Anaerobic ammonium oxidation mediated by Mn-oxides: from sediment to strain level

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Anaerobic ammonium oxidation mediated by Mn-oxides:from sediment to strain level

Cedric Javanaud a, Valerie Michotey a,*, Sophie Guasco a, Nicole Garcia a, Pierre Anschutz b,Mathieu Canton b, Patricia Bonin a

a Laboratoire de Microbiologie, de Geochimie et d’Ecologie Marine, Centre d’Oceanologie de Marseille, Universite de la Mediterranee - Aix Marseille II,

13009 Marseille, France CNRS-UMR 6117 Campus de Luminy, Case 901, 13009 Marseille, Franceb Laboratoire Environnements et Paleoenvironnements Oceaniques Universite Bordeaux 1, CNRS-UMR 5805, Avenue des Facultes,

33405 Talence Cedex, France

Received 10 September 2010; accepted 2 January 2011

Available online 1 February 2011

Abstract

Nitrite and 29N2 productions in slurry incubations of anaerobically sediment after 15NO3 or15NH4 labelling in the presence of Mn-oxides

suggested that anaerobic Mn-oxides mediated nitrification coupled with denitrification in muddy intertidal sediments of Arcachon Bay(SW Atlantic French coast). From this sediment, bacterial strains were isolated and physiologically characterized in terms of Mn-oxides andnitrate reduction as well as potential anaerobic nitrification. One of the isolated strain, identified as Marinobacter daepoensis strain M4AY14,was a denitrifier. Nitrous oxide production by this strain was demonstrated in the absence of nitrate and with Mn-oxides and NH4 amendment,giving indirect proof of anaerobic nitrate or nitrite production. Anaerobic Mn-oxide-mediated nitrification was confirmed by 29N2 production inthe presence of 15NO3 and 14NH4 under denitrifying conditions. Anaerobic nitrification by M4AY14 seemed to occur only in the absence ofnitrate, or at nitrate levels lower than that of Mn-oxides. Most of the other isolates were affiliated with the Shewanella genus and were able to useboth nitrate and Mn-oxides as electron acceptors. When both electron acceptors were present, whatever their concentrations, nitrate and Mn-oxide reduction co-occurred. These data indicate that bacterial Mn-oxide reduction could be an important process in marine sediments with lowoxygen concentrations, and demonstrate for the first time the role of bacteria in anaerobic Mn-mediated nitrification.� 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Anaerobic ammonium oxidation; Manganese; Nitrogen cycle; Denitrification

1. Introduction

Preferential use of the electron acceptor that yields the highestamount of free energy in the terminal step of bacteriallymediatedoxidation of organic matter constitutes a long-standing paradigmin biocheochemistry (Froelich et al., 1979; Postma and Jakobsen,1996). In undisturbed sediment, this paradigm is reflected in

establishment of theoretical depth zonation of redox reactions inwhich oxygen is reduced near the sedimentewater interface,followed by reduction of nitrate, manganese oxides, iron oxides,sulfate and carbon dioxide. In a classical view, the nitrogen cyclein marine sediments is therefore simplified using a two-layermodel with an oxic uppermost zone producing nitrate throughnitrification, under which, in an anoxic layer in which denitrifi-cation occurs, the concentration of nitrate decreases exponen-tially with depth (Fenchel, 1998; Hulth et al., 1999). The recentdiscovery of ammonia-oxidizing archaea, following that ofanammox a decade ago, and its implication in nitrogen removal inmarine environments have underlined the possible occurrenceof alternative processes even in well-known biogeochemicalcycles such as the nitrogen cycle. Nitrification, corresponding to

* Corresponding author. Tel.: þ33 4 91 82 93 36; fax: þ33 4 91 82 96 41.

E-mail addresses: [email protected] (C. Javanaud), valerie.

[email protected] (V. Michotey), [email protected] (S.

Guasco), [email protected] (N. Garcia), [email protected]

bordeaux1.fr (P. Anschutz), [email protected] (M. Canton),

[email protected] (P. Bonin).

Research in Microbiology 162 (2011) 848e857www.elsevier.com/locate/resmic

0923-2508/$ - see front matter � 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.resmic.2011.01.011

Author's personal copy

oxidation of ammonium into nitrate and nitrite, has long beenregarded as a strictly aerobic metabolism. However, geochemicalstudies have frequently described unexpected nitrate/nitrite peaksin anoxic zones of sediment corresponding toMn-oxide reductionareas. This finding was interpreted by Mortimer et al. (2004) asthe result of an anaerobic nitrate/nitrite-producing process linkedto Mn-oxide reduction (anaerobic lithotrophic nitrification)(Bartlett et al., 2008, 2007; Hulth et al., 2005, Hulth et al., 1999;Luther et al., 1997).Despitegeochemical data, the biotic aspect ofsuch putative anaerobic nitrification, although thermodynami-cally favorable, is still under debate and anaerobic nitrification isoften described as rare, random and very difficult to estimate bydirect nitrogen flux quantification (Bartlett et al., 2008).Furthermore, noorganismharboring thismetabolismhas yet beencultivated.

Together with nitrogen, manganese is probably one of thekey players in biogeochemical cycles in marine sediments dueto its abundance, its large surface of distribution and its highreactivity (Anschutz et al., 2005; Bratina et al., 1998; Dellwiget al., 2007; Roitz et al., 2002; Sundby, 2006; Tebo et al.,2005). In marine systems, manganese is essentially undera reduced soluble form (MnII) and oxidized particulate forms(MnIV-oxides and transitory MnIII-oxihydroxides (Anschutzet al., 2005; Van der Zee and Van Raaphorst, 2004)). Themanganese cycle can be summarized as simple alternating ofsoluble and solid states according to physicochemical andbiological conditions (Bratina et al., 1998; Van Cappellen et al.,1998) that favor either manganese oxidation or reduction (Aller,1994; Sundby, 2006; Thamdrup and Dalsgaard, 2000). Amongphysicochemical parameters, oxygen and pH are the maintriggers of the Mn oxidation state. Anaerobic Mn-oxidereduction can be realized according to a large variety of directand indirect processes (Anschutz et al., 2005; Bratina et al.,1998; Johnson et al., 1995; Nealson, 2006; Van Cappellenet al., 1998). For some bacteria, MnII can be an energy source(Tebo et al., 1997), whereas MnIII and MnIV can be terminalelectron acceptors of dissimilatory manganese reduction(Lovley and Phillips, 1988; Myers and Nealson, 1988).

As is the case for the N-cycle, with Mn-oxides beingproduced in oxic environments, the boundary between oxicand anoxic layers is probably also a hot spot for microbial Mnreduction activity due to direct diffusion of fresh Mn-oxidesfrom an oxic to an anoxic part of marine sediments. In marineMn-rich systems, Hulth et al. (1999) hypothesized stronginteractions between Mn-oxides and nitrogen compounds;putative coupling between anaerobic ammonia oxidation andMn-oxide reduction has been suggested.

The present study aimed to investigate the occurrence ofanoxic nitrate/nitrite production (anaerobic nitrification) andits further putative reduction in marine sediments (fromArchachon Bay, France) using an original integrative approachbased on the isotope pairing method (Nielsen, 1992) enablingdistinction of the origin of denitrified nitrate (from a watercolumn or from coupled nitrification). The capacity to oxidizeammonium under anaerobic conditions in the presence ofMn-oxides was investigated on a bacterial set isolated fromthis sediment, and interrelations between nitrate and Mn-oxide

respiration were analyzed. Our results suggested expression ofanaerobic Mn-oxide-mediated nitrification coupled withdenitrification in muddy intertidal sediments of Arcachon Bay,and the occurrence of this new process was confirmed for thefirst time by its expression in bacterial isolates.

2. Materials and methods

2.1. Synthesis of Mn-oxides

Since the crystallinity of MnO2 provided by manufacturersdoes not correspond to MnO2 freshly formed from oxidationof dissolved Mn(II) or particulate Mn(III) oxihydroxides (Teboet al., 2004), preparation of biologically active MnO2 wasperformed in the laboratory from 0.1 M KMn04, 0.1 M NaOHand 0.1 M MnCl2 (with ratio volume of 2/4/3, respectively)(Laha and Luthy, 1990). Mn-oxides were collected bycentrifugation and washed several times with distilled water.This method of synthesis produces Mn-oxides structurallysimilar to naturally occurring mineral birnessite. Moreover,since the reactivity of manganese oxide suspensions maychange with long storage, they were used for growth experi-ments within 24 h after preparation. To reduce heterogeneityamong subsamples of the resulting solid suspension, doses forindividual culture were prepared. As the recovery yieldpresented variability among doses, colorimetric Mn-oxideassays were performed at the beginning of each culture.

2.2. Incubation experiments on sediment slurries

Five mL from the 2 uppermost centimeters of sediment ofArcachon Bay (SWAtlantic French coast, sampled in October2007) were transferred to 120 mL head space vials containing70mL of sulfate-free artificial sea water (SF-ASW that contains20 mM NH4) (Baumann and Baumann, 1981) amended or notwith Mn-oxides (1 mM). All incubations were for 60 days induplicate in the dark at in situ temperature (18 � 1 �C) and inanaerobiosis under an He atmosphere to avoid nitrate produc-tion from aerobic nitrification. Argon (1% of head spacevolume) was added as internal standard for mass spectrometry.

Two kinds of labelling were performed with 15NH4

(20 mM, 10% labelling) or with 15NO3 (1 mM, 99.8% label-ling). The amounts of 14NO3 provided by sediment in theslurries were negligible (few mM). In order to monitor bioticanaerobic nitrification, living controls without Mn-oxides andheat-killed sterile controls amended with HgCl2 (finalconcentration, 70 mg L-1) were prepared.

2.3. Bacterial isolation and growth media

Bacterial isolations were performed after 60 days of slurryincubation. SF-ASW with 3 mM of molybdate was used forisolation to avoid sulfate reduction and fermentative processes.Mixtures of acetate, succinate and pyruvate (1 g L-1 finalconcentration each) were added as carbon sources. Serumflasks (120 mL) containing 70 mL of medium were inoculatedwith 5 mL of sediment slurry. Cultures were serial diluted and

849C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

appropriate dilutions were spread onto agar plates containing5 g of yeast extract (Merieux), 5 g of Biotrypcase (Merieux)and 15 g of agar (Merieux) per liter of synthetic sea water thatcontained 20 mM NH4 (ASW, (Baumann and Baumann,1981)). Microbial colonies were picked out and re-plated tocheck for purity on the basis of their colony morphology andmicroscopy observations. Twenty-six bacterial strains wereselected. All isolates were stored in liquid nitrogen in thepresence of glycerol (20%).

The isolates were screened for their ability to reduce nitrate(1 mM) and to produce nitrite and nitrous oxide in anaerobichungate tubes containing ASW supplemented with the samecarbon sources as above and with nitrate (1 mM) or Mn-oxides(1 mM) as electron acceptors. After inoculation, tubes wereincubated 7 days at 20 �C and chemical analyses of gaseousand liquid phases were performed at the end of incubation. Totest nitric oxide accumulation by denitrifiers in the presence ofnitrate, acetylene was added (10% of the head space) in a setof tubes. The ability to perform anoxic nitrification wasdetermined by quantification of nitrate and nitrite produced inthe presence of Mn-oxides as sole source of electron acceptorsunder anaerobic conditions.

Growth experiments were performed under the sameexperimental conditions as for screening. One-hundred-twentymL serum flasks containing 70 mL of the same growthmedium were amended with various 15NO3 and/or Mn-oxideconcentrations. Due to the difficulties in adding preciseamounts of fresh synthesized Mn-oxides, their concentrationswere quantified at the beginning of each experiment. Anaer-obic conditions were obtained by flushing with He for 30 min,Ar was added as internal standard for mass spectrometryanalyses and flasks were incubated at 20 �C and agitated ona reciprocal shaker (96 rev.min-1.5 cm amplitude).

2.4. Chemical analysis

Nitrite and nitrate from culture or pore water weremeasured automatically by colorimetry with a Techniconautoanalyzer (Treguer and Le Corre, 1975) (Cv ¼ 3%).Amounts of 29N2 and

30N2 produced in incubation vials duringincubation were measured with a mass spectrometer (Quad-rupole mass spectrometer Anagaz 100, MKS, England)(Minjeaud et al., 2008). Nitrous oxide was measured by gaschromatography (Shimadzu) (Bonin et al., 2002a) and dis-solved manganese by flame atomic absorption spectrometry(Perkin Elmer AA 300), on supernatant of samples centrifuged5 min at 15 000 g using an external aqueous standard forcalibration (Cv < 5%) (Anschutz et al., 2005). Mn-oxide wasmeasured by colorimetry (Boogerd and Devrind, 1987).

Student’s t-test was performed to evaluate differencesbetween concentrations and activities.

2.5. Denitrification and nitrification/denitrificationcoupling rates

In the presence of 15NO3 labelling, D15 corresponded todenitrification of the added 15NO3 and was calculated from

formation of single-labelled (14N15N) and double-labelled(15N15N) dinitrogen assuming random isotope pairing ofuniformly mixed natural 14N03 and added 15NO3 (Nielsen,1992).

Naturally occurring denitrification (Dtot) based on unla-belled nitrate available for the denitrifier was calculated as:Dtot ¼ D15 � [(14N15N)/2(15N15N)]

Dtot was supported by Dw (based on nitrate in the watercolumn) and Dn (based on the nitrate produced in the nitrifi-cation process): Dw ¼ D15 � [14NO3]/[

15NO3]Then, Dn was simply calculated from the difference Dtot-

Dw where [14NO3] and [15NO3] represented the concentrationof naturally occurring 14N and added 15N in the NO3 in theoverlaying water, respectively (Nielsen, 1992).

In the presence of 15NH4 labelling, D15 production was anindicator of production of 15NO3 from NH4; however, sincethe percentage of NO3 labelling was not determined, Dn couldnot be calculated.

2.6. Molecular analysis DNA extraction, PCR anddenaturing gradient gel electrophoresis (DGGE)analysis

Cell lysis and DNA extractions were performed asdescribed by Zhou et al. (1996) from 1 mL of sedimentsuspension for long-term incubation or growth culture at thebeginning and end of the incubation time (Zhou et al., 1996).

PCR-amplified fragments of the bacterial 16S rRNA genesuitable for subsequent genetic fingerprinting analysis wereobtained with the universal bacterial primer combinationGMF5 (50- GCCTACGGGAGGCAGCAG -30) with a 40-nucleotide GC-rich sequence attached to the 50 end (50-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCG-CCC -30) and 907RM (50-CCGTCAATTCMTTTGAGTTT-30)which amplifed a fragment approximately 585 bp long, aspreviously described (Bonin et al., 2002b)

Separation of PCR fragments was performed with a DGGED-Code system (Bio-Rad, Vienna, Austria) running for 3.5 hat a constant voltage of 150 V and 60 �C in a 30e50% verticaldenaturating gradient (a 100% denaturating agent was 7 murea and 40% deionized formamide). After electrophoresis,gels were stained with ethidium bromide and photographedunder UV transillumination (GelDoc, 2000 system, Bio-Rad).

2.7. Bacterial numeration

In culture without Mn-oxides, bacterial growth was fol-lowed by determination of optical density (450 nm). In thepresence of Mn-oxides, optical density (OD) was not directlycorrelated with bacterial numeration due to the presence ofbrown particles of Mn-oxides. Under these conditions, bacte-rial growth was followed by quantification of the 16S rRNAgene copy number by real-time PCR from 1 ml culture at thebeginning and end of growth using primer sets GMF5 (Boninet al., 2002b) and Uni516R (Takai and Horikoshi, 2000).Real-time quantification were performed using a Light cyclerapparatus (Roche, Mannheim, Germany) with all reactions

850 C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

proceeding with an initial denaturing step for 10 min at 95 �C,followed by 38 cycles of denaturing for 10 s at 95 �C, 10 s ofprimer annealing at 55 �C and 12 s of primer extension at72 �C. Calibration of gene copy numbers was performed usingreal-time standard curves generated with known copy numbersranging from 102 to 107 copies of ribosomal of Pseudomonasstutzeri Zobell generated during the same PCR.

2.8. Sequencing of 16S rRNA gene fragment andcomparative analysis

PCR-amplified fragments of bacterial 16S rRNA gene forbacterial identification were obtained with universal primers27f (50AGAGTTTGATCMTGGCTCAG30) and 1547Rd(50AAGGAGGTGATCCAGCC30) (Winker and Woese, 1991).

PCR products of partial 16S rRNA gene from the isolateswere sequenced by GATC (Konstanz, Germany). 16S rDNApartial sequences were aligned with the same region of theclosest relative strains available in the GenBank databaseusing the BLASTN facility (http://www2.ncbi.nlm.nih.gov/BLAST/). Sequence alignment was achieved using ClustalWimproving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gappenalties and weight matrix choice (Thompson et al., 1994).The phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei, 1987). Bootstrap analysiswith 500 replicates was carried out to check the robustness ofthe tree. Finally, the tree was plotted using the Mega 4program (Tamura et al., 2007). The sequences obtained in thisstudy are available from EMBL under the accession numbersFR669247 and FR669248.

3. Results and discussion

3.1. Anaerobic nitrate and 29N2 production in sedimentslurries

Nitrate and nitrite concentrations were followed over a 60-day period in sediment slurries incubated in closed systemsunder anaerobic conditions with or without Mn-oxides andcompared with sterile controls (Fig. 1). In incubation flaskswith Mn-oxides, significant nitrate (from 5 mM to 750 mM)and nitrite (10 mMe90 mM) quantities were rapidly produced(t-test, p < 0.05), whereas the concentration of thesecompounds remained stable in both sterile and Mn-unamendedcontrols. The production of nitrate/nitrite observable solely inthe presence of Mn-oxides showed that its production wasprobably microbially mediated and linked to the presence ofmanganese oxide. Several hypotheses could explain this: (i)Mn-oxides inhibited nitrate reduction enzymes, (ii) Mn-oxidesacted as additional electron acceptors, outcompeting nitrateand leading to a lower nitrate reduction rate, (iii) the rate ofnitrate production was higher than its consumption in thepresence of Mn-oxides, suggesting an additional nitrateproduction pathway. The occurrence of an additional pathwayof nitrate production was also suggested in several studies andwas interpreted as anaerobic nitrification of ammonium to

nitrate or nitrite coupled with Mn-oxide reduction (Bartlettet al., 2008, 2007; Hulth et al., 2005, Hulth et al., 1999;Mortimer et al., 2004). Indeed, anoxic incubations of surfacemarine sediment showing anoxic nitrate production duringMn-oxide reduction were interpreted by Hulth et al. (1999) asevidence for anaerobic coupled nitrification-denitrification.Anaerobic ammonia oxidation in the presence of Mn-oxides toproduce nitrate was also suggested in natural marine sedimentson the basis of co-occurrence of peaks of MnII and NO3

(Bartlett et al., 2008, Hulth et al., 2005, 1999). However, nostoichiometric effect was demonstrated using direct measure-ments of nitrogen species in relation to Mn-oxide amendmentin sediments of the Humber estuary (Bartlett et al., 2008) andthese atypical processes seemed to be strongly affected byspatial heterogeneity (Bartlett et al., 2008). Thus, we canassume that such production of nitrate/nitrite was transient.The absence of apparent net nitrate or nitrite production couldindicate that nitrite or nitrate formed through anaerobic nitri-fication may provide substrates for subsequent dissimilatorynitrate reduction processes such as nitrate ammonification(Bonin et al., 1998), denitrification (Anschutz et al., 2000) oreventually anammox.

In the present study, to determine the nitrate origin and tofollow subsequent transformation of nitrate putatively generatedby anaerobic nitrification, 29N2 and 30N2 production wasmeasured in sediment slurries after Mn-oxide and 15NO3

amendments (Fig. 2A). Under anaerobic conditions, when 15NO3

was added, there was strong production of N2 from nitrategenerated by nitrification (Dn) up to 0.6 mmol mL-1.day�1 inincubation flasks containing Mn-oxides. This production wassignificantly higher than that detected in control flasks (t-test,p < 0.05, Fig. 2A). The same tendency was also confirmed in

Fig. 1. Concentrations of nitrate (A) and nitrite (B) during incubation of

Arcachon Bay sediments. Three kinds of anaerobic incubations were per-

formed: heat-killed control with Mn-oxide amendment (gray dot), live control

without Mn-oxide amendment (white dot) or with Mn-oxide amendment

(black dot) (n ¼ 3).

851C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

incubation flasks with 15NH4 plus14NO3 amendment (Fig. 2B).

Indeed, significant 15N-labelled dinitrogen production (D15, t-test, p < 0.05) was noted, indicating that 15NH4 was oxidized in15NO3/NO2 by nitrification and subsequently denitrified. Thepartial implication of anammox in 29N2 production cannot beexcluded at this stage. NO3/NO2 accumulation together withsignificant rates of Dn and D15 in anaerobiosis indicate theexistence of anaerobic ammonium oxidation in the presence ofMn-oxides and its subsequent reduction by denitrificationcoupled to nitrification (Dn). These results constitute indirect butclear-cut evidence of anaerobic NO3/NO2 production withensuing reduction by denitrification.

3.2. Isolation and bacterial screening

To confirm the hypothesis of bacterial anaerobic nitrification,a collection of bacterial strains was isolated from the samesediment slurry incubation flask described above. All 26bacterial isolates were tested for their utilization of Mn-oxidesas electron acceptors, and for their capacity to exhibit N-respi-ration or produce nitrogen oxides in the presence of Mn-oxides(Table 1). Sixteen isolates were able to reduce Mn-oxides. The14 strains showing N-respiration were subsequently classifiedinto 2 groups: (I) denitrifiers, able to achieve denitrification up to

production of gaseous compounds (1 strain); (II) nitrate and/ornitrite reducers able to reduce nitrate only (1 strain) or able toprogressively reduce nitrate and then nitrite without nitrousoxide accumulation (12 strains). The anaerobic production ofnitrogen oxides (nitrate, nitrite or nitric oxide) in the presence ofMn-oxides as sole electron acceptors was observed for 6isolates, which were alsoMn-reducers. For 5 among 6, nitrate ornitrite accumulation, although at low levels, were observed.These low levelswere not surprising, sincemost of these isolateswere also nitrate reducers, and nitrate or nitrite accumulationswere probably transient. The final isolate was a denitrifyingstrain. In the presence of Mn-oxide and acetylene and withoutnitrate amendment, an accumulation of nitrous oxide instead ofnitrate or nitrite was observed in the end point culture experi-ment. No nitrous oxide accumulation was observed in theabiotic control. This accumulation was the proof of transientproduction of NO3/NO2 which was denitrified as soon as it wasproduced. Acetylene is well-known to be an inhibitor of aerobicnitrification (Kester et al., 1996; Bonin et al., 2002a). Theaccumulation of nitrous oxide resulting from anaerobicammonium nitrification and its subsequent denitrification intoN2O in the presence of acetylene by strain M4AY14 indicatedthat, in contrast to aerobic nitrification, anaerobic nitrificationseems unaffected by this inhibitor.

In parallel, the genetic diversity of the 26 isolates was alsostudied by DGGE and was compared to the community from

0

200

400

600

800

1000

14NH4+15NO3Living control

14NH4+15NO3+ Mn-OxidesHeat Killed Control

14NH4+15NO3+ Mn-Oxides

Dn

(n

mm

olN

2.m

L-1.d

ays

-1)

15NH4+14NO3Living control

15NH4+14NO3 15NH4+14NO3+ Mn-OxidesHeat Killed Control + Mn-Oxides

0

50

100

150

200

250

D15 (n

mo

l N

2.m

L-1

.d

ays -1

)

+ +-

+-

(-

.-

A

+

B

-

2

Fig. 2. A: Rate of anaerobic nitrification-denitrification coupling (Dn,) and

anaerobic D15 production rate (B) of sediment slurry incubations (n ¼ 3). In

the presence of NO3 labelling, Dn corresponds to denitrification rate of nitrate

produced in the nitrification process. In the presence of 15NH4 labelling, D15

corresponds to dinitrogen production containing 15N and originating from15NH4.

Fig. 3. Comparison of 16S DNAr DGGE profiles of isolated strain and sediment

incubation communities. LaneMcorresponds to standard ofmigration (from top

to bottom, Clostridium perfringens, Marinobacter hydrocarbonoclasticus,

uncultured bacteria SR283, Micrococcus luteus, uncultured crenarchaeata

ARC9S25). Lanes T0 and Tf correspond, respectively, to intitial and final

community structures of sediment incubationwith 15NH4 andMn-oxides. For all

strains, phylotypes are indicated in brackets.

852 C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

sediment slurry harboring anaerobic nitrification (Fig. 3, Table 1).Among the isolates, 7 different phylotypes were observed whichwere unequally distributed among the strains. Phylotype #E wasthe most frequently encountered (15 strains), whereas #B and #Gwere represented by two and five strains, respectively. Phylotypes#F, #H, #I and #K were represented by only one strain each. Asobserved in a previous study (Bonin et al., 2002b), strains withthe same DGGE phylotype might present differences in metab-olism, suggesting that nitrate, nitrite and Mn-oxide reductiongenes did not belong to the core genes of these species.Among the15 strains showing phylotype #E, only 9 were able to reduceMn-oxides, 8 were nitrate or/and nitrite reducers and only 3 werenitrate and/or nitrite producers. The presence of the strains amongdominant members of the slurry communities from which theywere isolated was checked by DGGE and pattern comparison.Drastic changes in the structure of the slurry community werenoted between the initial and final incubation times, highlightingmodifications in the structure of the community in the presence ofmolybdate and Mn-oxides under anaerobic conditions. Smearsillustrated the maintaining of a complex community with fewmore intense bands. However, no intense band presentinga pattern similar to that of the isolates was observed, suggestingthat our isolates were not predominant in the community. It isprobable that our medium used for isolation was not adapted toisolating some major components of the community revealed bymolecular tools.

3.3. Physiological characterization of two selectedisolates

Among the isolates, two strains, M4AY14 and M4AY11,detected in preliminary screening as positive for nitrogen oxideproduction under anaerobic conditions and Mn-oxide amend-ment, were selected for further detailed physiological studies.

The first strain, M4AY14, the sole representative of phy-loptype #H, was phylogenetically affiliated with Marinobacterdaepoensis (Fig. 4). Marinobacter are Gram-negative aerobicrod-shaped cells and can often grow anaerobically by deni-trification. Marinobacter colonizes a large variety of world-wide marine ecosystems ranging from psychrophilic tothermophilic environments, and cells tolerate a broad range ofsalinity and pH that demonstrate its exceptional adaptationcapacities (Duran, 2010). During anaerobic growth with 1 mMnitrate as sole electron acceptor, M. daepoensis strainM4AY14 rapidly reduced nitrate without nitrite accumulationduring the exponential phase of growth (Fig. 5A). In thepresence of acetylene, nitrate was stoichiometrically reducedto nitrous oxide, confirming its capacity to denitrify. Mn-oxides can also be used as sole electron acceptors for growth,as shown by an increase in cellular rrn-16S copy numbers/ml(from 1.6 106 initially to a final 1.15 107) and production ofdissolved MnII (Fig. 5B). Under these growth conditions, inthe presence of acetylene and without nitrate addition, nitrous

Table 1

Summary of metabolic capacity and phylogenetic characteristics of isolated strains. Capacity for Mn-oxide reduction and anaerobic nitrogen oxide production was

determined under anaerobic conditions with Mn-oxide as sole terminal electron acceptor. N-respiration was determined under anaerobic conditions with NO3 as

sole terminal electron acceptor in presence of acetylene.

Electron

acceptor

NO3 N-respiration Mn-oxides DGGE

phylotypeMn-oxide

reduction

N-oxide

production

M4A222 e e e B

M2A461 e e e E

M2A121B e e e E

M2A142 e e e E

M2A15 e e e E

M2A321L e e e E

M2A322L e e e E

M4AY25 e þ NO3 E

M4A46 e e e F

M4AY110 e þ NO3 G

M2A21 e e e I

M2A232L e e e K

M4AY14 denitrification D N2O H

M4AYT24 reduction of NO3 þ e E

M2A56L reduction of NO3 þ NO2 þ NO3 þ NO2 B

M4AY12 reduction of NO3 þ NO2 þ e E

M4A26 reduction of NO3 þ NO2 þ e E

M4AYT11 reduction of NO3 þ NO2 þ e E

M4AY11 reduction of NO3 D NO2 D NO3 D NO2 E

M4AY321 reduction of NO3 þ NO2 þ e E

M4AYT312 reduction of NO3 þ NO2 þ NO3 E

M4AYT14 reduction of NO3 þ NO2 þ e E

M4AYT23 reduction of NO3 þ NO2 þ e G

M4AY15 reduction of NO3 þ NO2 þ e G

M4AY16 reduction of NO3þNO2 þ e G

M4AYT21 reduction of NO3 þ NO2 þ e G

Bold strains were selected for further physiological studies.

853C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

oxide accumulation which attained about 150 mM wasobserved. Since strain M4AY14 is a denitrifier, accumulationof nitrous oxide gave indirect evidence for anaerobic nitrate ornitrite production in the presence of Mn-oxides.

Since strain M4AY14 is a denitrifier and an Mn-oxidereducer and is able to carry out anaerobic ammonium oxida-tion, experiments were planned to determine the balance andtemporal succession of the 3 processes. Growth experiments

E.coli (ATCC 11775T) ( X80725)

Shewanella colwelliana (AY653177)

Shewanella abyssi (AB201782)

Shewanella aquimarina (AY485225)

Shewanella hanedai (X82132)

Shewanella algae (FJ210800)

M4AY11

Shewanella alga (AF005249)

Shewanella haliotis strain DW01 (EF178282)

Marinobacter hydrocarbonoclasticus

(ATCC49840) (X67022)Marinobacter sp. CAB (U61848)

Marinobacter daepoensis strain SW-156 (NR025800)

M4AY14

Marinobacter vinifirmus strain FB1 (DQ235263)

Marinobacter maritimus (DSMZ16561) (AAJ704395)

Pseudomonas fluorescens (DSMZ 50090) (Z6662)

100

100

93

69

100

100

91

96

92

100 100

77

100

0.02

E.coli (ATCC 11775T) ( X80725)

Shewanella colwelliana (AY653177)

Shewanella abyssi (AB201782)

Shewanella aquimarina (AY485225)

Shewanella hanedai (X82132)

Shewanella algae (FJ210800)

M4AY11

Shewanella alga (AF005249)

Shewanella haliotis strain DW01 (EF178282)

Marinobacter hydrocarbonoclasticus

(ATCC49840) (X67022)Marinobacter sp. CAB (U61848)

Marinobacter daepoensis strain SW-156 (NR025800)

M4AY14

Marinobacter vinifirmus strain FB1 (DQ235263)

Marinobacter maritimus (DSMZ16561) (AAJ704395)

Pseudomonas fluorescens (DSMZ 50090) (Z6662)

100

100

93

69

100

100

91

96

92

100 100

77

100

0.02

Fig. 4. Phylogenetic position of strains M4AY14 and M4AY11. This Neighbor-joining tree is based on partial 16S DNAr fragments. The bootstrap numbers are

indicated at the node number after 500 resampled data sets. Branch length corresponds to sequence differences as indicated on the scale bar.

200

0

50

100

150

200

0 20 40 60 80 100

Incubation Time ( hours )

Mn

- o

xid

es / M

nII ( µ

M)

0

50

100

150

200

N O

( µ

M)

0

50

100

150

0 20 40 60 80 100

Incubation Time ( hours )

Mn

II ( µ

M)

0

50

100

150

200

N O

( µ

M)

0 200 400 600 800

1000 1200 1400

0 20 40 60 80 100 Incubation Time ( Hours )

NO

NO

- N

0 (µ

M)

0.001

0.01

0.1

1

O.D

. (450n

m)

0 200 400 600 800

1000 1200 1400

0 20 40 60 80 100

Incubation Time ( Hours )

NO

-N

O-N

0 (µ

M)

0.01

0.1

1

O.D

(450n

m)

initial concentration 1180 µ M NO - 0 µ M Mn - ox initial concentration 0 µ M NO - 394 µ M Mn - ox

initial concentration 1100 µ M NO - 0 µ M Mn - ox initial concentration 0 µ M NO - 168 µ M Mn - ox

A

C

B

D

Fig. 5. Growth kinetics of strains M4AY14 (A, B) and M4AY11 (C, D) in anaerobiosis in the presence of nitrate (A, C) or Mn-oxides (B, D) as sole electron

acceptor. Optical density (black diamond), nitrate (gray dot), nitrite (white dot) and nitrous oxide in the presence of acetylene (black dot), Mn-oxides (black

triangle) and soluble MnII (white triangle).

854 C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

were performed with various combinations of electronacceptors: the addition of 15NO3 or Mn-oxides or both (Fig. 6).Nitrate, nitrite, 29N2,

30N2 and dissolved MnII were followedduring growth and cellular rrn-16S copy numbers/ml werequantified at the beginning and end of culture. Under theseconditions, 30N2 production corresponds to direct denitrifica-tion of 15NO3 added (30N2 ¼ 15N þ 15N), whereas 29N2

production (29N2 ¼ 14N þ 15N) is the result of anaerobicammonium oxidation (leading to 14NO3/2 production) withfurther denitrification. In the presence of nitrate as sole elec-tron acceptor, the strain denitrified stoichiometry 15NO3 to30N2 and no 29N2 or MnII productions were observed(Fig. 6AeB). In contrast, in the presence of a similar range ofnitrate and Mn-oxides, concomitant production of 30N2,

29N2

and MnII was noted (Fig. 6CeD) resulting from concomitantactivities of denitrification and anaerobic nitrification by thestrain. In contrast, when nitrate and Mn-oxide amendment wasunbalanced in favor of nitrate, 29N2 production decreaseddrastically (Fig. 6EeF), suggesting inhibition of anaerobicnitrification under some conditions. The apparent inhibition of

anaerobic nitrification by high nitrate concentrations couldexplain the absence of a linear correlation between initial Mn-oxide concentrations and 29N2 production of sediment, asdescribed by Luther et al. (1997)

The second strain, M4AY11 (representative of the domi-nant phylotype #E), able to reduce nitrate and nitrite withoutnitrous oxide accumulation, was chosen to represent nitratereducers. This strain was phylogenically identified as Shewa-nella sp. (Fig. 4). The genus Shewanella is a member of theclass Gammaproteobacteria and comprises a group of Gram-negative, motile, rod-shaped, oxidase-positive, non-fermenta-tive and facultative anaerobic aquatic and marine bacteria(Hau and Gralnick, 2007). Strains of the genus Shewanellahave been isolated from a variety of ecosystems includingmarine environments. Among these bacteria, Shewanellaputrefaciens has been described for its capacity to reducemetal particles and its great capacity to present a versatilemetabolism (Hau and Gralnick, 2007; Krause et al., 1996).Indeed, S. putrefaciens, in addition to manganese oxides, isable to use oxygen, nitrate, nitrite, thiosulfate, sulfur, iron

0

200

400

600

800

1000

1200

0 20 40 60 80 100 120 140

Incubation Time (Hours)

NO

3-N

O2 (µ

M)

0

0.2

0.4

0.6

0.8

1

Dis

so

lv

ed

Mn

II

pro

du

ctio

n

(µ

M)

0

50

100

150

200

250

0 20 40 60 80 100 120 140

IncubationTime (Hours)

(µ

M)

0

20

40

60

80

100

120

140

Dis

so

lv

ed

Mn

II

pro

du

ctio

n

(µ

M)

0100

200300

400500

600700

800

0 20 40 60 80 100 120 140

Incubation Time (Hours)

(µ

M)

0

0.2

0.4

0.6

0.8

1

Dis

so

lv

ed

Mn

II

pro

du

ctio

n

(µ

M)

0100

200300

400500

600700800

0 20 40 60 80 100 120 140

Incubation Time (Hours)

N2-

N2 (µ

M)

0

200

400

600

800

1000

1200

0 20 40 60 80 100 120 140

IncubationTime (Hours)

N2-

N2 (µ

M)

initial concentration 1100µM NO - 0µM Mn-ox Initial concentration 1100µM NO -0 µM Mn-ox

40 60 80 100 120 140

Incubation Time (Hours)

50

100

150

200

250

N2-

N2 (µ

M)

00 20

initial concentration 200 µM NO - 130 µM Mn-oxinitial concentration 200 µM NO - 130 µM Mn-ox

initial concentration 700 µM NO - 90 µM Mn-ox initial concentration 700 µM NO - 90 µM Mn-ox

A B

C D

FE

NO

3-N

O2

NO

3-N

O2

Fig. 6. Growth kinetics of strain M4AY14 in anaerobiosis in the presence of nitrate (15NO3) and Mn-oxides as electron acceptors in various ranges. Nitrate (gray

dot), nitrite (white dot), soluble MnII (white triangle), 30N2 (black square) and 29N2 (white square). A and B, C and D, and E and F correspond, respectively, to the

same culture.

855C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

oxides and fumarate as terminal electron acceptors (Hau andGralnick, 2007).

Shewanella sp. M4AY11 cultures were grown anaerobicallyin the presence of nitrate as sole electron acceptor (1 mM)(Fig. 5C). Nitrate was rapidly consumed and stoichiometri-cally reduced in nitrite during the exponential phase of growth(Fig. 5C). After depletion of nitrite, when acetylene waspresent, no nitrous oxide was produced and thus nitrite wasprobably reduced to ammonium by dissimilative nitratereduction (DNRA). As S. putrefaciens, in addition to nitrateShewanella sp. M4AY11, was able to use Mn-oxides as soleelectron acceptor for growth, detectable by an increase incellular rrn-16S copy numbers/ml ( from 3.2 105 initially to9.9 105 at the end) and the production of dissolved MnII(Fig. 5D). Unexpectedly, no nitrate-nitrite accumulation wasobserved under these growth conditions after 80 h of incuba-tion, in contrast to preliminary data obtained with an end pointscreening test after 10 days and with slurries which weremaintained for 60 days in the presence of unknown organicmatter. Experiments were planned to determine the temporalsuccession of nitrate, nitrite and Mn-oxide reduction by anadditional combination of 15NO3 and Mn-oxides (nitratealone, similar concentration of nitrate and Mn-oxides or nitratein excess in comparison to Mn-oxides). In all cases, nitrate andnitrite reductions were consecutive and Mn-oxide reductionoccurred concomitantly with nitrate and nitrite reduction, asillustrated by the example shown in Fig. 7.

In conclusion, our results provide evidence of a close rela-tionship between nitrogen and manganese cycles in marinesediment via the activity of several bacterial processes.Manganese influences the quantity of N2 produced by couplingnitrification-denitrification, as illustrated by results obtainedwith strain M4AY14. Indeed, our results demonstrate theexpression, in sediment from the Arcachon Bay, of anaerobicnitrification mediated by Mn-oxides. The isolation of M. dae-poensis strain M4AY14 able to perform anaerobic nitrificationprovides evidence for the first time of the role of bacteria in thismetabolism. Strain M4AY14 expressed anaerobic nitrificationonly when Mn-oxides were present at ranges equal to or higherthan those of nitrate, and the resulting nitrate/nitrite productionwas subsequently denitrified. Our results also highlighteddifficulties in measuring this metabolism due to the dynamics of

production and consumption of nitrate by different processes, asshown with Shewanella sp. strain M4AY11. Further studies areneeded to evaluate the occurrence of bacterial anaerobic nitri-fication in environmental samples. Better evaluation of theintensity of this process is important, since it could drasticallymodify the nitrogen budget in marine sediment by additional N2

production, from up to 3e8% of the ammonium concentration(Thamdrup and Dalsgaard, 2000).

Acknowledgements

We thank Amelie Cabon, Christelle Cotteaux and KevinNoblet for technical assistance, and the CNRS (CentreNational de la Recherche Scientifique), Universite Aix Mar-seille II eMediterranee, grants from the INSU -EC2COeMicrobien 2010 program and ANR Protidal for financialsupport.

References

Aller, R.C., 1994. The Sedimentary Mn cycle in long-island sound - its role as

intermediate oxidant and the influence of bioturbation, O2, and C(Org) flux

on diagenetic reaction balances. J. Mar. Res. 52, 259e295.

Anschutz, P., Dedieu, K., Desmazes, F., Chaillou, G., 2005. Speciation,

oxidation state, and reactivity of particulate manganese in marine sedi-

ments. Chem. Geol. 218, 265e279.

Anschutz, P., Sundby, B., Lefrancois, L., LutherIii, G.W., Mucci, A., 2000.

Interactions between metal oxides and species of nitrogen and iodine in

bioturbated marine sediments. Geochim. Cosmochim. Acta 64, 2751e2763.

Bartlett, R., Mortimer, R.J.G., Morris, K., 2008. Anoxic nitrification: evidence

from Humber estuary sediments (UK). Chem. Geol. 250, 29e39.

Bartlett, R., Mortimer, R.J.G., Morris, K.M., 2007. The biogeochemistry of

a manganese-rich Scottish sea loch: implications for the study of anoxic

nitrification. Cont. Shelf Res. 27, 1501e1509.Baumann, P., Baumann, L., 1981. The marine gram negative eubacteria genus

Protobacterium, Beneckae, Alteromonas, Pseudomonas and Alcaligenes.

In: Mortimer, P.S., Heinz, S., Truper, H.G., Balows, A., Schlegel, H.G.

(Eds.), The Prokaryotes: A Handbook on Habitats, Isolation and Identifi-

cation of Bacteria. Springer, Berlin Heidelberg New York, pp. 1302e1330.

Bonin, P., Omnes, P., Chalamet, A., 1998. Simultaneous occurence of deni-

trification and nitrate ammonification in sediments of the French medi-

terranean coast. Hydrobiologia 389, 169e182.

Bonin, P., Tamburini, C., Michotey, V., 2002a. Determination of the bacterial

processes which are sources of nitrous oxide production in marine

samples. Water Res. 36, 722e732.Bonin, P.C., Michotey, V.D., Mouzdahir, A., Rontani, J.F., 2002b. Anaerobic

biodegradation of squalene: using DGGE to monitor the isolation of

denitrifying bacteria taken from enrichment cultures. FEMS Microbiol.

Ecol. 42, 37e49.

Boogerd, F.C., Devrind, J.P.M., 1987. Manganese oxidation by Leptothrix-

Discophora. J. Bacteriol. 169, 489e494.

Bratina, B.J., Stevenson, B.S., Green, W.J., Schmidt, T.M., 1998. Manganese

reduction by microbes from oxic regions of the Lake Vanda (Antarctica)

water column. Appl. Environ. Microbiol. 64, 3791e3797.

Dellwig, O., Bosselmann, K., Kolsch, S., Hentscher, M., Hinrichs, J.,

Bottcher, M.E., Reuter, R., Brumsack, H.J., 2007. Sources and fate of

manganese in a tidal basin of the German Wadden Sea. J. Sea Res. 57,

1e18.

Duran, R., 2010. Marinobacter - Microbes and communities utilizing hydro-

carbons, oils and lipids. In: Timmis, K.N. (Ed.), Handbook of hydrocarbon

and lipid microbiology. Springer-Verlag, , Berlin Heidelberg, pp.

1725e1735.

Fenchel, T., 1998. Formation of laminated cyanobacterial mats in the absence

of benthic fauna. Aquat. Microb. Ecol. 14, 235e240.

200µM NO3-220µM Mn-ox

050

100150200

250300

0 10 20 30 40 50 60Incubation Time (Hours)

NO

2-N

O2 (µ

M)

020

406080

100120

Disso

lved

M

n II

pro

du

ced

Fig. 7. Growth kinetics of strain M4AY11 in anaerobiosis in the presence of

nitrate and Mn-oxides as electron acceptors. Nitrate (gray dot), nitrite (white

dot), soluble MnII (white triangle).

856 C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857

Author's personal copy

Froelich, P.N., Klinkhammer, G.P., Bender, M.L., Luedtke, N.A., Heath, G.R.,

Cullen, D., Dauphin, P., Hammond, D., et al., 1979. Early oxidation of

organic-matter in Pelagic sediments of the Eastern Equatorial Atlantic -

Suboxic diagenesis. Geochim. Cosmochim. Acta 43, 1075e1090.

Hau, H.H., Gralnick, J.A., 2007. Ecology and biotechnology of the genus

Shewanella. Annu. Rev. Microbiol. 61, 237e258.

Hulth, S., Aller, R.C., Canfield, D.E., Dalsgaard, T., Engstrom, P., Gilbert, F.,

Sundback,K.,Thamdrup,B., 2005.Nitrogen removal inmarine environments:

recent findings and future research challenges. Mar. Chem. 94, 125e145.

Hulth, S., Aller, R.C., Gilbert, F., 1999. Coupled anoxic nitrification/manga-

nese reduction in marine sediments. Geochim. Cosmochim. Acta 63,

49e66.Johnson, D., Chiswell, B., Ohalloran, K., 1995. Microorganisms and manganese

cycling in a seasonally stratified fresh-water dam.Water Res. 29, 2739e2745.

Kester, R.A., deBoer, W., Laanbroek, H.J., 1996. Short exposure to acetylene

to distinguish between nitrifier and denitrifier nitrous oxide production in

soil and sediment samples. FEMS Microbiol. Ecol. 20, 111e120.

Krause, B., Beveridge, T.J., Remsen, C.C., Nealson, K.H., 1996. Structure and

properties of novel inclusions in Shewanella putrefaciens. FEMS Micro-

biol. Let. 139, 63e69.

Laha, S., Luthy, R.G., 1990. Oxidation of aniline and other primary aromatic-

amines by Manganese-Dioxide. Environ. Sci. Technol. 24, 363e373.

Lovley, D.R., Phillips, E.J.P., 1988. Novel mode of microbial energy-metab-

olism - organic-carbon oxidation coupled to dissimilatory reduction of iron

or manganese. Appl. Environ. Microbiol. 54, 1472e1480.

Luther, G.W., Sundby, B., Lewis, B.L., Brendel, P.J., Silverberg, N., 1997.

Interactions of manganese with the nitrogen cycle: alternative pathways to

dinitrogen. Geochim. Cosmochim. Acta 61, 4043e4052.

Minjeaud, L., Bonin, P.C., Michotey, V.D., 2008. Nitrogen fluxes from marine

sediments: quantification of the associated co-occurring bacterial

processes. Biogeochemistry 90, 141e157.

Mortimer, R.J.G., Harris, S.J., Krom, M.D., Freitag, T.E., Prosser, J.I.,

Barnes, J., Anschutz, P., Hayes, P.J., et al., 2004. Anoxic nitrification in

marine sediments. Mar. Ecol. Prog. Ser. 276, 37e51.Myers, C.R., Nealson, K.H., 1988. Bacterialmanganese reduction and growthwith

manganese oxide as the sole electron-Acceptor. Science 240, 1319e1321.

Nealson, K.H., 2006. The Manganese-Oxidizing Bacteria. In: The Prokary-

otes. Springer, New York, pp. 222e231.Nielsen, L.P., 1992. Denitrification in sediment determined from nitrogen

isotope pairing. FEMS Microbiol. Ecol. 86, 357e362.

Postma, D., Jakobsen, R., 1996. Redox zonation: Equilibrium constraints on

the Fe(III)/SO4-reduction interface. Geochim. Cosmochim. Acta 60,

3169e3175.

Roitz, J.S., Flegal, A.R., Bruland, K.W., 2002. The biogeochemical cycling of

manganese in San Francisco Bay: temporal and spatial variations in

surface water concentrations. Estuar. Coast. Shelf Sci. 54, 227e239.

Saitou, N., Nei, M., 1987. The neighbor-joining method - a new method for

reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406e425.Sundby, B., 2006. Transient state diagenesis in continental margin muds. Mar.

Chem. 102, 2e12.

Takai, K., Horikoshi, K., 2000. Rapid detection and quantification of members

of the archaeal community by quantitative PCR using fluorogenic probes.

Appl. Environ. Microbiol. 66, 5066e5072.

Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular

evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol.

Evol. 24, 1596e1599.

Tebo, B.M., Ghiorse, W.C., vanWaasbergen, L.G., Siering, P.L., Caspi, R.,

1997. Bacterially mediated mineral formation: insights into Man-

ganese(II) oxidation from molecular genetic and biochemical studies. In:

Banfield, J.F., Nealson, K.H. (Eds.), Rev. Mineral. Geomicrobiology:

Interactions between Microbes and Minerals vol. 35, 225e266.

Tebo, B.M., Bargar, J.R., Clement, B.G., Dick, G.J., Murray, K.J., Parker, D.,

Verity, R., Webb, S.M., 2004. Biogenic manganese oxides: properties and

mechanisms of formation. Annu. Rev. Earth Planet. Sci. 32, 287e328.

Tebo, B.M., Johnson, H.A., McCarthy, J.K., Templeton, A.S., 2005. Geo-

microbiology of Manganese(II) oxidation. Trends Microbiol. 13, 421e428.Thamdrup, B., Dalsgaard, T., 2000. The fate of ammonium in anoxic

manganese oxide-rich marine sediment. Geochim. Cosmochim. Acta 64,

4157e4164.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal-W - improving the

sensitivity of progressive multiple sequence alignment through sequence

weighting, position-specific gap penalties and weight matrix choice.

Nucleic Acids Res. 22, 4673e4680.Treguer, P., Le Corre, P., 1975. Manuel d’analyse des sels nutitifs dans l’eau de

mer. Universite de Bretagne Occidentale.

Van Cappellen, P., Viollier, E., Roychoudhury, A., Clark, L., Ingall, E.,

Lowe, K., Dichristina, T., 1998. Biogeochemical cycles of manganese and

iron at the oxic-anoxic transition of a stratified marine basin (Orca basin,

Gulf of Mexico). Environ. Sci. Tech. 32, 2931e2939.

Van der Zee, C., Van Raaphorst, W., 2004. Manganese oxide reactivity in

North Sea sediments. J. Sea Res. 52, 73e85.Winker, S., Woese, C.R., 1991. A definition of the domains archaea, bacteria

and eucarya in terms of small subunit ribosomal-Rna characteristics. Syst.

Appl. Microbiol. 14, 305e310.

Zhou, J.Z., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of

diverse composition. Appl. Environ. Microbiol. 62, 316e322.

857C. Javanaud et al. / Research in Microbiology 162 (2011) 848e857