Untersuchung
Bakterien
der
dem
zellularen
Doktors
aus
Fachbereich
Erlangung
geboren
Universität
kalten,
Martin der
-
vorgelegt
Dissertation
Dr.
Juli
Fettsäuren
Naturwissenschaften
des
in
rer.
zur
Biologie/Chemie
Könneke
2001
Wolfsburg
marinen
Grades
Bremen
nat. von
—
Max-PanckntItLit
1entar
eines
MarIne
von
Sedimenten
der
Nr
sulfatreduzierenden
für
C&siusstr.
Bibliothek
:
Mikrohlologle
Max-Panck-
3rernrnb
Marine
/9
BibHothe 1
e
c
Mkrobkog
D-28359
Insitt
für Bremen Die vorliegende Doktorarbeit wurde in der Zeit von September 1997 bis Juni 2001 am Max
Planck-Institut fur Marine Mikrobiologie in Bremen angefertigt.
Gutachter: Prof. Dr. Friedrich Widdel
2. Gutachter: Prof. Dr. Bo Barker Jørgensen
Tag des Promotionskolloquiums: 20. August 2001
Teil Zusammenfassung
Abkürzungen
Inhaltsverzeichnis
B
A
1:
3.
2. 4.
Ergebnisse
7. 5. 3.
6. Einleitung Darstellung
2.
1.
1.
phenolica
Lebendzellzahlen
3.2. 3.1. Charakterisierung 2.3. 2.2.
Sedimenten 2.1.
bulticiim Zellulare
Neueinordnung
Einfluß Zielsetzung
Physiologische
marinen Einfluß Mikrobieller
1.2. SRB
Bakterien
Psychrophile
1.1.
Wachstumstemperaturen hydmgenophilus psychrotoleranten
Wachstumsabhängige Zellulare
Physiologische
in
Chemotaxonomische
Tiefenprofile
Abschätzung Fettsäureanalyse
Analyse
Phylogenetische
und
marinen
der
der
sulfatreduzierenden
gen.
Fettsäuren
comb.
Diskussion
der
Temperatur Temperatur
der
mittels
Abbau
und
nov.,
Fettsäurenzusammensetzung
Merkmale Arbeit
von
Sedimenten
Ergebnisse
nov.
psychrotolerante
der
der
der
Phospholipidanalysen sp.
Desiilfrbacterium
von
von
und
und
Mikroorganismenzusammensetzung
Charakterisierung
bei
Bakterienzahlen
Phospholipidfettsäuren
nov.
und
auf auf
organischem
SRB
morphologische
wechselnden
psychrophiler
Beschreibung
Einordnung
Fettsäuremuster
die die
mesophilen
Bakterien
Lipidfettsäurenzusammensetzung
zellulare
im
SRB
Gesamtzusammenhang Material
phenolicinn
mittels
Temperaturen
mittels
SRB
Fettsäurenzusammensetzung
SRB
von
mittels
Merkmale
und
von von
Desulfotignuin
bei
Bestimmung
zellularer
der
in psychrophilen,
Desuljobacter
1
verschiedenen
6S
marinen
MPN-Methode
als rRNA
Desuijobacula
Sedimenten
von
Gensequenz
der
marinen
von
von
21
23
21
21 20
20 20
18
19 16
18 ii
18
7 6
9 4
3
3 1 3.3. Phylogenetische Einordnung der neuisolierten Reinkulturen aus Svalbard 23 4. Anreicherung und Isolierung von methanogenen Archaea aus permanent kalten. marinen Sedimenten (Svalbard) 25 4.1. Anreicherung und Isolierung 25 4.2. Morphologische und physiologische Charakterisierung 27 4.3. Phylogenetische Einordnung 27
C Literaturverzeichnis 30
Teil II: Publikationen 38
A Publikationsllste mit Erläuterungen 38
B Publikationen 40
1. Effect of temperature on the composition of cellular fatty acids in sulphate-reducing bacteria 40 2. Reclassification of Desiiift.bateriu,i, phenoluii,n as Desulfibucitla phenolwci“ comb. nov. and description of strain SaxTas Desiilfrtigzum balticuin gen. nov.. sp. nov. 56 3. Aerobic and sulfate-reducing bacterial communities of Arctic sediments characterized by phospholipid analysis and cultivation rnethods 72 4. Community structure and activity of sulfate-reducing bacteria in an intertidal surface-sediment: A multi-methods approach 90
Danksagung Abkürzungen
A Arrheniuskonstante ACP Acyl carrier protein AVS Acid-volatile sulfide C-irm MS Combastion interfaced isotope ratio mass spectrometry CFA Cyclopropanfettsäure(n) d Tag
D Diffusionskoeffizient
DAPL 4 ‚6- Diamidino-2-phenylindol DGGE Denaturing gradient gel electrophoresis DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen dwt dry weight eV Elektronenvolt
Ed Aktivierungsenergie FAME Fettsäuremethylester FID Flammenionisationsdetektor FISH Fluoreszenz-in situ-Hybridisierung GC Gaschromatographie GC-C-IRMS Gaschromatography combustion interfaced isotope ratio mass spectrometry GC/MS Gaschromatographie/Massenspektroskopie h Stunde HPLC l-lighperformance liquid chromatography ICBM Institut für Chemie und Biologie des Meeres ICP-OES lnductively coupled plasma optical emission spectroscopy kPa Kilopascal MPN most-probable number MS Massenspektroskopie mlz Masse/Ladung OD Optische Dichte
PCR Polymerase cham reaction PLFA Phospholipidfettsäuren R Allgemeine Gaskonstante = 8,31 JK‘rnol‘
SRB Sulfatreduzierende Bakterien SRR Sulfatreduktionsraten
T Absolute Temperatur Maximale Wachstumstemperatur 1T Minimale Wachstumstemperatur Tri Optimale Wachstumstemperatur TOC Total organic carbon V-CDT Vienna Canyon Diablo Troilit VFA Volatile fatty acids v Reaktionsgeschwindigkeit oder Wachstumsrate Zusammenfassung
Im Mittelpunkt der vorliegenden Dissertation standen Analysen der zellularen Fettsäurenzusammensetzung von verschiedenen sulfatreduzierenden Bakterien (SRB) aus marinen Habitaten. In vergleichenden Analysen wurde erstmals der Einfluß der Wachstumstemperatur und der Wachstumsphase auf die zellularen Fettsäurenzusammensetzungen von verschiedenen SRB in Reinkulturen untersucht. Ferner wurden in situ Analysen der Phospholipide an marinen Sedimenten der Arktis (Svalbard) und des Wattenmeers (Deutschland) durchgeführt, um durch den Nachweis von Biomarkern Hinweise über die Häufigkeit und die Verteilung unterschiedlicher Mikroorganismengruppen zu erhalten. Die Bestimmung der Lebendzellzahlen verschiedener stoffwechselphysiologischer Bakterien in Sedimenten aus Svalbard wurde mit der selektiven Isolierung von neuen Mikroorganismen in Reinkulturen bei konstant niedrigen Temperaturen verbunden.
1. Erstmalig konnte gezeigt werden, daß Angehörige der Gattung Desulfobacter die Fähigkeit besitzen, ihre Fettsäurenzusammensetzung an wechselnde Temperaturen anzupassen. Dadurch regulieren sie die temperaturabhängige Fluidität ihrer Lipidmembranen.
Bei niedrigen Temperaturen (4—12 °C) besitzt Desulfobacter hydrogenophilus, wie die meisten psychrophilen oder moderat psychrophilen SRB, einen konstant hohen Anteil an fluiditäterhöhenden, cis-ungesättigten Fettsäuren. Dieser Anteil verringert sich bei der Erhöhung der Wachstumstemperatur auf Werte, wie sie in anderen mesophilen SRB gefunden wurden. Durch wachstumsabhängige Untersuchungen mit C-markiertem Substrat wurde gezeigt, daß diese Temperaturanpassung durch de novo Synthese3 der zellularen Fettsäuren stattfindet. Bei niedrigen Temperaturen konnte neben einer ‘Neusynthese von ungesättigten
Fettsäuren, eine Inhibierung der Synthese von cis-9, 10-Methylenhexadecansäure (cyc17:0) und 10-Methyl-hexadecansäure (1OMe16:0) nachgewiesen werden.
2. Ein sulfatreduzierendes Bakterium, das aus marinem Küstensediment der Ostsee isoliert wurde, stellt hinsichtlich der 16S rRNA Gensequenz, der Morphologie und des zellularen Fettsäuremusters eine neue Gattung innerhalb der 6-Proteobakterien dar. Als Bezeichnung für den vollständig-oxidierenden Typenstamm wurde Desulfotignum balticum vorgeschlagen. Aufgrund der großen Ähnlichkeit der 16S rRNA Gensequenz, der Morphologie, der Physiologie und der Fettsäuremuster in Desulfobacterium phenolicuni und Desulfobacula Desuijobacula toluolica
Fettsäure Bezeichnung Spezifität
3. marinen
Sedimenten dominierten Nachweis Schicht nur obersten
die
Übereinstimmung
Verdünnungsserien Verwandtschaft rRNA Reinkulturen,
höchsten Konzentrationen 4.
Bedeutung Trimethylamin
isoliert ihrer
taylorii beschriebenen Verwandten isoliert
in
über
Die
Aus
Morphologie
geringen
Gensequenzen
gezeigt
werden. wurden. Sedimenten.
bzw.
wurden
lOMel6:O
spezifischer dieser
in
MPN
permanent
dieser
die
Sedimentschicht.
(Svalbard)
oberen
situ
lediglich
und für
Methanococcoides
denen
werden.
Methanogenen,
Konzentrationen
methanogene most-probable Zwei
der
zu
Fettsäure
Verdünnungsreihen
Gattung
beide
Desulfr)tignum
mit
D.
Analyse
Sedimentschicht
bekannten nachgewiesen
für
große konnten
Wasserstoff
der
der
phenolietim
kaltem.
neue Fettsäuren
zeigte
Desiilfovibrio
Methanol
Die
Stämme
sieben
Desti1/vibrio
Tiefenverteilung
als
Ähnlichkeiten
Gattungen
für
der
verschiedene ein
Archaea
marinem
Biomarker
psychrophilen
die
SRB
Die
number
Isolate
nachgewiesen
konnte
Phospholipidfettsäuren
konnte
burtonil.
als
in für
und
werden.
wurde
aus
zu
der
Elektronendonator spezifischen
mannes
spezifischen
Phospholipidfettsäuren-Profile
weisen
einer
innerhalb
selektiv Trimethylamin.
in
zeigen
permanent
die
Sediment
ein
Gattung
(MPN)
diesem
für
zu
Desulfobacula Die
von
Dieser
für
Reinkulturen
Wechsel
von
2
Sediment
den
Angehörige
aufgrund
Bakterien
auf
werden
isolierten Desiilfiibacter
kultivierbaren
bei
anaeroben
kalten
der
Biomarker Biomarker
Methode
Desulfobaciila
(Svalbard)
Befund
methylotrophen
eine
kalten,
niedrigen
einer
-Proteobakterien
und
Habitat
ihrer typisches
Die diente,
nur
Stämme
zeigten.
in
isoliert
der
widerlegt
marinen
zeigten
phenolica
von
Bakterien
ermittelt
Reinkulturen und
il7:l permanent
16S
untergeordnete Arten
aeroben
Temperaturen
konnten
Gattung
hin.
aeroben
stellen
deren
rRNA-Gensequenzen
verwerten
Tiefenprofil.
Drei
werden,
und
maximale
Archaea
als
Sedimenten
vereinigt.
die
dominierten
Bakterien
spezifisch
wurden.
aufgrund vorgeschlagen.
Abwesenheit
mit
lOMel6:O
sulfatreduzierende
Desulfobucter
waren Mikroorganismen dar.
oft
kalten,
sind
Methanol
die
wie
Methanolobus
beschriebene
in
Die
Werte
ökologische
Durch
Als
eine
Reinkultur
ihre und
der die
in
Aus
ihrer
geltende
marinen
geringen
konnten
tieferen
in
in
neue
guter
Arktis ersten
SRB,
nahen
enge
den oder
den
den der 16S
und
In
in Teil 1: Darstellung der Ergebnisse im Gesamtzusammenhang
A Einleitung
1. Mikrobieller Abbau von organischem Material in marinen Sedimenten
In den Meeren findet die Primärproduktion von organischem Material durch phototrophe
Organismen in den oberflächennahen Bereichen der Wassersäule statt, in denen ausreichend Licht für die Photosynthese eindringt. Während das für die Kohlenstofffixierung notwendige
Kohlendioxid im Meerwasser in gelöster Form im Überschuß enthalten ist, limitiert meistens die Verfügbarkeit der Elemente Stickstoff, Phosphor und Eisen die Bildung von Biomasse
(Sommer. 1998: Smetacek, 1998). Das in den euphotischen Zonen gebildete organische Material dient heterotrophen Organismen als Energie- und KohlenstoffqueLle und wird von diesen in unterschiedlichem Umfang beim Herabsinken in der Wassersäule abgebaut. Der wichtigste Elektronenakzeptor ist dabei Sauerstoff. Nur ein Teil des organischen Materials sedimentiert auf den Meeresboden und wird anschließend in den Sedimenten unter oxischen und anoxischen Bedingungen überwiegend durch Mikroorganismen remineralisiert
(Jørgensen, 1983). Der Transport des in der Wassersäule nur in geringen Konzentrationen gelösten Sauerstoffs in die Sedimente verläuft überwiegend durch Diffusion. So ist in Schelfsedimenten die Verfügbarkeit von Sauerstoff infolge der Atmungsaktivität aerober Organismen und durch die chemische (abiotische) Reaktion mit Sulfid auf die oberste, nur wenige Millimeter starke Sedimentschicht begrenzt (Revsbech et al., 1980; Jørgensen, 1982). In den darunterliegenden anoxischen Sedimenten erfolgt der Abbau des organischen Materials schrittweise durch anaerobe Mikroorganismen unter Verwendung verschiedener terminaler
Elektronenakzeptoren (Abb. 1). Die in diesen Schichten noch enthaltenen, komplexen organischen Polymere werden zuerst durch fermentierende Bakterien abgebaut. Die Endoxidation der Fermentationsprodukte findet durch Bakterien über die Reduktion von Nitrat, Eisen(I[E), Mangan(IV) und Sulfat statt (Canfield, 1993). Aufgrund der hohen Konzentration von SuLfat im Vergleich zu der von Nitrat und die an vielen Orten eingeschränkte Verwertbarkeit von schwerlöslichem Eisen(IlI) stellt die dissimilatorische Sulfatreduktion den wichtigsten anaeroben Prozeß für die Mineralisation von organischem Kohlenstoff in marinen Sedimenten dar (JØrgensen, 1982). Die methanogenen Archaea spielen infolge der Konkurrenz um Abbauprodukte der fermentierenden und acetogenen Bakterien (z.B. Acetat, Formiat, Wasserstoff) mit den energetisch begünstigten SRB in den sulfatreichen marinen Sedimentschichten eine untergeordnete Rolle. Ausnahmen bilden
3 Bereiche, (z.B. Archaea Methylsulfide) werden
Sedimenten
2.
SRB besitzt,
Sulfid
Abbildung
in Bakterien:
marinen
faulendes
bilden
Energie können.
SRB
können
über
in
Acetat
(4)
Sedimenten: 1: mit
denen
innerhalb
1/
Methanogene
in Vereinfachter
Elekironentransport
verwerten,
den Pflanzenmaterial)
zu
marinen
Dadurch
Methylgruppen
durch
konservieren.
SRB
(1)
der
Lactat,
Zucker.
Bakterien.
koexistieren
Polysacchande.
Kohlenstofffluß
Fermentative die Sedimenten
die
können
Organische
Prokaryoten
Mono-
und
von
Umsetzung
Aminosäuren,
Propionat,
und
andere
C02+HS
enthaltende
die Sie den
die
und
ij, während
Alkohole
(Oremland
Bakterien;
Sulfatverfi.igbarkeit
sind
SRB
methylotrophen
Oligomere
Polymere
Fettsäuren Proteine.
des
eine
Butyrat
großer
Peptide.
anaeroben
in
nicht
heterogene
der
der
Verbindungen
4 etc.
(2)
et
dissimilatorischen
oder
etc.
Mengen
Lage
Homoacetogene
al.,
mikrobiellen
nur
1982;
methanogenen
eine
Organismengruppe.
limitiert
sehr
an
‚
Zinder,
Vielzahl
(Methanol,
schnell
Abbaus
langsam
Bakterien:
ist.
Reduktion
E 1993).
abbaubarem
von von
Archaeen
Einige
Methansulfide
Methylamine, als
Methylamine
(3)
organischem
CO,+CH
niedermolekularen
Substrat
4
die Sulfatreduzierende
methanogene
von
die
in
Material
marinen
Fähigkeit
Sulfat
genutzt
Material
oder
zu organischen Verbindungen und auch Wasserstoff unter anoxischen Bedingungen als Elektronendonatoren zu nutzen (Rabus et al., 2000). In anoxischen marinen Habitaten, in denen Sulfat aufgrund der hohen gelösten Konzentration im Meerwasser (28 mM) meistens keinen limitierenden Faktor darstellt, wurden Acetat, Propionat und Butyrat sowie Wasserstoff als die wichtigsten Substrate von SRB bestimmt (SØrensen et al., 1981; Balba und Nedwell, 1982; Boschkeret al., 2001).
Das Wachstum von SRB ist nach bisherigen Erkenntnissen an anoxische Bedingungen gebunden. Jedoch wurden wiederholt hohe Zahlen SRB und hohe Sulfatreduktionsraten (SRR) auch in oxischen Zonen von marinen Sedimenten und mikrobiellen Matten nachgewiesen (Teske et al., 1996. 1998: Wierenga et al., 2000), wofür es noch keine vollständige Erklärung gibt. In Reinkulturen der Gattung Desulfivibrio wurde die Reduktion von Sauerstoff zu Wasser festgestellt, jedoch konnte dabei nur ein sehr geringes oder gar kein Wachstum beobachtet werden. Es wird vermutet, daß diese aerobe Atmung eine Schutzfunktion gegen Sauerstoff für die dazu befähigten SRB darstellt (Cypionka, 2000).
Ein wichtiger Faktor, der alle biologischen und chemischen Reaktionen in einem Habitat beeinflußt, ist die Temperatur. Durch in situ Messungen konnte eine mikrobielle
Sulfatreduktion in polaren marinen Sedimenten unterhalb von 0 °C (Sagemann et al., 1998) und in von geothermischen Quellen erhitzten Sedimenten oberhalb von 100 °C (Jørgensen et al., 1992) gemessen werden. Küstennahe Sedimente innerhalb der gemäßigten Breiten unterliegen jahreszeitlichen Temperaturschwankungen, die die Aktivität der dort vorkommenden mikrobiellen Gemeinschaften beeinflussen. Die 5ulfatreduktionsraten liegen daher in den Wintermonaten unterhalb der Werte des wärmeren Sommers (Jørgensen, 1977; Abdollahi, 1979). Generell konnte in lnkubationsversuchen mit marinen Sedimenten verschiedener Herkunft bei unterschiedlichen Temperaturen gezeigt werden, daß die optimalen Temperaturen für die höchsten Sulfatreduktionsraten um etwa 20 °C oberhalb der in situ -Temperaturen der jeweiligen Sedimente liegen (Nedwell, 1989; Isaksen und Jørgensen, 1996; Sagemann et al., 1998). Im größten Teil des Meeresvolumens (90 %) herrschen permanent niedrige Temperaturen ( 5 °C) (Morita, 1975), da in den offenen Ozeanen nur ein sehr langsamer Austausch zwischen dem kalten Tiefenwasser und dem hauptsächlich von der Sonnenstrahlung erwärmten Oberflächenwasser stattfindet. Weitere permanent kalte, marine Habitate befinden sich in den Polarregionen, die etwa 14 % der Erdoberfläche einnehmen. Aufgrund der im Vergleich zu Sedimenten gemäßigter Breiten niedrigeren Temperaturoptima (18-28 °C) der Sulfatreduktion in der Arktis und Antarktis
5 Lebensgemeinschaft
3. vermutet
Obwohl dortigen nur su[fatreduzierende hydrogenophilus.
Mittelmeers eingeordneter
und
vacuolatus, Wachstumstemperatur
Erst kalten, Fünf Lactat) 1996). arctica)
(Desuijojaba
Abundanz Sedimenten
2000). ebenfalls niedrigen
Desuljovibrio
letzten
(DeLong Methoden Franzmann
methanogene
Noch
wenige
kann
1999
neue
Psychrophile
Dieses
marinen Daneben
die
Abbaus
in
Jahren
oder
daher
geringer
man
Temperaturen
et gelang
noch Arten
Reinkulturen
SRB
gezeigt. dieser Sulfatreduktion
isoliert
wurde
al., und
vollständig
Stamm
gelida.
an
unvollständig-oxidierende.
als
hier
Reinkultur
bei
zeigt
von Sedimenten
haben
von
gibt
1994,
bekannt,
(Sass
es Mitarbeitern
ist
psychrotolerant
auch und
wurde (Isaksen
Bakterium,
eine
niedrigen
und
organischem
Knoblauch
daß
die
das
es
SRB
wurde
Desulfofrigus von
et
1999;
jedoch ebenfalls
nur
noch
zu
durch
Anzahl
psychrotolerante
al.,
zu Archaea
vollständig-oxidierende
isoliert.
Vorherrschen
(Widdel.
wurden
die
19
isolieren.
in
sehr
und
aus
Kohlendioxid vor
selektiv
1998; Sahm
°C.
Temperaturen
anderer,
noch
marinen
molekularbiologische gelungen.
Studien
das und
von Jørgensen,
der
wenige
der
wohingegen
Material
eingeordnet
mit
Organische
auch
Montamedi Wachstum
und
1987.
bei
bekannten Mitarbeitern.
Küste noch
fragile.
Lediglich
bei
Antarktis
bekannten
Sedimenten
Berninger,
bisher
einer
mittels
in
Studien,
10 einer
eine
D. verantwortlich
(Desu/fofrigus
bei SRB
sulfatreduzierende
Svalbards
1996; kalten,
°C
(4
Desu/fotalea
kein Idivgenop/iilus
6
Temperatur
werden.
moderat und drei
0
psychrophilen
Substrate
aus
nicht
°C)
bei an
biochemischer
Fermentationsprodukten zu
°C
Sagemann
in
obligat
Wachstum
Petersen,
Süßwasserisolate 1998.
marinen Art,
der
denen der
die
wachsen.
isolieren.
Methoden
Wachstum
0
zu
kultivierter
Ein
°C
Ostsee
psychrophile
Polarregionen
isolieren die
Kälte
Boetius, ist
werden
psychrophile
oceanense)
versucht
von
weiterer
psvchrophila
et
und (Glud
1998). Standorten
oberhalb aus
methanogenen
Sie al.,
wächst Bakterium.
isoliert
nachgewiesen
Dabei
angepaßten
und
zeigt,
4
marinem
(Knoblauch 2000). besitzt
1998).
gehören
unvollständig
et
SRB
als °C
wurde,
sind
al..
molekularbiologischer
und
umgesetzt.
optimal
von
für
moderat (lsaksen
handelt wachsen.
SRB
war
häufig
in
(Acetat.
1998),
und Bisher bisher
eine
bis
24
Desulfr.rlzopciliis
SRB
eine alle
den
Archaea.
Schlamm
aus
Desiilfr.bacter
mikrobiellen
Desti (Ravenschlag. °C
zu
bei
et
psychrophile psychrophil
der
bekannt, sind
und
es
vorkommen
ist
selektiv
permanent
stattfindet.
arktischen al.,
Das 42
zu
Propionat,
Die
optimale
28-30
/fotalea
es
sich
Gattung
Teske,
%
bisher
Acetat
1999).
In erste
große
einzig
des
des
°C
den
bei
die
um Methanococcojdes burtonii 0(T = 23 °C) bzw. Methanogeniurn frigidtun 0(T = 15 °C). Sie wurden aus methanhaltigem, anoxischem Wasser des Ace Lake isoliert, das eine konstant niedrige Temperatur von 1-2 °C besitzt (Franzmann et al., 1992, 1997).
Tabelle 1: Psychrophile und psychrotolerante SRB und ihre beschnebenen Wachstumstemperaturbereiche
Stamm Wachstumstemperatur (°C) 1-labitat Literaturverweis
Bereich Optimum
Des,tt‘fobacter hvdrogenophi1ii. 0-32 28-30 Mariner Schlamm Widdel, 1987
Deziiforhopaliis vucuo/a[ij.v 0-24 19 Mannes Sediment lsaksen et al., 1996 Desulfovibrio avpoeensis 4-35 25-30 Grundwasser Motamedi et al., 1998
Desitlf‘wihrio cuneatlis 0-33 28 Süßwassersee Sass eI al., 1998
Desu1foihrio litoralis 0-33 28 Süßwassersee Sass eI al., 1998
Destilfofliha gelida -1,7-10 7 Mannes Sediment Knoblauch et al., 1999
Desiilfofrigusfragile -1.7-27 18 Mannes Sediment Knoblauch et al., 1999
Destilfofrigtis oceanense -1,7-16 10 Mannes Sediment Knoblauch et al., 1999 Desulfotalea urctiLa -1,7-26 26 Mannes Sediment Knoblauch et al., 1999
Desulfotalea psvchrophila -1,7-19 10 Mannes Sediment Knoblauch et al., 1999
4. Physiologische Merkmale psychrophiler SRB Bakterien können anhand von Temperaturkardinalpunkten (Temperaturbereich, in dem Wachstum erfolgt sowie optimale Wachstumstemperatur) in Gruppen eingeordnet werden. In der vorliegenden Arbeit wurde in Anlehnung an die Definition von Wiegel (1990) die in
Tabelle 2 dargestellte Einteilung verwendet. Der Begriff “temperaturtolerante Mesophile“, der als synonyme Bezeichnung zu “psychrotroph“ verwendet wird, wurde durch den Begriff “psychrotolerant“ ersetzt. Weiterhin wurde die Einteilung durch den Begriff “moderat psychrophil“ ergänzt.
Tabelle 2: Einteilung von Bakterien hinsichtlich ihrer Temperaturanpassung aufgrund von Temperaturkardinalpunkten (in CC)(nach Wiege!. 1990, modifiziert). ,,T01 T,.
Psychrophile Bakterien 0 < 15 < 20
Moderat psychrophile Bakterien 0 < 20 20
Psychrotolerante Bakterien < 5 > 15 > 20
Mesophile Bakterien > 5 < 45 < 50
7 Wachstumsrate die Organismus Temperatur
(v
Aktivierungsenergie;
absoluten
ist Reaktionen organismenspezifische detinierbaren
E1 werden. Destilforhopalus wurden
optimalen Wachstumstemperaturen
Wachstumsraten 1982). Mechanismen Wachstumsrate
Reaktionen Wachstumsertrag.
Die
Graphisch
E4 = SRB,
für
Eine
E.
ursprünglich
aus
Reaktionsgeschwindigkeit
einen
optimale
sofern
d.h. somit
die
Temperatur
diesem weitere
bei
die
Temperaturen
zusammen
aus
Stamm
die
wird
Eigenschaft. seine
chemischen
der
Aktivierungsenergie und
als
auf
kaltem
fällt vacuolatus
Kulturen
sogenannten
Wachstumstemperatur
den
von
häufig
physiologische
Wert
psychrophil
der
Der
der
R
höchste
ändert,
dargestellt.
häufig
Zusammenhang =
Größe,
(Feiler
Temperatur Bakterien
arktischem
Ebene Allgemeine
Wachstumsertrag
(Isaksen
im
der
An
akklimatisierten
Reaktionen
ihrer
besitzen
wenn
suboptimalen
nicht Logarithmus
verschiedenen
et
Wachstumsrate korreliert
Arrheniusgraphen“
biochemischer
bzw.
al.,
und
Sulfatreduktionsraten
oder
ergeben
die
Sediment
dar. wird
mit
Größe,
1994).
Gaskonstante;
lnv=lnA
ihre
Jørgensen,
moderat
darstellt:
Zellen
zwischen v
jedoch
In
dem Wachstumsrate;
häufig
höchsten =
ist
der
der
sich
A
sich ist Temperaturbereich
Bakterien
die
isoliert
Temperaturoptimum definiert
bei
8
Anwendung
e Wachstumsrate
die
zu psychrophil
durch
besitzt. Reaktionen. an EJR‘F
aus
1996;
temperaturabhängig
verschiedenen
E
der
keiner
Wachstumsraten
T
stellt
die
Biomasse,
wurden =
vielen
die
Reaktionsgeschwindigkeit
wurde
absolute
Temperaturen
Knoblauch
1
als
bei
physiologischen
Die
um
Arrheniusgleichung
A
auf
die
der
eingeordnet.
(Knoblauch
geschwindigkeitsbestimmenden =
gezeigt,
Das
2-9
das
Beziehung
als
die
Temperatur)
Arrheniuskonstante;
bestimmt Anwendung Temperatur.
Temperaturen
und
°C Wachstum
Temperaturoptimum
Funktion
einzelner
pro
unterhalb
daß (Reichert
Jørgensen,
über
sein
umgesetzter
et
sich oder
Jedoch
wird,
al.,
zwischen
der
den
auf
von
enzymatischer
kann, von
beschrieben,
bei
der
biochemisch
1999),
vorinkubiert
und
reziproken
chemische
zwar
optimalen
1999).
20
Bakterien
lagen
Wert
und
der
Morita.
°C
ist
Menge
E
sowie
eine
der
der ein
von
und
Die
die
der
der = Substrat von einem Organismus gebildet wird. Der Wachstumsertrag wird üblicherweise in
der Einheit g Trockenmasse pro mol Substrat dargestellt. In den psychrophilen und moderat psychrophilen SRB sowie in den psychrotoleranten Süßwasserisolaten D. cuneatus und D. litoralis lagen die Temperaturen, bei denen der höchste Wachstumsertrag ermittelt wurde, unterhalb der jeweiligen optimalen Wachstumstemperaturen (Knoblauch und Jørgensen, 1999; Isaksen und Jørgensen, 1996: Sass et al., 1998). Die unterschiedlichen Kurvenverläufe des Wachstumsertrags in Abhängigkeit von der Temperatur einzelner SRB lassen jedoch kein generelles Prinzip erkennen.
5. Zellulare Fettsäuren von SRB
In Bakterien. die keine Neutralfette in Form von Trigylceriden enthalten, findet man zellulare Fettsäuren in den Lipiden der Cytoplasmamembran sowie in Gram-negativen Bakterien zusätzlich in geringeren Anteilen in den Lipopolysacchariden der äußeren Membran. Die Zusammensetzung dieser Fettsäuren bietet häufig ein Gattungs- oder sogar Art-spezifisches, chemotaxonornisches Merkmal, das bei der Einordnung und der Identifizierung von Reinkulturen oder bei der in situ Identifizierung von Bakterien hilfreich sein kann. Typische bakterielle Fettsäuren, ihre chemische Struktur und die in dieser Arbeit verwendeten
Kurzbezeichnungen sind in Tabelle 3 dargestellt. Die phylogenetische Einteilung von Bakterien beruht überwiegend auf vergleichenden Analysen der 16S rRNA Gensequenzen (Ludwig und Klenk, 2001). Innerhalb der SRB decken sich die über zellulare Fettsäuremuster ermittelten chemotaxonomischen Gruppen häufig mit den phylogenetischen Gruppen. Charakteristisch für viele SRB sind die oft hohen
Anteile verzweigter Fettsäuren, die in relativen Anteilen von über 50 % gefunden wurden (Vainshtein, 1992). Die in iso-Position verzweigte, ungesättigte Fettsäure i17:l wurde bisher nur in SRB detektiert. Den größten Anteil an i17:1 besitzen Angehörige der Gattung Desulfovibrio. So enthält Desu(frvibrio desulJiiricans beispielsweise einen relativen Anteil
i17:1 von bis zu 33 % (Kohring et al., 1994). Weiterhin wurde i17:1 in einem Anteil von 11 % in Arten der Gattung Desulfotornaculum nachgewiesen. In Desulfosarcina variabilis und
Desulfococcus nzultivorans wurde i 17:1 nur in sehr geringen Anteilen gefunden (1 %)‚ jedoch besitzen diese die anteiso-verzweigte Fettsäure a17:l in Anteilen von 4 bzw. 10 % (Kohring et al., 1994). Eine weitere für einige SRB charakteristische Fettsäure ist lOMel6:0, die bisher nur in wenigen Bakterien gefunden wurde (Kroppenstedt und Kutzner, 1978; Tsitko et al., 1999). Die Fettsäure lOMel6:0 wurde innerhalb der SRB nur in einigen Angehörigen
9 der autotrophicuni Desulfobacter
al., Merkmal SRB
Einzigartig Glycerol-Diethern sulfatreduzierenden und
Temperaturbereich Bakterien
Archaea der Fettsäuren
diese
Parkes, Verbindungen, über von Anwendungsbereich auf Tatsache.
können, Fettsäurezusammensetzung von
Reinkulturen Wachstums Fettsäurenzusammensetzung
1998).
Aufgrund
Aufgrund
Gattung
1985;
marine Familie
in
Fettsäuren
die
Fettsäuren
beiden
lOMel6:0
den
In
Art
(Dowling
1985; ist
Vainshtein
daß
vorherrschend)
vorkommt,
vielen
mit
innerhalb
Habitate
Desulfotomaculu,n
eine
Lipopolysacchariden
bei und
der
Desulfrbacteriaceae
der
Fettsäuren
gemessen
in
diese
Acylketten
die
nachgewiesen, Findlay
konstanter nur
z.T. Seltenheit
die unterschiedlichen
exakte
jedoch
Bakterien,
et
direkt
in
(Dowling
Gehalte
angesehen Thermodesuljobcicterium
Häufigkeit
einen
dieser
und der
höheren et
al.,
Actinomyceten
al..
wird
häufig
(Taylor
Quantifizierung
Bakterien und
1986;
in
nicht
aus
stabilere
Monoethern
von
Anteil Temperatur Fettsäure
den 1992).
von
jedoch
bei
Anteilen
et
Dobbs,
bis
von die nachgewiesen als
Parkes
als
lOMel6:0
vorkommender
(Langworthy,
Lebensräumen
al.,
möglich. und
Änderung
physikalischen
zu
identifiziert.
Gehalte
von Hydroxygruppen
Diese spezifische
der
ist
Anpassung
Etherbindung. 1986).
SRB
noch
Parkes, 34
als
und
das
in
1993).
äußeren
7,6
ändert
Kohlenstoffatomen
Biomarker
sind von
als Kombination
in
und
nicht Vorkommen Taylor,
durch
einzelner
So
der
%
(Rezanka
1983;
10 den
Bakterienpopulationen
commune.
charakteristisch
terrestrische der Als
(Gounot
Wachstumstemperatur Biomarker
ausmacht,
wurden
nachgewiesen
Organismengruppen 1983).
und
Während in
Membran
an
noch
unterschiedliche 1983,
SRB,
Biomarker Lipidmembranen Dowling
die
für
enthalten
physiologischen Fettsäuren
das
Für et
mit
von Desuljobacter
und
höheren
sonst
zum
1985).
al.,
wurde Die
für bildet
cycl7:0 lOMel6:0
Wachstum
Bakterien
lokalisiert
nicht-isoprenoiden,
wurden Russe!,
et
1990).
werden
SRB
(Boon Beispiel
Bakterien
nur im
für
Weiterhin in
ah,
bezeichnet
Spezifität
nachgewiesen,
sie
diversen
Gram-negative Vergleich
in
beschrieben
1986;
ein
geben.
1999; in
et
in
Kohlenstoffquellen Faktoren durch
oder
können
den
und
in
ungewöhnlich
Gram-positiven
(Wilkenson, gattungsspezifisches
al.,
des
Veränderungen
Arten
im
Aeckersberg Desiilfribacteriiim wurden
sogar
Cronan Desu[Jobacteriutn von
beschränkt Membranen
Bakterien
Das
1977;
man
in
thermophilen
thermophilen zu
und
situ
abhängig
i17:l der
verzweigten
Vorkommen
(Taylor daß
während
Estern
chemische
in
Edlund
Bakterien Aufschluß
Jr.,
Analysen
Gattung
einigen
wurden
sich
und
1988).
lange
1968).
et
SRB
von
und
sein den
(in
der der
des
al., die et
in Tabelle 3: Nomenklatur und die in dieser Arbeit verwendeten Abkürzungen sowie Strukturformeln bakterieller Fettsäuren. IUPAC Name Abkürzung* Strukturformel (Trivialname in Klammern)
Hexadecansäure 16:0 (Palmitinsäure) H0
0 cix -9-Hexadecensäure c9 16:1 — H0 (Palmitoleinsäure)
trans -9-Hexadecensäure t9 16:1 H0
cis-9. 10-Methylenhexadecansäure cyc 17:0 H0
14-Methylpentadecansäure i16:0 HO,_L_%/\./\__-\,._-\/\ 13-Methylpentadecansäure a16:0 1 lO-Methylhexadecansäure lOMel 6:0
15-Methyl-cis9-hexadecensäure ii 7:1 / HO/““““=\“sV\(/ 2-Hydroxyhexadecansäure 20H 16:0 11-10 *Die Abkürzung xn Y:Z enthält folgende Informationen: Y steht für die Anzahl der Kohlenstoffatome, Z für die
Anzahl an Doppelbindungen, deren Stellung in der Acylkette, ausgehend von der Carboxylgruppe, durch n
gekennzeichnet ist. Der Buchstabe x kennzeichnet eine cis (c)- oder rrans(t)- Doppelbindung. Die Vorsilbe cyc steht für einen Cyciopropanring in der Fettsäurenkette, während i(iso), a(anteiso) bzw. lOMe(10-Methyl) die Stellung der Methylgruppe innerhalb verzweigter Fettsäuren angibt.
6. Einfluß der Temperatur auf die Lipidfettsäurenzusammensetzung von Bakterien
Biologische Membranen bestehen aus einer Doppelschicht von amphiphilen Phospholipiden. In dieser fluiden Lipidmatrix befinden sich mosaikartig verteilt integrale und periphere Proteine. die in lateraler Richtung beweglich sind (Singer und Nicolson, 1972). Zur Beschreibung der Beweglichkeit von Molekülen innerhalb von Membranen wird gewöhnlich der Ausdruck Membranfluidität verwendet (Russe!, 1988).
11 Phospholipide liegen in der Natur in zwei unterschiedlichen Zuständen vor. Bei niedrigeren Temperaturen besitzen Phospholipide einen geordneten, gelartigen Zustand» während sie sich bei höheren Temperaturen in einem ungeordnetem, flüssig-kristallinen Zustand befinden. Die Temperatur, bei der die Phospholipide durch thermische Einwirkung von dem gelartigen in den flüssig-kristallinen Zustand i.ibergehen. wird als Phasenübergangstemperatur bezeichnet (Silvius, 1982). Diese Phasendbergangstemperatur ist abhängig von der chemischen Zusammensetzung der Phospholipide. In stoffwechselaktiven Zellen, in denen eine fluide Membran essentiell ist, müssen die Phospholipide somit überwiegend im flüssig-kristallinen Zustand vorliegen. d.h. die Phasenübergangstemperaturen der vorherrschenden Lipide müssen unterhalb der Wachstumstemperatur liegen (Jackson und Cronan, 1978). Eine Veränderung der Fettsäurezusammensetzung wirkt sich deutlich stärker als eine Veränderung der polaren Kopfgruppe auf die Phasentibergangstemperatur von Phospho!ipiden aus (Russe!, 1988). Die Phasenübergangstemperaturen künstlicher Lipide (Phosphatidylcholin). die aus unterschiedlichen Fettsäuren synthetisiert wurden, sind in Tabelle 4 dargestellt. Viele Eukaryoten und Prokaryoten sind in der Lage. ihre Lipidfettsäurenzusammensetzung bei wechselnden Umgebungstemperaturen zu verändern. Dies befähigt sie. die stark temperaturabhängige Membranfluidität in einem gewissen Temperaturbereich konstant zu halten. Beispielsweise reagieren viele Bakterien auf Temperatursenkungen mit einer Erhöhung des Anteils von Fettsäuren, die zu geringeren Phasentibergangstemperaturen der Lipide führen (Abb. 2). Dadurch wirken sie einer Abnahme der Membranfluidität bei niedrigeren Temperaturen entgegen (Gounot und Russe!, 1999).
Tabelle 4: Der Einfluß der Fettsäurenzusammensetzung in künstlich synthetisiertem Phosphatidylcholinen auf die Phasenübergangstemperatur. Das kunstliche Phosphatidylcholin besitzt jeweils zwei identische Fettsäuren.
Fettsäure Phasenübergangstemperatur (°C ) i16:0 22,0 a16:0 -3,0
16:0 41.5 cis9 16:1 -35,5 cyc 17:0 -19,9
17:0 48.8 18:0 55,8
‘Daten von Silvius (1982).
12 Die häufigste in Bakterien gefundene Regulation der Membranfluidität bei wechselnden Temperaturen ist eine Veränderung des Verhältnisses von gesättigten zu ungesättigten Fettsäuren (Gounot and Russel, 1999). Während die geraden Kohlenstoffketten gesättigter Fettsäuren optimal miteinander in hydrophobe Wechselwirkung treten können, verringern ungesättigte Fettsäuren mit eis-Doppelbindungen infolge des starren Knicks in den Kohlenstoffketten (Abb. 2) die Packungsdichte der Lipidschichten und erniedrigen so die Phasenübergangstemperatur. Einen ähnlichen, jedoch deutlich geringeren Effekt auf den Ordnungszustand der Lipide entsteht durch den Einbau von verzweigten Fettsäuren. Dabei besitzen Lipide mit anteiso-Methylverzweigungen niedrigere Phasenübergangstemperaturen als Fettsäuren mit iso-Methylverzweigungen (Tabelle 4; Russel, 1988). Ein weiterer temperaturabhängiger Regulationsmechanismus. der in einigen Bakterien zur Erhaltung der Membranfluidität dient, ist der Einbau von Fettsäuren mit unterschiedlich langen Kohlenstoffketten (Gounot und Russel, 1999). Kürzere Kohlenstoffketten besitzen untereinander geringere hydrophobe Wechselwirkungen als längere Ketten und führen somit zu geringeren Phasenübergangstemperaturen. Die Auswirkung der Kettenlänge von Lipidfettsäuren auf die Membranfluidität ist jedoch geringer als die Auswirkung des Sättigungs- oder Verzweigungsgrads (Tabelle 4). Ob und in welcher Art eine temperaturabhängige Veränderung der Lipidfettsäuren Zusammensetzung in Bakterien stattfindet, hängt von der jeweiligen enzymatischen Ausstattung des Organismus ab. Viele aerobe Bakterien besitzen sauerstoffabhängige Desaturasen, wodurch sie in der Lage sind, gesättigte Phopholipidfettsäuren in der Membran zu oxidieren und somit Doppelbindungen in diese einzufügen (Schweizer, 1989). Durch diese postsynthetische Modifikation können sie den Anteil von ungesättigten Fettsäuren direkt in der Membran erhöhen. Eine weitere Regulation der Membranfluidität, die direkt in den
Lipidschichten erfolgt, wurde in Angehörigen der Gattung Pseudomonas gefunden . Diese Bakterien besitzen Isomerasen, mit denen sie trans-Doppelbindungen in die fluiditätserhöhenden eis-Esomere umwandeln können (Okuyama, 1996). Isomerasen können die Reaktion auch von eis- zu trans-Doppelbindungen durchführen. Diese Umwandlung von eis- in trans- Isomere von ungesättigten Lipidfettsäuren wurde als Anpassung gegen sich in die Membran einlagernde, fluiditätserhöhende Substanzen beschrieben (Keweloh, 1995). Für die Regulation der Fettsäurenzusammensetzung durch Desaturasen oder Isomerasen ist kein Wachstum erforderlich.
13 Hohe Wachstums- temperatur
Abbildung
säuren-Synthase. Regulation temperatur
Syntheseweg Acylkette “Anaeroben
Hydroxydecanoyldehydratase
Die
meisten
2:
der
Beschriebene
Fettsäure
von
Fluidilät
verläuft
Bakterien
CFA-Synthase
Cyciopropan
cis-ungesättigte
Fettsäuren
10
Fettsauren M5
bei 3
Biosyntheseweg‘
Ä
Veränderungen die
Kohlenstoffatomen
unterschiedlichen
Isomerase
bilden
Bildung
den
/
/
ihre
trans-ungesättigte
weiteren
der
gesättigter
Desaturase
Neusynthese
Kurzkettige
Fettsäuren
Fettsäuren
Phospholipidfettsäuren
(Schweizer,
Lipidfettsäurenzusammensetzung
Wachstumstemperaturen.
Verlauf
14
gemeinsam,
und
1989).
zu
ungesättigter
Neusynthese Fettsäuren
Gesättigte
Verzweigte
Fettsäuren
einer
In
diesem
bevor
jedoch
gesättigten
CFA-Synthase —
von
Fettsäuren
sauerstoffunabhängigen
Hydrophobe
Polare
der über
ett
Bakterienrnembranen
sauren
oder
den
Enzymkomplex =
Kopfgruppe
Cyciopropanfett
ungesättigten
bis
sogenannten
zu
einer
zur
Veränderung
Kaneda, langsame gesättigten
(Schweizer, anschließend
Phospholipiden
Fettsäure
1991).
determiniert.
Anpassungsreaktion
in
1989).
des
in
die
in
die
Anteils
die
Man
jeweilige
Membran
Membran
Die
kann
kurzkettiger
jeweilige
daher
integriert,
ungesättigte
und
bezeichnen,
diese
somit
oder
Fettsäure
in
häufig
verzweigter
an
der
Fettsäure
15
Wachstum
die
keine
temperaturabhängige
wird
an
nachträgliche
Fettsäuren den
in
(oder
gekoppelt
die
Einbau
umgekehrt)
Phospholipide
(Gounot
von
ist.
Umwandlung
Veränderung
Gleiches
neusynthetisierten
und
stattfinden
eingebaut
Russel,
gilt
von
als
für
1999;
kann
einer
eine
und die 7.
Teil SRB
gemäßigten SRB dadurch stellen.
Bestandteile zellularen Sedimenten
sollte
Fettsäurenzusammensetzunge 1. Temperaturen verschiedenen, anderen
eingesetzt Anpassungsmechanismen 2.
Fettsäuren Fettsäuremuster phylogenetischen 3.
Organismengruppen
verschiedener die werden. Sedimente
Jadebusen). psychrophilen
der
Verteilung
spielen
isoliert,
an
Zielsetzung
Der
Zur
Bisher
Durch
marinen
wechselnde
psychrophilen,
Als
Fettsäuren
sollte werden.
Einfluß
verschiedener
als Charakterisierung Svalbards
in
Breiten
ein
und
die
untersucht, ist
marinen
der
in
ein
überwiegend
Klimabereiche
SRB) weiterer
ein
Habitate
jedoch
bei
Einordnungen
Reaktionen
situ
der
mikrobiellen
der
Gerade
der
chemotaxonomisches
Anforderungen
Gattungs-
weisen
nachgewiesen
von
solch
höhere
Arbeit
enthalten,
Analysen Temperatur Sedimenten
moderat
Einfluß
noch
Aspekt
durch
Kl
marinen permanent
die
innerhalb
niedrigen
imabereiche
dagegen
in
marinen
Anteile
nicht
der
von
oder
nach
wachstumsbegleitende
Lebensgemeinschaften
psychrophilen über
sollte den
sollten
als
SRB
der
von SRB
werden.
eine neuartigen
auf
an
bekannt,
sogar
jeweiligen
niedrige
die
der
der
Temperaturen
fluiditätserhöhender
untersucht
jahreszeitliche
die
die
Standorten
die
Phospholipidfettsäuren
sowohl
bedeutende
untersucht
16S
hat. Familie
wechselnden
spezifische
Kultivierung
Art-spezifisches
vorherrschende
Sedimente Merkmal
zellulare
Durch
rRNA
welchen
16
Temperaturen
Als
und
SRB
Wachstumstemperaturbereichen
in
werden,
DesuUobacteriaceae
diese
essentielle
mesophilen Reinkulturen
werden.
isoliert
Gensequenzen
ökologische
Fettsäurenzusammensetzung
sollte
wachsen.
für
Fettsäure-Analysen Einfluß
Fettsäuren
Temperaturschwankungen
in
gemäßigter
bei
Untersuchung
den
ob
Temperaturen
die
mikrobielle
Fettsäuren
wurden.
die Muster,
herrschen,
unterschiedlichen,
die
jeweiligen
phylogenetische
die
Zellbestandteile
SRB
Küstennahe
Bestimmung
permanent
als
Rolle.
in
deckt. Temperatur
von
Breiten gezeigt
das
auch
Zum
marinen
(wie
sollten Lebensgemeinschaft
bilden
wurden
Obwohl
sich
Sedimenten
SRB
direkt
verfolgt
einen
kalten,
und beispielsweise
werden,
Sedimente
(Wattsediment,
häufig
der
Hinweise
die
auf
und
erst
Sedimenten
Einordnung
sollten
im
lagen. in konstanten
wurde
mögliche
zellularen
zellularen werden.
von arktischen
zellulare
auf,
marinen
größten
wenige
anderen die
mit
erhalten
SRB
Zum
der
von
die
über
die
den die
in
isoliert 5.
Svalbard niedrigen
methanogene Durch
verschiedener Organismen
Reihen 4.
most-probable
Bakterien Ergebnissen
von sulfatreduzierender
Bakterienpopulationen
eine
Die
werden.
Durch
dienten
ließ
Wachstumstemperaturen.
aus
Entwicklung
selektive
der
Archaea
der
auf
Elektronendonatoren
klassische
number
kalten,
der
in
eine
jeweiligen
situ
Isolierung
Bakterien
Kultivierung
aus
mögliche
(MPN)
arktischen
Phospholipidfettsäuren-Analyse
mikrobiologische
von
permanent
verschiedener
und
Sedimente.
Methode
in
Gasbiasen
Methanogenese
den
anschließenden
Sedimenten
in
und
kaltem,
marinen
anoxischem,
angewendet.
Kohlenstoffquellen
in
Die stoffwechselphysiologischer
Methoden
17
marinem
gasdicht
Sedimenten
(Svalbard)
selektive
unter
Identifizierung
sulfatfreiem
Die
kalten
Sediment
verschlossenen
sollte
verglichen
hohen
Isolierung
erfolgte
untersucht
sollten
Bedingungen
das
Verdünnungsstufen
der
Medium
von
Vorkommen
werden.
erstmals
dabei
Arktis
numerisch
von
Sedimentproben
werden
Gruppen
unter
unter Zur
(4
kälteangepaßten
angereichert
kälteangepaßte
°C)
Abschätzung
und
aerober
dominanten
der
permanent
wurde
schließen.
der
mit
Zugabe
MPN
und
und
den
aus die B
1.
Einfluß Die von
Studien Abdollahi Weiterhin Wachstumertrags
Jørgensen, die
ausgewählten,
Wachstumstemperaturbereiche
mesophile
Mit 1.1.
Desuijorhopalus und Fettsäuren bei
des
Fettsäuren-zusammensetzungen. niedrige
D. Anpassung
mit kürzerkettigen
Lipiddoppelschicht kalten
Temperaturen,
zellulare gelida
unter
unterschiedlichen
Ausnahme
Optimums
weniger
De.vul/otalea
Ergebnisse
Einfluß marinen
Zellulare mesophilen
natürlichen
in
der
0 Phasen-übergangstemperaturen
den
et
SRB
mit
1999).
zeigten
mit
°C
Temperatur
an
Fettsäurezusammensetzung
al.,
als natürlichen der
nur
Acylketten bis
von suifatreduzierenden
unterteilt
überwiegend
ihre
zeigten
Fettsiiurenzusammensetzung
Fettsäuren,
in 1979;
In
bei
Temperatur 40
16 über
Lebensraum
SRB vacuolatus)
psychrophila)
Desuijofaba
Studien und
der SRB
verringert,
permanent
%
Temperaturen
Kohlenstoffatomen denen
vorliegenden lsaksen
100
ungesättigten
auf bei
werden.
diese
Diskussion
(lsaksen
Habitaten
von
verschiedenen
°C
die
die
die
marinen
der
überwiegend in
angesehen Arten
auf
(Isaksen
kann
gelida
dissimilatorische
und SRB kalten
Temperaturabhängigkeit Sulfatreduktionsraten
psychrophile,
Die
und und
die
die
innerhalb
und
Bakterien
Jørgensen.
Arbeit
ebenfalls
keine Fettsäuren
extrem
besaßen
hohen
Jørgensen,
zellulare
moderat
Lebensräume
hydrophoben
SRB
und von in
besitzen
werden.
in
Wachstumstemperaturen
Reinkulturen
16-18
wurde
signifikanten
JØrgensen,
SRB
18
den
ihrer
Anteile können
hohe
moderat
alle von als
psychrophilen
Fettsäurenzusanimensetzung
einen
1996;
Sulfatreduktion
Kohlenstoffatomen.
Acylketten.
(siehe
1996;
untersucht
erstmals besondere
Wachstumstemperaturbereiche
psychrophilen
psychrophilen,
Anteile
angesehen
an
hohen
aufgrund
konnte Wechselwirkungen
Knoblauch
psychrophile,
1996;
Sass gezeigt
Tabelle
cis-ungesättigter
Veränderungen
der
der
(>
Anteil
Anpassung (Publikation
et
JØrgensen
bereits
Der
Einfluß
(Desulfotalea
Wachstumsraten
70
al., werden
stattfinden
4),
(Desulfrfrigus werden.
ihrer
%)
psychrotoleranten
und
vermehrte
(>70
1998:
können
psychrotolerante
Nach
an
in
der
Jørgensen,
unterschiedlichen
(Jørgensen.
et
an
vorausgehenden
%)
cis-ungesättigten
Dagegen
Knoblauch
Temperatur
ihrer
dem
al,
Fettsäuren, 1).
kann,
den
innerhalb
als
an
arctica
von
1992).
Die Einbau
Wachstum oceanense
Fettsäuren,
permanent besondere zellularen
und
unterhalb
reichen
hierfür
1999).
besitzt 1977;
Der
und und
des
und
und
auf
von
die
der Die mesophilen SRB der Gattungen Desulfovibrio, Desulfococcus und Desulfosarcina, die die seltenen Fettsäuren i17:l und a17:l enthalten, zeigten ebenfalls nach dem Wachstum bei verschiedenen Temperaturen nur geringe Veränderungen innerhalb ihrer zellularen
Fettsäurenzusammensetzungen. Die relativen Anteile ungesättigter Fettsäuren sind mit 50 % in Desitlfovibrjo desulfuricans und 23-25 % in Desulfococcus multivorans und Desiilfosai-cinavariabilis deutlich unterhalb der Anteile, die in den psychrophilen SRB enthalten sind. Im Gegensatz dazu konnte in den mesophilen Arten der Gattung Desulfobacter (D. curvatus, D. hvdrogenophilus, D. latus und D. postagatei) mit Erniedrigung der Inkubationstemperatur eine Erhöhung der Anteile an cis-ungesättigten Fettsäuren gefunden werden (1-2 % pro °C). Die signifikantesten Veränderungen der zellularen Fettsäuren- Zusammensetzung bei unterschiedlichen Temperaturen wurden in D. hydrogenophilus nachgewiesen, das als einziges der untersuchten mesophilen SRB in der Lage ist, noch bei 0 °C zu wachsen. Es kann daher als psychrotolerant eingestuft werden.
1.2. Wachstumsabhängige Fettsäuremuster von Desulfobacter hydrogenophilus bei wechselnden Temperaturen
Weil bei D. hvdrogenophilus offensichtlich die Fähigkeit zur Regulation der Fettsäuren Zusammensetzung besonders ausgeprägt ist, wurden deren mögliche Änderungen während des Wachstumsverlaufs bei mehreren konstanten Temperaturen untersucht. Die zellularen
Fettsäuremuster von D. hydrogenophilus in unterschiedlichen Phasen des Wachstums in batch-Kulturen zeigten, daß sich bei mesophilen Temperaturen beim Übergang von der Wachstumsphase in die stationäre Phase die Anteile von cycl7:0 und lOMel6:0 auf Kosten von cis9 16:1 erhöhen. Es handelt sich vermutlich um dieselbe postsynthetische Modifikation durch Methylierung mit S-Adenosylmethionin, die bereits in anderen Bakterien gefunden und aufgeklärt wurde (Grogan und Cronan Jr., 1997). Bei Herabsetzung der Wachstumstemperatur verringerte sich der Grad der Methylierung. Bei 4 °C nahmen die Anteile von cycl7:0 und lOMel6:0 bis unter die Nachweisgrenze ab. Die zellulare Fettsäurenzusammensetzung von D. hydrogenophilus war also nicht nur abhängig von der Temperatur, sondern auch von der Wachstumsphase. Infolge der Inhibierung der Synthese von lOMel6:0 bei niedrigen Temperaturen ist die Anwendung dieser häufig als spezifischer Biomarker beschriebenen
Fettsäure (Findlay und Dobbs, 1993) in kalten, marinen Sedimenten fragwürdig. Mittels Zugabe von C-markiertem Acetat konnte gezeigt werden, daß die Erhöhung des Gehalts an cis9 16:1 in D.‘3 hvdrogenophilus bei Erniedrigung der Wachstumstemperatur durch 19 eine
Metabolismus einer
2. Inhibierung
Vergleichende
(Drzyzga machten phylogenetisch
und sowie
2.1.
der Aufgrund
Sequenzähnlichkeit Desiilfobacteriurn
Sequenzähnlichkeit s
2.2.
wie Stamm
Gegensatz joergensenii. wachsen.
in Sulfit mit
unterscheidet der
ulfatreduzierendem
Stamm
verwandten
ö-Unterklasse
de
Fettsäuren Erhöhung
Fumarat
phylogenetisch
die
Neueinordnung
comb.
als
Phylogenetische
Physiologische
novo
Sax
es
zellularen
der
et
Eine
Elektronenakzeptor
Sax
der
notwendig,
zu
und
al., nov.
Synthese
16S
Untersuchungen
sich
des von
Synthese neu oder
nachgewiesen D.
Kohlendioxidfixierung
und
SRB
D.
1993)
und
rRNA
Gehalts
phenolicum
der phenolicunz einzuordnen.
Stamm
Fettsäuren D.
phenoIicun
aromatische
mit
Bakterium, mit
Malat
wurden
verwandten
Beschreibung
den und
von
Proteobakterien
erfolgt.
hydrogenophilus.
Charakterisierung
von
dem
Desuifobacteriurn und Gensequenz-Analyse
an
Sax
ursprünglich Desulfobacterium
morphologische
durch
lOMel6:O
die
fluiditätserhöhenden
werden. Desulfobacterium
nächsten
nutzen. als
der
weist
morphologisch
Dieser Desuijobacula
kann jeweiligen
sind Als
Verbindungen
chemotaxonomisches
16S
Gärung
SRB
weitere
98,8 in
von
Stamm
Stamm
Neben
über
rRNA
und
Befund
Verwandten
der
dar.
als
Desulfobacterium Desulfotignum
%
Bei
physiologischen
cycl7:O
den
phenolicum.
Lage, DesulJoarculus mittels
zu
Charakteristika
Merkmale
Stamm
Ähnlichkeit
Gensequenzen 20 Sulfat
phenolicurn
Sax
Sax
stellt
toluolica,
von
niedrigen
ist Kohlenmonoxid-Dehydrogenase-Weg
wachsen.
vollständig
eine
Fettsäuren.
war
phenoIiiiin
mit
im
den
16S
sowie Stamm
konnte
Desulfospira
Sax
Einklang
in
Vielzahl
Merkmal
Wasserstoff
ovalen
rRNA
auf.
balticum
der
mit teilt
Die die
Temperaturen
als
Durch
Sax
spec.
Stamm
und
zu
von
von
Lage
Neusynthese Desulfobacula
der
phenolicum
maximal
16S
Gensequenz-Analyse Zellen
Kohlendioxid
eine
morphologischen
von
Stamm
bestimmt
SRB mit
(Bak
eingeordneten
vom
die joergensenii
gen.
mit
Sax
rRNA
oder
neue organischen
dem
der
bzw.
gerade
nächsten
und
Pyruvat,
auch
nov.,
Sax,
94,1
ö-Proteobakterien führen
Gattung
Formiat
strikt
(Publikation
Gensequenz
von
und vibrioiden
Widdel,
phenolica
D.
Thiosulfat
abzubauen.
sp.
%
Stäbchenform
cis9
phenolicuni
Stamm
jedoch
Desulfrspira
und
verwandten
anaeroben
16S
nov.
Substraten,
also
Merkmale
innerhalb
autotroph
16:1
93.9
rDNA
konnte 1986)
Zellen
eine
nicht
Sax
2).
oder
von
zu
Im
% 2.3. Chemotaxonomische Einordnung mittels zellularer Fettsäureanalyse
Das zeflulare Fettsäuremuster von Stamm Sax mit den markanten Fettsäuren lOMel6:O und cycl7:O zeigte große Ähnlichkeit mit Fettsäuremustern von Angehörigen der Gattung Desitifobacula und Desulfrbacter und unterstreicht damit die phyLogenetischeVerwandschaft zu diesen. Das Fettsäuremuster des phylogenetisch nächsten Verwandten, Desulfospira joergensenii, unterschied sich vor allem durch das Fehlen von lOMel6:O und das Vorhandensein von 3-Hydroxyfettsäuren vom Muster von Stamm Sax. Die große Ähnlichkeit
der zellularen Fettsäurenzusammensetzung zwischen D. phenolica und D. toluolica stimmt
mit ihrer engen phylogenetischen Beziehung überein und unterstützt die Einordnung in eine gemeinsame Gattung. Basierend auf den 16S rRNA Gensequenz-Analysen und unter Einbeziehung der morphologischen, physiologischen und chemotaxonomischen Merkmale stellt Stamm Sax eine neue Art innerhalb einer neuen Gattung dar. Es wurde der Name Desiiljotignu,n balticuni vorgeschlagen. Unter den gleichen Aspekten muß Desulfobacteriurn phenolicum in die Gattung Desulfobacula eingeordnet werden. Es wurde die neue Artbezeichn ung Desuijobacula phenolica vorgeschlagen. Durch den Nachweis von lOMel6:O im marinen SRB Desulfotignurn balticurn und der Gattung Desulfobacula ist die Spezifität dieser Fettsäure als Biomarker für Angehörige der Gattung Desulfobacter in marinen Sedimenten nicht mehr gegeben.
3. Charakterisierung der Mikroorganismenzusammensetzung von marinen Sedimenten mittels Phospholipidanalysen und Bestimmung der Lebendzellzahlen
Die Zusammensetzungen der aeroben und sulfatreduzierenden Mikroorganismengemein schaften von vier Fjorden Svalbards (Arktischer Ozean) wurden mittels der kultivierungsunabhängigen Analyse der Phospholipide und der Bestimmung der Lebendzellzahl untersucht (Publikation 3). Um mögliche Einflüsse der im arktischen Ozean vorherrschenden permanent niedrigen Temperaturen auf die dort vorkommende Mikroorganismengemeinschaft darzustellen, wurden die Ergebnisse mit denen aus Untersuchungen von Wattsediment des Jadebusen (Deutschland) verglichen (Publikation 4).
3.1. Tiefenprofile der Phospholipidfettsäuren
Die Analyse der Phospholipide und der in ihnen enthaltenden Fettsäuren bietet die Möglichkeit, ohne Kultivierung und ohne Selektion von Bakteriengruppen Aussagen über das
21 Vorkommen Dobbs, Schichten Phospholipiden als im
Phosphatkonzentrationen Svalbards unterschiedlich Organismengruppen spezifischen
mehrfach-ungesättigten typische werden. diesen
vermutlich verzweigten zeigte Mikroorganismenpopulation zeigten
Die Maxima (siehe Konzentrationen Dieser
numerische Desulfobacter nicht Durch
Dagegen
Zur
Große
Wattsediment
Die
beiden
die
unbedingt
Abschätzung
Bereichen
Publikation sich
1993).
Verteilungen
die
Befund
Unterschiede
PLFA
Diese
(0-1
Ähnlichkeiten
der
in
wurden
zeigten
Biomarker
aus
in niedrigen
von
und
Häufigkeit
den
cm).
Phospholipidfettsäuren
starken
gebundene beiden
durch kalten
der aerober
auf bestätigt
lebenden
Dominanz
wurde
anderen
i17:1,
des
obersten
1), deutlich dabei
Dabei
Verfügbarkeit eine
der
und
Sediments.
zugeordnet
niedrige zwischen
konnten
weisen terrestrischen
[-labitaten Konzentrationen
deren
die
PLFA,
Organismen
dieser
lebenden Sedimente
geringe
Häufigkeiten für
zwischen
mit
die Organismen
unterschiedliche lagen
Phosphat
größte
zu
der
höhere fakultativ
Anteil
Sedimentschichten der
in
auf
Temperaturen
in
Gattung
einer die
den die
Anzahl
Mit
molekularbiologischen
eukaryotischen
den
Tiefe
mit
Biomasse Biomasse
eine
werden
von
charakteristisch
analysiert.
Werte
in beiden
Eintrag
Sedimenten
von
Konzentrationen
in
zunehmender
kalten
Svalbards.
zunehmender
Reinkulturen in
der
und
geringe
Sauerstoff,
den
ab.
in
von
anaeroben ihren
(PLFA). lOMel6:0,
des
für den
können.
obligat
Die
Habitaten
aus pro oberen Profile. Sedimenten
in
SRB
22
inhibiert
Wattsediments Sowohl
SRB
natürlichen
den
Anzahl Sedimenten
den
g
Sedimente
aus
der Trockengewicht
und
anaerobe
nachgewiesen Tiefe
organischem
für In
beiden
Bakterien
nicht Sedimentschichten spezifischen
die angrenzenden
So
Tiefe
Die
Svalbard
deren
Familie
in
wird
prokaryotischen zeigten
eukaryotische
dieser
SRB
beiden
konnten
nur
Untersuchungen erhöhte
den
verschiedenen
von
Ursachen
Habitaten
marinen
ein
aus
Svalbards (siehe
in Synthese Bakterien
der
Sedimenten
der etwa
dominierten Desulfobacteriaceae
Biomarker
geringen
und
Wechsel
auch
vier
Gattung
sich
PLFA
[labitaten Material
Temperatur
die
Gletschern.
Sediment
Publikation
um
werden.
denen
Habitaten
zu
verschiedenen
liegen
die
der
Organismen
größten
spezifische
in
das
(Ravenschlag,
(0-1 treffen
i17:1
Konzentrationen
Aerobier
von
Svaibards
relative
Tiefenprofile Desulfovibrio
ermittelte,
SRB
Gemeinschaft.
des
physiologischen
und
im
Zehnfache
vermutlich cm)
in
Die
beeinflußt
und
nahmen
einer
wurde
Wattenmeers.
Wattsediment
(Findlay
den
Anteile von
1),
der
festgestellt
Anteil
PLFA.
lOMel6:0
niedrigen
sind,
schließen. kann
resultiert
obersten
als
Licht Fjorden
aeroben
Gattung
geringe
das
höher
2000).
auch
und
wird
von
von
und die
man
von
hin. am
mit
in
So
in gefunden. Die Tiefenprofile der relativen Anteile von i17:l und lOMel6:0 zeigten eine weitgehend konstante Verteilung der Biomarker unterhalb der oxischen Sedimentschicht von 0,5 cm.
Die unterschiedlichen Temperaturen zur Zeit der Probenentnahme von etwa 0 °C in den
Sedimenten Svalbards und 17 °C des Wattsediments hatten offensichtlich auf die PLFA Zusammensetzung nur geringfügigen Einfluß. Die relativen Anteile an ungesättigten PLFA waren in beiden Habitaten in der obersten, oxischen Schicht mit etwa 60 % am häufigsten und nahmen mit der Tiefe ab. Unterschiede hinsichtlich der Länge oder der Verzweigung der Acylketten der PLFA konnten ebenfalls nicht festgestellt werden.
3.2. Abschätzung der Bakterienzahlen mittels der MPN-Methode
In guter Übereinstimmung mit den PLFA-Profilen wurden in den kalten Sedimenten Svalbards die höchsten MPN-Zahlen von aeroben heterotrophen Bakterien und von SRB (mit Formiat oder Wasserstoff als Elektronendonator) in der obersten Sedimentschicht gefunden.
Während die Zahl der aeroben Bakterien von 4,6 . l0 Zellen 3cm in der obersten Schicht auf 2,4 Zellen• 3cm in 5-6 cm Tiefe abnahm, zeigten die SRB nur eine geringe Abnahme mit zunehmender Tiefe. Die ermittelten SRB Zahlen bei 4 °C aus Svalbard mit einem Maximum von 2,4 io Zellen 3cm nach 18 Monaten Inkubation waren um etwa zwei Zehnerpotenzen geringer als die maximalen Werte aus dem Watt (20 °C, 10 Monate Inkubation). Die MPN Zahlen der SRB aus Svalbard liegen im gleichen Größenbereich wie vorhergehende MPN Bestimmungen in verschiedenen Sedimenten Svalbards, die mit den Substraten Lactat, Propionat oder Acetat durchgeführt wurden (Knoblauch et al., 1999b). Die Abschätzungen der Bakterienzahlen mittels der MPN Methode liegen jedoch unter denen, die mittels molekularer Methoden ermittelt wurden und wohl eher die natürliche Populationsgröße widerspiegeln. Die deutlich geringeren MPN Zahlen sind zum einen in den selektiven Kulturbedingungen und zum anderen in der Tendenz der marinen SRB zur Aggregatbildung zu sehen.
3.3. Phylogenetische Einordnung der neuisolierten Reinkulturen aus Svalbard
Aliquots aus den höchsten MPN Verdünnungsröhrchen, in denen Wachstum festgestellt wurde, dienten als Inoculum für Isolierungsansätze. Dadurch sollten die am Standort häufigsten vorkommenden Bakterien der jeweiligen stoffwechselphysiologischen Gruppe isoliert werden. Aus dem permanent kalten Sedimenten Svalbards konnten durch wiederholtes
23 werden. Ausstreichen
Pseudoinonas schon
(Morita, psvchrophila, Smeerenburgfjord als
Hybridisierung beiden 16S
psychrophil
diesem verschiedenen
Wasserstoff Sedimenten
sie Smeerenburgfjorden
Stamm singaporensis.
Svalbard Desulforhopalus
einer
zeigt D.
eingeordnet Desulfobulbaceae ein
nächsten
zeigt Bande,
1996).
c‘atecholicum Elektronendonator
rRNA
neue
Angehöriger
eine von
weiterhin
Klon-Sequenz
bereits
Die
Habitat
Dt-tAl
Stamm
2000).
die
Taxa
Verwandten
anderen
Gensequenz-Analysen
(Sahm
enge
aus
Isolate
werden.
eingeordnet
als
auf
stammen
zugeordenet
organischen
aus
nachgewiesen
Die teilt bekannten
Zusammen
innerhalb der JHAI
(Knoblauch
Verwandtschaft
98,6
Elektronendonator
vacuolatus
der
Agarpiatten
et
Arbeitsgruppen
wurden
y-Proteobakterien ist
Wassersäule
lsfjorden
dabei
mittels
(JHAI)
und
Der
al.,
Desulfobacteriaceae
%
stellt
Desulfobacula
bisher
wurden
aus
ebenfalls
Ähnlichkeit
Stamm
99,9 (Knoblauch
der könnte
1999)
Substraten
Arten
werden. in
Svalbard-Sediment bilden
aufgrund
isoliert
et
Fluoreszenz (Ravenschlag,
abtrennen
einem
der
Familien
21
%
(Stamm ausschließlich
al.,
des
mit
konnten
JHAI dieser und aus
Sequenzähnlichkeit
einzige
aus
gemeinsam
Reinkulturen
diese
werden. 1999b). Dabei
komplexen
Manager
der
inkubiert wurden
wachsenden
mit
phenolica. marinem
der
95,9
permanent
et
läßt.
ist
Gattung,
Desitlfobulbaceae
von
diese
DHAI),
einer
phylogenetischen
einen
al.,
handelt
aufgrund
vollständig-oxidierende
in 2000).
%
und
Aufgrund Aus
Die
ebenfalls
Desulfobacteriurn
24
Fjords
sita
SRB
den
Sequenzähnlichkeit 1999a).
Sediment
mit
wurden,
partiellen
Medium
(Sva0999,
aerober,
teilt Cluster,
Die
16S
den
kalten,
SRB
Desuijotczlea
Aus
Gattungen
es
Hybridisierung
Krossfjorden der
Stamm
seiner
(Dänemark)
rRNA
16S
MPN-Verdünnungsröhrchen,
sich
94,8 mit
ihrer
in
bestätigt den
Der
Gattung
bei
marinen
großer
konnten
der
rDNA einem
16S der heterotropher
um anaeroben
%
16S
16S
Untersuchungen
Gensequenz 4 97,3
erneute
G1HA
bzw.
Küste Moritella,
°C
Sequenzähnlichkeit
sich
rDNA y-Proteobakterien,
rRNA
ihre
rRNA
Häufigkeit
catecholicum
arctua
Sequenz Desulfotalea
Isolat
isoliert
%) kultiviert.
Habitaten
Desul[obacteraceae.
bisher
(Stamm
Vertreter
Svalbards vermutete
als
mit
und
in
Nachweis
(Ravenschlag.
Ansätzen
Sequenz
Gensequenz
Gensequenzen
(Stamm
eine
Psvchrotnonas
neue
wurde
Bakterien
Desulforhopalits
von
und
rRNA
drei von
in
Durch
isoliert
ebenfalls
Stamm
neue
Sediment
GIHA
innerhalb
Häufigkeit Gattung (97,2
Desulfotalea
Stamm
und isoliert.
einer mit
(Teske
Isolate
LSv53)
dieser
siot
die
partielle
eindeutig
Formiat mit sind
wurden
isoliert
Gattung
die
%)
GIHA
2000). bilden
DGGE
auch
JHA1
et
einen
blot und
Die des
und von
dem
Der
auf
von
mit
und
aus
als
der
al.,
in Kandidaten für die Etablierung einer neuen Gattung dar. Für eine vollständige Einordnung der neuen Isolate sind jedoch weitere, detailiertere Charakterisierungen notwendig.
4. Anreicherung und Isolierung von methanogenen Archaea aus permanent kalten, marinen Sedimenten (Svalbard)
Diese Arbeit liegt bislang nicht jn Form eines Manuskriptes vor. Einige Versuche wurden von Christine Selz in Rahmen eines Studienprojekts durchgeführt. Die phylo genetische Charakterisierung wurde gemeinsam mit Enrique Liobet-Brossa (MPJ für marine Mikrobiologie) durchgeführt. Methanogene Archaea sind vorwiegend in anoxischen Süßwasserhabitaten von ökologischer Relevanz (Cavicchioli et al., 2000). Jedoch haben Studien in den letzten Jahren mittels biochemischer und molekularbiologischer Methoden gezeigt, daß die Archaea auch in kalten marinen Standorten abundant und an verschiedenen geochemischen Prozessen, wie beispielsweise der anaeroben Oxidation von Methan, beteiligt sein könnten (DeLong et al., 1994, 1999; Sahm and Berninger, 1998; Boetius et al, 2000). Bisher ist es einzig Franzmann und seinen Mitarbeitern gelungen, eine moderat psychrophile und eine psychrophile methanogene Reinkultur aus der Antarktis zu isolieren. Dabei handelt es sich um
Methanococcoides burtonjj (Tmn= -2,5 °C, 0T = 23 °C) und Methanogeniumfrigidum (Tmrn -10 °C, 0T = 15 °C). Sie wurden aus methanhaltigem, anoxischem Wasser des Ace Lake isoliert, das eine konstant niedrige Temperatur von 1-2 °C besitzt (Franzmann et al., 1992, 1997). Weitere Standorte, in denen die Methanogenese bei niedrigen Bedingungen nachgewiesen wurde, sind kalte, sumpfige Böden in Russland und der Bodensee (Zhilina und Zavazin, 1991; Schulz und Conrad, 1996). Reinkulturen methanogener Archaea, die noch bei niedrigen Temperaturen wachsen, konnten aus letzteren Habitaten noch nicht isoliert werden.
4.1. Anreicherung und Isolierung
Mit einer Sedimentprobe aus dem Hafenbereich von Ny-Älesund (Svalbard) wurden Kulturen für die Anreicherung von Methanogenen beimpft. Das selektive Medium entsprach hinsichtlich der Zusammensetzung der gelösten Salze weitgehend dem Meerwasser. Zur Unterdrückung des Wachstums von SRB wurde lediglich das üblicherweise zugesetzte Magnesiumsulfat durch eine aquimolare Menge Magnesiumchlorid ersetzt. Als Energie- und Kohlenstoffquelle wurden Verbindungen gewählt, die von Methanogenen verwertet werden können (Wasserstoff + Kohlendioxid, Formiat, Acetat und Methanol). Nach vier Monaten Inkubation bei einer Temperatur von 4 °C konnte in den Ansätzen mit Methanol eine
25 Methanbildung
Verwendung erkennbar, Anregung
Abbildung
mit
erfolgreich.
bisher Hemmung
neben Propionat,
zugesetzt Substratverwertung nicht
2 untersuchten
gebildeten
Inkubation Menge,
Versuche
Methanol
Monaten
verwertet
sieben den
die
des 3:
die
wurden,
des bei
Capronat,
bei bereits Autofluoreszenzinikroskopische
Methans Mittels
nach
der
zur
Inkubation
überwiegend
Zeitraums
für
4
mittels
Reinkulturen
Wachstums
°C.
12
wurde,
Isolierung
Epi-Autofluoreszenzmikroskopie
Methanogene
9
Der
°C
zeigten,
untersuchten Monaten aufeinander
bestanden.
Lactat.
entsprach 1,2 gaschromatographischer
eingezeichnete
von
wurden
des
%
in
von
dieser
daß
9
der
isoliert
Aggregaten
gebildet
Palmitat,
Ansatzes
Monaten
Wähiencl
Bakterien spezifischen
die
folgender
zwischen
Menge.
Wasserstoff,
Substraten
Maßstab
Methanogenen
gebildete
werden.
wurde.
Aufnahme
Benzoat)
bei
mit
vorlagen
die
Benzoat
entspricht
zugesetzt
Fltissigverdünnungsreihen,
4
beiden
Methanol Co-Faktors
und
Menge
nach
In
Weitere verschiedene
26
Methoden
Trimethylamin
den der
oder
12
(Abb.
als
über
12
Temperaturen
9
Ani
wurde,
Ansätzen
(Doddema
Methan
um.
°C
Monaten
einziges
Trimethylamin
eicherungskultui
Anreicherungsexperimente,
Agarverdünnungsreihen bei
3).
keine
F42,
nachgewiesen
konnten
4 organische
nach
waren
mit
°C
der
Unterschiede
gebildet
und
und
vollständig
2
Triniethylamin entsprach
zugesetzten
Monaten
aus
fluoreszierende Methanol
von
als
denen
Vogels,
wurde.
Fettsäuren werden.
den
methanogenen
mögliche
Anreicherungen
umgesetzt.
hinsichtlich
Antibiotika
bereits
die
Verbindungen
innerhalb
Während
1978)
waren
Durch
entsprachen Menge
Substrate
in
(Butyrat,
53 Zellen
Aichaea
denen
nicht
zur
%
die
Nach
zur
des der
des
der
dci die Mengen Methan nach 2 Monaten bei 4 °C 16 % und bei 12 °C 56 % der Menge nach vollständigem Substratumsatz. Die methanogene Umsetzung der eingesetzten Substrate verläuft offensichtlich bei 4 °C langsamer als 12 °C, obwohl die in situ Temperatur der Probenentnahmestelle -0,5 °C betrug. Die optimale Temperatur der Methanogenese wurde in diesem Sediment nicht bestimmt.
4.2. Morphologische und physiologische Charakterisierung
Die neu isolierten methanogenen Archaea wiesen alle eine coccoide Zellform mit einem Durchmesser zwischen 0,8 und 1,7 um auf. Zwei der sieben Isolate (l6SvalB und l6Svall) wuchsen als einzelne Zellen, während die Zellen der anderen Stämme (4Svall, 4Sval2, l6Sval2, l6SvalA und I2TMAI) Aggregate bildeten. Bei Anregung des für Methanogenen spezifischen Co-Faktors )F47 zeigten alle Isolate eine deutlich sichtbare Autofluoreszenz. Die Untersuchung des Substratspektrums ergab, daß die Stämme l6Sval(A), l6Sval(B) und I2TMA1 lediglich Methanol und Trimethylamin verwerten können. Dimethylsulfid wurde von keinem der Stämme zu Methan umgesetzt. Die Umsetzung verläuft vermutlich, wie in anderen methylotrophen Methanogenen beschrieben, durch eine Disproportionierung gemäß folgender Gleichungen (Zehnder, 1993): 3OH4CH -‘ 3CH+CO,+2H,O 4 )3NW(CH +6 H,O -4 49 4CH +3 2CO +4 4NH Die Zugabe von Biotin war für das Wachstum von l6Sval(A), l6Sval(B) und I2TMAI essentiell, während Thiamin das Wachstum beschleunigte, jedoch nicht absolut notwendig war.
4.3. Phylogenetische Einordnung
Durch Klonierung und Sequenzierung von 16S rRNA Genen konnten die Isolate l6Sval(A) und l6Sval(B) zwei Verwandtschaftsgruppen innerhalb der Methanosarcinaceae zugeordnet werden (Abb. 4). Zu den anderen Isolaten lagen zur Zeit der Fertigstellung der Dissertation noch keine Ergebnisse vor. Der Stamm l6Sval(A) zeigt sehr große Ähnlichkeit (>99 %) mit dem mesophilen Archaeon Methanolobus taylorii. Alle bisher bekannten Angehörigen der Gattung Methanolobus stammen aus marinen Sedimenten und verwerten ebenfalls nur Methylgruppen-enthaltende Verbindungen. Sie sind mesophil ,0(T = 28—40°C) und im Gegensatz zu Stamm l6SvaI(A) nicht in der Lage, bei Temperaturen unterhalb von 7 °C zu
27 wachsen Stamm aus 23
theoretische auf 5.6
Methanogeniiimfrigidurn (Franzmann
sulfidreichen
Archaeon Kohlendioxid
Gensequenzen. Abbildung
Aminoverbindungen
Methylamin-Oxiden Substrate begünstigten (Oremland
°C
i‘1ethan
Methylamine
Methanol °C,
einem
A4ethanosczeta
(Franzmann,
l6Sval(B)
jedoch
A4ethanolohus
(Kadam
“Ivlethanolohus
olohus
in
permanent
4:
stehen
et
Tmn
Reinkultur,
Phylogenetische
Methanolohus
et und
al.,
(8
oder
SRB
wurde
homhaensis
Iv!
von
al.,
mM)
und
Methylamine
ist
1982; sind
ethanolohus
concilii /
1992).
die
mit
und
Methanococcoides —
tindarjus
kalten,
und
Boone,
1992).
(z.B. 2,5 mittels
oregone!;sis“
Ace
unter
Methanogenen
das
Zinder,
Formiat
können
(Franzmann
Die
in
°C
vulcani
Einordnung
noch —
Lake
marinen
terrestrischen Cholin
1995).
tavlorii
Das
anderem errechnet.
geringste
des
begrenzt.
1993).
bei
optimal
mit
(Antarktis) einzige
Quadratwurzel-Modells
Der
0
und
der
Sedimenten
°C
et
diesen
Temperatur,
Produkte
nicht
burtonii
Das
nächste
Stlimme Wachstum
al.,
wachsen
Betain),
bei
va Gewässer
bisher
Substratspektrum
28
1997).
in
in
15 1
isoliert
l6SvaI(A)
phylogenetisch
(Ähnlichkeit
sulfatreichen
°C.
Substratkonkurrenz
des
weit
kann.
sowie
bekannte
auf
bei
M.
der
M.
anaeorben
verbreitet.
Wasserstoff der
und
frigidurn
Metha,zosarci,za
Antarktis
und
frigidunz
der
Wachstum
(Ratkowsky
wächst
l6Sval(B)
Methanohalohiurn
>
“Methanococcoides
psychrophile
von PvI‘ethanococcoides
mikrobiellen
Lebensräumen
16
99
Verwandte
Abbaus wurde
Durch
Sval
M.
%).
und
wurde
ist
mit
burtonii
aufgrund
beobachtet
mit
M.
wächst
(B)
das
et bei
die
Wasserstoff
von
burtonii nicht
den
al., Organismus
bisher
Reduktion
Nutzung
8
Archaeon
ihrer
ei‘estigatuin
°C
ist
methylierten
optimal
co-existieren
burtonii
1983)
beobachtet
‚nethvlutens“
energetisch
wurde
ebenfalls
aus
16S
stammt
einzige
dieser
eine
dem
rRNA
und
bei
zu
von ist
ist Im zeitlichen Rahmen dieser Arbeit konnten keine weiteren Versuche zur Charakterisierung oder hinsichtlich möglicher Kälteanpassungen der neuisolierten Methanogenen durchgeführt werden. Die neuisolierten Stämme sind die ersten marinen methanogenen Archaea, die aus permanent kaltem, arktischem Sediment in Reinkultur isoliert wurden und die Fähigkeit besitzen, noch bei 4 °C zu wachsen.
29 Abdollahl, C
Aeckersberg,
Bak,
Balba,
Boetius,
Boon,
Boschker,
Canfield,
Cavicchioli,
Cronan Cypionka,
DeLong,
alkanes fatty
Desulfobacteriu,n
in
Amann, consortium
taxonomic Desulf(wibrio 1415-1422. 2001. bacterial in
and
(eds.).
Extremophiles
Bacteriol.
archaea
827-848.
F. anoxic
anoxic
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33 Ravenschlag, Reichert, Revsbech, Rezenka, Russel, Sagemann, Sahm, Sahm, Sass, Schulz, Schweizer, Silvius, psychrophilic oxygen 411. Desulfotomaculuni, pp. Microbial sulfate Ocean). structure of Desulfovibrio Microbiol. Lebensgemeinschaften Microbiol. Psychrotolerant Microbiol. production Unviversität Ratledge. their Griffith, psychrophilic H., 279-365. K. K. N.J. J.R. modification S. Berchthold, reduction und W. , T., in N.P., Marine Knoblauch, J., of OH. E. und 1989. lipids. C. marine 65: 21: and Ecol. Solokov, 1982. benthic Berninger, Jørgensen, K. in 1989. Bremen. and bacteria. Sørensen, (eds.) 3976-3981. 212-219. cuneatus Conrad, Functions the Ecol. sulfate-reducing Morita, sulfate-reducing Ratledge, 2000. in 20: Wilkenson, sediments Thermotropic Biosynthesis cold a prokaryotes M., by permanently New sulfate-reducing 1-14. C. M.Y. Progr. J. membrane Branke, U.-G. B.B, sediment und Gen. in J., sp. R. Molekularbiologische R.Y. of York: C. and marinen, Ser. measured Blackburn, lipids: and Amann, S.G. and 1996. Microbiol. nov. 1998. from 1982. of Springer phase J., bacteria 165: Viden, isolates Wilkenson, Greef, of (eds.) cold fatty proteins. Structural and Konig, Abundance, Svalbard, Influence permanently bacterium. 7 Temperature R. arktischen with transition 1-80. profundal acids London: 0. Desuljovibrio 128: in I. 1999. from T.H. Verlag, 34 J., marine microelectrodes. 1990. 1998. In: S.G. roles 565-568. and Arctic Cypionka, an und Phylogenetic of Lipid-protein FEMS vertical Analyse Academic Vol. of oxic cold Temperature related Sedimenten (eds.) Unusual temperature sediment and Arctic characteristics Lomholt, pure ocean. 2. marine freshwater membrane Microbiol. litoralis London: distribution, pp. compounds. sediments. lipids H. und Press, Geomicrobiol. and 241-28 affiliation Limnol. and of sediments J.P. interactions. dependence (Svalbard). in on very-long Lake sp. Academic Struktur Babenzien, sediment, functions. Vol.2, Ecol. of model 1. 1980. pathways AppI. and nov.. psychrotrophic Oceanogr. In: and Constance. 73: (Svalbard. pp. community Microbial Distribution membranes J. Environ. quantification and fatty Jost, Sytem. Press, 23 description Dissertation. 3-50. In: mikrobieller 15: H.-D. 1-238. of rates 25: 85-100. acids P.C. Vol. methan Arctic FEMS lipids, Appi. 1998. 403- of and and and of 2, in of Singer, S.J. und Nicolson, G.L. 1972. the fluid mosaic model of the structure of cell
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Sommer, U. 1998. Biologische Meereskunde. Springer-Verlag, Berlin, Heidelberg, New York.
Taylor, J. and Parkes, R.J. 1983. The cellular fatty acids of the sulphate-reducing bacteria,
Desuijobacter sp., Desu1Jbulbiis sp. and Desulfrvibrio desu1fii-icans. J. Gen. Microbiol. 129: 3303-3309 Taylor, J. and Parkes, R.J. 1985. Identifying different populations of sulphate-reducing bacteria within marine sediment systems, using fatty acid biomarkers. J.Gen. Microbiol. 131: 631-642. Teske, A., Wawer, C., Muyzer, G. und Ramsing, N.B. 1996. Distribution of sulfate- reducing bacteria in a stratified fjord (Manager Fjord, Denmark) as evaluated by most probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments. Appi. Environ. Microbiol. 62: 1405-1415. Teske, A. Ramsing, N.B., Habicht, K., Fukui, M., Kuever, J. und Jorgensen, B.B. 1998. Sulfate-reducing bacteria and their activities in cyanobacterial mats of Solar Lake (Sinai, Egypt). Appl. Environ. Microbiol. 64: 2943-2951. Tsitko, I.V., Zaitsev, G.M., Lobanok, A.G. und Salkinoja-Salonen, M.S. 1999. Effect of aromatic compounds on cellular fatty acid composition of Rhodococcus opacus. Appi. Environ. Microbiol. 65: 853-855. Vainshtein, M., Hippe, H. and Kroppenstedt, R.M. 1992. Cellular fatty acid composition of Desulfovibrio species and its use in classification of sulfate-reducing bacteria. Syst.
Appl. Microbiol. 15: 554-566.
Widdel, F. 1987. New types of acetate-oxidizing, sulfate-reducing Desulfobacter species, D. hvdrogenophilus sp. nov., D. latus sp. nov., and D. curvatus sp. nov.. Arch. Microbiol. 148: 286-291. Wiegel, J. 1990. Temperature spans for growth: hypothesis and discussion. FEMS Microbiol. Rev. 75: 155-170.
35 Wieringa,
Wilkenson,
Zhilina,
Zinder
approach. reducing
Wilkenson,
Doklady (ed.)
New
S.H.
T.N.
E.B.A.,
bacteria Akademii
SG.
Environ.
York,
1993.
und
S.G.
1988.
Zavazin,
Overmann,
(eds.) London:
Physiological
in
Microbiol.
Nauk
marine
London
Gram-negative
SSSR
Chapman
G.
oxic
J.
1991.
2:
Academic
ecology
und
(Moskva)
4 sediment
17-427.
Low
and
Cypionka,
bacteria.
Hall
of
temperature
Press,
5:
layers
methanogens.
36
1242-1245.
Inc..
H.
Vol.1,
by
In:
pp.
2000.
a
methan
Microbial
128-206. combined
pp.
Detection
In:
299-457
production
Methanogenesis,
cultivation
lipids,
of
abundant
Ratledge,
by
and
a
pure
Ferry.
molecular
sulphate-
culture.
C.
J.G.
and
Tell II: Publikationen
A Publikationsliste mit Erlauterungen
Die vorliegende Dissertation beruht zum groI3tenTeil auf den folgenden Publikationen. Die angefiigten Erläuterungen stellen den eigenen Beitrag an derjeweiligen Arbeit dar.
1. Effect of temperature on the composition of cellular fatty acids in sulphate-reducing bacteria.
Martin Könneke and Fnedrich Widdel
In preparation
Entwicklung des Konzepts und DurchfUhrung aller mikrobiologischen und analytischen
Arbeiten. Gemeinsame Erstellung des Manuskripts mit F. Widdel.
2. Reclassification of Desulfobacterium pheizolicuin as “Des ulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignurn balticuin gen.nov., sp. nov..
Jan Kuever, Martin Könneke. Alexander Galushko and Oliver Drzyzga (2001) hit. J. Syst. Evol, Microbiol. 51, 171-177
Durchfi.ihrungder mikrobiologischen und analytisehen Arbeiten zur Bestimmung der zellularen Fettsäuren. Redaktionelle Mitarbeit bei der Erstellung des Manuskripts.
3. Aerobic and sulfate-reducing bacterial communities of Arctic sediments characterized by phospholipid analysis and cultivation methods.
Martin Könneke, Jan Kuever and Bo Barker Jørgensen
Enpreparation lnitiierung des Projekts durch B.B. Jørgensen. Entwicklung des Konzepts und Durchfuhrung der mikrobiologischen und analytischen Arbeiten. Erstellung des Manuskripts unter redaktionellen Mitwirken von J. Kuever und B.B. Jørgensen.
38
Analyse.
Beteiligung
4.
Lininologv Community
Rudolf
Schrarnm. A
Enrique
intertidal
Redaktionelle
multi-methods
an
Amann
der
Liobet-Brossa,
Rikke
and
surface-sediment:
Entwicklung
structure
Oceanography,
Mitarbeit
L.
approach
Meyer,
and
Raif
des
bei
Niko
activity
Konzepts Rabus,
der
submitted
Finke,
Erstellung
Michael
of
39
und
sulfate-reducing
Stefan
Durchfuhrung
des
E.
Götzschel,
Böttcher,
Manuskripts.
bacteria
Martin
der
Ramon
Phospholipidfettsauren
Könneke,
Rosselló-Mora
in
an
Andreas and Fax
*For
Effect
(+49)
Max-Planck-Institiite
correspondence.
421
of
2028
temperature
acids
790.
Martin
for
in
Alarine
sulphate-reducing
Könneke
on
Microbiology,
En
the
Gertnanv.
preparation
and
1
40
composition
Friedrich
Celsiitsstrasse
Tel.
Widdel*
(+49)
bacteria
421
of
1,
D-28359
cellular
2028
702:
Bremen.
fatty
temperature,
acid,
niethylenehexadecanoic postsynthetic
anaerobic
with unsaturated
increases
but mesophile
composition
observed
was
decreased. four
(measured The
increases psychrophilic
in Abstract
twelve
also
effect
decreased.
Desulfobacter
psychrophiles.
presumably
during
in
species
(by
of
in
(growth
in
In
metabolism.
and
psychrophilic
but the
were
modification
late
the
<3%
contrast,
and
growth
The
saturated
growth
not
(of
from
growth
relative
mesophilic
species,
per
measured
range
Isotope
by
at
eight
in
cis-9-hexadecenoic
low
Desulfobacter
far
acid
10°C)
temperature
batch
phase)
0—35°C)
genera)
species
fatty
Furthermore,
the
temperature.
but
amount
highest
(‘ 3 C)
(a
species
of
SRB
with
cultures
to
cyclopropane
acids
significantly
cis-unsaturated
labelling
(>70%).
a
of
among
content
(including
D.
de
of
on
sulphate-reducing
species
in
mostly
at
hydrogenophilus,
the
cis-9-hexadecenoic
this
novo
41
acid,
constant
of
Time
the
D.
of
cellular
species
increased
of
D.
(by
synthesis,
fatty
cis-unsaturated
all
toward
examined
hydrogenophilus
marine
hydrogenophilus
courses
psychrophiles)
>14%
fatty
temperature;
fatty
acid)
varied
the
the
bacteria
acids
origin.
per
as
acid
of
and
species.
end
not
the
amount
generally
changes
acid
10°C)
composition
if
lO-methyihexadecanoic
fatty
only
of
most
With
the
this
(SRB);
exhibited
confirmed
growth
are
The
if
formed
with of
acids
temperature
was
of
psychrotolerant
the
the
observed
cis-unsaturated
not
ratio
the
they
temperature,
not
temperature
exception
at
was
was
only
due
fatty
moderate
that
observed
cis-9,10-
included
between
studied
always
minor
in
to
acid
that
was
an
of a Introduction reducing compounds By
progress based) 2000). reducing (www.tigr.org). and parameters. the
organisms
Effects reduction physiological
sediments
exhibits certain growth lsaksen and effects and cold-adapted Jørgensen,
as Bacillus The decrease
the
availability
well
An
Cellular
regulatory
growth Jørgensen.
cytoplasmic
systematics,
use
on
The
rates
suboptimal
of
important and
as
during typical
bacteria bacteria
subtilis
their
rate,
or the
is
temperature
of
1999). in in
yields e.g.
Jørgensen.
mechanisms
genome
cultures
in
the
SRB sulphate
efficiency
marine
microorganisms
fluidity
parameters. of
mechanisms
the
cultures However, 1999:
the
asymmetric
in
frequent (Grau
(for
electron (SRB)
membrane
do
oxygen environmental
has
growth
temperature growth
past
(Jørgensen
not
sediments
overview
Sass
sequence
(mobility
as
been et
1996;
on
(e.g.
of
play
few
reveal
of
an
only
donors
decrease
a!., et physiology,
energy
gradients
SRB
rate
The
cold-adaptation
recently
optimum abundant
in
a!., decades
lsaksen has
a
Sagemann
1994;
very
(Margesin
SRB
see
key
a
of (Jørgensen
range.
and
of
temperature
1977:
(Rabus
have
1998)
been
general
conservation
lipid
change
Widdel.
or Desul/vibrio
few
role
Graumann
(Johnson
the
revealed if
and
in
curves
electron increase
Abdollahi been
metabolism, given
but
In
these
molecules
studies et et our
in
growth
principle Jørgensen,
contrast,
and
al.,1993)
a!.,
rather which
the
1982;
have
1988;
examined
knowledge
42
much and
encounter
dependence
in eta!.,
acceptor
Schinner.
of and
1998;
have anaerobic
and
a
yield,
been and
indicates
Widdel
may temperature
Odom
within
study
(Lsaksen often
its vugaris
correlation protein
attention
Marahiel,
or
been
1996;
1997:
Knoblauch Nedwell,
regulation.
studied
electron
follow
so
viz,
(28
changes of
of
and
one
triggers
1999),
1988).
dealing mineralisation
far
complex Knoblauch
of
structures, the Cypionka.
and Arctic
mM
has
on
layer Singleton, in
in an
with
1996),
sulphate in
ecology, 1979; curves
acceptors.
Jørgensen,
the
such
There in
and
in various
been
The of
moderate
Arrhenius
with
sediment adaptative
or
particular
ocean
respect
level and
physical
JØrgensen,
Jørgensen
eventually
but
existence
studies.
and
and
2000).
between
adaptative
has
recently
diverse
1993:
reduction
molecular
animals
of
not
of
water),
genes
Jørgensen,
1996;
been
climate
natural
(Knoblauch
to
function
so
‘macroscopic”
or
and
in
responses Lipid
Rabus
the
of eta!.,
temperature
temperature
between
1999) changes far significant
elaborated of
E.
Knoblauch
and
responses
sulphate-
obligately
chemical
sulphate
rates (rRNA organic
regions. sulfate-
coli
in
bilayers
over et
plants,
1999)
1992:
or
SRB. a!.,
and
and
the
in
in
in
of
a two layers) with decreasing temperature, and undergo a transition from the liquid crystalline
state (the mobile in vivo state) to the gel (“solid) state at a certain temperature (phase transition temperature): membrane functioning is hampered below this temperature (Russel, 1989). Cold adaption in various organisms involves a decrease of the phase transition temperature: this can be achieved by an increased synthesis of cis-unsaturated, short-chain or alkyl-branched fatty acids (Gounot and Russel, 1999). To gain first insights into mechanistic principles of cold adaption in SRB, we examined how temperature influences the composition of lipid fatty acids in a number of psychrophilic and mesophilic species.
Results
Effect of growth temperature on cellular fatty acid composition of various SRB. Twelve species of SRB comprising strictly psychrophilic, moderately psychrophilic and mesophilic types (Table 1) were grown at different temperature and subsequently used for fatty acid analysis. Growth temperatures were never above the optimum to avoid damaging effects and responses to unusual stress. Hence, growth experiments with obligately psychrophilic species were only possible within a relatively narrow temperature range. Fatty acid analyses were carried out when cells were still in the course of growth and had reached 3/4 of the maximum (final) optical density. In this way, a certain standardisation was achieved that allowed comparison of analyses without too much possible influence of fresh inoculation or of ageing on fatty acids patterns (see next section). Measured fatty acids patterns were regarded to represent those of the cytoplasmic membrane, because storage fatty acids such as neutral fats have not been found in bacteria (Wilkinson, 1988). The outer membrane usually contributes around 20% to the fatty acids and exhibits nearly the same ratios between fatty acids as the cytoplasmic membrane (Wilkinson, 1988). The most frequent change observed in the examined species was an increase of cis unsaturated fatty acids with decreasing temperature. However, the change of the cellular fatty acid composition was relatively small in most SRB (Fig. I). Only four Desulfobacter species (D. curvatus, D. hydrogenophitus, D. 1as, and D. postgatei) exhibited significant variation of the cellular fatty acid composition over the examined temperature ranges (Fig. 1). In species analysed before (Taylor and Parkes, 1983; Vainshtein et at., 1992; Kohring et at., 1994; Knoblauch et al., 1999), the presently observed fatty acid patterns were in agreement with the previous results obtained at a single growth temperature. We confirmed high portions (around 70%) of fatty acids with less than 16 carbon atoms in Desulfofaba gelida,
43
medium;
7, 3 brackish and 1.2 20, medium; 0.1 0.15 in and salt medium; in 26, 5 and and marine 1.4 in medium. For deails see and Widdel Bak (1992).
cultures All c.
in
chemically grown were defined
media.
The amounts NaCI, of 6 H,O MgCl, CaCI 2 arid 11,0 2 added litre per as . follows: were 0, 1 0.4 and 0.1 in freshwater
b. Pi-ecmse temperature (especially ranges lowest growth temperawres) or temperature optima have been not determined in cases. all
DSMZ, a. Deusche Sammnlung Mikroorganismen (German von Collection Microorganisms of and Cultures), Cell Braunschweig.
Desulfococcus
,n,,Itir’orcur.v (2059)
Sewage digestor
15 -40 28-35 Acetate, salt Widdel and Pfennig, 1984
Desi,lfosarci,ma r’ariahilis
(2060)
Mediterranean sediment
15 38
28-33 Acetate, salt - and Widdel Pfennig, 1984
Desulfor’ibrio des,m/furica,rs
(642)
Wet soil
25 40
nr Lactate, freshwater - and Widdel Pfennig, 1984
Desulfohacterpo.vrgi’nei (2034)
Brackish sediment
10 -37
32 Acetate, salt and Widdel Pfennig, 1981
I)e.vis/fohmw, (338
la,u.v I)
Temperate
marine
se(limcrit rw 29-32 Acetate, salt Widdel, 1987
Desulfohacter curr’atu,c
(3379)
Temperate
marine sediment nr 28-31 Acetate, salt Widdel, 1987
Mesophi tic
Desulfobacter Irvdrogei;uplrilus
(3380)
Tcrnperatc
marine
sedIment 35 0 29-32 Acetate, salt - Widdel, 1987
Mesophmlic, psychrotolerant
Destrlforhopalu.r
i’acuolatti,s (9700)
Temperate
r-narine
sediment
0 24 18-19 Lactate, marine - lsaksen and Teske, 1996
Desitifotalea arc(jca
(12342)
Arctic marine sediment
-1.8 26
18 Lactate, - marine Knoblauch 1999 et a!.,
Moderately psychrophilic
(
Desulfotulea
ps)’c/rroplriIa 12343)
Arctic
marine sediment
-1.8 19
10 Lactate, marine - Knoblauch al. (1999) et
Desidfofrigti.c oceu,re,rxe(12341)
Arctic marine
sediment
-1.8
16
10 Acetate, - marine Knoblauch a!., er 1999
Destilfofaixi (12344) gelidu
marine Arctic
sediment
-1.8
10
7 Piopionate, - marine Knoblauch a!., 1999 et
psychrophilic Strictly
. Range
Optimum and
mediurnc
( DSMZ collection number)
growth substrate.
Gemrs arid species
Origin
Temperature for (°C) growth” Organic Reference
Table of Species sulphate-reducing 1 bacteria included in the study of temperature effects on fatty cellular acids.
species
fatty Fig.
Desulfofaba
Desulfofrigus
I.
Desulfotalea
Desulfotalea
acids Desulforhophalus
Desulfobacter
Desulfobacter
Desulfobacter
Desulfobacter
Oesulfovibrio
Des
Desulfococcus
Relative
of
psychrophila
oceanense
arctica
hydrogenophilus
vacuolatus
curvatus
latus
postgatei
desulfuricans
variabi/is
multivorans
sulphate-reducins
ulfosarcina
which
portions
were
ge/ida
found
of
unsaturated
bacteria
in
10°C
10°C
20°C
16°C
20°C
28°C
20°C 12°C
comparable 28°C
20°C 12°C
28°C
12°C
20°C
28°C
12°C
28°C
28°C
12°C
4°C
16°C
28°C 4°C
4°C
16°C
4°C
4°C
4°C
at
fatty
different
—
—* —
-
analyses.
acids
growth
Total
among
20 45
I
+
temperatures.
amount
total
fatty
of
40
+ acids
I
unsaturated
÷
The
+
-1-
in
bars
psychrophilic
60 indicate
I
fatty
+
highest
acids
and
80 +
I
+
mesophilic
variations
(%) 100
I of and the
Desulfococcus 9,10-methylenehexadecanoic unbranched only in
composition
growth
analyses.
was course Desulfrbacter
(Fig.
An (16:0) content
detail. formation different
study (rather
period amount and
the the
level cells
same label
De.su/.fobacter
Detailed
other
high
initial
In
temperature
following:
formed
not
another
2).
synthesised
after
to was
another occurred
extent.
The
at temperature than
of
of
portions
The psychrophiles.
‘3C-enriched observed
prove
Such
temperatures.
28°C increase
of
cis9
incorporated
the
chains)
cessation
experiment
study
under
to
of
limiting,
inoculuin
cvc9
mu1ti’orans,
hvdrogenophiliis
a
In
(a) time
a
simultaneously 16:1
Desuifobacter
variation
was
after
species;
was
change
(>70%)
postsynthetic
the these
mesophilic
of
At
17:0
in
of
in
decreased
(Fig. course
used
shifted
fatty
of
inoculation.
mesophiles cis-9-hexadecenoic acetate.
psychrophilic 12°C,
but
second
was
and Special,
fatty was
In
into growth.
of
however,
of
non-labelled
for
of
1),
this
acid
the
Desulfosarcina
from
cis-monounsaturated lOMe the
experiment
back
performed
acid
all
acids
the conditions. isotope
but
After
growth
markedly
hvdivgenophilus
modification),
fatty
whether
way,
fatty
characteristic in
cellular
changes
Subsequent
a
were
(cvc9
to
also
When 16:0 fatty
a
significant
stationary
(Fig.
growth
acid
28°C
growth
species
acids
the
labelling
phase,
iso-
on amount in
in 17:0) an
fatty
3A) we
acids
while
experiment
growth
composition
in D.
acid
and
the cvc9
one
increase
variabilis
and
had
examined experiment
Desulfobacter
hvdrogenophilus
as increase
(other
phase
and
acids
portions
fatty
similar
the
46 acetate
time
(cis9
that
culture
at
acetate
anteiso-branched
of
in
17:0
ceased,
and
began
fatty
10-methyihexadecanoic
amount
the cellular constant
acids of
during
culture
of
point
strains
16:1)
and
could
D.
and
as
cvc9 was
of
experiment
with
acids
cis9 of
with
was
the
to
the posrgatei
at
in
the (besides
lOMe cvc9
DesulJ’ovibrio
at
and
added cease
fatty
the the
of
other obvious
17:0
that
the be
added.
hydrogeizophilus.
16:1
temperature
so
subsequent with
temperature
temperature
which
cis9
(see
combined decrease
first optimum
17:0
growth
far
16:0 had
and
acids
is
due
a
anaerobes.
unsaturated
16
fatty
before
depended
third
After
preceding
due not
growth
16:1
and
been cells
effect
to
remained
lOMe
to
by
acids
state
18 examined).
to
acetate
desu/fliricans,
time. of
lOMe incubation
temperature
was
increased
growth
(Fig.
also to providing with
were
carbon
a
acid
was
hexadecanoic
of
16:0
phase
de
28°C
of
An with
not
fatty
changed
section)
Results
temperature
grown
The
at 16:0
a 2).
(lOMe
novo
batch
harvested
depletion.
examined
increased.
initial
ceased
‘IC-labelling
constant,
only
atoms
at
showed
(b)
acids
fatty were
due
periods
The
a
nearly
synthesis
at cultures
of
The revealed
limiting in
to
on 16:0)
and
growth
28°C.
again,
in
in
28°C
to
more
time acid
12°C
acid
the
for
the
3C- low that
on
in
the the
at
when
cultures (Fig.
already unsaturated temperature
Simultaneously,
hydrogenophilus
acid
inoculation Fig.
analysis.
2.
3B)
the
Time
a of
LL temperature ‘
0 0 relatively C-) ci) C)
CO
cd indicating
0 U CD 0 in C
Co
D.
B,
fatty
downshift
the
course
hvdrogenophiltis
Growth
50
40
3Q
after
10
0.4
0.3
0.2
0.1
0
deceleration
the
acids
0
inoculation
of
high
‘ 3 C-content
a
recorded
A
B
was
de
temperature-independent
such
(and
cell
novo
decreased
phase
as
eventually
as
and
density
optical
cis9
synthesis
(Fig.
in
(the
incubation
this
16:0
phase
density
in
1).
and
5
fatty
the
partly
This
in
of
hence
following
at
this
present
at Time
cis9
28°C.
acid
difference
changes
660
47
due
experiment
a
16:1
(days)
nm.
decreased
The
relatively
to
the
experiment.
of
the
preculture
from
exponential
is
characteristic
change
10
explained
was
unlabelled
much
high
was
somewhat
growth
caused
pool
more
also
by
fatty
acetate.
of
grown
phase).
16:0
cyc
cis
the
lOMe
than
by
saturated
acids
lower
916:1
fact
917:0
acetate
at
in
15
16:0
A,
The
28°C
in
that
any
than
Results
Desulfobacrer
fatty
portion
and
addition).
other
there
in
used
of
acids
other
was
one
fatty
of for 50
40 0 30 a) C 0 u 20 -o C-)
,10(I U-
0
1.5
1.4
C 1.3 C 0 C.) E o 1.2
(-) 1.1
0.4
0.3 2 0C 0.2(0 0 0.1
0 5 10 15 20 25 30 Time (days)
Fig. 3. Time course of the changes of characteristic fatty acids in Desulfobacter hydrogenophilus upon temperature shifts. In addition, synthesis of fatty acids was followed by stable isotope labelling. Medium with
°C-enriched acetate was inoculated with a preculture grown at 28°C and first incubated at 28°C. Upon depletion of C-enriched acetate (not shown) after 7 days, the culture was shifted to 12°C and non-enriched acetate was added.‘3 When acetate was agin depleted after 18 days, the temperature was shifted back to 28°C and non- enriched acetate was added again. A. Results of fatty acid analysis. B, L3C-content of fatty acids. C, Growth recorded as optical density at 660 nm.
48
advantage
change
temperature
(growth
over
Further for components
membrane. and environment.
little temperature. the
is
and
adapted
(Knoblauch
bacteria
investigated acid
unsaturated knowledge,
species
1978; influence Cellular
and
Dowling
Discussion
possible
The
Several
regulation
psychrophiles
it
patterns for
growth
response
a
is
Gounot
given
and
research
to
(Taylor
the
still
phase
more
(for
fatty
eta!.,
of
in
permanently
with
In
species
fatty the
and
to
identification
changes
environments
SRB
the
This
a
or in
overview
temperature
rates
contrast,
acids
of
and
or
matter
of
dependent)
regulate
SRB.
1986;
first
insufficiently
the
Jørgensen,
growth
is
the and
acids
less
may
the
with
Russel,
represents
needed
in
in
same
revealed
one
fatty
but
Vainshtein
Parkes,
of
lipid
SRB
comparison pronounced
the
were
indicate
decreasing
see
cold
its
discussion
that substrate
also
acid
fatty
range
Desuijobacter
with
of
to
1999).
or
Gounot
membrane
1999).
have
fatty
measured
environments,
reveals
little
these
elucidate
1983;
caused
by
composition a
understood
acid
that
fluctuation
to
eta!.,
special,
been
other,
acid
On
on
One
temperature
or
allow
as
increase
and
bacteria
membrane to
composition.
effects
Dowling
cellular
the
even
by
to
in
examined
fatty
1992;
composition;
mesophilic
may,
whether
Russel,
presently
which
species
the
chemical
“optimised”
full
other
of
mechanisms
to
as
no
of
49
acid
in
therefore,
psychrophilic
of
Kohring
temperature
temperature
fatty
membrane
suggested
et
temperature
functioning
changes
extent matches,
hand,
natural
cis-unsaturated
the
1999).
apparently
mostly
There a!.,
composition
unknown
acids
changes
species
pronounced
this
eta!.,
there
1986).
membrane
adaptation
assume
habitats
The
are
of
for
by
as
has
in
functioning
and
system
(e.g.,
species.
the
over and
employ
biomarkers
examples
relatively
principle,
might
1994;
at
in
by
temperature
factors
been
The
other
offers
that
fatty
the
of
the
Bhakoo
far
a
fatty
(Taylor
ability
fatty
certain
Boschker
the
to
is
be
present
demonstrated
medium
the
conditions.
These
a
highest
same
that
acid
not
rather
the
presently
(Jackson
a
growth
high
of
acids
observations
acids
fatty
for
special
of
and
other
and
may
only
permanently
temperature
adaptation composition
bacteria
low
Desulfobacter sulfate
study
chemotaxonomy
sensitive
portions
et
acids
Herbert, in
during
have
phase
Parkes,
be
al.
bacteria
triggered
and
most
unidentified
competitive
temperature
with
endogenic
is,
to
1998).
pattern
reduction
are
Cronan,
in
on
growth.
change
system
of
to
of
of
1980),
range
1985;
some
well-
other
with
fatty
with
cold
cis
our
the
the
An
by
in to cyclopropane has
lipid and
role
understood
reduction
potential 17:0
reducing presently Dowling temperature of inhibition
temperature.
desaturate anaerobic with
introduction formation
process when hydroxyacyl-ACP
Experimental
temperatures Cultivation.
1989).
strictly
A
high
also
The
Cronan,
of
bilayer is
conversion
17:0
a
in
cyclopropane the
strictly
anoxic
been
‘C-labelling
levels
resulting
bacteria
et
agreement
biomarker
step
of
bacteria
observed
from
fatty
(Grogan
acyl
of
a!.,
by
questions
the 1997).
of
fatty
documented
For
(Table
(Grogan
unsaturated
media procedures methyl
of
anaerobic
cis-double
formation
acids
cis9 1986).
of
chain
(Vainshtein
acids
in
analysis cannot
cis9
cis9
The
followed
and
with
very
for
fatty
3,4-dehydration
experiment
(Widdel
the 16:1 1), after
transfer
However, and
has
16: Cronan,
is simultaneous
16:1
Desulfobacter
SRB
such
convert applicability
of
a
metabolism.
acids in
bonds
low
1
is
synthesis
of
Cronan.
fatty
the post-synthetic
to cvc9
which
other
by
et
the
and
the
a
were from
cyc9
content
a!.,
and appropriate mechanism
1997).
reduction
is
a
with acids
the
formation
17:0
cellular
Bak,
bacteria
saturated
assumed
1992;
is S-adenosylmethionine
cultivated
1997).
17:0 their
of
decrease
finding of
of
particularly
and D.
An
1992) In
the
always
of
3-hydroxyacyl-ACP this
species
in
Kohring
influence
hvdrogenophiliis
modification
contrast
fatty
assumed
lOMe
leads
(Grogan carbon
lOMe
the lOMel6:0
also
to
to
of
50
length. fatty
with
of
of
an in
occur
late
acid lOMe
needs
this
in
to
in
16:0
the
chemically
unsaturated
acid
16:0
et
appropriate
chain
suited
D.
to
growth
biochemical
on
saturated
sediments
and
al..
composition
The fatty cis9
during
at
several
hvdrogenophilus.
as
16:0
de has
membrane
in
of
low
1994. Cronan,
by
a
to
cis-unsaturated
common
16:1
Desulfobater
and
acid novo
phase
reliable been
(Fig.
which
oxygen-dependent
temperature
chain
decrease
defined,
hydrocarbon
(ACP
lipid
salinity
aerobic
proton
Kuever
(Taylor with
in
variant
previously
synthesis.
as
3B)
1997).
upon
fluidity
elongation other
biomarker.
involves
fatty
in
2,3-dehydration =
the
and is elimination
bicarbonate-buffered,
D. the
acyl et
bacteria,
of
and
growth
formation
The
fully
genera
is
The
al., acids
hvdrogenophilus fatty
organic
the
chain.
is
phase
to
species
carrier
suggested
The
Parkes,
an
2001) physiological
insufficiently
the
formation
by
in
formation
The
acids
desaturases,
(Schweizer.
of
at
additional
which
agreement
a advantage
Anaerobic
anaerobic
transition
substrates (Grogan
complex
of
different
obvious
sulfate-
protein)
and
at
in
1985;
cvc9
of
as
low
the
can
the
of
of
3-
a
C, 6 H 54 )
reduction
IRMS) of
above)
transfer trap
analysis
(2.8
300°C with
(BioTrends). chromatography
based
acetate,
method source
suiphide; cell
50
overpressure
(Figs. hvdrogenophilus,
volume during
grown with
sealed
(Table
70
The ml.
Growth
Fatty
To
density,
(Finnigan ml
an
230
eV.
on
the
and
with
at
on
2
in
with
(3°C
monitor
1).
For
Optima
>99%;
of
line
min’).
‘ 3 C-content
was
was
the
the ml
and
combination
temperature
retention
acid
a
Every
late
Sasser
electron
stable
was
black
Finnigan
known
temperature
the
medium
carried
latter 2000
min 1 ,
GC/FID
same
(10
3)
growth
Sigma-Aldrich).
MAT).
The
analysis.
5
the
sample
(GC)
recorded
strain
kPa)
(1997).
fused
were
rubber
isotope
the
was
ml
temperature.
isotope
time
donor
times
finally
oven
out
the
under
MAT
analysis
and
phase
strain
and
of
determined
of with
was
Mass
volume
silica
taken
using
FAME
of
course
N,
stoppers.
Extracted
Cellular
temperature
940
quantified
was
and
by labelling
the
ratio
cultivated
20
280°C,
DELTA
15
combustion
from
+ was
capillary
spectra
measuring
the
was
ml
inoculum
CO 2
and
amended
from
mass
mill
was
Cells
were
of
was
essentially
each
of
same
photometrically
fatty
carried Precultures
changes
a
600°C,
fatty
and
200
of
isotherm).
plus
anoxic
via
the
spectrometry
source
used
were
measured were at
was
column
bottle
the
conditions
the
ml;
a
acids
with
a
interfaced
size
acid
both
instrument closed,
given
flame
out
as
cellular
harvested
run
respectively.
collected
head
use
in
it
temperature
51
grown
and
standards.
0.5 was
methyl
could
in
with
the
(Machery
the
for
from
The
of
growth
ionisation
using
separately space
%
centrifuged
anoxic
200
optical
inoculation
as
an
fatty
for
(MS)
fatty
isotope
an
as
of
be
FID
(Finnigan
esters
60 CuS
by
in
inserted
decribed
gas
Autosystem
the
ml.
gradually
gas
(N,
temperature
acid
centrifugation
to
acids,
full
of
und
CO,
using
temperature
bottles
(Cord-Ruwisch,
density
chromatography,
pure
detector
Samples
+
ratio
chromatography
(FAMEs)
subjected
140°C
1
80°C,
composition
CO 2
scan
Nagel).
cells
outlet
and
(12
MAT)
the
‘ 3 C-compound
above,
authentic
mass
decreased
by
[90:10,
at
ml
(10°C
mode
a
(Perkin (FID).
and
acetate
were
in
at
tube.
660
long-chain
The
to
were
application
with for
spectrometry
parallel
was
different
(20
however,
an
fatty
methylated
(mlz
each
min 1 )
carrier
vIvj);
nm
Identification
of
FAME
At
ionisation
1985).
an
Elmer)
added mm,
during
separated
350°C.
and
acid
Desulfobacter
(conditions
the
and
oxidation
batches,
bottles)
(sodium
30-400)
bottles
a
and time
10,000
gas
alkane
the
initial
analysis.
as
GCIQ
standards
growth
produced
of
equipped
GC/MS
(GC-C
then
was
carbon culture
energy
by
by
points
slight
were
were
each
x
at
‘ 3 C-
was
low
and
gas
ion
(n
the
Fl 2
as
g)
to
to a Acknowledgements
Jens We with
Chemischen
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The
that
and
and
D.
In of 2
Department 1.
Corresponding
Mailing Fax: Phone:
Electronic Max-Planck-Institute
“Desulfobacula
Fakultät
University
D-28359
strain
+49-421-2028-580.
Jan
Reclassification
+49-421-2028-734.
address:
für
Kuever
Bremen,
of
of
Sax
Biologic,
Marine
Bremen.
author.
address:
Celsiusstr.
*,
as
mt.
Germany.
Martin
for
Microbiology.
Universität
Desulfotignum
Center
J.
jkuever@
phenolica”,
Marine
Syst.
1,
Könnek&,
D-28359
for of
Evolution
Microbiology,
mpi-bremen.de Environmental Konstanz,
Desulfobacterium
Leobener
Bremen,
Alexander
Microbiol.
comb.
2
Postfach
56
balticum
Strasse.
Department
Germany
Research
Galuschko2,
(2001).
5560,
nov.
D-28359
and
of
D-78457
51,
gen.
and
Microbiology,
phenolicurn
Technology
Bremen.
171-177 and
nov.,
description
Oliver
Konstanz,
Germany.
(UFT),
Drzyzga3
sp.
Celsiusstra3e
Germany.
as
nov.
of Abstract
A mesophilic, sulfate-reducing bacterium (strain SaxT)was isolated from marine coastal sediment in the Baltic Sea and originally described as a “Desulfoarculus” sp. It used a large variety of substrates ranging from simple organic compounds and fatty acids to aromatic compounds as electron donors. Autotrophic growth was possible with H, and CO, and formate in the presence of sulfate. Sulfate, thiosulfate and sulfite were used as electron acceptors. Sulfur and nitrate were not reduced. Fermentative growth was obtained with pyruvate, but not with fumarate or malate. Substrate oxidation was usually complete leading to CO,, but at high substrate concentrations acetate accumulated. Carbon monoxide dehydrogenase activity was observed indicating the operation of the carbon monoxide dehydrogenase pathway (reverse Wood-pathway) for CO, fixation and complete oxidation of acetyl-CoA. The rod-shaped cells were 0.8-1.0 p.m in width and 1.5-2.5 p.m in length. Spores were not produced and cells stained Gram negative. The temperature limits for growth were between 10 °C and 42 °C (optimum
growth at 28-32 °C). Growth was observed at salinities ranging from 5 to 110 g of NaC1 per liter, with an optimum at 10 to 25 g NaCI per liter. The G + C content of the DNA is 62.4 mol%. Vitamins were required for growth. Based on the 16S rRNA gene sequence strain SaxT represents a new genus within the delta subdivision of the Proteobacteria. The name Desulfotignurn balticum is proposed. After the 16S rDNA sequences of all members of the genus Desulfobacterium were published (Stackebrandt, 1999), the need to reclassify most members of the genus Des ulfobacterinin became obvious due to their strong phylogenetic affiliation to other genera. Here, we propose to reclassify Desulfobacterium phenolicum as “Desulfobacula phenolica”. Desulfotign urn balticum, Des ulfobacteriurn ph enolicurn, and “Desulfobacula toluolica” contain fatty acids which were so far only found in members of the genus Desulfobacter.
57 Introduction
Among the sulfate-reducing bacteria of the &subdivision of the Proteobactena several marine
isolates are known to grow on a large variety of aromatic compounds including phenolic compounds and toluene. They were originally classified as members of the genera Desulfrbacteriurn and “Desiilfrbacula” (Bak & Widdel, 1986; l986a; Brysch et a!., 1987. Rabus et a!., 1993). Beside the use of various aromatic compounds, members of the genus “Desulfobacula” are characteristically restricted to the utilization of short chain fatty acids and simple organic compounds as electron donors: whereas members of the genus Desulfobacterium can also grow chemoautotrophically on H, and CO, (Bak & Widdel. 1986:
Bak & Widdel. 1986; 1986a: Brysch et a!., 1987: Rabus et a!., 1993; Schnell et a!., 1989). In contrast, members of the genera Desuijococcus, Desulfi)nema, and Desulfr’sarcina only use a limited number of aromatic compounds, mainly benzoic acid derivatives, but also long chain fatty acids as their sole electron donor and carbon source (Fukui et a!., 1999; Widdel, 1980: Widdel, 1988; Widdel & Bak, 1992. Widdel & Hansen. 1992, Widdel et a!., 1983). Recently obtained isolates with interesting degradation capacities might indicate that in general marine sulfate-reducing bacteria are more versatile than isolates obtained from freshwater habitats (Galushko et a!., 1999: Harms et at., 1999: Rueter eta!., 1994). However, there are many other sulfate-reducing bacteria (including spore-forming ones) isolated from freshwater habitats which use a large variety of organic compounds as electron donor and are able to grow in marine media (Kuever et a!., 1999). Another marine isolate which was isolated with benzoate and tentatively classified as a “Desu!fr)arcu!us” (the correct spelling would “Desulfarculus”) sp. could grow slowly on fatty acids with a chain length higher than butyrate (Drzyzga et a!., 1993). Therefore, it would resemble the physiological properties of Desu!fi.bacteriuni and “Desulfi)bacula” spp. In the present paper we describe this species as a new genus within the &-subdivision of the Proteobacteria. The genus Desulfr)bacteriurn was established by using mainly physiological properties (Brysch et a!., 1987). After the 16S rDNA sequences of all members of this genus were published (Stackebrandt, 1999), the need to reclassify most members of the genus Desu!fobacterium became obvious due to their strong phylogenetic affiliation to other genera. A comparative analysis using the sequences published by Stackebrandt (1999) indicates that the genus comprises only the type species of the genus Desulfobacterium autotrophicuni and
58 two other species; the not validly published “Desulfobacteriwn vacuolatuni” (so far not described, only listed in Widdel (1988)) and the originally as Desulfococcus niacini (Imhoff and Pfennig, 1983) described “Desu!fobacteriutn niacini”. That both species are members of the genus Desu!fobacteriurn was already demonstrated by several phylogenetic analyses (Rabus et al., 1993) and by use of the genus-specific oligonucleotide probe 221 (Devereux et al., 1992; Manz et al., 1998). All other members of the genus have to be reclassified, because they belong to other genera or represent new genera. Here we suggest that the former “Desulfobacteritim phenolicum” be incorporated within the genus “Desu!frbacula” (Rabus eta!., 1993).
Methods
Source of organism. Strain Sax was isolated from a benzoate enrichment culture inoculated with anoxic marine mud from Saxild, Denmark (Drzyzga, et al., 1993). Strain Sax was deposited in the DSMZ under the accession number DSM 7044. “Desu!fobacula to!uolica” (DSM 7467), Desuljobacteritim phenolicum (DSM 3384), Desulfobacteriiirn autotrophicum (DSM 3382), and Desulfr.thacterpostgatei (DSM 2034) were obtained from the DSMZ. Media and culture conditions. For enrichment and cultivation the medium was prepared as described previously using benzoate (2.5 mM) as electron donor (Drzyzga, et al., 1993). Pure cultures were obtained by repeated use of deep agar dilution series (Widdel and Bak, 1992). Substrate utilization was determined by adding the carbon and energy sources from sterile stock solutions; the cultures were incubated for about 3 weeks. In order to avoid possible toxic effects of the substances, toluene and xylene isomers were supplied to cultures following adsorption in the deaerated organic solvent 2,2,4,4,6,8,8-heptamethylnonane (HMN; 2 %, v/v) (Rabus, et al., 1993). A check was made to ensure that HMN did not inhibit growth of strain Sax on benzoate. To test the capability of autotrophic growth, cultures were grown under an headspace of
80% H,-20% CO, at an overpressure of 101.3 kPa. The temperature range for growth was determined by incubation in a temperature gradient block from 35 to 80 °C in increments of
2-4 °C. The pH range of growth was determined in mineral medium with pH from 5 to 9. The dependence of growth on the concentration of NaC1was determined in mineral medium with
NaCl concentrations from 0 to 130 g NaCI per liter. Strain Sax was routinely cultivated in a carbonate-buffered, sulfide-reduced medium for marine sulfate-reducing bacteria supplied
59 with incubation
denaturation. as
measurement
Desulfr)bacterium
mM) Desu/fi)bacteriunl centrifuged composition mM)
preparation previously
mineral Cells once
buffer MgCI. cells were was enzyme
potassium
several with
1
.5
described
Chemical
For
Preparation
ml
Enzyme
2.5
placed
in and as
were
microliter passed
from
(50 *
anoxic
fatty
medium glass
j.tl only
activities
6H,O,
mM
acetate
conditions
(Sasser,
removed mM;
of
phosphate the
of
in
and 2-3
of
by was acid
electron
assays.
0.05
cuvettes
and
of
Analysis
saline
late
a
the
sulfide
syringes.
11.2
pH
(Postgate,
supplied times
(20
of
small
benzoate
aiitotrophicurn
were used phenolictini methyl
determined
biochemical %
1997).
fatty
exponential
7.3)
cell
from
were
g*1’).
buffer mM), All
dithionite
donor
buffer
through
production
done
sealed
glass
for
extract.
supplied
of
One
enzyme
acid ester
the
with
as
respectively. Collected
as 1959).
(potassium
a
aromatic at
described
and
homogenate
(50
vial unit
by
with
methyl 2.5
comparative (FAME)
a the an
were
solution.
characterization.
growth
Strain
and
growth
carbon assays
with
means mM;
was anoxic
of
mM
under
day
The
butyl
enzyme
grown
Desuitobacter
cells
described
ester
2.5 before of
benzoate
compounds, Sax
phosphate
pH
mol%
phase analysis
were
Cells All source,
N.,
French of
preparation.
substrate.
by
rubber
mM
were
was
7.3)
in
capillary
gas was
additions
centrifugation FAME activity
done (Widdel
60
were
G
from
marine
MgCI,
grown
previously
under
which
Strain press resuspended
phase
and
carried +
stoppers.
50
The
under
postgatei
C
harvested
Salt the
was
mM; identification
analysis. sulfate
and GC *
cell
were
content
a
in
presence
mineral
Sax.
and
was
6H20 late
gas
strictly
composition 1200 out
li.tmoImin1
Bak,
pH at
and
(Drzyzga,
(30
Assays
done stored
phase
exponential
“Destilfobactila
slightly
(28
137
in
using
were 7.3;
by and
ml
was
mass 000
medium
1992).
The
of
anoxic
anoxic
mM)
from MPa.
centrifugation
bottles
of
2.5
containing desulfoviridin
on
were
grown
x
determined
a
of
whole-cell reduced
N2/C0 spectral
et
g.
and
ice. mM
method
as
anoxic
of
Cell
potassium
conditions
al.,
20 cytochromes,
with
growth
containing
performed
electron
the
mg
with
Measurements
dithiothreitol
mm).
1993).
debris
toluolica
(80/20
NaCI,
benzoate
protein1.
by
stock
analysis.
medium
as
butyrate
by
and
phase
was
The
addition fatty
phosphate described
at
acceptor.
and
thermal
solutions
26
1000 vol.
washed
30
in
tested
extract
were intact
g*li;
acid and
(2.5 and
‘C
The and
I
(10
%).
and
ml
ml
of
of
in The presence of 2-oxoglutarate: electron acceptor oxidoreductase was checked by following the reduction of benzyl viologen (2 mM) at 578 nm in the presence of 2- oxoglutarate (3 mM and CoA (0.2 mM). Activity of CO dehydrogenase was determined by following the reduction of benzyl viologen (5 mM) in the presence of CO. To perform the assay, cuvettes were flushed with CO until the assay buffer was saturated with CO.
Protein content in cell free extract was determined with bichinchoninic acid as a reagent by following standard protocol assay (BCA protein assay kit; Pierce, Germany) and with bovine serum albumin fraction V (Pierce, Germany) as a standard for calibration. Gases were purchased from Messer-Griesheim (‘Darmstadt, Germany) and Sauerstoffwerke Friedrichshafen (Friedrichshafen, Germany). PCR amplification and sequencing of the 16S rRNA gene. To amplify the almost complete 16S rRNA encoding gene (1,500 bp) of strain Sax, primers GM3F and GM4R were used in a 35-cycle PCR with an annealing temperature of 40 °C (Muyzer et al., 1995). PCR products were purified by using the QlAquick Spin PCR purification kit (Qiagen, Inc., Chatsworth, Calif.) as described by the manufacturer. The Taq Dyedeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster Cit, Calif.) was used to directly sequence the PCR products according to the protocol provided by the manufacturer. The sequencing primers have been described previously (Buchholz-Cleven et al., 1997). The sequence reaction mixtures were electrophoresed on an Applied Biosystems 373S DNA sequencer. Phylogenetic analyses of 16S rRNA gene sequence data. The sequences were loaded into the l6S rRNA sequence data base of the Technical University of Munich using the program package ARB (Strunk et al. 1999). The tool ARBALIGN was used for sequence alignment. The alignment was visually inspected and corrected manually. Tree topologies were evaluated by performing maximum parsimony, neighbor joining, and maximum likelihood analysis with different sets of filters. Only sequences with at least 1200 nucleotides were used for the calculation of different trees. The partial sequence of strain Sax (1462 nucleotides) was added to the reconstructed tree by applying parsimony criteria without allowing changes in the overall tree topology. The strain designations and nucleotide sequence accession numbers which were not included in the ARB database are as follows: “Desuijobacitla ”Ttoluolica DSM 7467, X70593; Desulfobacterium Tphenolicum DSM 3384,
61 AJ237606 (submitted by E. Stackebrandt); Desulfispira joergensenhl’, DSM 10085 X99637; Desulfrtignuin balticiitn (strain Sax), DSM 7044, AF 233370; Clone SB-9, AF029042; Clone Sva0605, AF230098.
Nucleotide sequence accession number. The nearly complete 16S rRNA sequences of strain Sax (DSM 7044) is AF233370.
Results
FAME analysis. The fatty acid composition of strain Sax and its phylogenetically closest relatives are listed in Table 1. Significant amounts of unidentified fatty acids (ECL16.07, ECL 18.09) were found in “Desulfobac u/a to/au/wa” (7.5 %) and “Desu1fr.bacu1a phenolica (16.6 %) and also in traces in strain Sax. The FAME analysis for “Desu/Jobucula toliiolica” grown on ethanol was very similar to our results (van der Maarel et al., 1996).
Enzyme activities. In cell free extracts of strain Sax active CO dehydrogenase was found (2.6 U * mg protein’). whereas 2-oxoglutarate dehydrogenase (a key enzyme of the citric acid cycle) was not detected. This finding indicated the presence of the CO dehydrogenase pathway for the oxidation of acetyl-CoA (Thauer, 1988).
Table 1 Major cellular fatty acids in % of strain Sax and some phylogenetically related species.
Fatty acid strain Sax “Desulfobucirla “Desulfrthacula Desulfospira Desulfohacteriuni Desulfohacter phenolica toluolica joergensenii antotrophicum postgatei 14:1 - 1.4 - - - - 14:0 3.7 8.7 8.3 13.9 2.8 15.4 i15:0 - - - 1.8 - 0.7 15:1 c9 0.8 - - - 6.5 - 15:0 0.2 2.0 1.6 1.3 5.1 3.1
3OHl4:0 - 1.3 - 1.6 - - 16:1 c9 8.5 16.5h 19.7 38.9 30.7 10.0 16:0 24.2 20.4 31.2 28.4 13.2 20.9 lOMel6:0 14.0 17.2 17.6 - 7.4 11.2 17:1 cli - - - - 10.5 - 17:0 cyc 19.5 3.9 2.6 2.4 - 31.5 17:0 0.4 0.7 0.7 0.6 2.9 2.3 301-116:0 - - - 2.3 0.5 - 18:1c9 3.7 - - - - - 18:1 cli 7.4 3.9 6.0 5.3 2.7 1.2 18:0 9.8 2.1 3.1 0.7 1.1 0.5 i19:0 1.7 - 2.5 - - - 19:Ocyc 1.1 - - - - -
Percentage of total fatty acids are shown. Fatty acids present in all strains at less than 1 % are not listed. c, cis; i, iso; cyc, cyclopropane: OH, hydroxy; Me, methyl.
62 Physiological and morphological properties. All physiological and morphological properties of strain Sax, Desulfobacterium phenolicurn and “Desulfrthacula toluolica” are listed in Table 2 and compared to the closely related species Desulfospira joergensenii. For a more detailed description the original publications should be used (Bak & Widdel, 1986; Drzyzga, et al., 1993; Finster et al.. 1997; Rabus, et al., 1993).
Strain Sax did not grow on toluene, or o-, m-, p- xylenes supplied adsorbed in HMN, nor did this organic solvent inhibit growth of the bacterium on benzoate,
Phylogenetic analyses. The I6S rRNA gene sequence of strain Sax shares less than 94.1 % identity with the 16S rRNA gene sequences of other sulfate reducers of the delta subdivision of the Proteobacteria (data not shown). The phylogenetic position of strain Sax and its closest relatives is shown in Figure 1. The closest affiliation was found with a similarity value of 96.6 % to clone SB-9 (Phelps et a!., 1998), followed by Desulfospira joergensenhi (94. 1 %) (Finster, et al., 1997) and Desuijobacterium phenolicum (93.9). As can be seen in Figure 1 the sequence of clone Sva 0605 obtained from permanently cold sediments at Svalbard falls in close proximity to these organisms (Ravenschlag et al., 1999). Clearly, they are all members of the delta subdivision of the Proteobacteria. Furthermore, the comparative phylogenetic analyses indicated Desulfobacterium phenolicuni to be a member of the genus “Desulfobacitla”. It was closely related to “Desulfobacula toluolica” (98.8 % similarity of their 16S rRNA sequence), which is the only species of this genus so far (Figure 1). Based on the sequence of the 16S rRNA, strain Sax could not be affiliated with any of the other genera and was therefore proposed as a new genus in its own right.
63 Table 2 Comparison of selected characteristics of ‘Desitifobucula toluolica , ‘Desulfohacula phenulica’ (formerly Desulfohacterium phenolicunt). DesiilJspira joergensenhi, and strain Sax.
Characteiistic “Desulfohaculu “Desulfihacu/a Desuifospira Strain Sax toluolicu phenolica joergen.’.en Morphology Oval Oval to curved rod Vibrio Rod Width x length (urn) l.2-l.4x 1.2-2.0 l-l.5x2-3 0.7-0.8 x 1-2 0.5-0.7 x 1.5-3.0
Motility +(sp) +(sp) G ÷ C content (molf) 42 41 50 62 Desulfoviridin Major menaquinone NR MK-7(H) MK7 and MK-7(H) NR Salinity optimum (gil) 20 20 12-20 20 Optimal temperature (°C) 28 28 26-30 28-32 Oxidation C C C C Electron donors: H + (+) Formate (÷) + (+j Acetate (+) (÷)
Fatty acid : C atoms 4 (4) 4,8. 12, 14 4-(l0. 12, 16. 18) Isobutyrate 2-Methylbutyrate 3-Methylbutyrate Ethanol + (+) Lactate + + Pyruvate + + + + Fumarate + (+) + + Succinate + (+) + (+) Malate + (+) + Benzoate + + + 4-Hydroxybenzoate + + + Phenol + (+) Phenylacetate + (+) toluene + (+) Others Propanol, butanol, Propanol, butanol, Crotonate. glutarate. Crotonate, p-cresol, glutarate 2-hydroxybenzoate, maleinate, maleinate p-cresol, glutarate glycolate, glycerol, betaine, proline, yeast extract Fermentative growth on: NR NR - Pyruvate Electron acceptors: Sulfate + + + + Sulfite NR - + + Sulfur NR - + NR Thiosulfate NR + + + Nitrate NR - - - Growth factor requirement Vitamins - Biotin Vitamins *Cells are motile during exponential growth phase. but motility can rapidly decline during growth. NR, Not reported; +. good growth; H-). poor growth; -, no growth; , autotrophic growth: sp. single polar flagellum. Data obtained from Bak and Widdel (1996a), Drzyzga et al. (1993), Finster et a!. (1997) and Rabus et al (1993). All strains were negative for desulfoviridin and for growth on isobutyrate, 2-methylbutyrate and 3-methylbutyrate and could use sulfate as electron acceptor. Substrate oxidation was complete for all strains.
64 Bdeijov,br,o stOIpu Desu!foborulus sapovoafls OesulfonemalOesulfococcus
Oesulfure.IIa acetivo cans
Desuifomon,Ie riedje
Clone 5va0605 DesulFobacca ace toxidans’ Desulfobacuia roluolica Oesulfarculus bears,’ Oesul(obacteriurn phenolcum
Desu!forhabduslDesuifacinum I Desulfobu!buc
Figure 1 Phylogerietic tree showing the affiliations of 16S rDNA sequences from Desulfobacula phenolica and De.culforignunsba1tjciu to selected reference sequences of the delta subdivision of the Proreobacteria. The tree was calculated by neiahbor-joining analysis and corrected with filters which considered only 50% conserved regions of the 16S rRNA of _-Proteobacteria. The sequence of Desulfurella acetivorans was used as out group. The bar represents 10% estimated sequence divergence.
Discussion
The physiological and morphological properties (Table 2) in combination with the comparative 16S rDNA and FAME analysis (Figure 1 and Table 1) clearly demonstrate that
Desulfobacterjtinz phenolicitni is a member of the genus “Desulfobactila”. It can be clearly distinguished from “Desulfobacula toluolica” by its motility, morphology, missing vitamin requirement and use of different electron donors for sulfate-reduction. Therefore, we propose to rename it as Desulfobacula phenolica.
In view of the 16S rRNA gene sequence analyses presented in this study strain Sax should be regarded as belonging to a new genus, since it was not closely related to other genera (a maximum of 94.1% 16S rRNA sequence identity). The distinct morphological and physiological properties (Table 2) in accordance with the FAME analysis (Table 1) argue in the same direction. Both “Desulfobacula” species and strain Sax contain relative high amounts of the fatty acid IOMel6:O, whereas this fatty acid is completely absent in Desulfospira joergensenii (Finster, et al., 1997). The finding of this fatty acid in
65 “Desuijobacula toluolica” is consistent to the results obtained by van der Maarel and coworkers (1996). Strain Sax can be distinguished from both “Desulfobacula” species by the high amount of the cyclopropane fatty 17:0, which is also a dominant fatty acid in Desuijobacter spp. Therefore, the designation Desulfotignuin balticum is proposed for strain SaxT. Ecological relevance of members of the genera “Desulfobacula” and Desulfotignurn. The cellular fatty acid composition of strain Sax shows the highest similarity to that of
Desulfobacter postgarei. It is distinguished from the composition of “Desulfobacula tolitolica” and “Desulfobacuta phenolica” by the presence of cycl7:0. Nevertheless strain Sax, “Desitifrihacula phenolica” and “Desuijobacula toluolica contain significant amounts of lOMel6:0 which was previously described as a biomarker for Desu/ftbacter spp. (Dowling et al., 1986) and was also found in lower amounts (< 10 %) in Desulfobacterium autotrophicum (Vainshtein et at., 1992). Therefore, the use of this fatty acid as a specific biomarker for microorganisms belonging to the genus Desuifobacter should no longer be considered reliable. The presence of this fatty acid in marine sediment could also account for sulfate-reducing bacteria belonging to the aforementioned genera.
Emended description of the genus Desulfobacula (Rabus, et al., 1993). The sentence about the motility should be changed into: Oval cells which may be motile or nonmotile.
Description of Desulfotignum gen. nov.
Desulfotignuin (De.sul.fo.tig’num. L pref. de from, L. n. sulfur sulfur; M.L. n. tignum stick;
M.L. neut. n. Desulfotignum sulfate-reducing stick). Cells are straight sometimes slightly curved rods that are motile. They are strict anaerobes, using sulfate as the terminal electron
acceptor that is reduced to sulfide. Aromatic compounds, fatty acids and a number of low- molecular-weight aliphatic acids may be utilized as electron donor. Autotrophic growth on 2H plus CO, and formate. Electron donors are completely oxidized to CO, via the carbon monoxide dehydrogenase pathway, Desutfotigizumbelongs to the delta subdivision of the Proteobacteria; the closest relative are Desulfobacula and Desulfospira spp. The type strain of
the genus is Desulfotignuni balticurn (=DSM 7044).
66 Description of Desulfotignum balticurn sp. nov.
Desulfrtignuni balticuni (bal.ticum, M.L. neut. adj balticurn from the Baltic Sea, pertaining to location of the sampling site). Cells are short rods, 0.5-0.7 by 1.5-3.0 p.m.Spore formation is absent. Cells are motile. Gram stain reaction of cells is negative. Strict anaerobe. Growth on 2H/CO,. formate, acetate, butyrate, crotonate, straight long chain fatty acids up to ,C18 lactate, pyruvate, fumarate, succinate, maleinate, malate, benzoate, 4-hydroxybenzoate, phenol, phenylalanine. and phenylacetate. Substrate oxidation is usually complete leading to .2CO but at high substrate concentrations acetate can accumulate. Electron acceptors used are sulfate, sulfite and thiosulfate. Not used: nitrate and nitrite. Slow fermentative growth on pyruvate.
Addition of at least 10 g/l of NaCI is necessary. Optimum NaCl concentration for growth is 20 gil. NaCI is tolerated up to 110 g/l. Vitamins are required for growth. Temperature requirements: 10 °C; 0(.T 28-32 °C; Tm, 42 °C. The pH range for growth is 6.5 to 8.2; pH optimum at 7.3. The G + C content of the DNA is 62.4 mol% (Tm). The GenBank accession number for the 16S rRNA gene sequence is AF233370. The type strain is SaxT (=DSM 7044).
Acknowledgement
We thank Ingrid Kunze for technical assistance, Hans-Georg Truper, Karl-Heinz Blotevogel for advice and help, and Geoff Mattison for linguistic improvements to the manuscript. This work was in part funded by the Max-Planck Society, Munich (Germany).
References
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Desulfobacterium phenolicuin. Arch Microbiol 146, 177-180. Brysch, K., Schneider, C., Fuchs, G. & Widdel, F. (1987). Lithoautotrophic growth of sulfate-reducing bacteria, and description of Desuijobacterium autotrophicurn gen. nov., sp. nov. Arch Microbiol 148, 264-274.
67 Buchholz-Cleven, B., Ratunde, B. & Straub, K. (1997). Screening for genetic diversity of isolates of anaerobic Fe(El)-oxidizing bacteria using DGGE and whole-cell hybridization. Svst Appi Microbiol 20, 301-309. Devereux, R., Kane, M.D., Winfrey, J. & Stahl, D.A. (1992). Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria. Svst App! Microbiol 15: 601-609. Dowling, N. J. E., Widdel, F. & White, D. C. (1986). Phospholipid ester-linked fatty acid biomarkers of acetate-oxidizing sulphate-reducers and other suiphide-forming bacteria. J Geti Microbiol 132, 1815-1825. Drzyzga, 0., Kuever, J. & Blotevogel, K.-H. (1993). Complete oxidation of benzoate and 4- hydroxybenzoate by a new sulfate-reducing bacterium resembling Desu1frarcu1u.s.Arch Microbiol 159, 109-113. Finster, K., Liesack, W. & Tindall, B. J. (1997). Desulfospira joergensenii, gen. nov., sp. nov.. a new sulfate-reducing bacterium isolated from marine surface sediment. SystApp? Micmbio! 20. 20 1-208. Fukui, M., Teske, A., Assmus, B., Muyzer, G. & Widdel, F. (1999). Isolation, physiological characteristics, natural relationships. and 16S rRNA-targeted in situ detection of filamentous, gliding sulfate-reducing bacteria, genus Desulfonema. Arch Microbiol 172, 193-203. Galushko, A., Minz, D., Schink, B. & Widdel, F. (1999). Anaerobic degradation of naphtalene by a pure culture of a novel type of marine sulphate-reducing bacterium. Environ Microbiol 1,415-420. Harms, G., Zengler, K., Rabus, R., F, A., Minz, D., Rossello-Mora, R. & Widdel, F. (1999). Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria. App?Environ Microbiol 65, 999-1004. Imhoff, D. & Pfennig, N. (1983). Isolation and characterization of a nicotinic acid-degrading sulfate reducing bacterium, Desulftcoccus niacini sp. nov. Arch Microbiol 136:194-198. Kuever, J., Rainey, F. & Hippe, H. (1999). Description of DesulJbtomaculutn sp. Groll as
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Widdel, F., Kohring, G.-W. & Mayer, F. (1983). Studies on dissimilatory sulfate-reducing
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De,ciilftnierna liniicola gen. nov. sp. nov., and Desuitonenia magnum sp. nov. A rch Microbiol 134, 286-294.
70 IL Max-Planck-
Arctic
Phone: Fax: E-mail:
*For
Aerobic
correspondence
sediments
Institute
+49(0)421-2028-746 +49(0)421-2028-580
Martin
and
for
sulfate-reducing
Könneke*,
Marine
and
characterized
Microbiology.
cultivation
Jan
In
Kuever
preparation
3
Celsiusstr.1,
72
and
bacterial
by
methods
Bo
phospholipid
D-28359
Barker
communities
Bremen,
Jørgensen
Germany
analysis
of Abstract
Phospholipid fatty acid (PLFA) analysis in permanently cold sediments from fjords of Svalbard (Arctic Ozean) showed that the total microbial biomass determined as lipid-bound phosphate decreased with depth. Polyunsaturated PLFAs of eukaryotes and PLFAs generally characteristic of aerobes had highest concentrations in the top 5 cm of the sediments. Branched-chain PLFAs indicative of facultatively and obligately anaerobic bacteria were most abundant below that depth. Two specific biomarkers for sulfate-reducing bacteria (i17:1 and lOMel6:O) were present at low concentration with an absolute maximum in the top 0-1 cm but with the highest relative amounts in a depth of 10 cm. High amounts of unsaturated PLFA (>60%) typical for cold-adapted organisms were found in the surface sediment and decreased with depth in the anoxic sediment. A reduced diversity of PLFAs with depth reflects a decrease of the microbial diversity in the deeper sediment, probably associated with the limited quality and quantity of electron donors and electron acceptors. The abundance of bacteria as estimated by most probable number (MPN) counts over different horizons correlated well with the PLFA profiles. The cell numbers of aerobic heterotrophic bacteria and of sulfate-reducing bacteria (SRB) were highest in the top layer. Isolates obtained from the highest dilutions representing the most abundant culturable organisms among these physiological types were identified as members of the genera Moritella, Pseudomoitas, Psychroinonas and Desulfotalea. Pure cultures isolated with hydrogen as electron donor represented new taxa of the families Desulfobacteraceae and Des ulfobulbaceae within the 8-subclass of Proteobacteria. All isolates showed fastest growth below 20 °C which indicated the dominance of cold-adapted bacteria in this permanently cold environment.
73 Introduction
been
acetate, psychrophilic common
have organic
isolated psychrophiles
marine to depend
compound
reported different 2-methyl-hexadecenoic
(10 20]. but cultures temperature
physiological membrane the prokaryotes
membrane sediments total
sediment
bacteria,
hydrogen
Measurements
molecular Phospholipid
In
In
The
the
amount me
found
These
shown
viable
the
sediments
addition
16:0) lactate
matter on
before
practical
can substrates
for
PLFA
habitats
present
two
layers.
to
the
specific
of
composition
to
and
[25,
a biomass of
a
is
[Könneke
be
for be
methods
sulfate-reducing
[2].
north-western
high and
number in
be
cultivation
the features
[5-7].
of
unsaturated
influenced
comparable analysis
26]. profiles
application formate of
to
members
the
were
study,
[10-16].
Members
analysed the
regulation
propionate
for abundance
fatty the
molecular
cold
was
These
most
for
of only
and
SRB sulfate
acid
of
we
acids
to
of
were
eukaryotic
of
sea were
estimated
the
Genus-specific
of
by microbial Widdel,
environmental
fatty investigated changing
of detected
abundant
[9].
(i17:I)
is with
findings
organisms
[4].
of
Svalbard
the
floor, [2,
reduction
were bacteria
the
the quantification
of
limited
the
compared
community
used
acids
Specific
Further
genera
those
SRB:
3]
genera
type
fluidity
for
often
unpublished
and
from
at
which
and
temperature.
indicate
communities
bacterial
because
or
as by (SRB)
low Desulfrn’ibrio
of
of
and
the most rate
Desulft)bacrer, that
prokaryotic Des11)ovibrio described
an
studies
signature
the
fatty samples
electron the
carbon temperate
with
of
numbers
are
microbial
that in
of
structure
the
incorporation
of
their
the total
use were
permanently
the
groups.
typical
acid
the
data].
them
is
MPN-counts
74
in
SRB
importance
source
cellular
membrane
of
A
has
free as
amount
in
the
donors.
microbial
selectively
patterns
[8]
phopholipid
sediments
organisms
species
communities
well
cultivation-independent
are SRB
products situ,
community
and often
cold
analyses,
and
For of
Destilfobacterium
unknown [17,
fatty
described
potential
of cold
Desulfrbac
biomarkers
of
were
the
sediments,
of such
been
and Both
to
of
community
short-chain
of
lipid
24],
isolated
[1].
acid
SRB
low
and
sediments
of
cultivation
SRB
not
the
fatty
SRB
used
of
as PLFA
of
the
are
species, From
by
bound
temperatures
selections compositions can
among
adaptation
have
four
lO-methyl-hexadecanoic
the
the
ter
for
in
anaerobic
the acids
as at
using
common
and
be
permanently .
the marine fatty
Arctic structure
and
low
been
adaptation an the
profiles
of
that
phosphate
growth
and
used
the
which
aerobic
PLFA the
same
alternative
(PLFA)
molecular
remineralization
Desulft.bacula
of
temperatures
are
acids
isolates
found,
of
isolation
is
as
Arctic habitats
by
food
the
end-products of
often
eukaryotes
have
phase, dominated location,
that
biomarkers
can
an analyses.
SRB
into
heterotrophic
biochemical of
for
cold
have
chain
obtained.
containing
increase
Sea
approach
does
not
found methods
the
elucidate
[21-23],
of
the
different
in
and
marine
have
with been
new
been
SRB,
and
pure
[17-
lipid acid
lipid
not
by The and
of
in by
in
in
of fermentativebacteria aiid can be utilized by methanogens, homoacetogens, or especially in marine environments due to the high sulfate concentration, by SRB [27]. Hydrogen together with a low concentration (1-2 mM) of acetate as carbon source should be selective for the isolation of Desiilfrvibrio species [28]. Aliquots of the highest positive dilution of the MPN series were used for the isolation of the most abundant bacteria at low temperatures. The physiological properties and phylogenetic position were determined.
Material and Methods
Sampling site. Four permanently cold sediments located at the north-western coast of Svalbard, were sampled during a cruise in July of 1998: [sfjorden (78°10907N, 14°34124E), Kongsfjorden (78°55259N, 12°17222E), Krossfjorden (79°16535N, 1l°58065E), and Smeerenburgfjorden (79°42’815N, I l05 189E). Water depths of the sampling sites were between 100 and 246 m, with bottom water temperatures around 0 °C. The sediment was collected with a Haps corer, subsampled with acrylic cores and plugged with rubber stoppers. The subcores were transported at 1-4°C over 1-3 days, until further processing. PLFA analysis. The analytical procedure involved one-phase extraction, fractionation on silicic acid, derivatization to methyl-esters, and analysis by capillary gas chromatography. Total lipids from the sediment were extracted as described by Bligh and Dyer [29] with some modifications. The total lipids were seperated on a silicic acid column (Isolute SPE column, 1ST Mid Glamorgan, UK). The eluents, having different polarity, were dichiormethane, acetone, and methanol. The methanol fraction contained the polar phospholipids, which were used for further analysis. A mild alkaline methanolysis was used to transmethylate the ester linked fatty acids of phospholipids to methyl esters [30]. Nonadecanoic acid methyl ester was used as an internal standard. A 2.0 p1 sample volume was injected splittless onto a fused silica capillary column (Optima-5- MS, Macherey und Nagel, Dtiren, Germany). The fatty acid methyl esters were identified by a mass spectrometer (GC-Q, Finnigan. Bremen, Germany) and quantified by a flame-ionisation detector (GC-Autosystem, Perkin Elmer, Oberlingen, Germany). The GC-temperature-program was described previously [30]. The mass spectra and retention times of 68 fatty acid methyl esters were determined by injection of authentic standards (Supelco, Deisenhofen, Germany and BioTrend, Köln, Germany). We used a fatty acid nomenclature in following form: x A:B, where A designates the total number of carbon atoms, B the number of double bonds, and x the distance of the closest double bond to the carboxyl group. The suffixes c for cis and t for trans refer to the geometric isomers of the double bond, whereas i (iso) and a (anteiso) indicate the position of a methyl group in branched fatty acids.
75 of
dilution artificial sampling ratio bacteria. physiology with carbon (20
formation from Broth determined
organisms,
streaked Association cultures rRNA
the and
into
package parsimony, inspected sequences nucleotide
follows: balticumT,
all
Determination
Cultivation
The
A
The Isolation
Phylogenetic PCR
use mM)
GM4R
of
hydrogen
the
complex samples the
2216,
gene SRB
1:9
source
of MPN
seawater
series
after
products out.
highest ARB 1
Desulfobacula
All
site,
as
6S
measured
an
and (inoculum:media).
of
with sequence
AF
(5-TACCTTGTTACGACTT-3)
by an
neighbor
electron was
[33].
were
Colonies
cultures
rRNA automatic
were Difco).
centrifugation
the
calculations organic
mixed
(0,75
[35]. of
to
for aliquot
233370:
measuring
manually
dilution
and
at
amplified
medium
organisms.
were
cultured inoculate
nucleic analysis
used
growth.
least of
by sequence
bar The
donor.
for
joining, accession
During
isolation medium
were
were
of
biomass
the
sequenced phenolicaT, sequencer
for
overpressure
that
several
Desulforhopalus
tool
1300
the
with
corrected.
were
in
method calorimetric
turbidity
by
transferred
triplicate
(5mm, incubated
Acetate
Growth acid
showed
of
a ARB_ALIGN
For
growth data
highest
and
was
nucleotides
PCR
no
defined
numbers
based
minutes
16S
by
of
most (Li-Chor
maximum
by organic
and base
used
described
AJ237606;
10000
at
using
growth bacteria.
phosphate
and
of of
Tree
MPN-series
rRNA
AMODIA
diluted
the
a at
at on
saltwater
phosphate
probable
of
a
for sequencing.
final
SRB
and
least
4
which mixture
vacuolatumT,
by
cultures
the
x
substrates
the
were primers topologies
4200,
MPN
°C
was
using
g)
likelihood
transferred
by
concentration
positive as
gene
microscopy.
three
method Technical
Desulfrspira
was
as
over
76
described Bioservice
For
Cord-Ruwisch
number
were
used medium
used
MWG-Biotech analysis
counts
of
analysis
described the
of
were
GM3F
times
sequence
determined
90%
liquid
was
different
aerobic
deep
tube
for
published
were not for
analysis
Nucleic
L42613;
into
University
of
(MPN) shaken
before as
(v/v)
inoculated
sequence
previously
to
the
agar
(5 included
of
was master
GmbH of joergenseniiT;
For
aerobic
by described
the determine
evaluated
‘-AGAGTTTGATC(AJC)TGGC_3’)
heterotrophic
1
PLFA. calculation
H,
time
data.
Isaksen dilution
[32]. with
by
transferred acids mM
considered
isolation
next
AG,
estimates,
by two
Desulfobacterium
and
dilution
(Braunschweig,
heterotrophic
of
alignment.
microscopy
with
the
in
periods,
was
[34]. different
Ebersberg).
dilution
Pure
times
The
were Munich
10%
by
the
by
the
and technique After
American
3
added
X99637;
of
Widdel
of
viable
sequences
cultures
series,
performing we
pure. (v/v)
onto ARB
ml
and
Teske
a
isolated
extraction,
the
different
flask
sets depending
day.
The
using
used
sediment
as
CO,) and
sulfate-reducing
bacteria
biomass
agar
database
most
[28].
and
27
of
[34].
Desulfotignurn
alignment
Public
catecholicurnT,
an
were by
Germany)
Growth
these
the
were from
filters.
via
ml
or
plates
Bak alternative
a
trees.
maximum
abundant
The
aliquots
of
program formate
(Marine volume
isolated
on [31].
anoxic
master sulfide
Health
loaded
are
liquid
[281,
each
Only
was
and
was 16S the
The
by
as AJ237602; Desii1fi.rhopa1us singaporensis, xxxx; DesulJxe1la halophila AF022936; Desulfofaba Tgelida AF099063; Desii1frjitstis glvco/icus’, X99707; Desulfocapsa1 sitlfiexigens Y13672; Desu1fin’a1ea T ; arLa’, AF099061; Strain LSv53, AJ241014; ,Strain LSv23, AF099059; Strain LSv24, AF099060; Strain DI-IAI, xxxxx; Strain GIHA, xxxxx; Strain JHA1,, xxxxxx; Clone SVA0632, AJ241014; Clone SvaOl 13, AJ240982; Clone Sva0999, AJ241013; DGGE band h, L40787.
Results and Discussion
Estimation of the viable biomass. For all four fjords the highest content of lipid-bound phosphate, as a measure of the total microbial biomass, was found in the top layer of the sediments (Figure 1). The phospholipid phosphate decreased with depth and changed from 70 to 10 nmollg sediment dry weight, which is in the range observed also from other sedimentary environments
[36]. Based on the assumption that I nmol phosphate is equivalent to 3.4 x io cells [31], a total cell number for eukaryotic and prokaryotic microorganisms of 1.5 x i0 cells per gram dry weight sediment in the top layer can be calculated, which is in the range of cell numbers determined by molecular methods [8]. The total biomass for different depth intervals varied between the four sampling sites. A very pronounced decrease with depth was found for samples from Isfjorden and Krossfjorden, which are both located near the open sea, whereas sediment samples from Kongfjorden and Smeerenburgfjorden showed less decrease with depth and are more strongly influenced by terrestrial input of organic and inorganic matter from the neighboring glaciers. Vertical distribution of signature PLFA for functional microbial groups. The more detailed PLFA profiles of the sediment from Krossfjorden is shown in Figures 2-4, however, similar tendencies were found at all other sampling sites. Specific signature PLFA, previously described for aerobic organisms and for anaerobic bacteria were grouped together into different plots (Figure 2). Polyunsaturated fatty acids, c5 20:4 and c5 20:5 which have often been found in significant amounts in diatoms and other eukaryotes [37], were abundant in the upper sediment layers. The c4 22:6, a typical fatty acid of eukaryotes [9] decreased with depth. The same fatty acid has been detected in psychrophiliclbarophilic bacteria that have been isolated from different cold marine environments [38]. The polyunsaturated PLFAs were extremely low in the deepest horizon. In contrast to the polyunsaturated PLFAs, the relative amounts of iso and anteiso branched PLFAs, specific for facultative or strict anaerobic bacteria, increased with depth and reached a maximum at 10-11 cm. The bacterial specific fatty acid a15:0 [39], was the most abundant branched PLFA with a relative amount of up to 10%. All together, the data imply that a large fraction of the biomass of the upper sediment layer originated from eukaryotes and aerobic bacteria, either actively growing in the sediment or deposited from the overlaying water column, and
77 Figure
(+),
aerobic
dominated
specific
comparison low environments.
lower
horizon.
A studies
temperatures
with
same
SRB
quantification
Krossfjorden
amounts
depth
concentrations 1
amount
bacteria
Depth-profiles
signature
have
These
specific
by
to
in
facultative
[Könneke
shown
c) E at
PLFA
(I)
association The of
data
feeding
a
of
PLFAs and
depth
both
highest biomarker.
SRB
agree
profiles
at
of Smeerenburgijorden
that
10
15 20
5
depths
on lipid-bound
and
biomarkers or
of
0
by
for
with
the
with
obligate
20
them.
absolute
Widdel,
Lipid
of
specific
Desulfobacter
synthesis cm,
from
previous
temperate
the
Below
The
phosphate
whereas
bound
anaerobic
2
total
abundance
unpublished
show
20
to biomarkers
absolute
(A),
molecular
of
10
a
biomass
phosphat
estuarine
depth permanently
the
cm, as
that
lOMel6:O
and
bacteria.
indication
maximum
yet
and
was
78
these
of
40
data].
Desuifovibrio
is,
studies and
with
sediments
(nmol
6
relative
detected
however,
cold
cm
for
in
MPN-counts,
SRB
The
a
relative
relative Arctic
the viable
g from Desulfrbacter
absolute
abundances
dry
play 60
microbial in
from
the t]ords
biomass
spp.,
the
not weight)
abundance
maximum
a same
different
of
amount top
as
minor
whereas
are
north-western
in
community
reliable
environment
species
sediment
of
80
lsfjorden
shown
at was
IOMeI6:0
locations
of role
10
i17:1
lOMel6:0
since
found
inhibited
cm.
(0).
in
in
Svalbard.
and
seemed
was
Figure
cold
[8].
pure Kongsfjorden
[9,
and
in a
five
the
found 40],
declined
by
marine
culture
i17:l,
to
3.
deep
fold
low
the
be
In
in ______I ______
PLEA (molqc) PLFA (mol%) 0 2 4 6 8 10 0 2 4 6 8 10
I I 1 N ‘S -, ;‘i.
2-3 N N N NN NJ -= I 2”t_”
5-6 •22:6 S N N N 51 •cycl7:0 c-i D20:5 Dal7:0 in D 20:4 N a15:0 lJ 18:2 l)i15:0 10-Il ‘S 5 N N N S N N N S N ‘S N ‘S S ‘I. I
-ci
19-20 A B
Figure 2 PLFA profile of selected biomarLers for (A) aerobic eukaryotes and prokaryotes. and (B) facultative and obligate anaerobic bacteria. The relative amounts of PLEA are shown in different horizons of sediment sampled from Krosst]orden.
Absolute PLFA abundance (nmol g-1 dryweight) Relative PLEA abundance (mol%)
0 0.1 0,2 0,3 0,4 0,5 0,6 0 0,4 0,8 1,2 0-I
2-3
5-6 C-) • i17:1 0 N D lOMeI6:0
10-11 V
-ci ‘I-)
19-20 A B
Figure 3 PLEA profiles of I0-methyl-pentadecanoate (lOMe 16:0) and 2-methyl -hexadecenoate (117:1) as specific
biomarkers of SRB of the Genera De.vulfohucter/Destilfobactei-iunz/De.vulfohciculoand Deculfovibi-jo, respectively. The
signature PLEA are shown in (A) absolute abundance and (B) relative abundance in different horizons of sediment sampled from Krossfjorden.
79 The largest amount of total unsaturated PLFAs (mono-unsaturated 50%, poly-unsaturated 12%) was detected in the top layer of the sediment (Fig 4A). The relative amounts of total unsaturated PLFA decrease with depth to 55 qc between 2 to 6 cm. and to about 40% below a depth of 10 cm. The total amount of branched PLFAs, originate from bacteria, increase with depth from 7% in the top to 16% in the depth of 20cm (Fig. 4A). The most of the PLFA consist of even-numbered acyl chains (>80%). A slight increase of acyl chains with [4 carbon atoms were found with increasing depth. The fatty acid composition is an important factor that determine the membrane fluidity. Eukaryotic and prokaryotic microorganismsgenerally respond to a decrease in growth temperature by increasing acyl chain unsaturation or branching, or by decreasing the acyl chain length. These changes decrease the liquid-cristalline to gel phase transition temperature of the phospholipids. Therefore the organisms regulate their membrane fluidityto changing temperatures. The portions of unsaturated, branched and short chain PLFAs from Svalbard sediments match with those reported previously for shelf sediments of temperate regions.
PLFA (mol%) PLFA (mol%)
0 10 20 30 40 50 60 0 20 40 60
I I I I
. , ... .,“
2-3
- 5-6 z C IS C 10-Il 6 B branched Cl) B ci8 D polyunsaturated Dcl6 mono-unsaturated c14 saturated 19-20 A B
Figure 4 PLEA proliles of sediment from Krossfjordenof (A) total relative amounts saturated, mono-and poly
unsaturated PLFA. Additionally the total relative amount s of branched-chain PLFA are presented. The vertical
distribution of PLFA with even-numbered acyl-chains (14 to 18carbon atoms) is shown in (B).
80 * Sulfate-reducing Aerobic Table
MPN
important methods. These
high
Psychrornonas
tubes cell
availability heterotrophic
lower.
with
cells
modifications
the
PLFA lts
bacteria
unsaturated unsaturated
95%
MPN-counts
One
In
same
numbers also
series
adundance
depth. in After After 1 After6
confidence
were
bacteria
contrast, heterotrophic compositions
Vertical
The
can
the
originate
possible
fatty role
were All 18
18
of identified
weeks
upper
In
speculate
and
abundance (70-90%) months
months
in bacteria bacteria
MPN
of
of
oxygen
identified belong
cultivated
acid bacteria
limits.
or
the
psychrophilic
in
short-chain
cold-adapted
the
from that
bacteria
of layer
Pseudomas, Svalbard
counts
depth composition with with
(cells
lipids and
by
aerobic
to
that
were
only
as the
at
other
or of hydrogen
formate
the
of
strains therefore
of
(cells 16S
4 of electron
cm’)
membrane
the (Gounot
°C.
changes short
aerobic
found
a
aerobes
‘y-
2-3
fatty
sediments
permanently
minor
rRNA aerobic
cm 3 )
SRB
sediment
subclass
bacteria.
which
cm
or
were
chain
acids
in acceptor.
heterotrophic
are
about
are
and
were
isolated
part
the
in
genesequenze-analyses
functioning
bacteria
active
previously
have
with
usually
have
fatty
oxic
of
the
Russel,
might of 4.6 4.6 2.1 0-1
Sediment
other
two
cold
The
Proteobactena,
the Pure (0.71-24) cm
(0.35-4.7) (0.7124)*107
deeper
often
a
surface from
under
also acids
and
in
and
maximum
also
habitats
highest
achieved recent
cold
81
1999).
this cultures
sulfate-reducing is
horizons
been
been Svalbard
in
described
these
be
(Knoblauch
possible
sediment
of
adaptations
permanently a
iO microbial
l0
the
(Gounot
sediment
depth
found
MPN-counts
isolated
by
in
cold
obtained
which reason 4.6 4.6 4.6
2-3
the
de
sediments
as over
of
as in
(0.71-24) (0.71-24) conditions. (0.71-24)
cm
layers
bacteria novo
upper
and
members
community
to
5-6
from
psychrophilic
from
et
psychrophiles,
have
cold
a
for
from
regulate
certain
Russel, al,
cm
(4.6
synthesis
is
Krossfjorden
first
the sediment.
from cold
•iO
been exhibited jO 5
10’
1999).
three
the
probably
x
of
changes
A cm
Krossfjorden.
temperature
the
exhibited
1999).
marine
iO
previously
highest high 7.5
2.4
2.4(0.36-13) 5-6
the
orders
by
and
or membrane (1.4-23)
(0.36-13)
cm per
High
genera
that
high
density
by
using
of
limited
psychrotolerant
(Table
environments.
ml)
positive
of
postsynthetic
cold
indicate
unsaturation amounts
The
portions
.1t3 range
detected
magnitude
of
Moritella, molecular iO
iO
of
fluidity.
triplicate adapted
1).
aerobic
by
active
MPN
with
The
the
the
in
of of MPN-counts of SRB. In agreement with the absolute abundance of the SRB specific biomarkers the highest cell numbers of SRB grown with hydrogen or formate were found in the top layer of the sediment (Table 1). The estimates for SRB were in the range of those reported previously for permanently cold marine sediments at low temperatures with other substrates like acetate, propionate or lactate [2]. Ten fold lower numbers were found at a depth of 2-3 cm and 5-6 cm depth. In contrast to Lsfjorden(Table 2). no substrate-depending differences in the cell numbers were found in Krossfjorden and Smerenburgfjorden. but we observed faster growth of SRB with formate than with hydrogen. In Smeerenburgfjorden, the estimated numbers of SRB were significant lower than those detected by molecular approches (> 5 lO SRB per 3).cm Differences between cultivation and molecular methods have often been described. In the present study the selective media and the low growth temperature, combined with the microscopic observation that all obtained pure cultures form aggregates might enhance the differences between both approches. The formation of cell aggregates could be a strategy of the SRI3 to escape oxygen stress in the oxic-anoxic sediment surface where the highest MPN numbers of SRB were found.
Table 2 MPN counts of sulfate-reducing bacteria from different fjords of Svalbard. The triplicate MPN series were incubated at 4 C.
lstjorden Kongst)orden Smeerenburgijorden (5-6 cm) (8-9 cm) (5-6 cm)
Sulfate-reducing bacteria (cells cm)
After 18 months with hydrogen 2.4 (0.3613)*.103 2.4 (0.36-13) 1O 2.4 (0.36-13) •lO After 18 month with formate 2.4 (0.36-13) i3 2.4 (0.36-13) i0 2.4 (0.36-13)
* 95% confidence limits.
SRB isolates and phylogenetic characterization. From the MPN series with formate as electron donor, seven isolates were obtained that could be identified by I6S rRNA gene sequence analysis as Desulfotalea psychrophila or D. arctica. These are psychrophilic SRB from permanently cold Arctic sediment which are able to grow on a variety of organic substrates [3]. The repeated isolation of these strains indicates the abundance of Desulfhtaiea species in the sediment around Svalbard. Until now, three SRB were isolated in pure culture with hydrogen as electron donor from Isfjorden (strain DHAI), Krossfjorden (strain G1HAI) and Smeerenburgfjorden (strain JHAI).
According to its I6S rRNA gene sequence strain DHA I forms a cluster together with clones and isolates from Svalbard and the previously described Desulfrrhopalus singaporensis [53] within the
82
“Desulfobulbaceae”
genus LSv53,
identity).
members
bases
using
Desitlfobacterjuin using
strain {2j.
presents Fig.
species Svalbard
16S
I
The
It
rRNA
parsimony JHAI
of
neighbor-joining
5.
Desuljbrhopalu,c
shares
the
closest
an
that
Phylogenetic
another
This
of
(97.3%
to
16S
isolate
gene),
selected
shows
this
rRNA
criteria.
might
-
between
relative
new
cluster,
obtained
catecholjciun
an
identity);
reference
gene
analysis
tree
complete
(Fig.
indicate
The
genus.
organism
of represented
95.9
showing
sequences. bar
-
5).
whereas
strain
with
corrected
sequences
f represents
-
that
—
According
and
all substrate
--
r - —
-
the
lactate
is
that
-— G1HA
- The
10%
other
the 99.9 ______
—
unique
affiliations
by
with
L
_j- Desulforhopalus
of
10%
shorter
aforementioned
has its
—-—
the
as
%
a
sequences L..
n-—
—
oxidation. to
-
is
estimated
position
type
to
delta electron Strain
---—------—
identity
within 16S
Desulfofustis
Desulfobacterium sequence Desulfobacterium
-
be of De.sulfota!ea
Clone
strain. subdivision
—-
rRNA 16S
G1HA
reclassified,
83
variability sequence
Desulfocapsa the
Sva0999
donor
--—--—-—-
with The
Oesulforhopalus
showed
for
rDNA
[—
vacuolatus cluster Clone Strain
glycolicus
sequences. “Desulfobulbaceae”,
Strain the arcilca Clone
Strain
cluster the
Strain
of
and
divergence.
DGGE sequences catecholicum and Sva0632 DHA1
LSv53 the
Desulforhopa/us
forms
16S lower
sulfoexigens and SvaOll3
LSv23
a
carbon catecholicuni
LSv24
Proteobacteria.
termini containing
Desulfobacterium band
sin
is strain
Desulfotignum
rRNA a
Desulfofaba from
Desulfobacula gaporensis
a
clone
only
identities
h
source
new
filter was
Desulfospira
strain DHA1
vacuolatus
gene
distantly
sequence
genus
d
which strain Desulfocella
gelida
because
(97.2%
The
— from
Strain DHAI, balticum autotrophicum Desulfobacler
phenolica
sequences is
(< to
DGGE
tree
considered separate
joergensenil identical
JHA1
the
G1HA
the
95%).
related
strain
was
Sva0999
identity it
band existing
halo
same
is calculated
phila Gil-IA, postgatei
h
from
only
probably of
the
to
(93.6%
So
habitat
of
tree
strain
other
from
only
1255
far,
the
the
ari
by by its Strain JHA 1 is a member of the Desulföbacteraceae with Desuifi)bacula phenolica being closest described relative (94.8% identity). As can be seen in Figure 5, the 16S rRNA gene sequence of DGGE band h obtained from a stratified marine water column of Manager Fjord, Denmark [49] falls in close proximity to this organisms (98.6% identity). Based only on the sequence of the 16S rRNA gene, strain JHAI probably represents also a new genus within the family De.culfibacteraceae. A detailed characterization has to reveal whether these strains indeed represent new genera. The utilization of hydrogen as electron donor combined with acetate as carbon source were often described as selective substrate for the isolation of members of the genus DesuUovibrio from marine habitats. The absence of Desulfrvibrio species in high dilutet MPN tubes and the low concentration of the Desu/Jovibriospecific biomarker i17:1 confirm the low abundance of the genus in Svalbard sediment which have been previously reported [8]. In conclusion, the microbial population of the permanently cold marine environment along the coast of Svalbard might be dominated by psychrophilic bacteria as indicated by newly isolated bacteria of different physiological groups, which grow optimally below 20 °C. Most of them are highly related to known psychrophiles from other cold marine habitats. A significant indication for a cold adaptation of the PLFA profiles was not observed.
Acknowledgement We thank Ruth Meincke for help in the isolation of the aerobes. This work was supported by the Max-Planck-Society, Germany.
References [1] Sagemann, J., Jørgensen, B.B. and Greef, 0. (1998) Temperature dependence and rates of sulfate reduction in cold sediments of Svalbard. Geomicrobiol. J. 15, 85-100. [2] Knoblauch, C., Jørgensen, B.B. and Harder, J. (1999) Community size and metabolic rates of psychrophilic sulfate-reducing bacteria in Arctic marine sediments. Appl. Environ. Microbiol. 65, 4230-4233. [3] Knoblauch, C., Sahm. K. and Jørgensen, B.B. (1999) Psychrophilic sulfate-reducing bacteria isolated from permanently cold Arctic marine sediments: description of De.vulftjrigus oceanense gen. nov., sp. nov., De.vulfrJrigusfragile sp. nov., Desulfofaba gelida gen. nov., sp. nov., Desulfrtalea psychrophila gen. nov., sp. nov. and Desulfrnalea arctica sp. nov.. mt.J. System. Bacteriol. 49. 1631-1643 [4] Sørensen, J., Christensen, D. and Jørgensen, B.B. (1981) Volatile fatty acids and hydrogen as substrate for sulfate-reducing bacteria in anaerobic marine sediment. Appi. Environ. Microbiol. 42, 5-11.
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the
hybridization
T.
Y,
Y.
Lake
2-aminoethanesulfonate
Syst.
A.,
A,,
R.
K.
J., Mariana compounds
Y.
isolate.
Kato,
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bacteria
a
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N. DNA
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Ramsing, Evol. Clawson,
(1998) Mac,
isolated
Kato.
and
S., (1975)
0.,
C. vertical
antarcticus
(1991)
23 of Extremephiles
E..
fragments.
implications.
Trench
and
Microbiol.
in counts
Kroder, Russel, Gregor,
from
Rainey,
in
Egypt)
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Sulfate-reducing bacteria
C.
bacteria.
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a
a
Psychrophilic Veronck,
DesulJbrhopalus
Horikoshi,
from N.
M.
coastal
distribution
stratified
(1999)
ajapan
reclassification
Bacterial Muyzer,
and
inorganic
B.,
and
L.,
Appi.
N.J.
iiscosu.c
gen.
F.
M.,
B.
Appi.
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from
description
Arch.
50,
A..
Habicht,
Godchaux,
J.
marine (Taurine)
denaturing
Taxonomic
3,
J., (1990)
trench.
L..
Dilling.
nov.,
Environ. Appi.
fjord
479-488.
K.
7
Burghardt. Antarctica. G.
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Microbiol.
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Rev.
activities
Kuever,
electrophoresis
B.
2943-2951. Denmark) H.F.
a bacteria aerotolerant
Microbiol.
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morphologically
Stahl,
(1996)
Moritella
39,
S.
H.
Ice
and
E.
to
physiological
sp.
sp.
barophilic
44.
AppI.
136.
144-167.
J.,
and
in
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quantified
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at
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Distribution 289-295.
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1669-1673.
anaerobic,
Swings,
vi.ccosa
65-74.
A.
evaluated
Environ.
a
of glucose
Antarctica.
Oxidation
a
novel
bacteria
(1999)
of
new
unusual
by PCR-amplified
Sulfidogenesis
geographical
aspects
comb.
0,,
rRNA
J.
barophilic of
B.
barophilic
E.
uptake
Microbiol.
halophilic
by
or
Sulphate isolated
mats
sulfate-
(2000)
(1998)
B.
sulfate-
of
nov.. Arch.
most-
nitrite
slot-
and
and
H.,,
in
of 68 SAarhus Hans-Frisch-Str. Present Denmark (CSIC-UIB). * ‘Corresponding Enrique Schramm, Present Max-Planck-Enstitute
Community University, address: address: Liobet-Brossa,
bacteria Crtra. Rikke author: 1-3, Lehrstuhl Area Valldemossa Institute L. D-95440
structure [email protected] for Meyers, de
A Limnology
in Microbiologia, RaIf Marine für
multi-methods for
an okologische Bayreuth, Rabus’, Biological Niko Km Microbiology,
intertidal and and 7.5, Finke,
and Rudolf Michael Germany Oceanography, E-07071 Departament Sciences. Mikrobiologie,
4 Stefan 90
activity Amann Celsiusstr. E. Palma
surface-sediment:
approach Grötzschel, Böttcher, Dept. de submitted de Recursos
of BITOK. for Mallorca, I,
sulfate-reducing Microbial D-28359 Martin Ramon Universität Naturals, Spain Könneke, Bremen, Ecology, RossellóMora*, EMEDEA Bayreuth, Germany Andreas Aarhus, Dr. Abstract
The community structure of sulfate-reducing bacteria (SRB) in an intertidal mud flat of the German Wadden Sea (Site Dangast) was studied and related to sedimentary biogeochemical gradients and processes. Below the penetration depths of oxygen (-3 mm) and nitrate (—4 mm), the presence of dissolved Fe and Mn and the absence of dissolved sulfide indicate suboxic conditions within the top 10 cm of the sediment. Moderate to high bacterial sulfate reduction rates were measured with radiotracers throughout the sediment and dissimilatory sulfate reduction was also demonstrated by the presence of acid-volatile sulfides (AVS, essentially iron monosulfide). Stable sulfur isotope S/S)3432 discrimination between dissolved sulfate and AVS was dominated by sulfate reduction,( but a contribution from anaerobic metabolism of sulfur intermediates is likely. The diversity of SRB was studied using denaturant gradient gel electrophoresis (DGGE) of 16S rDNA and counting viable cells with the most probable number (MPN) technique. Phylogenetic groups of SRB identified with these two techniques were evenly distributed throughout the vertical profile (0-20 cm) of the studied sediment. However, application of fluorescence in situ hybridization (FISH) demonstrated a maximum of the Desulfovibrio and Desulfosarciiza-Desulfococcus-Desulfofrigus groups between 2 and 3 cm depth. These two groups encompass acetate and lactate utilizing SRB. The coincidence of this SRB maximum with a local maximum of sulfate reduction rates and the depletion of acetate and lactate reflects the biogeochemical processes related to sulfate reduction.
91 Introduction
coastal
sinks
continental the receiving Up
they
different performed oxidants
and that
sulfate electron
mM,
intertidal of (Trudinger
sediments sulfate-reducing
organic completely (SRB)
to in was
Wieringa Ravenschlag
1990) different
Continental
The identify reactive to the
oceans
finally
up
A encompass
it
to
30%
applied
ranges. and
anoxic
reduction may
to
key isolated
importance
the
substrates
acceptor
is
yield
up
about
et sediments
activity
(Gibbs of
is
by
the
S042 usually
margin and
1992).
organic oxidize
question
sediment
still
to
al.
reflected
the
et
microorganisms
zones
These
to
introduction
90% of margin
quantify
2000).
50%
from be
bacteria
a!.
oceanic
(Froelich
only
(Jørgensen
marine (Fenchel
free
SRR
1981).
sediments
such
observed
and abundant
of
acetate
of
compounds 1999:
of
sediments
in may
of
(Wollast
this
sulfate
also
energy.
the
10% sediments
are
marine
microbial nutritional
as the
SRBs
(Sahm
primary
Since
sediments
global
Sahm
et
vary
environment.
generally
by short
et
to
of
organic
of
in
in
l982b). al.
play
reduction
al. CO-
the
16S
in
that
En
1991). sediments. the most are
deeper
the
et
considerably
(Schubert
1979).
chain
et
their flux production 1998).
accordance
variety
include a!.
a ecology
(Widdel
rRNA
generally
vertical
employ al.
total properties
prominent
matter
controlled
of
Since
1999:
of
(Devereux
In natural
layers
fatty
1999;
Using the
for
The
particles
Pure addition,
of ocean
based
shelf
Phylogenetic
et
in is
deposited sediment
Böttcher the
sulfate
1988;
an
acids,
metabolic
takes
characterized
area!
of
Bowman
92
the
al.
habitat. marine
the
with between
cultures
role by array
on remineralization
the
molecular
area.
sediments
2000),
and
influence
Rabus
et
temperature
place
5S042 rates
alcohols the
the
concentration
the
sediment,
in
et
profiles:
of
al.
sediments
matter
organic
Only
the
The
deltaic capacities
al.
biogeochemical
et
decreasing
about of
electron
in of
analysis
and
et
radiotracer
1996:
2000). al.
marine
by the
tools
SRB
dissimilatory
al.
oxidation recently
and
of and
is
correlated
2000:
and a
matter
0-,.
areas shelf
0.2
2000):
(Vosjan
sulfate-reducing
remineralized
high
Llobet-Brossa (Amann aromatic is
of intertidal
have
acceptors.
of
of
in
carbon
consequently
NO3,
and mineralized
yields,
organic
areas.
Ravenschlag
sulfate-reducing
ocean
of
this
isolates
input
technique
transported
all
been
of
the
1974).
104
to
Fe(EIl)
gradients type
important
sulfate et
cycle,
organic
compounds,
From
of
a
mud
water
the shelf
at. carbon
each
shown
mmol
zonation (Devereux
organic
(Berner
of
the
1995) abundance
via it
the
and
even
bacteria this
et flats
investigation
reduction
by
regions
is
was
et
allowing
availability
carbon
microbial
in in substrates dominant
al.
to
about
rivers
Mn(IV),
a!. 25-50%
m2
bacteria
matter.
allowed though
of
shown marine
1982),
of
marine
utilize
and
2000; 1998;
et
the
with
are
the
28
to
d’
is in a!. of
to
a sediments. The present study focuses on the diversity, abundance and activity of SRB and their relation to the biogeochemical processes in a tidal sediment of the southern North Sea, (Wadden Sea, Site Dangast). To advance our understanding on the relationship between the community of sulfate-reducers and biogeochemical gradients, we combined cultivation dependent and independent microbiological approaches with biogeochemical and stable isotope analyses.
Materials and Methods
Study site. The river Weser is one of four major rivers draining into the German Bight of the southern North Sea. The Jade Bay, a meso- to macrotidal embayment, is situated in the coastal area to the west of the Weser estuary in the northern part of Lower Saxony (Germany). The Jade Bay is under influence of the fluvial input of the river Weser, and the mean tidal range in the southern part reaches 3.75 m (lrion 1994). The sampling station, ‘Site Dangast”, is located 2 km west to the small village Dangast, about 25 m west of a tidal creek (“Dangast
Tief’), connected to a freshwater outlet (“Dangast Siel”) and about 15 m north from the shore line (Fig. 1). Thus the site represents a highly dynamic system.
The sediment experienced tides which exposed it to the air for about 5 h and left it inundated for about 7 h, with some variability due to the wind velocity and direction (Liobet Brossa et al. 1998). Processing of sediment samples. Sediment cores were obtained on June 28, 1999 at low tide between 7 and 9 am with polycarbonate tubes (diameter 8-10 cm; length 50 cm). The sediment cores were closed with air-tight rubber stoppers on both ends and transported cool
(approx. 4°C) and dark to the laboratory for further processing within about 3 hours. Sediment cores were sliced by extruding them from the polycarbonate tube and cutting with a thin aluminum plate into the following layers: 0-0.5, 0.5-1, 1-2, 2-3, 3-4, 4-5, 5-10, 10
15 and 15-20 cm (top-down). Sediment samples for enumeration of viable cells (MPN) and molecular analysis (DGGE and FISH) were taken from the same sediment layers of a single core. For geochemical measurements of pore water and sediments, parallel cores were used and sliced in 1 cm layers under inert gas (N,) in a temperature controlled room (4 °C). All cores were taken from an area of I by 2 m. In September 1999 additional samples of Wadden Sea surface sediments (2-5 cm depth) were taken from the same site in Dangast and from Horumersiel, located approx. 20 kin north of Dangast.
93 dot
is Figure
properties
investigation.
constance. sediments carbon
carbon carbon.
were derivatization Dyer
Glamorgan,
phospholipids methanolysis
was silica
Germany) methyl
Elmer,
indicated
Sediment
(•)
(I)
utilized
1: Characterization
(1959). marks
capillary
fractionated
(TOC)
on
Phospholipid Map esters
Oberlingen,
by
and
were
a
Total
and
the
the
of
CM
as
characterization.
UK)
The
to
biological location the
arrow. were contents
to
column
procedure
quantified an
determined
methyl-ester,
carbon
5012
sampling
methyl
internal
with procedure
by
The identified
of fatty
Germany).
coulomat
(Optima-5-MS, of
the
were
the
river
chloroform,
was
activities
ester
site.
organic
by
standard. acids city
was
gravimetrically
use
Weser
obtained
measured
Site
the
involves
and Bremen.
by
(Palojärvi
A
of
(PLFA)
used
Dangast. with
use
the is
variety
material
analysis
related
The
indicated
silicic
2.0
of
acetone
use
(B)
from
a
to
Macherey a
using
il
a
CM GC-temperature-program
were
(A)
one-phase
Position
and of
of
transmethylate
to
flame-ionization-detector
sample
acid
by
by by
and
the
methods
Position
a
sulfate-reducing
5130
94
Albers a (2).
extracted
capillary
and
mass drying
difference
Leco
sulfur
of
columns
und
volume the
acidification
methanol
of
extraction,
spectrometer
were
1998). sampling
Nagel, sediment
the
specialion_Pore
induction
gas
essentially
German
(Isolute of
was
applied
the
chromatography.
Nonadecanoic
Düren,
total
site,
bacteria as
injected
ester sections
fractionation
Bight
modul
eluents.
furnace
Site
(GC-Q,
carbon
to
as
SPE
Germany).
(GC-Autosystem,
is
in
Dangast
study
described
linked
of
the water
described (UIC).
splittless
at
columns,
the
and
and
The
Finnigan,
North
70°C
physicochemical acid
in
sediment
The
on
contents
fatty total
total
the
Total
mild
The
by
Sea.
until methyl
onto silicic
polar Jade
elsewhere
Bligh
inorganic
inorganic
1ST
The
fatty
acids
Bremen,
alkaline
organic
weight
Bay
a
under
in
Perkin
lipids
black
fused
acid.
ester
Mid
acid
and
the
(I)
of (Palojärvi and Albers 1998). The mass spectra and retention times of 68 fatty acid methyl esters were determined by injection of authentic standards (Supelco, Deisenhofen, Germany and BioTrend, Köln, Germany). Unsaturated trans -isomers were not determined. Biomass was determined by phosphate analysis of PLFA. After the Bligh and Dyer (1959) extraction, aliquots of all samples were used for calorimetric phosphate analysis to determine the viable biomass (Findlay et al. 1985; Findlay Ct al. 1989). The fraction of acid volatile sulfide (AVS) was separated from the wet, Zn-acetate preserved sediment by the reaction with cold 6N HC1containing 2SnCl (Duan et al. 1997) in a stream of nitrogen. The addition of SnCl, increased the recovery of the AVS fraction within the first 5 cm, but no further influence was observed at greater depths (Böttcher, unpublished data). Sulfur isotope ratios (see below) of the AVS fraction recovered by both methods agreed within 1%c (Böttcher, unpublished data). The sum of (essentially) pyrite and (minor) elemental sulfur (fraction ‘Cr-lI’) was obtained by the distillation with hot acidic Cr(ll)chloride solution (Fossing and Jørgensen 1989). 2SH was trapped as Ag,S in a 3AgNO solution and quantified gravimetrically. For stable sulfur isotope analysis S/3432S), pore water sulfate was precipitated from filtered Zn-acetate preserved samples as 4BaSO( carefully washed and dried. Sulfur isotope ratios of the AVS, Cr-lI, and pore water sulfate, fractions were measured by C-irmMS (Pichlmayer and Blochberger 1988). Samples were converted to SO, using a Eurovector elemental analyser which was connected to a Finnigan MAT Delta gas mass spectrometer via a Finnigan Conflo
[I split interface. Isotope ratios are given in the -notaEion versus the SO,-based Vienna- Canyon Diablo Troilite (V-CDT) standard. International standards EAEA-S-l, IAEA-S-2, IAEA-S-3, and NBS 127 were used to calibrate the mass spectrometer. (ii) Pore i’aters_Air and pore water temperatures were measured with a digital sensor (GTH 1150 digital thermometer) at the beginning and the end of the sampling session. After transport to the laboratory. subcores (3.6 cm diameter) for pore water analyses were taken in a temperature-controlled room. Pore waters were separated from the sediment by centrifugation in closed centrifugation vessels under inert gas. Prior to analyses, pore waters were filtered through membrane filters (0.45 aim; Sartorius) and acidified with nitric acid (reagent grade quality) into pre-cleaned PE bottles. Concentrations of dissolved iron, manganese and sulfate were analyzed after appropriate dilution by means of ICP-OES (Perkin Elmer Optima 3000 XL). It is assumed that dissolved iron essentially consisted of Fe(II), although a contribution from complexed Fe([lI) can not be completely ruled out (Luther III et al. 1996). Dissolved sulfate was additionally quantified gravimetrically as 4BaSO from the pore water of a parallel
95 sediment core which was immediately cut into sections and preserved in 20% zinc acetate solutions. The results of the two methods agreed very well. H,S was measured in selected samples preserved with 2% ZnCI, solution according to Cline (Cline 1969). Salinity of filtered samples was measured with a hand refractometer. Pore waters for the analysis of volatile fatty acids (VFA) analysis was extracted by centrifugation of the sediment in precombusted glas centrifuge tubes. 2 ml of the supernatant were sampled with a glas syringe into precombusted borosilicate vials (4 ml ) with teflone lined caps and frozen (-20°C) until analysis. The VFA were measured by HPLC as 2- nitrophenyl hydrazin derivats as described by Albert and Martens (1997). A Sykam Sl2ll HPLC pump combined with a linear UV/VLSdetector and a Gilson 232XL autosampler were used for separation and measurement of the acids. The detected signal was recorded and
integrated by a Knauer Eurochrom 2000 integration software. For the separation of the acids a 25 cm LiChrosphere RP8 column with a 1.5 cm LiChrosphere RP8 guard column from Knauer was used. 2.5cm polymeric reversed phase (PRP-l) cartridge (Hamilton) installed in the sample loop was used as a sample concentrator. Differing from the original method. the
flow rate was reduced to I mi/mm, the concentration of tetrabutylammonium hydroxide of
solvent A was reduced to 1 mM and the concentration of tetradecyltrimethylammonium
bromids in solvent B to 25 mM. Additionally, the pH of the solvent was adjusted with HCI instead of phosphoric acid. The detection limit for glycolate and lactate was 0.2 IIM, for acetate and propionate 0.3 jiM and formate 0.6 jiM. (iii) Microsensor measurements- Profiles of oxygen, hydrogen sulfide, and nitrate concentrations at the sediment-water interface were measured in the laboratory with microelectrodes. The determination of oxygen profiles was done with Clark-type 0 sensors with guard cathodes (Revsbech 1989). The 02 sensors had tip diameters between 10 jim and
20 jim. a stirring sensitivity <2 %, and a 90 % response time t)< I s. To calibrate the sensors. readings in the overlying air saturated water and in the anoxic zone of the sediment were taken. A two point calibration curve was calculated with oxygen solubility values for different temperatures and salinities based on equations from Garcia and Gordon (1992). Amperometric H,S microsensors were constructed, calibrated and applied as described by KUhlet al (1998). Nitrate was measured with a microbiosensor in which nitrate and nitrite are reduced to N,0 by bacteria and N,0 is detected electrochemically (Larsen et a!. 1997). The nitrate sensor showed no detectable stirring sensitivity and a 90 % response time t)< 45 s with a tip diameter of 70 jim. Calibration was done by measuring sensor readings in nitrate solutions with salinity and temperature identically to the sample.
96 From steady-state oxygen profiles area! fluxes of oxygen were calculated based on Fick’s first law of one dimensional diffusion J = D dC(z)/dz (Ktihl et al. 1996) with Deas effective diffusion coefficient and dC/dz as concentration gradient for oxygen. Volumetric production and consumption rates were calculated based on Ficks second law of diffusion using the second derivative of the measured concentration profile. Nitrate values were calculated correspondingly. Furthermore, it was also determined if consumed nitrate originated from the water phase (Dw) or was produced by nitrification (Dn) by calculating back a concentration profile from an activity profile with no nitrate production (Meyer et al. 2000). Sulfate reduction rates (SRR). Bacterial sulfate reduction rates (SRR) were measured using the whole-core incubation technique with the injection of a carrier-free 4235S0 tracer (Jørgensen 1978; Fossing and Jørgensen 1989). Although the sediment temperature during sampling varied only slightly and was close to 17°C, much higher changes in pore water temperatures were found on a daily base between 26th and 30th of June, 1999 (Böttcher, unpublished data). Therefore, sediment cores were equilibrated after sampling in the laboratory at 10 and 20°C for several hours and subsequently incubated with the radiotracer
(—200 kBq per injection along 1 cm intervals) for 4.5 h in the dark. Activities counted with a Packard liquid scintillation counter were corrected for blank contributions derived from the counting and the distillation procedures. The apparent activation energy calculated from an Arrhenius equation using the depth-integrated SRR for the 10 and 20°C incubations gives a value of 66 kJ 1mo! which is similar to observations on a seasonal base in tidal mudflats (Kristensen et a!. 2000) and general findings on microbial sulfate reduction (Westrich and Berner 1988). Nucleic acid extraction and DGGE amplification. In addition to the sediment core used for MPN counts and VFA analysis, a second core was processed for molecular analysis. Replicate sediment cores were analysed to avoid changes in the community composition caused by handling and containment as described before (Rochelle et al. 1994). DNA and
RNA of each sediment horizon were extracted from 1.5 ml wet sediment by bead-beating, phenol extraction and isopropanol precipitation as described previously (Sahm and Berninger 1998). PCR amplification specific for SRB of the ö-subclass of Proteobacteria was carried out with the forward primer SRB385 carrying a GC-clamp and the reverse primer 907 (Sass et al. 1998). DGGE, excision of bands, reamplification, and sequencing were performed as previously described (Muyzer et al. 1996). DGGE partial sequences were added to an alignment of about 15,000 homologous bacterial 16S rRNA genes (Maidak et al. 2000) by
97 Table 1iUB338 SR11385 l)NMA657 DSV698 DSVl292 I)SV2 I)SV107 l)S1)13 1)S139X5 I)SS65% 66(1 221 I)S130224 l)SMA$%% l)TM229 I)SR65 N0N338 SVAI.428
using sequences
counting
(Snaidr stained oligonucleotide with 1998). situ processed
Percentage
Escherichia
Probe
In 14
1.
hybridization I I
Cy3
situ
the
Oligonucleotide
Accession
et cells 1)acteria Desulfocibrio Desulfoneina of Desu/frv’ihuio
Desulftu’ibrio/D’nzkrobiu,n Desu/fothrio De.wi!tovthrio
De.vuifrthorrer/D’hacufo De.suIfr.so,cinoID’coccu.v/D’fr,u., De.vuljobcicterium 16S
De,vulfohu1bu’ 16S 16S 16S 16S Desulfobonilus
De.oitforiIirio/D’tnonile 16S Desulforhopalu.s
Desultoro,naciilwn 16S 16S
DesuIfiito1eii/D’ti.vris 16S 16S (necative 16S none 16S 16S
fluorochrome 16S
aligning 16S 16S
al.
were 6S
hybridization as
of
hybridized rRNA. rRNA. rRNA. rRNA. rRNA. rRNA. rRNA. rRNA. rRNA. rRNA. rRNA. rRNA, rRNA. rRNA. rRNA, rRNA,
were rRNA.
1997;
coli
described
formamide
inserted
probes control)
numbers positions position position position position position position posItion position position position position position position position position position
numbering.
analyzed (FISH)
tool vp.
Llobet-Brossa
probes (16 (3 (13 (1 of
Target species) species Proteohacteria species) species)
used
cells
of 385-402 657-676 698-7
at 407-424 within 658-675
before 2)4-230 985- 660-679 221-238 1292-1311) 488-507 224-242 13 65) 33S355 428-446 229-246
used
(FA)
and
of 1-148
of the
the -668 1003
and 17
in the
and
in
sediment
5’
cell a
ARB
in
this this
(Liobet-Brossa
partial
counted;
stable
end.
total
hybridization
et
study.
counts.
study
al.
program GCT CGG TTC GTT CA,\ (Ki CCU CCC CAT TCC CAC T(iC
Probes (IAA (ICC (lUG GG CCC CC\ .\AT ACT
cell
sequences tree
samples
1998), CGY CCT GCC
were GAT CCI CUT
duplicates ACT TCC AAG GCG AUG CCT GUT CCT GUG TTC TCT ACG
counts
by
Hybridizations,
98
Sequence
used CCA TTC TCC CGG TCC CUC UGA CUT CCA
For CAC UCC GAC GAC
package ACG GCT .TG
using CGG ACG
buffer.
et
purchased of
two
are CCT GAT CGT CTC probe TGC
a!. CTG ACG
each CTG TTT GT1\ Tft’ TCC T(’A GG.\ TCA AUG
are were C(iG ACT
(5-3’) the
were
replicate
XXX CTC
1998). TCC AUG ATC (‘CC GGC GTc’ 1TC (IGA AA iTt CTC
listed TTC AAC GGC
ATTTrA CAT AXC
sample (Strunk
ARB
performed TGC CCA (‘AT TAC CTC CCT GUC AGT
from AUG AUG carried AAA ,\AG CGC CC,\
and CCT AGC CAT
in
microscopic From TA
parsimony TU
sediment C (‘ (1
between
YYY,
Table
et Interactiva in
out 0-35
al.
MPN-tubes ((-35 35 35
35 I 35 5(1
as 20 60 2(1 35 60
situ 60 6)) 35 25 15
IA 0
and
1.
for
1998-2000).
previously
700
cores For
tool
Rclcrence
deposited
each
examination
(Ulm, 27 27 27 29 29 29
and 29 29 29 29 29 29 29 29 29 29 36
fluorescence 27
(Ludwig
were
sample.
that
1000
Germany)
described
Aligned
at
cut showed
ZZZ. DAPI
et
The
and
and
al.
in growth, aliquots of 1-2 ml were withdrawn with N-flushed syringes. Cells were sedimentated by centnfugation and washed once with lx PBS. Subsequent fixation and hybridization were performed as previously described (Snaidr et al. 1997). Media and enumeration of viable cells. A defined, bicarbonate-buffered, sulfide-reduced
(1 mM) mineral medium, essentially having the same salt composition as natural seawater was used for cultivation experiments (Widdel and Bak 1992). Na-dithionate at a final concentration of 10 ig/ml was applied as an additional reductant. Organic substrates were added from concentrated stock solutions. Gaseous substrates 2(H/CO were supplied by applying an overpressure of I atm to the headspace of the culture )tubes. Viable sulfate-reducing bacteria were enumerated using an MPN technique with liquid media and with agar shakes. Samples from each layer of the sediment core were transferred via a funnel to glas bottles (250 ml volume) and mixed 1:1 with substrate-free, anoxic media under a steady stream of NiCO, (90:10 {v/v]),yielding approximately 100 ml of homogenate. The bottles were then anoxically sealed with butyl-rubber stoppers and screw caps. These sediment slurries were diluted in steps of 1:10 by transferring aliquots to substrate-free, anoxic media under N/CO, (90:10 [v/v]) in butyl-rubber sealed glass-bottles (250 ml volume of bottle, 100 ml total volume of dilution). The transfer of sediment suspensions between the glass-bottles was carried out with 2N-flushed syringes. Prior to transfer, the sediment suspensions were shaken vigorously to achieve optimal mixing. These “master”-dilutions were used to inoculate culture tubes for MPN-counts in liquid media. Similarily, “master”- dilutions were prepared for the inoculation of MPN-counts in agar-shakes. However, in this case not every single sediment layer was diluted but rather a homogenous mix of the top 5cm of the sediment core. (i) MPN-counts with liquid media_These MPN-counts were carried out in glass tubes (160 x 16 mm) sealed with butyl-rubber stoppers and screw caps that contained anoxic media with or without substrate under an N,/CO-atmosphere (90:10 [v/vl). Substrates used were as follows: 2H,/C0 (90:10, [vIvj, applied2 with 1 atm to the gas head space); H,/CO, + 2 mM acetate; 15 mM formate; 7.5 mM acetate; 5 mM propionate; 5 mM lactate. Each tube contained 9 ml medium and was inoculated with 1 ml from the corresponding “master” dilution. Three replicates were prepared for each substrate condition. To sustain reduced conditions, 0.5 mM Na,S were added after about four weeks of incubation. The tubes were incubated at 23 °C for 10 months. Growth of sulfate-reducing bacteria was determined by measurement of the optical density (660 nm) and sulfide using the semiquantitative method described by Cord-Ruwisch (1985) and by macroscopic and microscopic examination.
99 conducted
approximately
prepared
observing media. Bak Replicates Such and
Results °C
20.2
accumulation part greyish
investigated geochemical indicated polychaetes
surface Hespenheide. of 2000).
sampling 2000). The surface
showed processes AVS
(ii)
Sediment.
mud
during
Acid
transferred
was
1992).
TOC °C
colonies
should
il’IPN-counts
Agar
An Correspondingly,
diffuse (without
(0.5
(clay
sediments
a brown
volatile as
by in
(Fig.
sampling formation
in
maximum to
and
increase
The
described
were
June
cm
sediment
the
shakes
a
obtain
Downcore of were
and
9 unpublished essentially parameters.
to
diffuse
streaks. incubation 2E) which
ml
depth)
tubes
iron sulfur
wind).
sulfides
liquid
found
1999.
silt
of
picked
decreased
of
was with
were
of in
single
at monosulfide
anoxic
previously
turned grain
section.
the were
blackening
which cycle
medium
At The
for
brownish
around down
agar-shakes
temperatures
carried
corresponding
the
(AVS:
mirror
sand
by incubated
data)
about
conditions
instance, The
size
colonies. sediment
inoculated
(Böttcher
within
pore media
decreased means
with
to
These
grain
8
containing
and
sediment
essentially
about fraction
9-12
that
for out
cm
colonies
water
(acid-volatile
of
depth
with
the
of maximum
showed
at the
fraction
vertical depth the cores
on
and
of
Butyl-rubber cmbsf
in were
et
finely 16
first with
23
to
air
contents
molten
in
isolation
the
a!. <
HS
sediment,
sediments
cmbsf.
52%
was
the
°C.
displayed isolations_MPN-counts parallel which 63im;
temperatures
iron (Fig.
as
1-2
1998b).
that
sediments 100
(cm I
changes drawn
was
which
corresponding
water
ml
Growth
described
and
cm
characterized agar
sulfide,
monosulfide)
the
2C).
An
decreased
below
are of observed
essentially from
with
into
remaining
29%
stopper
Pasteur The
and first analysis
recovered contents
sulfate-reducing
is 2
were typical
The
distinct
was varied
a
derived
the
the
AVS)
surface),
AVS between at
substrates
darker
10
above
stable
10
also
water
continuously below
pipettes
corresponding
determined
cm
sealed
substrate.
of
of
phyllosilicates
for
were
and fraction
between
black
with
by color
the in almost
olive 67% reflected
from
for
sulfur
content. sulfate-reducing the
16.0
29
that
bioturbation,
as
May
grain found
decreasing
glass
(Widdel
changes:
MPN-counts until
in
coupling
cm were
described green
bacteria
the
(4S
°C
depth
completely
about
isotopic
by
agar
depth.
with 1998
size
tubes
the in
(in
overall by
observed
master
microscopically
color
values
all and (Böttcher
(Böttcher
the
biological
the
distribution
bottom shakes
16.0
depth.
(Widdel
of
porosity
(Böttcher
respectively.
above
sections
containing signature
and
Bak
oxic
with wind)
increasing
in metabolic
dilution. between bacteria.
and consisted
near
During
liquid
of
living 1992)
upper were
were
some
et
et
17.5 and
was
and
and
and the
the
al. al.
of
&
of - 21.1 and -26.2%) was significantly enriched in the lighter sulfur isotope compared to coexisting pore water sulfate S(ö34 values between +20.4 and 4+25%c: Fig. 2D). Apparent sulfur isotope enrichment factors between -41 and 2%-S are calculated. from the data given in Fig. 2D which are at the upper end or even exceed the results obtained in experiments with pure cultures of sulfate-reducing bacteria (Kaplan and Rittenberg 1964; Chambers et a!. 1975). The isotopic composition of the Cr-Il fraction which consists essentially of pyrite was more or less constant in the top 11 cm S(634 of —16±1%o) but decreased at greater depths (Fig.2D). Pore waters. The salinities of the pore waters during sampling in June 1999 were rather constant with 28%. This corresponds well to earlier data obtained during sampling on a seasonal base over a two year period, where it has been found that salinities in the pore waters of the mud flat at Site Dangast varied between 22 and 30%, averaging about 26% (Böttcher, unpublished data). During sampling in June 1999 the microsensor measurements were impeded by high bioturbation activity in the upper 1.5 mm of the sediment and, therefore, no steady state 02 profiles could be gained. During this measurement only the oxygen penetration depths (3.1 mm ± 0.17 mm) could be determined. Under similar environmental conditions but slightly less bioturbation a sediment core from the same site was investigated in June 1998 and the results are presented in Fig. 3. In the upper 0.7 mm the sediment was significantly supersaturated with respect to oxygen as it has been found previously for a situation in April 1998 (Böttcher et al. 2000). The maximum concentration occurred at 0.3 mm depth with 0.43 mM. Below 0.7 mm the oxygen concentration declined constantly and the oxygen penetration depth was 2.7 mm. The volumetric rate calculations also showed a distinct area of oxygen production in the upper 1.5 mm of the sediment sample (Fig. 3), with a maximum in the upper
0.5 mm. Below 1.5 mm oxygen was consumed. Nitrate concentrations from measurements in June 1999 are plotted in Fig. 4, as a mean of two profiles. During the measurements the sediment was heavily bioturbated. Down from the water/sediment interface the nitrate concentration increased to a maximum value of more than
10 IIM at 1 mm depth. At 2 mm the concentration was still higher than in the water phase but declined in deeper layers. Nitrate could be measured down to 3.6 mm. The calculated activity rates showed a clear separation between nitrate production in the upper 2.3 mm of the sediment and nitrate consumption below that layer (Fig. 4). Highest production rates occurred concomitant with the maximum nitrate concentration.
101
Isotopic
fractionation
ofsttlfur
species; E,
total
organic
carbon (TOC);
F,
acetate; G, and lactate propionate; I-I. Mn(1I) Fe(ll). and
Fig.
2.
lliogcoclieinical
aiialysis
of
vertical the
sediiiierit
profile.
SO. 2 ;
A,
13,
sulfate reduction
Jate (SRR);
C,
Cr(Il)—IedLicihle
siiltiir
(Cr—Il)
and ;icide volatile siillidcs (AVS), I),
Mn; Fe
(pM) Acetate (pM) Lactate, propionate
(pM) TOC (dwt.%)
0 40
0
60 20
120 40
60 0 80
20 0 1 2 40 3
4
20
20 I
201 I 20 I I I I I I
Ho’
H.
F. G.
• Fe
. .1.
Mn
16
16 16 /
0
0 0 •, .—,
0
0
0
12 12
12
a)
a) a,
a)
0.
ci. 0.
• 0
I’.
-C -C
U U
U
U
E
E
E
E S., 8- 8
8
-
a’ II
It.
4
.“ 0•
.
El
0 • Proplonae
0
•
•
De
•
0 0
0 —
o 34 s SRR (mM) SO 4 (%.) cm 3 d) (nmol dwt.% S
0 5 0.0 0
10
0.2 15 20 -30 25 500 -20 30 -10 0.4 0.6 0 0.8 10 1000 30 20
20
H
I I
. I . .
. .
. B. LI -
A. 0.
Cr-li so.
.
• -
. •
El
cr-il
16 16 16 16 —
.
It] •D •
.
AVS
•o
•o
+0
.
.
•0
0
0 0
0
12
12 12 —
12
— a)
a)
a)
a)
o.
. •0 0.
0 0. • 0 0
.
•o
-c
-C
•
0 . 0 + 0
.
C)
U
C)
U
.
•
. 0 E 0
0 - .-. E E
E . a
8
• 8+0
0 .
•0
.
El • +0 D
.
. •
0 +0 0
.
. . 0
0
0 - -.
.
4+
0 . • 0 0
.
r
. 0 • 0
.
• El I+0 0
.
I
0 •
Ló
0 0
0
______No hydrogen sulfide could be detected in the sediment cores within the first 20 mm, the maximum depth reached by our microsensor measurements. Sub-oxic, non-sulfidic conditions were indicated by the presence of dissolved Fe(I1) and Mn(I1) (Fig. 2H) and absence of sulfide at concentrations exceeding 5 jiM between about 5 mm down to at least 12 cm depth (data not shown). Sulfate remained essentially constant within the first 4-5 cm and decreased further downcore (Fig. 2A), associated with an enrichment in S.34 Despite of minor changes in dissolved sulfate, moderate to high microbial sulfate reduction rates were measured with radiotracers through the whole sediment core and showed a maximum with up to 485 nmol •3cm d’ in the sub-oxic zone at around 7 cmbsf (Fig. 2B). The apparent activation energy of 66 kJ mol’ was used to relate the measured SRRs at 10 and 20°C to the mean temperature of 17°C measured during field sampling. The concentrations of volatile fatty acids (VFA; acetate, propionate, and lactate; Fig. 2F and 2G) were determined in the pore waters of two sediment cores. In both cases concentrations of VFA except for acetate were below 5 p.M in the top 5 cm of the sediment. Acetate concentration increased with depth to concentrations of about 50-60 p.M. Concentrations of glycolate and formate on the other hand did not exceed 5 p.M throughout the entire core. Elevated concentrations of lactate and propionate ranging between 10 and 30 p.M were detected in the deepest horizon of 15-20 cm. This accumulation of organic acids occurred only below the maximum of the sulfate reduction rates (Fig. 2B, F and G). PLFA analysis. Three different depth-depending PLFA patterns were observed in the studied sediment cores (Table 2). The first 0.5 cm are dominated by high amounts of polyunsaturated PLFA. In the depth between 0.5 and 10 cm, the PLFA patterns did not show any pronounced variations. The PLFA patterns included many branched fatty acids, cyclopropane fatty acids and a high amount of cli 18:1. The deepest layer is distinguished from the upper layers by the low amount of c9 16:1 and a higher amount of 18:0. The content of phospholipid phosphate decreased with depth (967 nmol g’ dry sediment at 1 cm depth to 228 nmol g’ dry sediment at 15-20 cm depth). DGGE profiles of sediment samples. Changes in the diversity of the SRB population with depth were analyzed by DGGE of PCR-amplified 16S rDNA fragments and of the reversely transcribed 16S rRNA fragments. In the same sediment sample, the electrophoretic profiles of the 16S rDNA fragments were more complex than the ones observed after reverse transcription of the 16S rRNA (Fig. 5).
103 -0.002 -0,001 0.000 0.001 0.002
0
—1
E E
a0 -2
-3
-4 Activity —.— Concentration
00 0.1 02 03 0.4 05
Fig. 3. Vertical sediment profile of oxygen concentrations determined with an °: microsensor and calculated oxygen production/consumption rates in a sediment core from Dangast (6/98). Profile and rates represent the mean of two data Sets
6-lOxlO 8-5x10 0 5x10 6lOxlO Activity L Concentration 0
—1
E E
0a -
-3
-4
0 32x10 34x10 36x10 38x10 10x10 12x10
Fig. 4. Vertical sediment profile of nitrate concentrations determined with a 1NO microsensor and calculated nitrate production/consumption rates in a sediment core from Dangast (7/99). Profile and rates represent the mean of two data sets.
104 Table 2. PLFA profile of site Dangast. PLFA were analysed as fatty acid methyl esters and are shown in relative amounts (mol). PLFA methyl esters present in all horizonts less than 0,5 % are not listed. Not detectable PLFA are abbreviated with nb. Sediment horizons (cm)
PLFA 0.0.0.5 0.5.1.0 1.0-2.0 2.0-3.() 3.0.4.0 4.0-5.0 5-10 10-15 15-20 methyl esier
i 14:0 1.4 0.9 1.0 1.1 1.0 0.8 1.1) 0.4 0.3 c7 14:1 0.7 nd 0.1 0.3 nd nd 0.3 0.2 0.6 14:0 6.3 3.6 3.5 0.4 3.1 0.9 3.3 1.6 1.6 i15:0 2.() 3.2 3.8 4.1 3.8 3.9 3.8 3.1 3.2 a15:0 2.8 5.5 6.6 7.3 6.9 7.3 7.6 7.3 8.0 15:0 3.6 2.8 2.1 2.2 2.0 2.0 2.1 1.8 1.4 16:3b 8.4 nd nd nd nd nd nd nd nd 116:0 id 1.6 1.7 1.8 1.7 1.8 1.6 1.7 1.6 c7 16:1 1.2 1.8 1.6 1.8 1.7 1.8 1.5 1.5 1.3 c9 16:1 16.5 17.2 17.8 19.2 18.3 19.5 17.9 14.0 9.4
ci I 16:1 4.1 1.6 2.1 2.4 2.3 2.7 2.6 2.2 1.4 cl3 16:1 1.3 2.4 2.2 2.5 2.4 2.9 2.5 2.8 2.7 16:0 10.5 19.2 17.6 18.2 17.! 17.4 16.7 15.7 17.4 i17:l 0.3 1.2 1.2 .1 1.4 1.1 0.8 1.2 2.9
lOmcl6:() 0.4 I .3 1.7 1.9 1.9 2.3 2.4 2.3 2.1
a17: 1 ((.5 (1.2 0.7 0.7 0.7 0.5 0.5 0.6 nd
i [7:0 0.5 ((.8 0.9 1.0 1(1 1.0 0.9 1.1 1.2 a17:() 0.4 1.1 1.2 1.3 1.3 1.4 1.4 1.8 2.0 c9 17:1 3.2 1.5 1.3 1.4 1.5 1.4 1.4 1.2 0.5 cyc 17:0 0.7 1.3 1.5 1.6 1.6 1.6 1.6 0.3 0.2 17:0 1.0 1.6 1.6 1.6 1.6 1.5 1.5 1.5 1.2 c6 18:2 1.9 ((.8 0.7 0.6 (1.5 0.5 0.5 0.4 0.1 c6 18:3 nd ((.6 0.6 0.5 0.5 0.5 0.6 0.5 nd c9 18:2 1.4 ((.4 (1.7 0.7 0.8 0.7 0.4 0.6 0.7 c9 18:1 3.4 4.4 3.6 4.1 3.8 3.5 4.0 4.3 4.1 cli 18:1 3.9 10.7 11.6 [3.3 13.3 13.3 12.7 15.6 14.6 c13 IX:! 0.5 4.2 3.0 (1.7 2.2 2.4 3.3 4.1 8.0 18:0 2.4 3.2 2.8 3.3 2.6 2.4 2.7 3.8 8.2 cyci9:0 nd 0.4 (1.6 0.3 0.5 (1.1 nd 0.5 0.7 c5 20:4 1.6 0.9 ((.8 1.0 0.9 ((.8 0.7 1.4 0.5 c5 20:5 13.7 2.1 1.4 1.2 1.0 0.8 0.8 2.3 0.4 c9 20:2 nd 0.8 0.2 nd ((.8 0.2 0.2 1.6 nd c4 22:6 5.7 ((.7 ((.7 0.5 0.2 0.2 ((.2 0.2 nd 24:0 nd 0.2 0.2 nd 0.! 1.6 0.3 0.2 0.1
hranched 8.8 16.7 20.2 22.1 20.6 20.7 21.2 20.3 22.9 saturatedd 32.5 47.1 47.7 46.6 47.0 46.6 47.8 43.9 49.9 unsaturate& 67.5 53.0 52.3 53.4 53.0 53.5 52.2 56.1 50.1 used short-hand nomenclature: i=iso-branched; a=anteiso-branched; c—cis;cyc=cyclopropane; me=methylgroup; x:y x= number of carbon atoms; ynumber of double bonds hposition of double bond not determined include all iso, anteiso and lOMe branched PLFA-methyl esters dalI PLFA methyl esters without a double bond in the carbon chain call PUFA methyl esters with one or more double bonds in the carbon chain
105 DGGE of 16S rDNA showed between 6 and 9 bands of different intensities. There were no major changes between different sediment layers, except for the deepest layer at 15-20 cm depth. DGGE of 16S rRNA showed only two to four bands in all the samples. In contrast to the 16S rDNA-based DGGE, these were not evenly distributed but appeared as distinct patterns. It was not possible to get any PCR product from the deepest layer. As rRNA-based
DGGE is influenced by the “activity-regulated” ribosome contents, this pattern might better indicate the identity and distribution of the active SRBs in the sample.
A B $C çj c ‘ ‘.
1’...-‘ — - — 3,....
5.’...- -
. ,‘.
Fig. 5. DGGE profiles of 16S rDNA (A) and reversely transcribed 16S RNA (B) by using the SRB specific forward primer SRB385 which carries a GC-clamp and the reverse primer 907 (Sass et al. 1998). DGGE bands 6
and 7 affiliate with the Desulfonenia group. It has to be noted, that probe DSS658 does not target this group.
Identification of the most prominent DGGE bands by sequencing. All the bands analyzed by sequencing were shown to originate from members of the _-subcLass of Proteobacterici. the taxonomic group that encompasses most gram-negative SRB (Fig. 6).
Bands 1 and 2 were affiliated with a sequence similarity of 99% to Desulftbulbus sp. (accession numbers L40786 and L40785, respectively). DGGE bands 3 and 4 were found to
be closely related to Desiilfovibrio species. Band 3 was related to Desulfovibrio caledoniensis (accession number U53465) sharing similarities of 97%, and band 4 was related to De.cutfovibrio sp. Ac5 (accession number AF2281 17) with 99% similarity. Band number 5 was identified as Desiilfrbacter sp. with 96% similarity (accession number L40787). Bands 6
and 7 were related with 99% similarity to Desulfonema liniicolci(accession number U45990),
106 and to Destilfonenia ishimotoei (accession number U45991), respectively. The band number 8 had 98% sequence similarity with Desulfumnionaspalmitatis (accession number U28172). MPN counts of SRB. A variety of defined substrates was used to meet specifc substrate preferences of different SRBs possibly occurring in the studied sediment. For example, chemolithoautotrophic SRBs, such as Desulfobacteriuni autotrophiciin, can grow with H, and CO, as the sole sources of energy and carbon, respectively. The bacterial numbers from MPN
counts with liquid media are shown in Table 3.
Table 3. Total cell counts of SRB from different sediment horizons from Site Dangast after 1.5 and 10 months of incubation at 23 °C’.
Horizon (cm)
0-0.5 0.5-I 1-2 2-3 3-4 4-5 5-10 10-15 15-20 1-lydrogen2)*(C0 1.5 months 1.1 x 10’ 4.6x 10’ 4.6x 10’ 1.1 x 10’ 1.1 x 10’ 1.1 x LO’ nd 1.1 x iO’ nd l0months 4.6x 106 1.1 x 10’ 1.1 6x10 4.6x 106 1.1 x 106 4.6x 106 4.6x 106 4.6x 106 1.1 x 106 Hydrogen (CO,+Acetate)* 1.Smonths 4.6x IO 1.1 x 10’ 1.1 x IO’ 1.1 x 10’ 1.1 x 10’ 4.6x 10’ 4.6x 10’ 1.1 x 10’ 1.1 x 10’ 10 months 1.1 x 10’ 1.1 x 10’ 1.1 x 10’ 1.1 x 106 1.1 x 106 1.1 x 106 4.6x 106 4.6x 106 4.6x 10’ Formate 1.5 months nd nd 1.1 x io 1.1 x 10 4.6x iO 4.6x iO’ 1.1 x IO 1.1 x iO 4.6x 10’ 10 months 1.1 x 10 nd 4.6x 10’ 1.1 x 106 4.6x 106 4.6x 106 1.1 x 10’ 1.1 x 106 4.6x 10’ Acetate 1.5 months 4.ôx 10’ 1.1 x iO 1.1 x l0 1.1 x iO 1.1 x iO 1.1 x 4i0 4.6x l0 1.1 x 10’ 1.1 x 1O I0months 1.1 x 10’ 4.6x 10’ 1.1 x iO’ 1.1 x 106 1.1 x iO’ 4.6x iO’ 1.1 x 10’ 3.9x 106 4.6x 10’ Lactate 1.5 months 1.1 x 10’ 1.1 x 10’ 4.6 x 10’ 4.6 x iO’ 4.6 x 10’ 4.6 x 10’ 1.1 x iO’ 4.6 x iO’ 4.6 x iO’ 10 months 1.1 x 10’ 3.9 x 10’ 4.6 x iO’ 4.6 x 106 4.6 x 106 4.6 x 106 1.1 x iO’ 4.6 x 106 4.6 x iO Propionate 1.5 months 1.1 x 10’ 1.1 x 10’ 1.1 x 10’ 1.1 x 10’ 1.1 x 10’ 1.1 x 10’ 1.1 x iO 1.1 x 10’ 4.6x 10’ 10 months 1.1 x iO I.! x 10’ 4.6x 10’ 1.1 x 106 1.1 x 106 4.6x 106 4.6x iO’ 1.1 x 106 4.6x 3i0 nd = not detected; * carbon source
a To confirm the low numbers of bacterial cells observed after the short incubation period a control experiment was performed. A comparative MPN study was carried out with sediment (2-5 cm depth) from the same location at Dangast and from sediment at Horumersiel, north of Jadebusen in September 1999. The latter site is characterized by a locally high organic input and a high sulfidogenic activity. The MPN tubes were incubated for 2.5 months at 23 °C. MPN counts with all substrates tested (H + ,2C0 H, + CO, + 2mM acetate, 7.5 mM acetate, 5 mM lactate and 5 mM propionate) were between 1.1 x 10’ an 1.1 x 10’ per ml for the Dangast sediment. In contrast. MPN counts for the Horumersiel site were between 1.1 x 106 and 1.1 x I0 cells per ml.
During incubation of MPN tubes first a fast growing population and then after prolonged incubation a slow growing population developed. After about six weeks of incubation only
107 only 4.6 low
substrates this
to profile slowly contrast
medium, packages indicated
Table numbers
Hydrogen
Hydrogen Formate
Acetate
Lactate
Propionate b nd
Probes Lower
Positive
20 =
10 1.5
10 1.5
10 1.5
10 1.5
1.5 1.5 10 10
cell x
first
exception
not
4.
months months
months cm. months
months
months months
months
months
months
months
months
10’
of
growing
Identification
brightness
are
detected;
numbers
to
(C02)*
(CO+Acetate
hybridization
only
but population
0
that
known
tested,
Prolonged
described the cells
to
rather
in
cells
15 faster
0-0.5
DSB985 221 DSB985/h
DSV698C DSV698
nd nd were DSB985 DSB985/ DSV698 DSS658
DSV698 660
nd
*carbon
but
of
the per
ranging
cm.
rather
from
the
of
in
formed
were
last with
more
MPN )*
growing
of
ml.
SRB
Table
incubation Cell
signal
source
0.5-1
DSB985 nd
DSV698C DSV698
nd
nd fast DSB985/ DSB985 DSS658 Desulfisarcina DSVÔ98
DSV698 660
nd
similar horizon.
more
between
In by evenly
abundant
cultures
numbers I.
after
flocs
FISH
growing
general,
than
SRBs.
8.5
cell
221 1-2 221 DSV698 DSV698C
nd (Fig. DSVÔ98 DSB985 DSB985
DSV698
DSV698 660 nd
distributed (10
one
after
months
1.
with
decreased
SRBs.
1
numbers here
probe.
months)
SRBs
cells
1.5
x 7
c
spp.
IO H,/C02/acetate
and
incubation.
2-3
221 221
DSV698 DSV698 nd DSV698 DSB985 cells DSB985
DSV698
DSV698 and 660 660
Counts
grew
and
Horizon
appears
ID
throughout
As
d)
were
108
by
of
did
months
4.6
and
with
the
about
homogeneously 3-4
22!
221
DSV698 DSV698
DSV2I4 DSV698/ DSV698 DSB985 nd
DSV698
DSV698 nd nd
not
increased
(cm)
obtained x
aggregates, to
of MPN
1
grow
the
o
incubation.
one
be
as
the
cells
4-5
substrates
221
DSV698 nd fast
DSV698C
DSV698/ evenly DSV2I4 DSV698 DSB985 DSB985
DSV698
DSV698 nd nd
cultures
order
homogeneously
horizons,
for
up
per
growing
sometimes
to
all
of
ml
distributed
in
5-10
nd 221
nd
DSV698
DSV698 1.1 DSB985 DSV698 DSB985
DSV698 DSV698
660 660
horizons allowed
magnitude
yielding
could
the
with
x
SRBs.
io
medium.
a
be similar
221
221 10-15 distributed
DSV698 nd
nd DSV698 DSB985 DSB985
DSV698 DSV698
660 660
the
decrease
cells analyzed.
counts
across
observed.
in
MPN
detection
horizon
per
of
With
of nd
DSV698 221 15-20
nd
DSV698/ DSB985 DSB985 nd DSV2I4
DSV698
660 a nd 660
the
counts
in
ml.
depth
in
up
Thus
The
cell cell
the
all
15 to
of
In From the highest dilution steps that still showed growth, samples were recovered for FISH analysis, the results of which are summarized in Table 4.
MPN in agar shakes yielded numbers of colony forming units somewhat higher than those observed with MPNs in liquid media. Colonies from the highest dilution were transferred to fresh liquid media containing the same substrates as used in the corresponding agar shakes. Subsequent analysis of growth cultures by FISH revealed no other phylogenetic groups as already identified in MPNs with liquid media. FISH of sediment samples. Total cell counts of the upper 20 cm of the sediment were determined microscopically (Table 5). From top to bottom DAPI counts strongly decreased from 53.9 x l0 in the first cm to 1.0 x 10 cells per 3cm of sediment in the zone below 15 cm. The microbial community dwelling in Dangast sediments was dominated by Bacteria. In the top 0.5 cm of the sediment, up to 82.3% of the total microorganisms hybridized with the probe EUB338 (Table 5). Archaeal and eucaryal counts remained below the detection limit of 0.1% of the DAPI stained cells. Recently, the bacterial probe EUB338 was shown not to detect all members of the domain Bacteria (Daims et a!. 1999). Some bacterial phyla, most notably the Planctomycetales and Verrucornicrobia, are missed by this probe. Consequently, total bacterial numbers monitored in this study were most probably underestimated. Initial experiments with the newly designed additional probes EUB338-1I and -III (Daims et a!. 1999) indicated that this underestimation may amount to 4-10% of the total microbial community. Detection rates with the probe EUB338 decreased exponentially over the vertical profile. Thus, not only the absolute cell numbers decreased with depth, but also the percentage of detectable cells with our FISH protocol.
Group specific probing. A set of 15 different probes specific for SRB of the y-subclass of
Proteobacteria were tested (Table 1) with sediment samples from Dangast. Only five of these probes (i.e. 221, DSB985, DSR6512, DSS658, and DSV698) gave counts above the detection limit set at 0.1% of total DAPI counts (Table 5). The target groups of the other probes were either not present in high abundance (>0.1 % of the total cell counts) or not detectable by FISH, e.g. due to a low ribosomal content per cell. The most abundant SRB present in the sediments were members of the Desulfosarcina
Desulfococcus-Desulfofrigus group (probe DSS658, Fig. 7 a and b) and Desulfovibrio spp. (probe DSV698) with maximum values of 2.9 x 108 and 2.8 x 108 cells per 3cm of sediment, respectively. The counts of these two groups decreased strongly with depth to values of 1.0 x 106and 0.6 x 106 cells per 3cm of sediment at 20 cm, respectively.
109 decreasing 0.5 between
relatively per the between
The with
number cells Table
DAPI ‘7
Percent hNumber CNot standard
SRB
Desulfr.bacter of
Probe EUB338 221 Probe Probe DSB985 D5R651 Probe DSS658 Probe cm3
Probe DSV698 Sum detection cm SRB
total
hybridized
numbers
detected. per
5.
absolute
Quantification
detected to
of detection
probes of
deviations
throughout
0.5
15 of
cm3
SRB
low
2.7
SRB
cells numbers
and
x
cell
(or
ceIls
limit
up
abundance.
community
IO
x
82.3±14 53.9±2.2 0.21±0.070.18±0.1
2.5±1 per
2.5±) 4.3±0.9
8.6
20
compared
cells
no.
with
nd to
were as 0-0.5
iO
and
cni3.
(mean±SD) cm
spp.
(x1O
of
in 6.6
low
the
from
detected
cells
the
calculated SRB
the depth
1.0
x
69.7±13 0.22±0.04 53.7±0.9
sediment.
as
(probe
12.3
to
4.7±1.3
5.3±1.1
1.9±0.7
probe
0.5-1 i0
top
The x in
sediment
was
DAPI.
per
1.7%
Dangast
i07
were
cells
to
by
from
cell
found
cm3
DSR65I
cells
DSB985)
bottom
Numbers
56.5±1.3 69.8±5
of
FISH
11.3
2.1±0.7
5.3±0.9 below
3.9±0.8
Desulfobacteriuni
per
1-2
numbers
the
sediments
total
n& nd
except
sediment
per
to
counts
cmi,
was
be
from
have cells)
the
cm3
specific
53.1±3 53.5±2.3
10.0
0.15±0.03
4.4±0.4 3.8±0.8 were
1.7±0.6 maximum
2-3
accounting nd decreasing
of
for
by
were Horizon
detection
been
were
two I
110 FISH at
between .
the
1
found 20
corrected for
parallel
decreasing x
45.6±4
33.1±1.5
0.1±0.08
found.
2.4±0.9
4.2±0.7 2.4±0.7
9.1 (cm) layers
3-4 cm
I
Desulforhopalus
0
detected
within
nd
limit.
over
for
depth. cells
over
cores.
10
by
8.6 between
25.5±2.3
37.4±6
and 11.5
subtracting
3.5±0.9 the
0.1±0.09
6.2±1.2
the 1.7±0.8
4-5
per from
nd
the
with
to
vertical
upper
15
cm3
12.3%
whole
1.1
the
cm
25.0±3
17.0±2.1
1
5-10
0.7±0.3
2.2±0.4
3.3±0.3 6.3
N0N338
nd
nd of were
and
x
3
probe
profile
depth. of
cm
108
sediment
sediment
total
2
also
of
to
cm
17.0±2
10-15
counts. 221
9.0±3.3
0.3±0.4
3.0 1.7±0.5
1.0±0.7
the
nd
nd
0.1
to
Cell
cells.
counted
depth
were
1.7
sediment
in x
profile
Means
numbers
x 106
the
The
13.6±6
15-20
0.1±0.04
1.0±4.6
0.6±0.4
1.0±0.6
1.7
where below
106
nd
nd
with
cells
first
and
in Z— (Maidak parsimony tree Fig. without 6. Phylogenetic et analysis al. modifying 2000). including The 10% affiliation its bu1brhabdoformis topology topology only of complete during of ___LesugoEEE ,—. the ______the sequenced tree sequence Desu!forhopaiu.s ______or Desulfoniicrobium resulted Desulfurella almost DGGE1 Des DesulfoWbrio DGGEZ De.sulfobulbus ulfoco positioning Desulfowibrio DGGE DesulfobuThus DGGE3 - Desutfofuslis DesulfoWbrio DesulfoWbrio DGGE6 Des Des Des DGGU 111 Des Desuifovibrio from Dexulfovibrio complete ccus acelivorans ulfobulbus ulfuromonas Desulfonema ulfonemo DGGE1 vacuo(alus ulfovibrio Desulfonema Desulfosarcina . gracilis the fragments. propionicus DGGE4 AF desulfurican glycolicus (Strunk sp. !edonieig profundus magnum insertion elongatus 141454, africanus halophilus 16S longus palniliatia Geabacter ishimaioei limicola BdeI!oWbrio Desulfobulbus rRNA uncultured et — The Nitrospina Myxobacleria of al. Ct Peiobacser-Deruifuromonas the sequences at. 1998-2000). tree bacieroivorus eubacterium partial et Ct Ct at. is a!. a!. based DGGE of representative on sequences the result bacteria into of the the Fig. MPN
for Discussion
sediment sediment
sand especially
Mayer described
Böttcher
is measurements organic
sediment
under states
with
indication
preferential
visualization
Biogeochernistry
of
7.
The
enrichment
grain Microscopic
depth
marine
anaerobic
(essentially
1994).
matter
sulfur
depth
caused
et
layers
srnectite, earlier
fraction
variations
that
al.
of
intracellular,
origin,
culture
on
cells A
is
(Böttcher
isotopic
image
1998;
by is
conditions
microbial
for due parallel
TOC
pyrite
a
stained
ate
a
(essentially
(C
clear
decreases
of
tidal
of
mixing
essentially
to
and
Böttcher
able
at
sediment
the
changes
discrimination
with
with
et
Site
enzymatic d).
indication decrease
sediments
(e.g..
to a!.
sediment.
dlissiTnilatory
Epit1uonscence
of
the
Dangast
minor adsorb
2000).
with samples
minerals
Cy3-labelled
quartz).
et
decoupled
in
Morse
al.
the
of
reduction depth
for
elemental
of
significant
The
(a
2000;
have
pore
The sedimentological
the
et
the
between
and
In of
al.
image
accumulation
piobe
due sulfate
from
the German
dissirnilative contrast
additionally
observed
b) water
(1987).
Volkman
112
of
and
sulfur)
to
mud
DSS658
using
amounts
the
325042
of
sulfate
the
and
reduction
cells AVS
Wadden
fraction
to
UV
Solid
preferential decrease
were
(b
TOC
quartz,
et
compared
shown
of
in filter and
activity
and
of
trend
and
al.
a
phase
AVS
organic
found
d). 10
with
mineralogical
for
contents
Sea
took
2000).
the
that Scale (Fig.
phyllosilicate
in
dilution
DAPI
throughout
sulfur
of
(Delafontaine
increasing
to biodegradation
through pore
AVS-fractions
the
place.
sulfate-reducing
bar
matter
2C).
SO12
Stable
(a
with
from
=
labile
with
and
water
10
This the (Keil
properties
tIm.
an c),
(e.g.,
depth
higher
the
amounts
carbon
fraction,
minerals, acetate
and
content
sediment
et investigated
et
is
Kaplan green
of
has
is
al.
al.
oxidation
due
utilizing
bacteria
isotope
marine
a of
of
which
1994;
1996;
with
filter been
deaL’
and
core
the
to the
and
a Rittenberg 1964). The isotope enrichment factors observed in the sediment partly exceeds the maximum found in pure cultures. The enhanced sulfur isotope discrimination may be due to the activity of sulfate reducers in the Dangast sediments that have not been available in previous experiments on isotope discrimination and/or a contribution from the oxidative part of the sulfur cycle. H.,S is to a significant portion reoxidized and sulfur species with intermediate oxidation states may be formed (Jørgensen 1982a). It has been shown experimentally that bacterial disproportionation of the latter leads to the formation of S-32 enriched 2SH (Canfield et al. 1998; Cypionka et al. 1998; Böttcher and Thamdrup 2001) which may contribute to the observed overall isotope effect. Bacteria which are able to disproportionate elemental sulfur, for instance, have been isolated from the Dangast sediments and their ability to discriminate sulfur isotopes has been confirmed experimentally, too (Canfield et al, 1998). Compared to the AVS fraction, the physicochemically most stable phase pyrite from the top ii cm was generally enriched in S34 (Fig. 2). In the deeper more sandy sediment layers the isotope data of both fractions are close to each others. This may indicate the influence of particle mixing by bioturbation on the pyrite pooi in the upper sediment section. Additionally, the pyrite fractions have preserved a change in the depositional environment and the near surface sulfur cycle of the Jade Bay sediments in the last century (Böttcher et al., in prep.). Pore water composition. The compositions of interstitial waters sensitively mirror the biogeochemical processes in the sediment. According to the typical zonation scheme as proposed by Froelich et al. (1979), organic matter oxidation should be related to the consumption of oxygen followed by nitrate, the build up of Mn(II) and Fe(II) due to the reduction of Mn(lV) aid Fe(1II) (oxyhydr)oxides, and later to the depletion in sulfate. Highest oxygen concentration in the photic zone are not directly linked to maximum nitrate production (Fig. 3 and 4). In the top 0.7 mm, where oxygen is produced via photosynthesis and oxygen concentrations are high, nitrate production rates seem to be relatively low. The nitrate concentration profile in the upper part of the sediment is influenced by an advective transport of nitrate resulting from a macrofauna mediated bioturbation. Since an assumption for activity calculations is a diffusion regulated transport and steady state concentrations, the upper part of the profile should be considered as non-informative. Nitrate production is taking place in the depth of 1 mm to approx. 2 mm. Net nitrate consumption was observed below 2 mm depth, where little or no oxygen was present. Sulfate concentrations at the sediment-water interface correspond well to the measured salinities, indicating that dilution of seawater with sulfate-poor freshwaters took place in the
113 enrichment
surface exceeds
of
site
essentially
(BOttcher and
sulfate-reduction variations concentrations 1990:
most
sulfate Böttcher by
concentrations (Burdige
the reaction
in Thamdrup reactions
this (1994)
to
therefore, substrates
The in still
with
sulfide
The
the
Microbial interstitial SRR (Böttcher
degrade
composition result
phenomenon
reactions
not
likely
Kristensen
low
measured
reduction
reaction
the observed
waters
with
(l-Iartman
understood. near and
et
with
sulfate
of identical
of 1993,
in also
supply
et of
related
al.
polymeric
et microbial
34S.
Hespenheide sulfate overall
sulfide at.
waters,
sulfate-reducers
and
responsible reactive the
of
linked
of
2000).
al.
with
sulfate
within
can
et
of
5-fold Thamdrup
This
1994).
reduction
dissolved
mainly
of
2000)
the limited
sediment-water and
to
to
at.
SRB
reduction
react sulfate
Fe(I1l)
produced
In
boundary
results
therefore,
to
is
reoxidation
2000).
coastal metal Hydrogen Nielsen
the reduction
reduction higher
substances
During
the expected
the
and
to
(Böttcher
accumulation with
for
unpublished
suboxic
rates
the
Fe(l1)
by
present
production
or
obtained
et
(oxyhydr)oxides
in
The
the
depends
SRR
during
reoxidation transport
(e.g.,
region. iron 1969). Mn(IV)
other
short-time al.
depend conditions
(Fig.
quantitative
for rates of
sulfide
and
downcore
of
zone,
et
interface
1994: compounds
to
study at
the
a
Sørensen
sulfide
fine-grained
during
2).
dissimilatory
Mn(tl)
at.
short
system,
are
smaller data).
on
rate
The
compounds
on
of
from
respective
together
This
which 2000).
high
the
Böttcher within
of
incubations dissolved
the
of through
compared
summer
decrease
(Moeslund variation
114 The
H,S observed
balance the in is
activity
is
VFA.
where
balance
et
concentrations
molecules
to
the
in
most
is
This
sediment
the
same with
during tidal al.
form
agreement
produced
oxyhydroxides
sulfate
surface to
The
the and
sulfide
time
range
the
1981;
in
of
indicates
of
to
enhanced
intermediate likely
in
of sediments
in
of
is
iron
different et
years.
fermentative
concentrations incubation
consumption
long SRR
Thamdrup
the
also
coastal
sulfate on
production
water
reduction.
like al.
observed
sediments
Christensen
has
sulfdes
the
pore-waters
during
superimposed
with
incubation
found
of
found
1994:
The
a
been VFA.
dissolved
same
interface
of acetate
sediments,
environments,
similarity
was
but observations
with
sulfur
the
and
maximum for
Thamdrup
previously
bacterial
in
2001).
observed
The
and
bacteria
(Moeslund
mudflat of
which
may
associated
the North
of
the
time.
1984).
may
sulfate
and
times.
are
consumption
observed
or
Fe(ll)
species
volatile
present
community
also
in
Moeslund
by
Therefore.
by
not lactate
be
Sea are
which
dissimilatory
earlier 1
the
in reoxidation The
at They by et
however, by
year
reoxidation
re-oxidized and
result
necessarily
et
(Oenema the
or the
fatty
al. bacterial
with
seasonal
bacteria Novelli
study
al. decrease
coincide
SRR
are
Mn(Il)
before
sulfate
and
related
same
1994,
actual
1994:
et
from
rates.
size acids
able
an
the
is
are
al.
is
is,
et al. (1988) and Holmer and Kristensen (1996). One may speculate that this correlation may be due to changes in sedimentological parameters (e.g., decreased water content), the TOC composition and an overall decrease in the availability of substrates.
The sulfate reduction rates per cell observed in these sediment samples were around 1 fmol
42S0 1cell . 1day (as calculated from FISH and MPN results). This result is in agreement with previous reports (Sahm et al. 1999; Böttcher et al. 2000; Ravenschlag, 2000). They are at the lower end of cellular sulfate reduction rates determined for pure cultures of different psychrophilic, mesophilic, and thermophilic sulfate reducing bacteria (Canfield et al. 2000). Sulfate reducing bacteria. The distribution of SRB in the vertical profile of the sediment was studied with four independent techniques. First, the phospholipid fatty acids (PLFA) can be used as biomarkers for specific groups of organisms. The top 0.5 cm of the sediment was dominated by polyunsaturated PLFAs, which are typical for eucaryotic organisms (Vestal and White 1989; Findlay and Dobbs 1993), including protozoa, algae and higher plants. The sediment layers between 0.5 and 15 cm typically contained the PLFA Cli 18:1, characteristic of anaerobic bacteria (Findlay et a!. 1990; Findlay and Dobbs 1993; Vestal and White 1989). Thus the shift in PLFA pattern around 0.5 cm depth correlates well with the oxygen penetration as observed by the microsensor study (Fig. 3). In the deepest studied horizon (15-20 cm) a pronounced decrease in the diversity of PLFA indicated also a general decrease in microbial diversity. This is in good agreement with the changes of DGGE patterns (Fig. 5) and decrease in viable cell and
FISH numbers (Table 3 and 5). Throughout the studied sediment horizons PLFA markers characteristic of SRB genera were identified pointing to an even distribution of SRB in the sediment profile. We found the marker fatty acids lOMel6:0 for Desulfobacter (Taylor and Parkes 1983) and il7:l for DesulfovibriolDesulfornicrobiurn (Vainshtein et al. 1992) in significant quantities in all horizons. We also found a17:l, a fatty acid which can be detected in Desutfrcoccus and Destilfosarcina (Kohring et al. 1994). The highest relative percentage of i17:1 was discovered in 15 to 20 cm depth. Whereas the most relative amount of lOMel6:0 was found between 4.0 and 15 cm depth. Second, DGGE analysis was carried out to study the diversity of SRB (Fig. 5). A similar electrophoretic pattern was observed in the different horizons within the upper 15 cm of the sediment, showing an even distribution of SRB. Phylogentic identification of the most prominent bands showed diverse phylogenetic affiliation within the 6-subclass of Proteobacteria. Interestingly most sequences were closely related to SRBs originating from marine environments, e.g. Desulfonema ishimotoei (isolated from Dangast, the present study
115 area,
Hole, identified the substrates
dilution properties substrates as
addition, growth predominantly
utilization of
(i.e. During time The
but should
may as
performed
cells, DGGE 6) (DGGE discrimination evenly
based
that 3
Third,
substrates
cm)
compared
Fourth, the
vertical
were
formed
with
more
ref.
the of
result
USA.
rendering
DGGE
of
be genera
incubation
distributed
about profiles
as
showing
Desulfr’bacter/Desuljobacula
4
Fukui
the every
H,/CO,,
also
the
used
of
and
added
of
noted
abundant
and
known
in profile ref.
flocs
the using
typical
known
to which
sediment, viable
an six
profiling.
quantified
found
or
6; Deculfrbacter
Rabus
substrate et members FISH
of
from analysis
this
that
(Fig.
to growth underestimation
weeks
Fig. the
FISH
the
al.
generally of
throughout
detectable
from
requires
the
for
SRBs.
cells
population
method
this
Length reversly
the
(Table
in 1999)
et
5) 7 utilization
Desul,tovibrio.
if
MPN
c
a
al. In and
was
the
all Desulfobacteriuni used
sediment
of
population
monitored and
in
by of
more
For
contrast,
1993).
of
and of
the
two
5). the
the
a
the DGGE DOGE
performed combining
and/or d). transcribed cultures,
and
bands
the
possible
the
those example,
required
rapidly capacity
Destilfrbacula
presence
active types
The
genus of
of
studied
with
incubation
was
this
which by
the
type
identified
analysis with
profiles
Desulfrbacula, observation
type
were
of
every
tool results
observed
growing
16S
Desulfobacteriu,n. actual SRB with
band cultures
of a
MPN
populations did sediment
16S
of
depth
prolonged
SRB
requires
complete
indeed
rRNA-based to
autotrophicuni
a
sampling 116 period,
FISH
not
of
rRNA could of
or
identify
were
cell
by
variety
and
represented toluolica population
was
with
reversty
(Table
16S those grow
that
DGGE
dominated
horizons numbers
(Table
FISH only
the
in
indirectly
an
oxidation,
incubation
found. could
either these
both rRNA.
active depth of
homogenously good
even
with
capacity
3).
DGGE
be
profiling
substrates
transcribed
(isolated
studies.
4).
known
SRB
of
Identification
be
lactate
detected if MPN-cultures
by determined
tested). agreement
For distribution
cells.
a
(Brysch
by
However, With
monitored
less
reflect
observed
DGGE
were
high
profiling.
time
predominantly
members
example,
for
abundant
of
or
to Regardless
Except
respect
from
typical
After
dominated
in
chemolithoautotrophic
the
ribosome
16S HJCO,/acetate of
possess
in
l6S
et
band
the
with with
under
about
in
with of the ribosome
Eel at.
of
rDNA
This
of
a
for
rRNA
with
to lower
DGGE
cells
the
5
short cells
for
SRB
culture
this
MPN 1987),
the
Pond,
the the
are
two
this
all
of
could 10
grow
latter by content
SRBs
acetate,
throughout
(Fig.
nutritional
species.
developed. part
most individual
16S
conditions in incubation
fragments. the
months.
content
sequences
capacity. members
band (Table
Woods
medium
highest yielded
known
in
indicate
rDNA
(below
MPN
case
5,
were
active
floes
was
Fig.
the
5
In
3)
of
It
is
a in the lower part of the sediment, even though they are also present in the upper part. The opposite was observed for DGGE band 3, representing Desiilfovibrio type SRB. The examination of the microbial community in Dangast by FISH showed that SRB account for a significant part of the detectable Bacteria. Up to 6.5 x lO cells per 3cm were identified as SRB. This number of cells was always higher than the number of viable SRB observed using MPN. Similar discrepancy between MPN counts and for instance DAPI based cell counts have been described (Sievert et al. 1999). The absolute numbers of the different physiological groups might be underestimated since typically less than 1% of the total bacterial population in natural habitats may be accessible by current cultivation dependent methods (Amann et al. 1995). In addition, floc formation and clumping of cells may also lead to an underestimation, since the MPN evaluation assumes that only a single cell is required to initiate growth at the highest dilution. The Desulfosarcina-Desulfococcus-Desulfofrigus group (targeted by probe DSS658) was identified as the most abundant SRB when sediment samples were directly analysed by FISH. This group was also identified by PLFA analysis of sediment samples. However, this group was not identified when MPN and DGGE methods were applied. Possible explanations could be on the one hand that the cultivation conditions used for MPNs do not select for SRBs affiliating with the Desulfisarcina-Desulftcocciis-Desulfofrigus group, and on the other hand that the primers used for DGGE are not targeting this group (Ravenschlag et a!. 2000). The second most abundant group of SRB identified in the sediment samples by FISH belong to the
Desulfiwibrio group. In this case, identification was possible with all three other techniques.
Conclusions A combined microbiological, molecular, biogeochemical and isotope geochemical approach was applied to gain new information on the relationship between abundance, community structure and activity of the sulfate-reducing bacteria (SRB) of a surface marine sediment. Dissolved oxygen and nitrate were only found within the first few mm of the top sediment with different microscaled zones of formation and consumption, as obtained from profile modeling. Bacterial dissimilatory sulfate reduction was measured through the whole investigated sediment section. However, further reaction and reoxidation of hydrogen sulfide led to the development of sub-oxic conditions in the pore water. Microbial and chemical reactions led to the accumulation of dissolved iron and manganese in the suboxic zone.
Maximum sulfate reduction rates (SRR) were found in the top 2 to 10 cm coinciding with a maximum of active SRBs as detected by FISH. Cellular SRR calculated from combined FISH
117 and sulfur
dissolved sulfate
sulfur pyrite, sedimentation
Thus microbiological
found
situ community SRB
Desuifofrigus DSV698
DesulJovibrio possess concentrations results Desul,toi.’ibrio terminal
Acknowledgments. We
the S.
measurements,
microsensors.
facilities. the mansucript.
Munich.
The
Fleischer,
volumetric abundance
laboratory.
wish
allowance
population,
presence
across species.
isotopic
seem
reduction
combined
we
mineralization
the
to
sulfate
and
We
in
suggest
to thank
We the
K.
capacity conditions
group,
spp.
the Contents
of are be
thus
of or
composition
and
The
to
SRR
H.-J.
(PLFA
Neumann, and
studied
wish a
with
cultivatable
cellular decoupled at
lactate
work
upper B.
grateful
good application -
Desuljm’ibrio authors
at
most
low
to that
by
data
Schnetger
to Brumsack
additional
the
and 0.
step
to
and
DGGE correlation
in vertical
and
thank
and sediment
cellular
activity.
the
basically
sampling
likely
to Eickert, J.
the
utilize
are
MPN
in
of sulfur
from
particle
acetate
Botzenhardt,
B.B.
SRBs
Desu1sarcina-Desu1fococcus-Desu1fofrigus
the the
tidal
indebted of
iron of
and
sediment
contributions
(ICBM
the
analyses) by rates
reversly
anaerobic
However,
Nationalparkverwaltung was molecular
isotopic
Jørgensen
acetate
was
A.
groups.
H.
stay
in
time flats.
in mono
mixing
vertical
members
Lüschen a
the
Eggers, dominated
observed under
to
given
constant
point
profile,
Oldenburg)
The
C.
T. transcribed upper
sulfides
compositions
and
tools
(bioturbation).
zone
with
as
Probian 118 Kjaer
profile
for
(DGGE
conditions
study
horizon
in
from
and
(ICBM
revealed
of
layers lactate.
constructive
between respect demonstrated
regardless
of
June
along
by
for
the
(AVS)
V.
the
of
the
was
and
SRBs
16S
of
providing
kindly
1999 cannot
Hübner of
Oldenburg) AVS
temperate
Desulfosarcina-Desulfococcus
the disproportionation
to
of
by
This D.
supported
essentially the PCR-amplified
the
rRNA.
Niedersächsisches
is
the
targeted
of
the -
Lange
vertical
and
FISH
sediment.
different
are in
suggestions
always
allowed
the
presumably
that
for
is
most
the agreement
significantly
the
It
intertidal
biogeochemical
in
(MPI for constructing
and
diverse is
by biosensors
by
sediment
key
stable
open
be
known agreement
technical
techniques.
Max
Based probes
access
in
Bremen) related
populations
and
16S
active
mud
with
the SRB
of
sulfide
with
Planck
Wattenmeer
that
influenced
on
reading
intermediate
profile.
for DSS685
to
rDNA)
case
support,
flat.
0, to
group
respect
are
the
part microbial
these
with for
The zonation.
analytical the
either
species,
and
Society,
evenly
present
of
help for
of
nitrate
of
SRB
The
SRB
and
low and
and
H,S
the
the
and
by
the to
the
in
for
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125 Wo1.I.AsT.
In change.
R.
F.
R. C.
John
Mantoura,
1991.
Wiley
The
&
coastal
J.-M.
Sons,
Martin,
New
organic
York.
and
carbon
R.
Wollast
126
cycle:
fluxes,
(ed.),
Ocean
sources,
margin
and
sinks.
processes
p.
365-381.
in
global
Mein
Verständnis
hier
Stefan
Dirk
hatten
haben.
Mein
aussprechen,
Meinen Danksagung
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Einen
nicht
Schüler.
groBer
allergroBter
und
Sievert.
besonderen
ersten
genannten
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bedanke
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Olaf
deren
stets
Dank
inir
seid
Dank
Dank
gilt
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Kniemeyer,
Kollegen
gestarkt
das
Fachwissen
nicht
mich
auch
rnöchte
wende
Thema
gilt
nur
bei
wurde.
Jens
jedoch
des
die
ich
der
dieser
ich
Udo
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Institutes,
Harder
besten
an
gesamten
häufig
Prof.
Susanne
Heyen.
meine
Arbeit
und
Kollegen
Fritz
profitiert
mit
Mitstreiter
Jan
uberlassen
Abteilung
Jan
und
deren
Detmers
Widdel
Küver,
sondern
meiner
habe.
Hilfe
Karsten
Mikrobiologie
und
die
und
und
diese
Mutter,
auch
deren
stets
natürlich
Prof.
Arbeit
gute
Zengler,
em
Durchführung
durch
Freunde.
Bo
offenes
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meinem
entstanden
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allen
Astrid
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anderen
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JØrgensen
Behrends,
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für
mich und