning-based app oaches, indica ing ha he de ec ed di e si y a ies be ween he 2
app oaches, likely because o he sho e agmen leng h ob ained om amplicon py-
osequencing as well as by a iabili y in DNA ex ac ion. The noise- emo al algo i hms
employed in he p esen s udy emo e e o s caused by py osequencing (Py oNoise) as
well as by PCR-ampli ica ion (SeqNoise) [31]. As PCR-ampli ica ion e o s a ec con-
en ional sequencing and py osequencing in a simial magni ude, i is mo eo e easible
ha ea lie cloning based sequencing e o s o e es ima e he gene ic di e si y o na G
because o hose PCR-ampli ica ion e o s. Sequences ela ed o Ac inobac e ial na G
domina ed amplicon lib a ies (Figu e 1). Ac inobac e ial p edominance is obse ed in
many o he we land soils including pea land soils [26, 28, 29].
Sequences o ni K and ni S a ilia ed wi h sequences o Alpha-,Be a- and Gammap o-
eobac e ial ni i e educ ases (Figu e 2,Figu e 3). ni K was domina ed by sequences
dis an ly ela ed o En e ococcus sp., indica ing ha deni i ie s ha bo ing new Ni K-
ype ni i e educ ases occu in Schl¨oppne b unnen en soil. ni S was domina ed by
sequences ela ed o B ady hizobium sp., indica ing he p esence o his common soil
bac e ium in Schl¨oppne b unnen en soil. De ec ed nosZ a ilia ed wi h Alphap o eobac-
e ial nosZ, con i ming he esul s o ea lie cloning based sequencing e o s [27].
4.2 Rela i e abundance o deni i ie s in he acidic en
The ela i e na G gene copy numbe anged be ween 6 and 13% (Figu e 5 A). The
de ec ed na G copy numbe s a e in he same ange as na G copy numbe s in pe -
ma os a ec ed pea pea soil in Russia, ag icul u al soils o he Bu gundy egion in
F ance, eshwa e habi a s such as i e s and loodplains as well as glacie o elands
[2, 3, 19, 26] In some ag icul u al soils lowe na G copy numbe s o abou 1% o e en
4 Manusc ip s
274
lowe a e de ec ed [3, 18]. This indica es a high abundance o ni a e educe s in he en
Schl¨oppne b unnen. Indeed, ni a e-dependen MPNs show simila numbe s o ni a e
educe s in he acidic en [27]. On he o he hand, ela i e nosZ copy numbe s anged
om 0.05% o 3% (Figu e 5 C), indica ing ha abou 1% o 10% o ni a e educe s in
Schl¨oppne b unnen en soil possess he gene ical po en ial o comple e deni i ica ion.
Indeed, cul u e-dependen s udies also e eal a much highe p opo ion o ni a e e-
duce s han o deni i ie s in soils [5], and many cul u e-independen s udies also show
ha ni a e educe s a e abou 100- old han deni i ie s [2, 26]. Deni i ie numbe s
om MPN coun s om he acidic en app oxima ed 0.02% o he o al cell coun [27].
The de ec abili y o deni i ie s by quan i a i e PCR is he e o e sligh ly highe han he
de ec abili y by MPN coun s. In glacie o eland soils ela i e nosZ gene copy numbe s
anged om abou 0.02% o abou 1% [19], he deni i ie abundance in he acidic en
is he e o e in he same ange as in o he soils o e en highe .
4.3 E ec o d ying and ewe ing on deni i ie communi ies in he
acidic en
Clima e change models p edic a highe equency o ex eme wea he e en s, includ-
ing p olonged pe iods o d ough [38]. Expe imen al d ough inc eases luxes o N2O
om he en, especially when a signi ican d awdown o he wa e able is ob ained [10].
Subsequen we ing o he soil inc eases N2O emission om p e iously d ained en soil
[10]. The inc ease in N2O p oduc ion a e ewe ing may be a ibu ed o egene a ion
o elec on accep o s such as ni a e due o highe soil ae a ion du ing he p eceeding
d ough [21]. N2O p oduc ion is mos s ongly a ec ed in he op soil o he en [10].
Long- e m wa e able d awdown does no lead o a s ong inc ease in he yea ly N2O
emissions in nu ien -poo pea lands, while i inc eases N2O emissions om nu ien - ich
pea lands [23], bu sho - e m d ainage o pea land mesocosms leads o inc eased N2O
emission om he soil [33]. Sho - e m d ainage mos ly e ec s uppe soil laye s o en
soil and leads o a mo e simila mic obial communi y composi ion in di e en soil laye s
[16]. Wa e able d awdown in we lands does no a ec he communi y composi ion o
deni i ie s, e en hough p onounced impac s on he deni i ica ion p ocess a e obse ed
[20, 35]. Ac inobac e ial communi y composi ion in a meso ophic pine en is ela i ely
d ough - esis an , while d ough inc eases he o e all biomass and C- u no e [17]. In-
deed, he communi y composi ion o na G did no change du ing he d ough ea men
(Figu e 6 A), as he na G communi y in he en Schl¨oppne b unnen is domina ed by
4.2 Manusc ip s in p epa a ion
275
Ac inobac e ial na G [27].
The esponse o deni i ie abundance o d ough di e s be ween di e en we land
ypes, anging om a dec ase in abundance in bogs and ens o no changes in ipa -
ian we lands and ma shes [20, 35]. Indeed, he obse ede inc ease in nosZ abundance
was smalle han he inc ease in na G abundance, sugges ing ha ni a e educe s a e
a ou ed mo e s ongly by d ough condi ions han deni i ie s. Deni i ie s a e acul-
a i e ae obes ha gene ally p e e oxygen as a e minal elec on accep o [41]. Thus,
he g ow h o deni i ie s is likely a ou ed when lowe ing o he wa e able allows he
di usion o oxygen in o he soil. Howe e , deni i ica ion can also occu in he p esence
o oxygen, and some o ganisms simul aneously use ni a e o oxygen as e minal elec on
accep o [37].
Wa ming expe imen s in a c ic soil show ha deni i ie communi y composi ion is
mo e s ongly in luenced by si e cha ac e is ics han by wa ming, e en hough wa ming
did no lead o signi ican changes in soil wa e con en [40]. Mo eo e , deni i ie
communi y composi ion in sal ma shes and ipa ian we lands is no signi ican ly a ec ed
by wa e able al e a ions [20, 35]. Long- e m wa e able d awdown in bo eal pea lands
does no signi ican ly a ec Ac inobac e ial communi y composi ion [17]. Indeed, mos
de ec ed na G om Schl¨oppne b unnen en soil a ilia ed wi h Ac inobac e ial na G
(Figu e 1), indica ing ha Ac inobac e ia in Schl¨oppne b unnen en migh be one majo
componen a ec ing he obse ed s abili y o he ni a e educe communi y composi ion
in Schl¨oppne b unnen en soil.
4.4 Conclusions and limi a ions
P olonged pe iods o d ough a e an icipa ed in u u e yea s due o clima e wa ming
[38]. Expe imen al d ough and subsequen inc ease in si u emissions o N2O om he
acidic en Schl¨oppne b unnen [10]. The abundance o ni a e educe s and deni i ie s
was inc eased a e he d ough pe iod and dec eased a e he ewe ing, indica ing
ha he mo e oxic condi ions a ou g ow h o deni i ie s. The composi ion o he
deni i ie communi y emained la gely unal e ed. Howe e , he di ec link be ween
in si u emissions and he molecula da a ob ained in his s udy is no possible, as N2O
emissions o he p ecise s op and imepoin a e un esol ed, and labo a o y incuba ions o
sampled soil a e likewise missing. Wi hin hese limi a ions i is s ill possible o conclude
ha he communi y composi ion o ni a e educe s and deni i ie s in he acidic en
Schl¨oppne b unnen is no se e ely a ec ed by expe imen al d ough .
4 Manusc ip s
276
5 Acknowledgmen s
Funding o his wo k was p o ided by he Deu sche Fo schungsgemeinscha (DFG
HO 4020/2-2), and he Uni e si y o Bay eu h. Wo k was also suppo ed by he DFG
Resea ch g oup FOR 562. We a e hank ul o Rol Daniel and And ea Th¨u me o
py osequencing, and S e en Kolb, Ma kus Nebel, Sebas ian Wild, Jus in Kuczynski
and Ch is ophe Quince o help wi h sequence analyses.
Re e ences
[1] Al schul, S. F., Gish, W., Mille , W., Mye s, E. W., and Lipman, D. J.
Basic local alignmen sea ch ool. Jou nal o Molecula Biology 215, 403–410 (1990).
[2] B u, D., Rame e, A., Saby, N. P. A., Dequie , S., Ranja d, L., Jo-
li e , C., A ouays, D., and Philippo , L. De e minan s o he dis ibu ion
o ni ogen-cycling mic obial communi ies a he landscape scale. ISME Jou nal 5,
532–542 (2011).
[3] B u, D., Sa , A., and Philippo , L. Rela i e abundances o P o eobac e-
ial memb ane-bound and pe iplasmic ni a e educ ases in selec ed en i onmen s.
Applied and En i onmen al Mic obiology 73, 5971–5974 (2007).
[4] Ch`
eneby, D., Halle , S., Mondon, M., Ma in-Lau en , F., Ge mon,
J., and Philippo , L. Gene ic cha ac e iza ion o he ni a e educing communi y
based on na G nucleo ide sequence analysis. Mic obiology Ecology 46, 113–121
(2003).
[5] Ch`
eneby, D., Pe ez, S., De oe, C., Halle , S., Cou on, Y., Bi-
zoua d, F., Iu e ig, G., Ge mon, J. C., and Philippo , L. Deni i ying
bac e ia in bulk and maize- hizosphe ic soil: di e si y and N2O- educing abili ies.
Canadian Jou nal o Mic obiology 50, 469–474 (2004).
[6] Con ad, R. Soil mic oo ganisms as con olle s o a mosphe ic ace gases (H2,
CO, CH4, OCS, N2O, and NO). Mic obiological Re iews 60, 609–640 (1996).
[7] Denman, K. L., B asseu , G., Chid haisong, A., Ciais, P., Cox, P. M.,
Dickinson, R. E., Hauglus aine, D., Heinze, C., Holland, E., Jacob, D.,
Lohmann, U., Ramachand an, S., da Sil a Dias, P. L., Wo sy, S. C., and
4.2 Manusc ip s in p epa a ion
277
Zhang, X. Couplings be ween changes in he clima e sys em and biogeochemis y.
In: Clima e Change 2007: The Physical Science Basis. Con ibu ion o Wo king
G oup I o he Fou h Assessmen Repo o he In e go e nmen al Panel on Clima e
Change, edi ed by Solomon, S., Qin, D., Manning, M., Chen, Z., Ma quis,
M., A e y , K. B., Tigno , M., and Mille , H. L., pp. 499–588. Camb idge
Uni e si y P ess (2007).
[8] Ege , M. and F ied ich, M. W. Fo ma ion o pseudo- e minal es ic ion
agmen s, a PCR- ela ed bias a ec ing e minal es ic ion agmen leng h poly-
mo phism analysis o mic obial communi y s uc u e. Applied and En i onmen al
Mic obiology 69, 2555–2562 (2003).
[9] Fo s e , P., Ramaswamy, V., A axo, P., Be n sen, T., Be s, R.,
Fahey, D., Haywood, J., Lean, J., Lowe, D., Myh e, G., Nganga, J.,
P inn, R., Raga, G., Schulz, M., and VanDo land, R. Changes in a -
mosphe ic cons i uen s and in adia i e o cing. In: Clima e Change 2007: The
Physical Science Basis. Con ibu ion o Wo king G oup I o he Fou h Assessmen
Repo o he In e go e nmen al Panel on Clima e Change, edi ed by Solomon, S.,
Qin, D., Manning, M., Chen, Z., Ma quis, M., A e y , K. B., Tigno ,
M., and Mille , H., pp. 129–234. Camb idge Uni e si y P ess (2007).
[10] Goldbe g, S. D., Kno , K. H., Blodau, C., Lischeid, G., and Gebaue ,
G. Impac o al e ing he wa e able heigh o an acidic en on N2O and NO luxes
and soil concen a ions. Global Change Biology 16, 220–233 (2010).
[11] Goldbe g, S. D., Kno , K. H., and Gebaue , G. N2O concen a ion and
iso ope signa u e along p o iles p o ide deepe insigh in o he a e o N2O in soils.
Iso opes in En i onmen al and Heal h S udies 44, 377–391 (2008).
[12] Go ham, E. No he n pea lands - ole in he ca bon-cycle and p obable esponses
o clima ic wa ming. Ecological Applica ions 1, 182–195 (1991).
[13] G i i hs, R. I., Whi eley, A. S., O’Donnell, A. G., and Bailey, M. J.
Rapid me hod o coex ac ion o DNA and RNA om na u al en i onmen s o
analysis o ibosomal DNA- and RNA-based mic obial communi y composi ion.
Applied and En i onmen al Mic obiology 66, 5488–5491 (2000).
[14] Hambe ge , A., Ho n, M. A., Dumon , M. G., Mu ell, J. C., and
4 Manusc ip s
278
D ake, H. L. Anae obic consume s o monosaccha ides in a mode a ely acidic
en. Applied and En i onmen al Mic obiology 74, 3112–3120 (2008).
[15] Hen y, S., Texie , S., Halle , S., B u, D., Damb e ille, C., Cheneby,
D., Bizoua d, F., Ge mon, J. C., and Philippo , L. Disen angling he
hizosphe e e ec on ni a e educe s and deni i ie s: insigh in o he ole o oo
exuda es. En i onmen al Mic obiology 10, 3082–3092 (2008).
[16] Jaa inen, K., F i ze, H., Laine, J., and Laiho, R. E ec s o sho - and long-
e m wa e -le el d awdown on he popula ions and ac i i y o ae obic decompose s
in a bo eal pea land. Global Change Biology 13, 491–510 (2007).
[17] Jaa inen, K., Laiho, R., Vuo enmaa, A., del Cas illo, U., Minkkinen,
K., Pennanen, T., Pen il¨
a, T., and F i ze, H. Responses o ae obic mic o-
bial communi ies and soil espi a ion o wa e -le el d awdown in a no he n bo eal
en. En i onmen al Mic obiology 10, 339–353 (2008).
[18] Kandele , E., B une, T., Enowashu, E., Do , N., Guggenbe ge , G.,
Lame sdo , N., and Philippo , L. Response o o al and ni a e-dissimila ing
bac e ia o educed N deposi ion in a sp uce o es soil p o ile. FEMS Mic obiology
Ecology 67, 444–454 (2009).
[19] Kandele , E., Deiglmay , K., Tsche ko, D., B u, D., and Philippo ,
L. Abundance o na G,ni S,ni K, and nosZ genes o deni i ying bac e ia du ing
p ima y successions o a glacie o eland. Applied and En i onmen al Mic obiology
72, 5957–5962 (2006).
[20] Kim, S. Y., Lee, S. H., F eeman, C., Fenne , N., and Kang, H. Compa -
a i e analysis o soil mic obial communi ies and hei esponses o he sho - e m
d ough in bog, en, and ipa ian we lands. Soil Biology & Biochemis y 40, 2874–
2880 (2008).
[21] Kno , K. H., Lischeid, G., and Blodau, C. Dynamics o edox p ocesses in
a mine o ophic en exposed o a wa e able manipula ion. Geode ma 153, 379–392
(2009).
[22] Kuma , S., Nei, M., Dudley, J., and Tamu a, K. MEGA: A biologis -
cen ic so wa e o e olu iona y analysis o DNA and p o ein sequences. B ie ings
In Bioin o ma ics 9, 299–306 (2008).
4.2 Manusc ip s in p epa a ion
279
[23] Ma ikainen, P. J., Nyk¨
anen, H., C ill, P., and Sil ola, J. E ec o a
lowe ed wa e - able on ni ous-oxide luxes om no he n pea lands. Na u e 366,
51–53 (1993).
[24] Mounie , E., Halle , S., Cheneby, D., Beniz i, E., G ue , Y., Nguyen,
C., Piu i, S., Robin, C., Slezack-Deschaumes, S., Ma in-Lau en , F.,
Ge mon, J. C., and Philippo , L. In luence o maize mucilage on he di e si y
and ac i i y o he deni i ying communi y. En i onmen al Mic obiology 6, 301–312
(2004).
[25] Muh , J., Hohle, J., O ieno, D. O., and Bo ken, W. Manipula i e lowe ing
o he wa e able du ing summe does no a ec CO2emissions and up ake in a
en in Ge many. Ecological Applica ions 21, 391–401 (2011).
[26] Palme , K., Biasi, C., and Ho n, M. A. Con as ing deni i ie communi ies
ela e o con as ing N2O emission pa e ns om acidic pea soils in a c ic und a.
ISME Jou nal 6, 1058–1077 (2012).
[27] Palme , K., D ake, H. L., and Ho n, M. A. Associa ion o no el and highly
di e se acid- ole an deni i ie s wi h N2O luxes o an acidic en. Applied and
En i onmen al Mic obiology 76, 1125–1134 (2010).
[28] Palme , K. and Ho n, M. A. Ac inobac e ial ni a e educe s and P o eobac-
e ial deni i ie s a e abundan in N2O-me abolizing palsa pea . Applied and En i-
onmen al Mic obiology 78, 5584–5596 (2012).
[29] Philippo , L., Piu i, S., Ma in-Lau en , F., Halle , S., and Ge mon,
J. C. Molecula analysis o he ni a e- educing communi y om unplan ed and
maize-plan ed soils. Applied and En i onmen al Mic obiology 68, 6121–6128 (2002).
[30] Pihla ie, M., Sy ¨
asalo, E., Simojoki, A., Esala, M., and Regina, K.
Con ibu ion o ni i ica ion and deni i ica ion o N2O p oduc ion in pea , clay
and loamy sand soils unde di e en soil mois u e condi ions. Nu ien Cycling in
Ag oecosys ems 70, 135–141 (2004).
[31] Quince, C., Lanzen, A., Da enpo , R. J., and Tu nbaugh, P. J. Re-
mo ing noise om py osequenced amplicons. BMC Bioin o ma ics 12, 38 (2011).
4 Manusc ip s
280
[32] Ra ishanka a, A. R., Daniel, J. S., and Po mann, R. W. Ni ous ox-
ide (N2O): The dominan ozone-deple ing subs ance emi ed in he 21s cen u y.
Science 326, 123–125 (2009).
[33] Regina, K., Sil ola, J., and Ma ikainen, P. J. Sho - e m e ec s o chang-
ing wa e able on N2O luxes om pea monoli hs om na u al and d ained bo eal
pea lands. Global Change Biology 5, 183–189 (1999).
[34] Sai ou, N. and Nei, M. The neighbo -joining me hod - A new me hod o econ-
s uc ing phylogene ic ees. Molecula Biology and E olu ion 4, 406–425 (1987).
[35] Song, K., Lee, S. H., Mi sch, W. J., and Kang, H. Di e en esponses o
deni i ica ion a es and deni i ying bac e ial communi ies o hyd ologic pulsing in
c ea ed we lands. Soil Biology & Biochemis y 42, 1721–1727 (2010).
[36] Takakai, F., Desya kin, A. R., Lopez, C. M. L., Fedo o , A. N., Desy-
a kin, R. V., and Ha ano, R. CH4and N2O emissions om a o es -alas
ecosys em in he pe ma os aiga o es egion, eas e n Sibe ia, Russia. Jou nal o
Geophysical Resea ch - Biogeosciences 113, G02002 (2008).
[37] Thomas, K. L., Lloyd, D., and Boddy, L. E ec s o oxygen, pH and ni a e
concen a ion on deni i ica ion by Pseudomonas species. FEMS Mic obiology Le -
e s 118, 181–186 (1994).
[38] T enbe h, K., Jones, P., Ambenje, P., Boja iu, R., Eas e ling, D.,
Klein Tank, A., Pa ke , D., Rahimzadeh, F., Renwick, J., Rus icucci,
M., Soden, B., and Zhai, P. Obse a ions: Su ace and a mosphe ic clima e
change. In: Clima e Change 2007: The Physical Science Basis. Con ibu ion o
Wo king G oup I o he Fou h Assessmen Repo o he In e go e nmen al Panel
on Clima e Change, edi ed by Solomon, S., Qin, D., Manning, M., Chen, Z.,
Ma quis, M., A e y , K. B., Tigno , M., and Mille , H., pp. 235–336.
Camb idge Uni e si y P ess (2007).
[39] Ven e ink, H. O., Da idsson, T. E., Kiehl, K., and Leona dson, L.
Impac o d ying and e-we ing on N, P and K dynamics in a we land soil. Plan
and Soil 243, 119–130 (2002).
[40] Walke , J. K. M., Egge , K. N., and Hen y, G. H. R. Long- e m expe -
imen al wa ming al e s ni ogen-cycling communi ies bu si e ac o s emain he
4.2 Manusc ip s in p epa a ion
281
p ima y d i e s o communi y s uc u e in high a c ic und a soils. ISME Jou nal
2, 982–995 (2008).
[41] Zum , W. G. Cell biology and molecula basis o deni i ica ion. Mic obiology
and Molecula Biology Re iews 61, 533–615 (1997).
4 Manusc ip s
282
4.3 P e ious pee - e iewed publica ions no included in he disse a ion
4.3 P e ious pee - e iewed publica ions no included in he
disse a ion
283
pa o he en deni i ie communi y is simila o p e iously
esol ed gene a. Communi ies o di e en soil laye s we e
phylogene ically simila , indica ing ha en deni i ie s we e
de i ed om he same pool o mic oo ganisms. TRFLP anal-
ysis o nosZ e ealed only mino di e ences be ween di e en
soil laye s. In con as , na G-associa ed di e ences we e mo e
p onounced, sugges ing ha he de ec ed dissimila o y ni a e
educe s (which ha e na G bu lack nosZ [60]) in he en a e
mo e dissimila be ween soil laye s han a e de ec ed deni i-
ie s. The la ges di e ence obse ed in he na G TRFLP p o-
iles was be ween he p o ile o 10- o 20-cm-dep h soil and
p o iles o he o he soil laye s, indica ing ha 10- o 20-cm-
dep h soil ha bo s a ni a e- educing popula ion ha is no
iden ical o he popula ions o he o he soil laye s.
In si u consequences o deni i ie ac i i y. Ni a e concen-
a ions in he Schlo¨ppne b unnen en a e gene ally low and
FIG. 5. Compa a i e TRFLP analyses o na G (A o C) and nosZ (D o F) ampli ied om di e en soil laye s o he acidic en. PCR p oduc s
we e diges ed wi h C oI (A), HaeIII (B), XhoI (C), B gI (D), NlaIV (E), and P uI and SacI (F). Mean alues o h ee eplica es a e shown.
De ec ed TRFs could be assigned o sequences om OTUs 1, 3, and 4 o na G and OTUs 1, 3, and 11 o nosZ. Soil laye s 1, 2, 3, and 4 e e
o soil dep hs o 0 o 10 cm, 10 o 20 cm, 20 o 30 cm, and 30 o 40 cm, espec i ely. Leng hs o de ec ed TRFs (in base pai s) a e gi en in boxes
nex o he diag ams.
VOL. 76, 2010 NEW FEN DENITRIFIERS 1131
by on Feb ua y 8, 2010 aem.asm.o gDownloaded om
4 Manusc ip s
290
o en below he de ec ion limi bu can be as high as 0.13 mM
in he uppe 20 cm o soil (42, 55). Unsupplemen ed en soil
p oduced mino amoun s o N
2
O, e lec ing he low in si u
ni a e concen a ions in he en (30, 42, 55). Supplemen al
ni a e caused a apid inc ease in he p oduc ion o N
2
O wi h-
ou appa en delay, indica ing ha (i) en deni i ie s a e
poised o espond apidly o ni a e and ha e a high po en ial
o deni i y, and (ii) in si u deni i ica ion is likely limi ed by
ni a e a ailabili y. Inc eased concen a ions o ni a e caused
an inc ease in he ela i e p opo ion o N
2
O in o al N gases,
a phenomenon ha has been obse ed wi h o he soils (3, 18).
The en Schlo¨ppne b unnen is a ne sou ce o N
2
O, and N
2
O
concen a ions o up o 100 ppm occu in he po e wa e (20).
The e is a la ge di e ence be ween ni a e inpu and de-
ec ed ni a e concen a ions in he en soil as nea by oxic soils
ecei e he same amoun o ni a e inpu bu ha e nea ly 100-
old highe ni a e concen a ions (42). This di e ence is sug-
ges i e o a high u no e o ni a e ha is due in pa o
deni i ica ion. The K
m
alues (⬍20 M) (Table 1) o ni a e
a e well below he maximum ni a e concen a ions ound in
si u, indica ing ha en deni i ie s ha e a high a ini y o
ni a e and can cope wi h low ni a e concen a ions. Highe
ni a e concen a ions inc eased he ela i e amoun o N
2
O
o med by 0- o 20-cm-dep h soils (see Fig. S1 in he supple-
men al ma e ial). Ni a e concen a ions abo e 40 M a e
a ely encoun e ed in si u (30, 42, 55). Thus, he comple e
educ ion o ni a e o N
2
migh occu unde mos in si u
condi ions, and he ela i e emissions o N
2
O e sus N
2
migh
inc ease when ni a e concen a ions a e pe iodically ele a ed.
The apid inc ease o N
2
O p oduc ion in esponse o ni a e
and he capaci y o en soil o consume supplemen al N
2
O
wi hou appa en delay (Fig. 1) sugges ha deni i ie s a e
ac i e in si u.
High deni i ica ion po en ials in uppe soil laye s (0 o 20
cm) a e coinciden wi h highe concen a ions o ni a e in
hose laye s (42, 55). Rain e en s likely con ibu e o he la ge
amoun s o ni a e in su ace soils. The pe cen age o N
2
Oin
o al N gases o med by en soils inc eased wi h inc easing soil
dep h, a end ha migh be due o he limi a ion o eadily
a ailable o ganic ca bon in deepe laye s in he Schlo¨ppne -
b unnen en (72). Elec on dono limi a ion can enhance he
pe cen age o N
2
O in o al N gases p oduced by pu e cul u es
o deni i ie s (53).
Ni i ica ion e sus deni i ica ion as possible sou ces o
N
2
O. Iso ope signa u es o he N
2
O indica e ha deni i ica-
ion is he main sou ce o he N
2
O emi ed om he Schlo¨p-
pne b unnen en (20). Al hough deni i ica ion ends o be he
dominan sou ce o N
2
O unde wa e -sa u a ed condi ions
(44), ni i ica ion likely occu s in he acidic en when oxic
condi ions a e augmen ed du ing d ye pe iods. Ni i ica ion
du ing d ye and mo e oxic condi ions would heo e ically p o-
ide addi ional ni a e o deni i ica ion in anoxic mic ozones
o subsequen o a ain e en . In his ega d, N
2
O emissions
om he en inc ease a e ewe ing e en s ollowing pe iods
o d ough (19).
Deni i ica ion as an N
2
O sink. We lands can consume N
2
O
(4). The capaci y o we lands o consume N
2
O is in luenced by
en i onmen al ac o s such as pH and empe a u e, as well as
he composi ion o he mic obial communi y (6). The capaci y
o Schlo¨ppne b unnen en soil o consume N
2
O o suba mo-
sphe ic le els unde anoxic condi ions and he pe iodic occu -
ence o N
2
O a suba mosphe ic concen a ions in en po e
wa e (20) a e indi ec e idence ha N
2
O consump ion occu s
in si u. Iso ope signa u es o N
2
O om he en indica e ha he
upwa d di usion o he N
2
O p oduced in lowe soil laye s is
subjec o educ ion o N
2
in he uppe soil laye s (20). Indeed,
N
2
O consump ion a es we e highe in uppe soil laye s han in
lowe soil laye s. These collec i e indings sugges ha he
Schlo¨ppne b unnen en unc ions as no only an N
2
O sou ce
bu also an N
2
O sink.
Ecophysiology o en deni i ie s. K
m
alues o deni i ica-
ion anged om 6 o 19 M ni a e, indica ing ha en deni-
i ie s had a high a ini y o ni a e. K
m
alues we e in he
same ange o lowe han hose o o he soil ypes (32, 37, 62)
and in he ange o hose o pu e cul u es ha display a high
a ini y o ni a e (e.g., species o Alcaligenes,Pseudomonas,
and Fla obac e ium) (2, 41, 64).
Deni i ica ion a es o di e en en soil laye s we e op imal
a 34 o 46°C, op imal empe a u es ha app oxima e hose o
many model soil deni i ie s (e.g., Pseudomonas deni i icans,
which deni i ies op imally a 38°C) (69). In con as , he high-
es deni i ie ac i i y and highes numbe s o cul u ed deni i-
ie s o di e en soils occu be ween 25 and 30°C (50, 51),
indica ing ha deni i ie s in he Schlo¨ppne b unnen en ha e
a empe a u e op imum ha is sligh ly highe han he op ima
o deni i ie s om o he soils. Enhanced deni i ica ion
capaci ies a empe a u es ha exceed mos in si u condi ions
a e a common phenomenon, and highe a es o deni i ica ion
in soils in summe can be a ibu ed o inc eased soil empe -
a u es (10, 24, 33).
The capaci y o en soil o consume N
2
O unde anoxic con-
di ions was blocked by ace ylene (Fig. 1B) and is he e o e
assumed o be due o he educ ion o N
2
O oN
2
by N
2
O
educ ase (74). The consump ion o N
2
O by di e en soil lay-
e s was highes be ween 6 and 30°C, empe a u es lowe han
hose o ni a e-dependen deni i ica ion. These con as ing
empe a u e op ima sugges ha di e en deni i ie subpopu-
la ions in he acidic en ha e di e en empe a u e and elec-
on accep o (i.e., ni a e o N
2
O) p e e ences. The e minal
eac ion o he deni i ica ion pa hway appea s o be mo e
adap ed o in si u empe a u es han he p eceding eac ions.
The deg ee o which N
2
O educ ase o soil deni i ie s is in-
hibi ed by lowe empe a u es a ies, wi h e ec s anging om
no inhibi ion o almos comple e inhibi ion (12).
Deni i ica ion a es we e highes a in si u pH, indica ing
ha Schlo¨ppne b unnen en deni i ie s a e well adap ed o
he mode a ely acidic en en i onmen . Deni i ica ion ac i i-
ies o acidic ag icul u al soils can be highes a in si u pH e en
hough deni i ica ion capaci ies migh be highe in mo e pH-
neu al soils (40), sugges ing ha soils o di e en pH alues
ha bo dis inc deni i ie communi ies adap ed o in si u pH.
Many pu e cul u es o deni i ie s (e.g., Pseudomonas sp.) ha e
nea ly neu al o sligh ly alkaline pH op ima (63). Deni i ica-
ion also occu ed a e y low pH by all en soil laye s, whe eas
only he uppe 10-cm soil laye was capable o deni i ica ion
unde sligh ly alkaline condi ions. The e o e, alkaline condi-
ions appea o be mo e limi ing o en deni i ie s han acidic
condi ions, which is consis en wi h he in si u condi ions en
deni i ie s a e subjec ed o. Acidic pH inc eases he pe cen -
age o N
2
O in o al N gases (57, 70). Deni i ica ion by Pa a-
1132 PALMER ET AL. APPL.ENVIRON.MICROBIOL.
by on Feb ua y 8, 2010 aem.asm.o gDownloaded om
4.3 P e ious pee - e iewed publica ions no included in he disse a ion
291
coccus deni i icans yields ni i e and ni ous oxide as ansien
in e media es a pH 5.5, whe eas he amoun s o hese in e -
media es a e low o no de ec able a pH 8.5 (64). Up o 5 M
N
2
O occu s in he en po e wa e (20), indica ing ha hose
in e media es occu in si u. The ela i e pe cen age o N
2
Oin
o al N gases o med by Schlo¨ppne b unnen en soil was high-
es a pHs o 2 o 3 bu was simila a in si u pH o alues
ob ained a pH 7, indica ing ha he N
2
O educ ases o en
deni i ie s a e no inhibi ed by he mode a ely acidic in si u
condi ions.
Conclusions. Schlo¨ppne b unnen en soil p oduces o ma e,
e hanol, and ace a e unde anoxic condi ions ia e men a-
ion (22, 72). Such subs a es a e u ilized by pu e cul u es o
deni i ie s such as Pseudomonas deni i icans,Pseudomonas
s u ze i, and Pa acoccus deni i icans (38, 61); o ma e and
ace a e a e de ec able in he en po e wa e (22, 30, 72); and
he augmen a ion o deni i ica ion in en soil mic ocosms by
hese subs a es sugges s ha en deni i ie s migh o m o-
phic links o en e men e s. Deni i ica ion op ima by en
deni i ie s a mode a ely acidic pH a e dissimila o hose o
model deni i ie s (such as hose lis ed abo e). Tha he em-
pe a u e op ima o en deni i ie s a e abo e empe a u es
usually occu ing in si u indica es ha he en deni i ie s a e
p one o espond o global wa ming wi h inc eased ac i i y.
Thus, he sou ce and sink unc ions o he en o N
2
O migh
be enhanced. These physiological indings and he no el phy-
logeny o deni i ie communi y membe s indica e ha he en
con ains he e o o e unknown deni i ie s ha a e adap ed o in
si u condi ions and a e in eg a ed in he in e media y ecosys-
em me abolism (i.e., p ocesses ha link inpu and ou pu ) o
he en (13).
ACKNOWLEDGMENTS
Suppo o his s udy was p o ided by he Deu sche Fo schungsge-
meinscha (DFG DR 310/3-3 and DFG HO 4020/2-2) and he Uni-
e si y o Bay eu h.
REFERENCES
1. Balch, W. E., G. E. Fox, L. J. Mag um, C. R. Woese, and R. S. Wol e. 1979.
Me hanogens: ee alua ion o a unique biological g oup. Mic obiol. Re .
43:260–296.
2. Be lach, M. R., and J. M. Tiedje. 1981. Kine ic explana ion o accumula ion
o ni i e, ni ic oxide, and ni ous oxide du ing bac e ial deni i ica ion.
Appl. En i on. Mic obiol. 42:1074–1084.
3. Blackme , A. M., and J. M. B emne . 1978. Inhibi o y e ec o ni a e on
educ ion o N
2
O oN
2
by soil mic oo ganisms. Soil Biol. Biochem. 10:187–
191.
4. Blackme , A. M., and J. M. B emne . 1976. Po en ial o soil as a sink o
a mosphe ic ni ous oxide. Geophys. Res. Le . 3:739–742.
5. B emne , J. M. 1997. Sou ces o ni ous oxide in soils. Nu . Cycling Ag o-
ecosys . 49:7–16.
6. Ca igelli, M. A., and G. P. Robe son. 2000. The unc ional signi icance o
deni i ie communi y composi ion in a e es ial ecosys em. Ecology 81:
1402–1414.
7. Con ad, R. 1996. Soil mic oo ganisms as con olle s o a mosphe ic ace
gases (H
2
, CO, CH
4
, OCS, N
2
O, and NO). Mic obiol. Re . 60:609–640.
8. Daniel, S. L., T. Hsu, S. I. Dean, and H. L. D ake. 1990. Cha ac e iza ion o
he H
2
- and CO-dependen chemoli ho ophic po en ials o he ace ogens
Clos idium he moace icum and Ace ogenium ki ui. J. Bac e iol. 172:4464–
4471.
9. Da idson, E. A. 1992. Sou ces o ni ic oxide and ni ous oxide ollowing
we ing o d y soil. Soil Sci. Soc. Am. J. 56:95–102.
10. DeKlein, C. A. M., and R. S. P. VanLog es ijn. 1996. Deni i ica ion in
g assland soils in The Ne he lands in ela ion o i iga ion, N-applica ion
a e, soil wa e con en and soil empe a u e. Soil Biol. Biochem. 28:231–
237.
11. DeMan, J. C. 1975. The p obabili y o mos p obable numbe s. Eu . J. Appl.
Mic obiol. 1:67–78.
12. Do sch, P., and L. R. Bakken. 2004. Low- empe a u e esponse o deni i-
ica ion: compa ison o soils. Eu asian Soil Sci. 37:S102–S106.
13. D ake, H. L., M. A. Ho n, and P. K. Wu¨s . 2009. In e media y ecosys em
me abolism as a main d i e o me hanogenesis in acidic we land soil. En-
i on. Mic obiol. Rep. 1:307–318.
14. Ege , M., and M. W. F ied ich. 2003. Fo ma ion o pseudo- e minal es ic-
ion agmen s, a PCR- ela ed bias a ec ing e minal es ic ion agmen
leng h polymo phism analysis o mic obial communi y s uc u e. Appl. En-
i on. Mic obiol. 69:2555–2562.
15. Enwall, K., L. Philippo , and S. Hallin. 2005. Ac i i y and composi ion o he
deni i ying bac e ial communi y espond di e en ly o long- e m e iliza-
ion. Appl. En i on. Mic obiol. 71:8335–8343.
16. Foken, T. 2003. Lu hygienische-bioklima ische Kennzeichnung des obe en
Ege ales Bay eu he Fo um O
¨kologie 100:1–69.
17. Fo s e , P., V. Ramaswamy, P. A axo, T. Be n sen, R. Be s, D. W. Fahey, J.
Haywood, J. Lean, D. C. Lowe, G. Myh e, J. Nganga, R. P inn, G. Raga, M.
Schulz, and R. VanDo land. 2007. Changes in a mosphe ic cons i uen s and
in adia i e o cing, p 129–234. In S. Solomon, D. Qin, M. Manning, Z. Chen,
M. Ma quis, K. B. A e y , M. Tigno , and H. L. Mille (ed.), Clima e change
2007: he physical science basis. Con ibu ion o wo king g oup I o he
ou h assessmen epo o he in e go e nmen al panel on clima e change.
Camb idge Uni e si y P ess, Camb idge, Uni ed Kingdom.
18. Gaskell, J. F., A. M. Blackme , and J. M. B emne . 1981. Compa ison o
e ec s o ni a e, ni i e, and ni ic oxide on educ ion o ni ous oxide o
dini ogen by soil-mic oo ganisms. Soil Sci. Soc. Am. J. 45:1124–1127.
19. Goldbe g, S. D., K. H. Kno , C. Blodau, G. Lischeid, and G. Gebaue . 2010.
Impac o al e ing he wa e able heigh o an acidic en on N
2
O and NO
luxes and soil concen a ions. Glob. Change Biol. 16:220–233.
20. Goldbe g, S. D., K. H. Kno , and G. Gebaue . 2008. N
2
O concen a ion and
iso ope signa u e along p o iles p o ide deepe insigh in o he a e o N
2
O
in soils. Iso opes En i on. Heal h S ud. 44:377–391.
21. G i i hs, R. I., A. S. Whi eley, A. G. O’Donnell, and M. J. Bailey. 2000. Rapid
me hod o coex ac ion o DNA and RNA om na u al en i onmen s o
analysis o ibosomal DNA- and RNA-based mic obial communi y compo-
si ion. Appl. En i on. Mic obiol. 66:5488–5491.
22. Hambe ge , A., M. A. Ho n, M. G. Dumon , J. C. Mu ell, and H. L. D ake.
2008. Anae obic consume s o monosaccha ides in a mode a ely acidic en.
Appl. En i on. Mic obiol. 74:3112–3120.
23. Ha igan, W. F., and M. E. Mc Cance. 1966. Labo a o y me hods in mic o-
biology. Academic P ess, London, Uni ed Kingdom.
24. Hol an-Ha wig, L., P. Do sch, and L. R. Bakken. 2002. Low empe a u e
con ol o soil deni i ying communi ies: Kine ics o N
2
O p oduc ion and
educ ion. Soil Biol. Biochem. 34:1797–1806.
25. Ho n, M. A., H. L. D ake, and A. Sch amm. 2006. Ni ous oxide educ ase
genes (nosZ) o deni i ying mic obial popula ions in soil and he ea hwo m
gu a e phylogene ically simila . Appl. En i on. Mic obiol. 72:1019–1026.
26. Ka s en, G., and H. L. D ake. 1997. Deni i ying bac e ia in he ea hwo m
gas oin es inal ac and in i o emission o ni ous oxide (N
2
O) by ea h-
wo ms and in i o emission o dini ogen by ea hwo ms ia deni i ying
bac e ia in he gu . Appl. En i on. Mic obiol. 63:1878–1882.
27. Kesik, M., S. Blagoda sky, H. Papen, and K. Bu e bach-Bahl. 2006. E ec
o pH, empe a u e and subs a e on N
2
O, NO and CO
2
p oduc ion by
Alcaligenes aecalis p. J. Appl. Mic obiol. 101:655–667.
28. Kuhne , C. H., H. L. D ake, E. Alm, and L. Raskin. 1996. Me hane p oduc-
ion and oxida ion by soils om acidic o es we lands o eas -cen al Ge -
many, p. 304. Abs . 96 h Gen. Mee . Am. Soc. Mic obiol. Ame ican Socie y
o Mic obiology, Washing on, DC.
29. Kuma , S., M. Nei, J. Dudley, and K. Tamu a. 2008. MEGA: a biologis -
cen ic so wa e o e olu iona y analysis o DNA and p o ein sequences.
B ie . Bioin o m. 9:299–306.
30. Ku¨sel, K., and C. Alewell. 2004. Ripa ian zones in a o es ed ca chmen : ho
spo s o mic obial educ i e p ocesses, p. 377–395. In E. Ma zne (ed.),
Biogeochemis y o o es ed ca chmen s in a changing en i onmen : a Ge -
man case s udy. Sp inge Ve lag, Be lin, Ge many.
31. Ku¨sel, K., and H. L. D ake. 1995. E ec s o en i onmen al pa ame e s on
he o ma ion and u no e o ace a e by o es soils. Appl. En i on. Mic o-
biol. 61:3667–3675.
32. La e man, A. M., P. VanCappellen, D. VanRo e dam-Los, C. Pallud, and J.
Abell. 2006. Po en ial a es and pa hways o mic obial ni a e educ ion in
coas al sedimen s. FEMS Mic obiol. Ecol. 58:179–192.
33. Malhi, S. S., W. B. Mcgill, and M. Nybo g. 1990. Ni a e losses in soils: e ec
o empe a u e, mois u e and subs a e concen a ion. Soil Biol. Biochem.
22:733–737.
34. Ma hies, C., A. F eibe ge , and H. L. D ake. 1993. Fuma a e dissimila ion
and di e en ial educ an low by Clos idium o micoace icum and Clos id-
ium ace icum. A ch. Mic obiol. 160:273–278.
35. Messing, J. 1983. New M13 ec o s o cloning. Me hods Enzymol. 101:
20–78.
36. Mille , M. N., B. J. Zeba h, C. E. Dandie, D. L. Bu on, C. Goye , and J. T.
T e o s. 2008. C op esidue in luence on deni i ica ion, N
2
O emissions and
deni i ie communi y abundance in soil. Soil Biol. Biochem. 40:2553–2562.
37. Mu ay, R. E., L. L. Pa sons, and M. S. Smi h. 1989. Kine ics o ni a e
VOL. 76, 2010 NEW FEN DENITRIFIERS 1133
by on Feb ua y 8, 2010 aem.asm.o gDownloaded om
4 Manusc ip s
292
u iliza ion by mixed popula ions o deni i ying bac e ia. Appl. En i on.
Mic obiol. 55:717–721.
38. Nishimu a, Y., T. Kamiha a, and S. Fukui. 1979. Ni i e educ ion wi h
o ma e in Pseudomonas deni i icans ATCC 13867. Biochem. Biophys. Res.
Commun. 87:140–145.
39. Palme , K., H. L. D ake, and M. A. Ho n. 2009. Genome de i ed c i e ia o
assigning en i onmen al na G and nosZ sequences o ope a ional axonomic
uni s o ni a e educe s. Appl. En i on. Mic obiol. 75:5170–5174.
40. Pa kin, T. B., A. J. Sexs one, and J. M. Tiedje. 1985. Adap a ion o deni i-
ying popula ions o low soil pH. Appl. En i on. Mic obiol. 49:1053–1056.
41. Pa sonage, D., A. J. G een ield, and S. J. Fe guson. 1985. The high a ini y
o Pa acoccus deni i icans cells o ni a e as an elec on accep o : analysis
o possible mechanisms o ni a e and ni i e mo emen ac oss he plasma
memb ane and he basis o inhibi ion by added ni i e o oxidase ac i i y in
pe meabilized cells. Biochim. Biophys. Ac a 807:81–95.
42. Paul, S., K. Ku¨sel, and C. Alewell. 2006. Reduc ion p ocesses in o es
we lands: acking down he e ogenei y o sou ce/sink unc ions wi h a com-
bina ion o me hods. Soil Biol. Biochem. 38:1028–1039.
43. Philippo , L., S. Piu i, F. Ma in-Lau en , S. Halle , and J. C. Ge mon.
2002. Molecula analysis o he ni a e- educing communi y om unplan ed
and maize-plan ed soils. Appl. En i on. Mic obiol. 68:6121–6128.
44. Pihla ie, M., E. Sy a¨salo, A. Simojoki, M. Esala, and K. Regina. 2004.
Con ibu ion o ni i ica ion and deni i ica ion o N
2
O p oduc ion in pea ,
clay and loamy sand soils unde di e en soil mois u e condi ions. Nu .
Cycling Ag oecosys . 70:135–141.
45. Pinay, G., B. Gumie o, E. Tabacchi, O. Gimenez, A. M. Tabacchi-Plan y,
M. M. He ing, T. P. Bu , V. A. Black, C. Nilsson, V. Io dache, F. Bu eau,
L. Vough , G. E. Pe s, and H. Decamps. 2007. Pa e ns o deni i ica ion
a es in Eu opean allu ial soils unde a ious hyd ological egimes. F eshw.
Biol. 52:252–266.
46. Ra kowsky, D. A., R. K. Low y, T. A. McMeekin, A. N. S okes, and R. E.
Chandle . 1983. Model o bac e ial cul u e g ow h a e h oughou he
en i e biokine ic empe a u e ange. J. Bac e iol. 154:1222–1226.
47. Reiche, M., G. To bu g, and K. Ku¨sel. 2008. Compe i ion o Fe(III) educ-
ion and me hanogenesis in an acidic en. FEMS Mic obiol. Ecol. 65:88–101.
48. Rich, J. J., R. S. Heichen, P. J. Bo omley, K. C omack, and D. D. My old.
2003. Communi y composi ion and unc ioning o deni i ying bac e ia om
adjacen meadow and o es soils. Appl. En i on. Mic obiol. 69:5974–5982.
49. Robe son, G. P., and J. M. Tiedje. 1987. Ni ous oxide sou ces in ae obic
soils: ni i ica ion, deni i ica ion and o he biological p ocesses. Soil Biol.
Biochem. 19:187–193.
50. Saad, O. A. L. O., and R. Con ad. 1993. Adap a ion o empe a u e o ni ic
oxide-p oducing ni a e- educing bac e ial popula ions in soil. Sys . Appl.
Mic obiol. 16:120–125.
51. Saad, O. A. L. O., and R. Con ad. 1993. Tempe a u e-dependence o ni i-
ica ion, deni i ica ion, and u no e o ni ic-oxide in di e en soils. Biol.
Fe il. Soils 15:21–27.
52. Sai ou, N., and M. Nei. 1987. The neighbo -joining me hod—a new me hod
o econs uc ing phylogene ic ees. Mol. Biol. E ol. 4:406–425.
53. Schalk-O e, S., R. J. Se iou , J. G. Kuenen, and M. S. M. Je en. 2000.
Ni ous oxide (N
2
O) p oduc ion by Alcaligenes aecalis du ing eas and
amine egimes. Wa e Res. 34:2080–2088.
54. Schloss, P. D., and J. Handelsman. 2005. In oducing DOTUR, a compu e
p og am o de ining ope a ional axonomic uni s and es ima ing species
ichness. Appl. En i on. Mic obiol. 71:1501–1506.
55. Schmalenbe ge , A., H. L. D ake, and K. Ku¨sel. 2007. High unique di e si y
o sul a e- educing p oka yo es cha ac e ized in a dep h g adien in an acidic
en. En i on. Mic obiol. 9:1317–1328.
56. Segel, I. H. 1993. Enzyme kine ics: beha io and analysis o apid equilib ium
and s eady-s a e enzyme sys ems. John Wiley & Sons, New Yo k, NY.
57. Simek, M., and J. E. Coope . 2002. The in luence o soil pH on deni i ica-
ion: p og ess owa ds he unde s anding o his in e ac ion o e he las 50
yea s. Eu . J. Soil Sci. 53:345–354.
58. Simek, M., L. Jiso a, and D. W. Hopkins. 2002. Wha is he so-called
op imum pH o deni i ica ion in soil? Soil Biol. Biochem. 34:1227–1234.
59. Smi h, M. S. 1983. Ni ous oxide p oduc ion by Esche ichia coli is co ela ed
wi h ni a e educ ase ac i i y. Appl. En i on. Mic obiol. 45:1545–1547.
60. S olz, J. F., and P. Basu. 2002. E olu ion o ni a e educ ase: molecula and
s uc u al a ia ions on a common unc ion. Eu . J. Chem. Biol. 3:198–206.
61. S ohm, T. O., B. G i in, W. G. Zum , and B. Schink. 2007. G ow h yields
in bac e ial deni i ica ion and ni a e ammoni ica ion. Appl. En i on. Mi-
c obiol. 73:1420–1424.
62. S ong, D. T., and I. R. P. Fille y. 2002. Deni i ica ion esponse o ni a e
concen a ions in sandy soils. Soil Biol. Biochem. 34:945–954.
63. Thomas, K. L., D. Lloyd, and L. Boddy. 1994. E ec s o oxygen, pH and
ni a e concen a ion on deni i ica ion by Pseudomonas species. FEMS Mi-
c obiol. Le . 118:181–186.
64. Thomsen, J. K., T. Gees , and R. P. Cox. 1994. Mass spec ome ic s udies o
he e ec o pH on he accumula ion o in e media es in deni i ica ion by
Pa acoccus deni i icans. Appl. En i on. Mic obiol. 60:536–541.
65. Tiedje, J. M. 1988. Ecology o deni i ica ion and dissimila o y ni a e e-
duc ion o ammonium, p. 179–244. In A. J. B. Zehnde (ed.), Biology o
anae obic mic oo ganisms. John Wiley and Sons, New Yo k, NY.
66. VanBeek, C. L., E. W. J. Hummelink, G. L. Vel ho , and O. Oenema. 2004.
Deni i ica ion a es in ela ion o g oundwa e le el in a pea soil unde
g assland. Biol. Fe il. Soils 39:329–336.
67. VanB emen, N. 1995. How Sphagnum bogs down o he plan s. T ends Ecol.
E ol. 10:270–275.
68. VanCleempu , O. 1998. Subsoils: chemo- and biological deni i ica ion, N
2
O
and N
2
emissions. Nu . Cycling Ag oecosys . 52:187–194.
69. Wang, J. H., B. C. Bal zis, and G. A. Lewandowski. 1995. Fundamen al
deni i ica ion kine ic-s udies wi h Pseudomonas deni i icans. Bio echnol.
Bioeng. 47:26–41.
70. Weie , K. L., and J. W. Gilliam. 1986. E ec o acidi y on deni i ica ion and
ni ous oxide e olu ion om A lan ic coas al plain soils. Soil Sci. Soc. Am. J.
50:1202–1205.
71. Well, R., J. Augus in, J. Da is, S. G i i h, K. Meye , and D. My old. 2001.
P oduc ion and anspo o deni i ica ion gases in shallow g ound wa e .
Nu . Cycling Ag oecosys . 60:65–75.
72. Wu¨s , P. K., M. A. Ho n, and H. L. D ake. 2009. T ophic links be ween
e men e s and me hanogens in a mode a ely acidic en soil. En i on. Mi-
c obiol. 11:1395–1409.
73. Yamulki, S., R. M. Ha ison, K. W. T. Goulding, and C. P. Webs e . 1997.
N
2
O, NO and NO
2
luxes om a g assland: e ec o soil pH. Soil Biol.
Biochem. 29:1199–1208.
74. Yoshina i, T., and R. Knowles. 1976. Ace ylene inhibi ion o ni ous oxide
educ ion by deni i ying bac e ia. Biochem. Biophys. Res. Commun. 69:
705–710.
75. Zum , W. G. 1997. Cell biology and molecula basis o deni i ica ion.
Mic obiol. Mol. Biol. Re . 61:533–615.
1134 PALMER ET AL. APPL.ENVIRON.MICROBIOL.
by on Feb ua y 8, 2010 aem.asm.o gDownloaded om
4.3 P e ious pee - e iewed publica ions no included in he disse a ion
293
4 Manusc ip s
Cu iculum i ae
294
Ka ha ina Palme
Dipl. Biol.
Leh s uhl ü Ökologische
Mik obiologie, Uni e si ä
Bay eu h
D .-Hans-F isch-S . 1-3, 95440
Bay eu h
H+49172-9314685
T+49921-555672
Bka ha ina.palme @uni-
bay eu h.de
Ausbildung
2008–2012 P omo ion,Leh s uhl ü Ökologische Mik obiologie (Be euung: PD D . Ma cus
A. Ho n),
Uni e si ä Bay eu h.
Thema: G eenhouse gas me abolizing p oka yo es in pea lands
2007–2008 Diploma bei ,Leh s uhl ü Ökologische Mik obiologie (Be euung: PD D .
Ma cus A. Ho n, P o . Ha old L. D ake,
Uni e si ä Bay eu h.
Thema: Phylogene ische und unk ionelle Di e si ä N2O-p oduzie ende und -
e b auchende P oka yo en in einem sau en, N2O-emi ie enden Niede moo
2003–2008 S udium de Biologie,
Uni e si ä Bay eu h.
2006–2007 Auslandssemes e (ERASMUS),
Oulun yliopis o, Oulu, Finnland.
2002–2003 S udium de Physik und Geowissenscha en,
Ruh uni e si ä Bochum.
Be u se ah ung
Juli-Augus 2010 Fo schungsau en hal im Ausland (Finnland),im Rahmen des EU-P ojek es
„Lapland A mosphe e-Biosphe e Facili y“ (LAPBIAT), Fo schungss a ion Ke o
(Uni e si y o Tu ku), Fo schungss a ion Oulanka (Uni e si y o Oulu),
Thema: E ec o high la i ude on deni i ica ion-dependen N2O- luxes and deni-
i ie communi y s uc u e in acidic mi es.
2006–2007 P ojek a bei ,Depa men o Biology, Plan physiology, D . Anna Ma ia Ma -
ila,
Oulun yliopis o, Oulu, Finnland.
Themen:
1. ENDIS: Disco e y and de elopmen o new an ibac e ial compounds om endophy es
2. Role o endophy ic Me hylobac e ium in plan issue
2004–2006 S uden ische Hil sk a ,Leh s uhl ü P lanzensys ema ik, Ab eilung Mykolo-
gie, P o . D . Ge ha d Rambold,
Uni e si ä Bay eu h.
1/4
4.3 P e ious pee - e iewed publica ions no included in he disse a ion
295
E hal ene Fö de ungen
Ku zs ipendium ü Dok o anden,Deu sche Akademische Aus auschdiens
(DAAD), Juli-Augus 2010:
Un e s ü zung des Fo schungsau en hal es in Finnland (Ausse zung de DFG-
S elle)
S ipendium zu Fö de ung on Kong ess eisen, Deu sche Akademische Aus-
auschdiens (DAAD), Ap il 2011:
Kong ess „Ecology o Soil Mic oo ganisms“, P ag
Reisekos enzuschüsse, F auen ö de ung aus den Mi eln des Zen alansa zes zu
Gleichs ellung, Uni e si ä Bay eu h:
Tagungs eisen: BAGECO-10 (2009), ISME-13 (2010), No dSIR-Mee ing (2010),
Go don Resea ch Con e ence on Applied and En i onmen al Mic obiology (2011)
Mi be eu e Abschlussa bei en
Schulz, K. 2009. Ein luss on Aus ocknungse eignissen au die Me hanogenenpo-
pula ion im sau en Niede moo . Bachelo -A bei , Uni e si ä Bay eu h.
Mundinge , A. 2010. Ein luss on Saue s o au die Me hanogenese im sau en
Niede moo . Bachelo -A bei , Uni e si ä Bay eu h.
Pe as, S. 2011. T ophische In e ak ionen im mik obiellen Nah ungsne z eines
Me han-emi ie enden pH-neu alen Niede moo es. Bachelo -A bei , Uni e si ä
Bay eu h.
Gu mann, T. 2011 (lau end). (Toxische) E ek e on Eisennanopa ikeln au Bo-
denmik oo ganismen. Bachelo -A bei , Uni e si ä Bay eu h.
2/4
4 Manusc ip s
296
Publika ionen
Palme , K., D ake, H.L., Ho n, M.A. 2009. Genome-de i ed c i e ia o assigning
en i onmen al na G and nosZ sequences o ope a ional axonomic uni s o ni a e
educe s. Appl. En i on. Mic obiol. 75:5170-5174.
Palme , K., D ake, H.L., Ho n, M.A. 2010. Associa ion o no el and highly di-
e se acid- ole an deni i ie s wi h N2O luxes o an acidic en. Appl. En i on.
Mic obiol. 76:1125-1134.
Palme , K., Biasi, C., Ho n, M.A. 2011. Con as ing deni i ie communi ies ela e
o con as ing N2O emission pa e ns om acidic pea soils in a c ic und a. ISME
Jou nal 6: 1058-1077.
Publizie e Abs ac s au Tagungen und Wo kshops
Palme , K., Ho n, M.A. 2012. Unknown deni i ie di e si y in a pH neu al en soil
in Finnish Lapland. Annual Mee ing Ve einigung ü Allgemeine und Angewand e
Mik obiologie, BioSpec um. Abs ac SMV003, S. 210
Palme , K., Ho n, M.A. 2012. Palsa pea s ep esen hi he o unde app ecia ed
ese oi s o new deni i ie di e si y associa ed wi h N2O luxes. In e na ional
Pola Yea 2012 Con e ence. Abs ac online.
Palme , K., Biasi, C., D ake, H.L., Ho n, M.A. 2011. C yo u ba ion a ec s deni-
i ie communi ies in N2O-emi ing a c ic pe ma os pea soil. Annual Mee ing
Ve einigung ü Allgemeine und Angewand e Mik obiologie, BioSpec um. Ab-
s ac EMP104, S. 116.
Palme , K., Biasi, C., D ake, H.L., Ho n, M.A. 2011. C yo u ba ion a ec s deni-
i ie communi ies in N2O-emi ing a c ic pe ma os pea soil. Annual Mee ing
Ve einigung ü Allgemeine und Angewand e Mik obiologie, BioSpec um. Ab-
s ac EMP104, S. 116.
Palme , K., Biasi, C., D ake, H.L., Ho n, M.A. 2011. Impac o c yo u ba ion on
deni i ie communi y s uc u e and ac i i y in N2O-emi ing a c ic pe ma os
pea soil. Ecology o Soil Mic oo ganisms. Abs ac 99
Palme , K., Schulz, K., Ho n, M.A., D ake, H.L. 2010. S abili y o he me hano-
genic communi y in an acidic en o expe imen al d ough . Annual Mee ing Ve -
einigung ü Allgemeine und Angewand e Mik obiologie, BioSpec um. Abs ac
ECV02, S. 81.
Palme , K., Schulz, K., Ho n, M.A., D ake, H.L. 2010. E ec s o enhanced d ough
on he di e si y o me hanogens in an acidic en. Bay eu h Cen e o Ecology and
En i onmen al Resea ch (BayCEER) Wo kshop 2010, Abs ac O 1.5.
Palme , K., Schulz, K., Ho n, M.A., D ake, H.L. 2010. Impac o a i icial d ough
on di e si y, abundance, and gene exp ession o me hanogens in an acidic en. 13 h
In e na ional Symposium on Mic obial Ecology (ISME-13), Abs ac on disk
Palme , K., D ake, H.L., Ho n, M.A. 2009. Ac i i y and di e si y o phyloge-
ne ically no el acid- ole an ni a e educe and deni i ie communi ies in an
N2O-emi ing en. Annual Mee ing Ve einigung ü Allgemeine und Angewand e
Mik obiologie, BioSpec um. Abs ac PN22, S. 127.
3/4
4.3 P e ious pee - e iewed publica ions no included in he disse a ion
297
Palme , K., D ake, H.L., Ho n, M.A. 2009. Ac i i y and di e si y o phyloge-
ne ically no el acid- ole an ni a e educe and deni i ie communi ies in an
N2O-emi ing en. Bay eu h Cen e o Ecology and En i onmen al Resea ch (Bay-
CEER) Wo kshop 2009, Abs ac O 1.7.
Palme , K., D ake, H.L., Ho n, M.A. 2009. No el and highly di e se acid- ole an
ni a e educe s a e associa ed wi h N2O- luxes o an acidic N2O-emi ing en.
10 h In e na ional Symposium on Bac e ial Gene ics and Ecology (BAGECO-10),
Abs ac P197, S. 279.
Sons ige Tagungsbei äge
Palme , K., Biasi, C., Ho n, M.A. 2011. Deni i ie communi iy composi ion im-
pac s N2O emission pa e ns in acidic und a pe ma os soils. No dic Ne wo k
o S able Iso ope Resea ch (No dSIR) Mee ing
Palme , K., D ake, H.L., Ho n, M.A. 2011. E ec o high la i ude on
deni i ica ion-dependen N2O- luxes and deni i ie communi y s uc u e in pea-
lands. Go don Resea ch Con e ence on Applied and En i onmen al Mic obiology
Palme , K., D ake, H.L., Ho n, M.A. 2010. No el and highly di e se acid- ole an
ni a e educe s a e associa ed wi h N2O- luxes o an acidic N2O-emi ing en.
No dic Ne wo k o S able Iso ope Resea ch (No dSIR) Mee ing
Schmid , O., Ho n, M.A., Palme , K., D ake, H.L. 2010. H2- A hidden link be -
ween e men a ion and me hanogenesis in he in e media y ecosys em me abolism
o a we land soil. Go don Resea ch Con e ence on he Molecula Basis o Mic obial
One-Ca bon Me abolism
4/4
4 Manusc ip s
298
