The Diversity and Abundance of Bacteria Inhabiting Seafloor Lavas

Environmental Microbiology (2009) 11(1), 86–98 doi:10.1111/j.1462-2920.2008.01743.x

The diversity and abundance of bacteria inhabiting seaﬂoor lavas positively correlate with rock alteration

Cara M. Santelli,1† Virginia P. Edgcomb,2 the basalt biome may contribute to the geochemical Wolfgang Bach3 and Katrina J. Edwards4* cycling of Fe, S, Mn, C and N in the deep sea. 1MIT/WHOI Joint Program in Oceanography and Ocean Engineering, 2Geology and Geophysics Department, Woods Hole Oceanographic Institution, MS #52, Woods Introduction Hole, MA 02543, USA. The uppermost few hundred meters of ocean crust formed 3Fachbereich Geowissenschaften, Universität Bremen, at mid-ocean ridges (MORs) is composed largely of glassy, Postfach 33 04 40, D-28334 Bremen, Germany. basaltic lava flows that are enriched in reduced species of 4Geomicrobiology Group, Department of Biological elements such as Fe, S and Mn. On exposure to oxygen- Sciences, Marine Environmental Biology, University of ated deep seawater (DSW), oxidation and hydration reac- Southern California, 3616 Trousdale Blvd, Los Angeles, tions occur on the surfaces and within the fractures and CA, USA. void spaces of the basalt, particularly in the glassy portions (Alt, 1995). This low-temperature, oxidative seawater Summary alteration occurs on sparsely sedimented ridge flanks at exposed basalt outcrops, which cover an estimated Young, basaltic ocean crust exposed near mid-ocean 1 000 000 km2 of the Earth’s surface, and in the subseaf- ridge spreading centers present a spatially extensive loor where large volumes of seawater circulate through the environment that may be exploited by epi- and endo- basalt aquifer (Stein and Stein, 1992; Fisher, 1998; Fisher lithic microbes in the deep sea. Geochemical energy and Becker, 2000; Hutnak et al., 2008). Because Ca is released during basalt alteration reactions can theo- released during basalt glass alteration, these reactions retically support chemosynthesis, contributing to a impose a negative feedback on atmospheric CO , with trophic base for the ocean crust biome. To examine 2 basalt alteration accounting for about 30% of the silicate- associations between endolithic microorganisms and weathering based CO drawdown globally (Dessert et al., basalt alteration processes, we compare the phyloge- 2 2001; 2003). netic diversity, abundance and community structure Recent studies have demonstrated a diverse and abun- of bacteria existing in several young, seafloor lavas dant epi- and endo-lithic microbial community on seafloor from the East Pacific Rise at ~9°N that are variably basalts (Lysnes et al., 2004; Templeton et al., 2005; affected by oxidative seawater alteration. The results Mason et al., 2007; Einen et al., 2008; Santelli et al., of 16S rRNA gene analyses and real-time, quantitative 2008) and in fluids emanating from ridge-flank crust polymerase chain reaction measurements show that (Cowen et al., 2003; Huber et al., 2003; 2006). Rarefac- the abundance of prokaryotic communities, domi- tion analyses show that the basalt biome appears to nated by the bacterial domain, positively correlates harbour bacterial diversity and richness levels com- with the extent of rock alteration – the oldest, most parable to some of the most diverse identified so far on altered basalt harbours the greatest microbial Earth (Santelli et al., 2008). The energetic underpinnings biomass. The bacterial community overlap, structure of this biotope are not established, but it has been sug- and species richness relative to alteration state is gested through both observation and thermodynamic/ less explicit, but broadly corresponds to sample char- bioenergetic calculations that basalt alteration reactions acteristics (type of alteration products and general may contribute to the energetic foundation of this habitat alteration state). Phylogenetic analyses suggest that (Bach and Edwards, 2003; Edwards et al., 2005; Santelli et al., 2008). This interpretation is consistent with infer- ences based on observations made in terrestrial subsur- Received 23 April 2008; accepted 20 July, 2008. *For correspon- face environments (Lovley and Chapelle, 1995; Pedersen dence. E-mail [email protected] Tel. (+1) 213 821 4390; Fax (+1) 213 et al., 1997; Edwards et al., 2000), but this potential has 740 8123. †Present Address: School of Engineering and Applied Sciences, Harvard University, 40 Oxford St. Rm. 228, Cambridge, only rarely been empirically examined in the deep sea MA 02138, USA. (Wirsen et al., 1993; Eberhard et al., 1995).

Here we examine the bacterial communities inhabiting portions directly exposed to seawater on the surfaces and young, basaltic seafloor lavas from the 9°N region of the in fractures have been replaced by palagonite–amixture EPR, a portion of the MOR system characterized by a of amorphous, hydrated glass and poorly crystallized fast spreading rate of ~11 cm year-1 (Carbotte and Mac- secondary minerals such as clays and Fe oxyhydroxides donald, 1992), active hydrothermal venting, and unsedi- (e.g. Stroncik and Schmincke, 2001). One sample, AlO, mented seafloor lava flows directly exposed to seawater. contains ‘slightly altered’ glass and is also coated by a thin In an initial study of seafloor-exposed basalts from two layer of aluminum hydroxide (from hydrothermal activity). geographically distinct environments (EPR and near The samples containing ‘more altered’ glass are all

Hawaii), we demonstrated that the basalt biome is domi- encrusted with Fe oxyhydroxides (samples FeO1 and nated (88–96% of total prokaryotes) by active, diverse FeO2), or a mixture of Fe oxyhydroxide and Mn oxyhy- bacterial communities. In this study, we provide a com- droxide ‘oxide’ minerals (MnO). prehensive phylogenetic analysis of epi- and endo-lithic bacteria from the EPR (discovered in the initial study) Phylogenetic diversity and distribution and further use statistical and comparative taxonomic approaches to examine the phylogenetic diversity, cell Clones from five different seafloor lavas and DSW sample abundance and community structure as a function of rock represent a total of 16 distinct bacterial taxonomic groups, alteration. We aim to establish relationships between bac- and the results showing the relative proportions of the terial taxa (and groups of taxa) and the products of alter- different groups in each clone library (sample) are sum- ation reactions on seafloor basalts to further our marized in Table 1. Clones from five subdivisions of Pro- understanding of the potential role this biome could play in teobacteria (a, b, g, d and e) are present and account for elemental cycling in the deep sea. the majority of sequences in each clone library, but not all subdivisions (e.g. b and e) exist in every sample. With the exception of the aluminum oxide-coated sample (AlO), Results sequences belonging to g-Proteobacteria dominate all clone libraries and account for 22–37% of each basaltic Samples library and 89% of the DSW library. The g-Proteobacteria Five samples of young, ocean floor basalts, described clones for each sample are quite diverse, distributed previously (Santelli et al., 2008) and in Table S1, were among at least 15 different families (Fig. 1). Many of selected from among several dozen samples collected the clones cluster within groups that are metabolically from the EPR in 2004 in the area between 9°28′N and diverse and ubiquitous in marine environments, such as 9°51′N. The samples were selected to represent a range Alteromonadaceae, Halomonadaceae and Pseudomona- in (i) extent of alteration and (ii) the type and thickness of daceae. Clones belonging to families that are more hydrothermal coatings. All lavas, representing a variety of physiologically constrained include the methane oxidiz- flow morphologies and ages, have a normal mid-ocean ing Methylococcaceae (e.g. EPR3968-08a-Bc20) and ridge basaltic composition (Sims et al., 2002) and typical sulfide-oxidizing Ectothiorhodospiraceae (e.g. EPR397- major element compositions – between 7.0 and 8.5 wt.% M01A-Bc32). Species most closely aligning with the MgO and 8.0 to 10.0 wt.% FeO (Perfit et al., 1994). Based psychrophilic Colwelliaceae and Idiomarinaceae (e.g. on the half spreading rate of 5.5 cm year-1 (Carbotte EPR3967-O2-Bc92 and EPR3965-I2-Bc80 respectively), and Macdonald, 1992) and distance to the ridge axis aerobic chemoorganotrophs primarily isolated from (maximum 1.0 km; Table S1), eruption ages can be esti- deep-sea environments, were also recovered in the clone mated at < 18 kyrs. These ages represent maximum esti- libraries. For example, clones EPR3967-O2-B1 and mates as lava is transported off-axis for considerable EPR3967-O2-B25 are 97% similar to clones recovered distances at the EPR (Soule et al., 2005). from Japan Trench cold seep sediments (Li et al., 1999); All basalts are generally characterized by the abun- several sequences including EPR3967-O2-Bc66 and dance of fresh, unaltered glass. The apparently youngest EPR3968-O8a-Bc21 are 97% similar to sequences from sample, fresh glass (FG), is a nodule of pure glass lacking gas-hydrate associated sediments (Knittel et al., 2003); any visible alteration that was collected from an unsedi- clones EPR3967-O2-Bc24 and EPR3968-O8a-Bc12 are mented lava flow within the axial summit trough, an elon- 97% and 98%, respectively, similar to Hawaiian seafloor gate volcanic collapse feature at the spreading axis where basalt clones (Santelli et al., 2008); and clone EPR3968- lava is erupted and hydrothermal venting is focused O8a-Bc61 is most similar to a deep continental crust (Fornari et al., 1998). Due to frequent volcanic activity in aquifer sequence (Lin et al., 2006) based on BLAST the sampling area (e.g. Tolstoy et al., 2006), we infer that analyses (Altschul et al., 1997; Table S2). As observed in sample FG is probably less than a few decades old. In the the phylogenetic tree (Fig. 1), many of these uncultured majority of samples collected off-axis, however, the glassy species (including those from this study) form their own

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 86–98 88 C. M. Santelli, V. P. Edgcomb, W. Bach and K. J. Edwards

Table 1. The distribution of phyla recovered from clone libraries of each sample.

% of clone library

Phylogenetic group FG AlO FeO2 FeO1 MnO DSW a-Proteobacteria 13 46 30 18 20 8 b-Proteobacteria 16 0 0 4 0 3 g-Proteobacteria 35 22 37 27 30 89 d-Proteobacteria 41 1 164 0 e-Proteobacteria 18 0 0 0 0 0 Plantocmycetes 19 7 11120 Bacteroidetes 0311220 Verrucomicrobia 00 4 2 0 0 Firmicutes 10 0 0 0 0 Actinobacteria 68 6 6 110 Acidobacteria 14 0 4 6 0 Nitrospira 00 1 0 1 0 Gemmatimonadetes 00 0 0 1 0 Spirochaetes 10 0 0 0 0 Chlamydiae 01 0 0 0 0 Candidate Division OP11 0 0 0 2 0 0 Unclassiﬁed 3 5 2 8 13 0 monophyletic group and are only distantly related to any of bacteria able to grow heterotrophically or chem- cultured microorganisms. olithotrophically such as through hydrogen oxidation. a-Proteobacteria sequences dominate clone library Clones representing the d-Proteobacteria have been AlO (46%) and comprise 8–30% of the other clone lib- recovered from all seaﬂoor lava samples, but not the raries (Table 1). As observed in Fig. 2, many of the a- DSW sample (Table 2). The highest proportion (16%) of Proteobacteria clones (e.g. EPR3965-I2-Bc68 and d-Proteobacteria is observed in one of the Fe oxide

EPR3967-O2-Bc83) are related to members of the family encrusted (FeO1) samples. The d-Proteobacteria contain Sphingomonadaceae, a physiologically and ecologically a number of species that are known as dissimilatory diverse group that are predominantly aerobic chemohet- sulfate and sulfur reducers, such as Desulfovibrio, erotrophs. Other clones (including EPR4059-B2-Bc68 however, most sequences from this study do not cluster and EPR3970-M01A-Bc67) cluster within the family with these organisms (Fig. 2) or any cultured bacteria. Hyphomicrobiaceae, a group often recovered from olig- Several clones from libraries FG (EPR3967-O2-Bc57), otrophic environments that are characterized by hyphal, FeO1 (EPR3965-I2-Bc1 and -Bc9) and FeO2 (EPR3968- prosthecate and budding microorganisms with either O8a-Bc67) were most similar (Table S2) to clones chemoorganotrophic (e.g. methane or manganese oxida- obtained from warm ridge-flank fluids emanating from tion) or autotrophic capabilities. Several a-Proteobacteria basaltic ocean crust (Huber et al., 2006) or environmental clones from Fe oxide-coated sample FeO2 (e.g. clones from Juan de Fuca Ridge basaltic lavas (GenBank EPR3968-O8a-Bc58 and -Bc72), the ferromanganese Accession No. DQ070830 and DQ070818). Sequences encrusted sample MnO (EPR3970-MO1A-Bc3, -Bc11, belonging to e-Proteobacteria have been recovered only -Bc18, -Bc25, -Bc49 and Bc67), and sample AlO from sample FG and make up 18% of the total library (EPR4059-B2-Bc27) are Ն 97% similar (Fig. 2 and (Table 1). All e-Proteobacteria clones (13 total) represent Table S2) to uncultured clones obtained from basaltic a single phylotype, which is 99% similar to another clone lavas near the Juan de Fuca mid-ocean ridge (GenBank isolated from basaltic glass recovered from the EPR in a Accession No. DQ070827 and DQ070833). Additionally, separate study (GenBank Accession No. DQ070790). some clones (e.g. EPR367-O2-Bc89; EPR3970-MO1A- In addition to Proteobacteria, clones belonging to a total Bc19 and -42; and EPR4059-B2-Bc13, -16 and -49) are of 11 other bacterial groups have been recovered from the most similar (Table S2) to clones from recovered from clone libraries (Table 1). Planctomycetes and Actinobac- seafloor lavas near the Hawaiian Islands (Santelli et al., teria sequences have been isolated from all basalt 2008). samples, ranging from 1% to 12% and 6% to 11% respec- b-Proteobacteria clones have been obtained from only tively (Table 1). Planctomycetes are budding bacteria that two basalt libraries (FG, FeO1) and the DSW library are ubiquitous in the environment but poorly understood (Table 1), and are most prevalent (16% of the clone with regard to physiology; however, species such as library) in the FG sample. Many of these sequences (e.g. P. bekefii have been observed to accumulate Mn and Fe EPR3967-O2-Bc21) cluster with the family Comamona- oxide particles (Schmidt et al., 1982). Additionally, one daceae (Fig. 2), which are a physiologically diverse group basalt clone (EPR3965-I2-B17) is 97% similar to Candi-

EPR4059-B2-Bc57 (AlO; EU491558) 51 EPR3970-MO1A-Bc29 (MnO; EU491614) 100 EPR3968-O8a-Bc12 (FeO2; EU491682) 52 EPR3967-O2-Bc20 (FG; EU491769) 97 EPR3965-I2-Bc18 (FeO1; EU491843) hydrothermal sed. clone AT-s3-1 (AY225632) Thialkalivibrio denitrificans (AF126545) EPR3970-MO1A-Bc32 (MnO; EU491618) EPR4059-B2-Bc67 (AlO; EU491567) EPR3970-MO1A-Bc15 (MnO; EU491600) 99 Coxiella burnetii (D89797) 93 EPR3968-O8a-Bc91 (FeO2; EU491758) EPR3967-O2-Bc86 (FG; EU491832) 72 Beggiatoa alba (L40994) 57 Thioploca ingrica (L40998) Nitrosococcus halophilus (AF287298) 97 Methylococcus capsulatus (X72771) deep continental crust clone BE16FW031601GDW_hole1-36 (DQ088732) EPR3968-O8a-Bc20 (FeO2; EU491691) 61 gas hydrate sed. Clone Hyd89-87 (AJ535246) 100 EPR3967-O2-Bc48 (FG; EU491795) EPR3968-O8a-Bc83 (FeO2; EU491751) 100 EPR3967-O2-Bc47 (FeO1; EU491794) 66 Methylomonas rubra (M95662) 65 EPR3968-O8a-Bc39 (FeO2; EU491707) 68 carbonate hydrothermal chimney clone LC1133B-99 (DQ270621) 60 Methylosphaera hansonii (U67929) 100 EPR4059-B2-Bc35 (AlO; EU491541) 92 EPR3968-O8a-Bc49 (FeO2; EU491716) Legionella brunensis (X73403) 96 Thiothrix sp. EJ1M-B (AB042544) EPR3968-O8a-Bc66 (FeO2; EU491733) 100 EPR3967-O2-Bc52 (FG; EU491798) 99 cold-seep sed. clone JT58-28 (AB189348) Kangiella aquimarina (AY520561) 93 Pseudomonas stutzeri (AY321589) 94 EPR4055-N3-Bc77 (DSW; EU491936) 100 Mn-oxidizing Pseudomonas sp. LOB-2 (DQ412061) Pseudomonas putida (AB038136) γ−Proteobacteria 98 EPR3967-O2-Bc38 (FG; EU491786) 100 EPR3965-I2-Bc36 (FeO1; EU491856) 74 EPR3968-O8a-Bc82 (FeO2; EU491750) 100 Microbulbifer sp. JAMB-A7 (AB107975) 97 Mn-oxidizing Microbulbifer sp. KBB-1 (DQ412068) 100 EPR4055-N3-Bc93 (DSW; EU491952) Alcanivorax sp. I4 (AB053125) 82 Halomonas variabilis (U85871) 76 EPR3967-O2-Bc56 (FG; EU491802) 100 EPR4055-N3-Bc74 (DSW; EU491933) 55 Mn-oxidizing Halomonas sp. LOB-5 (DQ412065) 53 100 EPR4055-N3-Bc12 (DSW; EU491893) 99 Mn-oxidizing Marinobacter sp. LOB-4 (DQ412064) Marinobacter aquaeolei (AJ000726) 63 Colwellia maris (AB002630) 73 EPR4055-N3-Bc42 (DSW; EU491914) Colwellia psychroerythrus (U85841) 93 Colwellia sp. BSi20520 (DQ007433) EPR3967-O2-Bc92 (FG; EU491838) 100 EPR3965-I2-Bc80 (FeO1; EU491881) 96 Idiomarina sp. PO-M2 (DQ342238) Idiomarina loihiensis (AY553079) 100 Pseudoalteromonas marina (AY563032) Mn-oxidizing Pseudoalteromonas sp. SPB-9 (DQ412078) 96 EPR3967-O2-Bc74 (FG; EU491820) 93 Cold-seep sed. Moritella marina (AB121097) 100 EPR4055-N3-Bc76 (DSW; EU491935) 80 Moritella marina (AJ297540) Shewanella oneidensis (AF039054) Alteromonas macleodii (Y18234) Mn-oxidizing Alteromonas sp. SPB-5 (DQ412075) EPR4055-N3-Bc50 (DSW; EU491918) 0.1

Fig. 1. Phylogenetic trees showing the relationships of select g-Proteobacteria sequences recovered from EPR basalts and deep seawater as determined by maximum likelihood analysis. Clones from this study are indicated in bold (full clone names as EPR Alvin dive # – sample # – bacterial clone #, e.g. EPR3965-I2-Bc10) with their short sample indicator (e.g. FeO1). Bootstrap values for nodes with > 50% support, based on 1000 replicates, are displayed as percentages. The scale bar represents 0.1 substitutions per nucleotide position.

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 86–98 90 C. M. Santelli, V. P. Edgcomb, W. Bach and K. J. Edwards

68 EPR3970-MO1A-Bc18 (MnO; EU491603) seafloor basalt clone JdFBGBact_40 (DQ070827) EPR3968-O8a-Bc54 (FeO2; EU491721) Prosthecate α-proteobacteria sp. PWB3 (AB106120) EPR3967-O2-Bc62 (FG; EU491808) EPR3970-MO1A-Bc5 (MnO; EU491636) continental shelf sed. clone LC1-25 (DQ289899) 54 94 EPR4055-N3-Bc14 (DSW; EU491895) 93 Mn-oxidizing Sulfitobacter sp. SPB-3 (DQ412073) 97 Sulfitobacter mediterraneus (Y17387) 100 Roseobacter sp. ANT9283 (AY167321) EPR3965-I2-Bc4 (FeO1; EU491858) 67 Agrobacterium kieliense (D88524) EPR4059-B2-Bc92 (AlO; EU491593) EPR4059-B2-Bc68 (AlO; EU491568) 90 EPR3970-MO1A-Bc67 (MnO; EU491654) α−Proteobacteria 96 EPR3968-O8a-Bc72 (FeO2; EU491739) 81 seafloor basalt clone JdFBGBact_73 (DQ070833) 100 Pedomicrobium manganicum (X97691) Hyphomicrobium sulfonivorans (AF235089) 98 EPR4059-B2-Bc17 (AlO; EU491529) 94 EPR3968-O8a-Bc27 (FeO2; EU491697) 100 Rhodospirillaceae sp. CL-UU02 (DQ401091) EPR3970-MO1A-Bc87 (MnO; EU491674) 59 99 EPR3965-I2-Bc30 (FeO1; EU491852) Tistrella mobilis (AB071665) Star-like microcolonies (AJ001344) EPR4055-N3-Bc71 (DSW; EU491931) EPR3965-I2-Bc68 (FeO1; EU491874) 100 EPR3967-O2-Bc83 (FG; EU491829) 70 Sphingomonas echinoides (AJ012461) 100 Methylobacterium fujisawaense (AJ250801) EPR3967-O2-Bc84 (FG; EU491830) 86 Desulfobacterium indolicum (AJ237607) EPR3965-I2-Bc72 (FeO1; EU491876) 96 Desulfomonile limimaris (AF282177) δ− 100 deep-sea sediment clone MBMPE81 (AJ567571) Proteobacteria 90 EPR3970-MO1A-Bc20 (MnO; EU491606) 100 EPR3965-I2-Bc9 (FeO1; EU491889) seafloor basalt clone JdFBGBact_43 (DQ070830) Aquamonas gracilis (AB109889) EPR3965-I2-Bc13 (FeO1; EU491841) EPR4055-N3-Bc38 (DSW; EU491912) EPR3967-O2-Bc21 (FG; EU491770) 99 MTBE-degrading bacterium PM1 (AF176594) EPR3967-O2-Bc18 (FG; EU491767) 67 Leptothrix discophora (L33975) β− EPR3967-O2-Bc49 (FG; EU491796) Proteobacteria Herbaspirillum lusitanum (AF543312) Thiobacillus denitrificans (AJ243144) 100 Nitrosospira multiformis (X90820) 90 EPR3967-O2-Bc73 (FG; EU491819) Nitrosomonas oligotropha (AF272422) 73 Gallionella ferruginea (L07897) 99 Thiomicrospira denitrificans (L40808) Sulfurimonas autotrophica (AB088431) 100 ε−Proteobacteria 100 EPR3967-O2-Bc64 (FG; EU491809) seafloor basalt clone 9NBGBact_3 (DQ070790) 0.1

Fig. 2. Phylogenetic trees showing the relationships of select a-, b-, d- and e-Proteobacteria sequences recovered from EPR basalts and deep seawater as determined by maximum likelihood analysis. Clones from this study are indicated in bold with their short sample indicator. Bootstrap values for nodes with > 50% support, based on 1000 replicates, are displayed as percentages. The scale bar represents 0.1 substitutions per nucleotide position. datus Scalindua brodae (Schmid et al., 2003), an anaero- Bc12 and -Bc27, Santelli et al., 2008) and EPR seafloor bic ammonium oxidizing (ANAMMOX) bacterium. Most basalts from an independent study (EPR3970-MO1A- Planctomycetes clones from this study (Fig. 3 and Bc77, GenBank Accession No. DQ070794). Although a Table S2) are closely related to clones from other deep few Actinobacteria clones are closely related (97–99%) marine environments, for example from warm basement to cultured species of Micrococcus (e.g. EPR3967-O2- fluids from the eastern Juan de Fuca ridge flank (e.g. Bc28) and Nesterenkonia (e.g. EPR3967-O2-Bc31), EPR3968-O8a-Bc45, Huber et al., 2006), young basaltic many of the clones form a monophyletic group with other lavas (EPR3965-I2-Bc5, GenBank Accession No. uncultured microorganisms (Fig. 3). This includes clones DQ070834), seafloor basalts near Hawaii (EPR3965- obtained from an inactive deep-sea hydrothermal

Table 2. Estimated and shared richness between bacterial communities as determined by the Chao1 and shared Chao1 (S1,2 Chao) estimators respectively.

Shared richness (S1,2 Chao)

Sample Chao1 95% CI FG AlO FeO1 FeO2 MnO DSW

FG 84 56–157 NA 6 23 7 14 7 AlO 74 53–131 NA 9 15 18 0

FeO1 157 91–320 NA 42 14 3

FeO2 145 93–268 NA 40 0 MnO 153 96–290 NA 0 DSW 12 11–20 NA

NA, not applicable.

chimney (Suzuki et al., 2004), and hydrothermal sediment in sample FeO1. Approximately 46 different OTUs are (Lopez-Garcia et al., 2003). The Bacteroidetes, although sampled from only 52 sequenced clones. The other ‘more not represented in the FG sample, accounts for 2–11% of altered’ samples FeO2 and MnO exhibit indistinguishable the clone libraries for the ‘slightly to more altered’ samples rarefaction curves with less richness than FeO1. Rarefac- (Table 1). The Bacteroidetes phylum includes Flavobac- tion curves for the ‘slightly altered’ sample AlO and the FG teriaceae, heterotrophic bacteria that cover widely diverse sample similarly cannot be differentiated (overlapping ecological niches and physiologies. 95% confidence intervals) but reveal the lowest relative A small proportion (0–6%) of the clones belongs to one species richness. or more of the following phyla (Table 1): Verrucomicrobia, Firmicutes, Acidobacteria, Nitrospirae, Gemmatimona- Bacterial community overlap and distribution estimates detes, Spirochaetes, Chlymdiaea and Candidate Division OP11. It is difficult to place most of these clones in any The similarities in the OTUs detected and their abun- specific rank past the phylum level with any confidence dance and distribution in each sample are compared because they are related primarily to other unclassified using a variety of statistical analyses implemented in environmental clones. One exception is the Nitrospirae the program SONS (Schloss and Handelsman, 2006). The clones (Fig. 3) in which sequences are 95% similar to number of shared OTUs (OTU0.03) between each sample, Nitrospira marina (Teske et al., 1994), a chemolithoau- calculated by the shared Chao1 richness estimator (Chao totrophic nitrite oxidizer. A significant number of clones, et al., 2006), is shown in Table 2. The results show that

2–13%, from each library could not be assigned a phyla samples FeO1 and FeO2 (the two Fe oxide-coated (unclassiﬁed; Table 1) because they have conﬁdence samples) share the greatest number of OTUs (42) com- thresholds lower than 80% as assigned by the Ribosomal pared with any other two samples. Sample FeO2 also Database Project, RDP (Cole et al., 2003). shares a large number of OTUs (40) with sample MnO (an Fe and Mn oxide encrusted sample). Shared richness is observed in all of the basalt samples, with the lowest Rarefaction analysis and richness estimates number of shared OTUs generally found relative to the FG

The relative species richness of bacteria observed within sample with the exception of sample FeO1, which shares each sample was determined previously (Santelli et al., approximately 23 types. In contrast, the DSW sample only

2008) through rarefaction analysis and is presented here shared richness with two samples, FG and FeO1, of which including 95% standard error for more accurate sample- the majority (7) was shared with FG. to-sample comparison. For rarefaction, the number of The overlap in bacterial community membership observed operational taxonomic units (OTUs) assigned between two samples, based on the fraction of sequences by DOTUR (Schloss and Handelsman, 2005) is compared that belong to shared OTUs, is estimated by the with the sequencing effort for each clone library. Here abundance-based Jaccard (Jabund) similarity index (Chao we deﬁne OTUs by a 0.03 distance level (OTU0.03), so et al., 2005) as shown in Table 3. The overlap measure- sequences with Ն 97% similarity are designated by a ments represent the probability that an OTU0.03 recovered single OTU. Rarefaction curves for all seaﬂoor lava in one community will also be recovered in the other samples have steep, near-linear trends, indicating that community, so an overlap index of 1.00 means there is a more OTUs would be obtained with greater sequencing 100% chance that a randomly chosen OTU will be repre- coverage (Fig. 4). In contrast, the DSW sample reaches sented in both communities. The trends in bacterial com- a near asymptotic value of 11 clones. Among basalt munity overlap between the several basalt-hosted and samples, greatest richness (number of OTUs) is observed DSW samples are similar to what is observed for the

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 86–98 92 C. M. Santelli, V. P. Edgcomb, W. Bach and K. J. Edwards

100 EPR3968-O8a-Bc71 (FeO2; EU491738) 100 deep-sea octacoral clone ctg_NISA088 (DQ396010) EPR4059-B2-Bc85 (AlO; EU491585) Pirellula marina (X62912) Pirellula staleyi (X819646) EPR3970-MO1A-Bc61 (MnO; EU491649) 98 EPR4059-B2-Bc54 (AlO; EU491641) 57 seafloor basalt clone 9NBGBact_17 (DQ070794) 96 EPR3968-O8a-Bc80 (FeO2; EU491748) EPR3970-MO1A-Bc77 (MnO; EU491663) 100 EPR4059-B2-Bc30 (AlO; EU491538) farm soil clone AKYG983 (AY922083) EPR3968-O8a-Bc45 (FeO2; EU491712) Planctomycetes ocean crust fluids clone FS142-21B-02 (AY704401) EPR3970-MO1A-Bc12 (MnO; EU491597) EPR3965-I2-Bc5 (FeO1; EU491864) seafloor basalt clone JdFBGBact_75 (DQ070834) 100 EPR3968-O8a-Bc62 (FeO2; EU491730) 59 ocean crust fluids clone FS275-22B-03 (DQ513104) 59 EPR3970-MO1A-Bc9 (MnO; EU491677) 99 Planctomyces brasiliensis (AJ231190) 100 EPR3967-O2-Bc55 (FG; EU491801) ocean crust fluids clone FS274-34B-03 (DQ513097) 100 Candidatus Scalindua brodae (AY254883) EPR3965-I2-Bc17 (FeO1; EU491842) 85 EPR4059-B2-Bc52 (AlO; EU491554) 100 ocean crust fluids clone FS266-75B-03 (DQ513088) EPR3965-I2-Bc66 (FeO1; EU491872) 79 86 EPR3970-MO1A-Bc31 (MnO; EU491617) Acidobacteria 100 cave clone wb1_A08 (AF317741) 57 Roman catacombs clone CAL7 (DQ139449) 100 EPR3970-MO1A-Bc79 (MnO; EU491665) 88 pumic fragment clone AT-pp27 (AY225642) EPR3967-O2-Bc60 (FG; EU491807) 84 EPR3970-MO1A-Bc27 (MnO; EU491612) 100 EPR3968-O8a-Bc32 (FeO2; EU491702) Nitrospirae 100 Nitrospira marina (L35501) Leptospirillum ferrooxidans (X86776) 100 deep-sea sediment clone A20 (AY373407) 99 EPR4059-B2-Bc40 (AlO; EU491546) 52 EPR3965-I2-Bc22 (FeO1; EU491565) 100 EPR3970-MO1A-Bc70 (MnO; EU491657) deep-sea sed. clone E17 (AJ966591) 100 EPR3970-MO1A-Bc30 (MnO; EU491616) hydrothermal sed. clone AT-s2-33 (AY225655) EPR3967-O2-Bc36 (FG; EU491785) extinct hydrothermal chimney clone IheB3-34 (AB099989) 100 EPR3965-I2-Bc74 (FeO1; EU491878) Micrococcus luteus (AJ409095) 79 EPR3967-O2-Bc28 (FG; EU491776) EPR3965-I2-Bc28 (FeO1; EU491851) 100 Micrococcaceae sp. KVD-unk-39 (DQ490457) Actinobacteria 100 Micrococcus antarcticus (AJ005932) 81 Nesterenkonia sp. YIM 70079 (AY226510) 100 Nesterenkonia aethiopica (AY574575) EPR3967-O2-Bc31 (FG; EU491780) 97 100 EPR3968-O8a-Bc68 (FeO2; EU491735) 57 ocean crust fluids clone FS266-92B-03 (DQ513090) Microthrix parvicella (X89774) 54 EPR3970-MO1A-Bc33 (MnO; EU491619) EPR3968-O8a-Bc19 (FeO2; EU491689) EPR4059-B2-Bc69 (AlO; EU491569) 93 EPR3967-O2-Bc16 (FG; EU491766) 64 EPR4059-B2-Bc83 (AlO; EU491583) 67 100 EPR3970-MO1A-Bc51 (MnO; EU491638) cave clone wb1_P06 (AF317769) Acidimicrobium ferrooxidans (U75647) Leptotrichia sp. DR011 (AF385518) 0.1

Fig. 3. Phylogenetic trees showing the relationships of select non-Proteobacteria sequences (recovered from EPR basalts and deep seawater as determined by maximum likelihood analysis. Clones from this study are indicated in bold (full clone names as EPR Alvin dive # – sample #

– bacterial clone #, e.g. EPR3965-I2-Bc10) with their short sample indicator (e.g. FeO1). Bootstrap values for nodes with > 50% support, based on 1000 replicates, are displayed as percentages. The scale bar represents 0.1 substitutions per nucleotide position.

Fig. 4. Rarefaction analysis comparing the 60 FeO2 (~6x107 cells per g basalt; 93% Bacteria)* relative richness of Bacteria among variably altered seafloor basalt samples and a deep MnO (~1x109 cells per g basalt; 50 FeO1 seawater sample (modified from Fig. 3C of (biomass not measured)* 95% Bacteria)* Santelli et al., 2008). Total bacterial plus AlO (~2x107 cells per g basalt; archaeal biomass (cells per g basalt) and 40 96% Bacteria)* community composition (% Bacteria) is shown in parentheses following the sample. Samples FG 6 (~3x10 cells per g basalt; denoted with ‘*’ were analysed previously 30 88% Bacteria)* using qPCR analysis (Santelli et al., 2008). Biomass estimates and community composition for deep seawater ‘#’ were 20 reported by Karner and colleagues (2001). OTUs observed OTUs DSW 4 Rarefaction curves are plotted with error bars (~1x10 cells/mL seawater; representing 95% confidence intervals. OTUs 10 # 50% Bacteria) are defined as a sequence similarity of Ն 97%. 0 0 102030405060708090 Clones sequenced

shared richness estimates; however, the Jabund calculated 2001; Lysnes et al., 2004; Mason et al., 2007; Santelli for samples MnO and FeO2 (0.27) is slightly greater than et al., 2008). Thorseth and colleagues (2001) recovered between FeO1 and FeO2 (0.24). The overlap between AlO, sequences belonging to three major bacterial taxonomic and MnO and FeO2 is similarly high (0.20 and 0.19 groups whereas Lysnes and colleagues (2004) revealed respectively), but is low compared with the other Fe oxide eight groups of the same rank. A recent review of publicly sample (0.05). The Jabund calculated for FG and DSW is available sequences from both culture-dependent and 0.17. culture-independent studies of the ocean crust biome Community overlap differs from community structure in (Mason et al., 2007) exposed bacterial phylotypes from that overlap considers only membership (OTUs) in each 13 different groups. Santelli and colleagues (2008) com- sample, whereas structure accounts for similarities in both pared seaﬂoor-exposed basalt diversity in samples from membership and abundance. Estimates of community two geographically distinct sites, the EPR and samples structure similarity (qYC) shown in Table 3 were deter- used in this study, and from around the big island of mined using the non-parametric maximum likelihood esti- Hawaii. This study revealed 16 and 21 different groups, mator by Yue and Clayton (2005) at an OTU0.03 deﬁnition. respectively, indicating greater bacterial diversity by com-

The similarity (qYC) measured for FeO1 and FeO2 is 0.65 parison with previous studies. In this study, we examine (SE = 0.04). In comparison, the next highest value is 0.19 the phylogenetic diversity of bacteria colonizing EPR

(SE = 0.06) for samples MnO and FeO2, and in general basalts and the factors that may influence community the values comparing the basalt samples range from 0.02 composition, distribution and abundances as a function of to 0.19 with the above exception. The basalt samples FG alteration state of the individual samples. and FeO1 have similar qYC values of 0.07 when compared Results show that bacterial biomass [as assessed by with the DSW sample. Santelli and colleagues (2008)] correlates well with the increase in age and alteration state of the rock, as demonstrated in Fig. 4. Specifically, a 10- to 1000-fold Discussion and conclusions increase in bacterial biomass is observed for the ‘slightly’ Few prior studies have examined the phylogenetic diver- to ‘more’ altered samples, respectively, relative to the FG sity of young crust at the ocean floor (Thorseth et al., sample. Rarefaction and community structure analyses

Table 3. Bacterial community overlap (Jabund) and structure (qYC) similarity estimates with 95% conﬁdence intervals (CI).

FG AlO FeO1 FeO2 MnO DSW

Sample Jabund qYC Jabund qYC (CI) Jabund qYC (CI) Jabund qYC (CI) Jabund qYC (CI) Jabund qYC (CI)

FG 1.00 1.00 0.04 0.02 (0.01) 0.20 0.10 (0.05) 0.10 0.10 (0.04) 0.09 0.04 (0.02) 0.17 0.07 (0.03) AlO 1.00 1.00 0.05 0.03 (0.03) 0.19 0.14 (0.05) 0.20 0.12 (0.04) 0.00 0.00

FeO1 1.00 1.00 0.24 0.65 (0.04) 0.08 0.06 (0.04) 0.03 0.07 (0.01) FeO2 1.00 1.00 0.27 0.19 (0.06) 0.00 0.00 MnO 1.00 1.00 0.00 0.00 DSW 1.00 1.00

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 86–98 94 C. M. Santelli, V. P. Edgcomb, W. Bach and K. J. Edwards are less robust, though do broadly correlate positively with all groups. Furthermore, Santelli and colleagues (2008) alteration. For example, an order of magnitude increase in demonstrate that there is little overall community overlap cell density is observed from samples FG to AlO, despite (for all bacterial phyla and subphyla) between EPR basalts similarities in species richness. Because Al is not redox and a hydrothermal white smoker spire also from the EPR reactive in this habitat, the presence of Al hydroxides is at 9°N. That study additionally showed significant commu- likely due to abiotic, hydrothermal precipitation. One pos- nity overlap between seafloor-exposed basalts from distal sible interpretation is that this non-redox reactive crust geographic locations (the EPR and Hawaii). As demon- may inhibit some community members at the basaltic strated earlier, many clones recovered from the EPR are glass surface. Given the overall high biomass and phylo- most similar to those recovered from the seafloor off genetic diversity, however, bacterial growth does not Hawaii, as well to other ocean crust sequences. Addition- appear suppressed. ally, several clones are Ն 98% similar to clones belonging Interestingly, all three community distribution analyses to the ocean crust-specific clades (specifically to

(SChao1,Jabund and qYC) suggest that there is very little a-Proteobacteria OCC II and Actinobacteria OCC V) overlap or similarity in the membership and structure deﬁned by Mason and colleagues (2007). These results between samples AlO and FG, but the microbial commu- support the interpretation that many of the phylotypes nities in AlO are more like those found in the other ‘more’ observed here are indigenous to the basalt habitat. altered samples (FeO1 and FeO2). So although fewer Santelli and colleagues (2008) hypothesize that basalt OTUs were obtained from the Al hydroxide-coated sample alteration reactions, in addition to the input of hydrother- relative to the other oxide-coated samples, the types and mal compounds, may supply energy to this ecosystem. abundances of OTUs recovered from these lavas imply Potential metabolisms of microorganisms linked to these that the microbial community is evolving to match the chemical redox reactions include the oxidation of S2–,S0, 2+ availability and variety of electron donors and acceptors Fe ,H2 and CH4 (Bach and Edwards, 2003; Edwards present on the basalt surfaces. et al., 2005). Here we examine the communities that Although one of the most altered basalt sample (MnO) occur on EPR seaﬂoor basalts and consider possible also harbours the greatest biomass, the alteration state physiological roles for these bacteria. Empirically, we note and species richness correlation is again inconsistent. the presence of bacterial clones EPR3970-MO1A-Bc32,

Instead, the ‘more’ altered sample FeO1 harbours the -58 and -80 closely related (92% similar) to sulfur- most bacterial OTUs (Fig. 4 and Table 2). It is possible, oxidizing Thioalkalivibrio thiocyanodenitrificans (Sorokin however, that the relative richness may change with et al., 2004). Additionally, e-Proteobacteria clones 95% greater sequencing as the microbial communities have similar to a chemolithoautrophic hydrogen- and sulfur- not been fully sampled. Because species richness does oxidizing bacterium (Takai et al., 2006) are observed. not explain differences in community complexity, compari- Several phylotypes related to bacteria that gain energy for sons of community overlap and structure may be more metabolic growth from the oxidation of methane and inter- beneficial to discern relationships with basalt geochemis- mediate 1-C compound species are also present. For try. For instance, the bacterial community structure and instance, g-Proteobacteria clones (EPR3968-O8a-Bc2, membership (Table 3) of sample FeO2 is most similar to -Bc10, -Bc25 and -Bc39) are 93% similar to a methan- both the other Fe oxide-coated sample (FeO1) and the otrophic bacterium Methylobacter luteus (Gulledge et al., ferromanganese encrusted sample (MnO). This may 2001) and a-Proteobacteria clone EPR3967-O2-Bc84 suggest that the communities are in some way controlled is 99% similar to the methylotroph Methylobacterium by the geochemical reactions occurring on the basalts, fujisawaens (GenBank Accession No. AJ250801). Evi- potentially contributing to the formation of these oxide dence for bacteria that may oxidize Fe(II) is problematic crusts. because this metabolic trait is so broadly distributed phy- The most frequently observed phyla or subphyla in logentically and poorly studied by cultivation. Many of this and other recent studies of the ocean crust these are also capable of more than one type of metabo- biome (Lysnes et al., 2004; Mason et al., 2007) are g- lism [e.g. Sulfobacillus acidophilus can oxidize inorganic and a-Proteobacteria. Despite the dominance of sources of Fe or S for growth (Norris et al., 1996)]. Still, g-Proteobacteria sequences in nearly all EPR basalt clone some clones that cluster within the Hyphomicrobiaceae libraries (except AlO) as well as the DSW clone libraries, (e.g. EPR3967-O2-Bc72; a-Proteobacteria, Fig. 2) are these phylotypes show little community overlap between observed and are related to neutrophilic Fe oxidizers that the basalts and background seawater. This observation promote basalt glass dissolution (Edwards et al., 2003; also holds true for the group with the second highest 2004). relative proportion of clones, the a-Proteobacteria. Indeed, As basalt alteration proceeds, biofilm formation, the dichotomy in bacterial diversity observed between the exopolymer production by epi- and endo-lithic microor- EPR basalts and surrounding seawater is rather large for ganisms, accumulation of secondary mineral precipitates

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 86–98 Bacteria inhabiting seafloor lavas 95 and sedimentary input produces a three-dimensional rine basalts and their role in elemental cycles. It is becom- redox-stratified crust. Thus, both heterotrophic and ing increasingly apparent that the ocean crust biosphere anaerobic metabolic pathways (e.g. sulfate reduction, is extensive and holds significant opportunity to affect the methanogenesis, anaerobic ammonia oxidation or geochemical exchange between crust and seawater. anaerobic iron oxidation) should be possible. Generally, evidence is present in this study that is consistent with this Experimental procedures hypothesis – clones highly related to known heterotrophic Actinobacteria include EPR3965-I2-Bc28 that is 99% Phylogenetic analyses similar to Micrococcaceae sp. (GenBank Accession No. Seafloor lavas (described in Table S1) were sampled for this DQ490457) and EPR3967-O2-Bc31 that is 97% similar study from the East Pacific Rise (EPR) between 9°28′N and to Nesterenkonia abyssinica (Delgado et al., 2006). Addi- 9°50′N as described previously (Santelli et al., 2008). Envi- tionally, many clones belong to families (e.g. Idiomari- ronmental DNA from seafloor lavas and DSW was extracted, naceae and Flavobacteriaceae, belonging to the amplified, cloned, sequenced and checked for chimeras using protocols and methods also outlined by Santelli and g-Proteobacteria and Bacteroidetes respectively) demon- colleagues (2008). Sequences used in this study were pre- strating generally heterotrophic metabolisms. Many well- viously submitted (Santelli et al., 2008) to the GenBank known Fe and S reducing bacteria are classified as Database with accession numbers EU491521–EU491952. d-Proteobacteria, yet the presence of d-Proteobacteria Sequence data were submitted to the online BLAST (Basic clones in the basalt libraries is unhelpful in providing Local Alignment Search Tool) (Altschul et al., 1997) program robust support for the inference of these metabolisms to be compared against the GenBank 16S rRNA database for because they are only distally related to cultivated identifying closely related environmental clones and cultured isolates. The phylogenetic affiliation of each clone was members possessing these traits (Fig. 2). However, the assigned using an 80% confidence threshold by the taxo- prevalence of d-Proteobacteria with these physiologies nomical ‘Classifier’ program available through the Ribosomal suggests that targeting these groups from this biome for Database Project, RDP (Cole et al., 2003). cultivation studies may result in novel members of Fe- and Sequence were aligned using the computer program ARB S-reducing bacteria from the deep sea. (Ludwig et al., 2004). Aligned sequences from the clone Surprisingly robust evidence for an active nitrogen libraries, along with closely related environmental clones and cycling community within seafloor basalts is also cultures, were selected for phylogenetic tree construction. observed in this study. In addition to the clone closely Tree topology and branch lengths were determined using GARLI (Zwickl, 2006), which uses maximum likelihood crite- related to the ANAMMOX bacterium, b-Proteobacteria rion for distance analysis. MODELTEST (Posada and Crandall, clone EPR3967-O2-Bc73 is 97% similar to an ammonia- 1998) confirmed the use of a General Time Reversible model oxidizing Nitrosospira sp. (Purkhold et al., 2003) and with gamma rate heterogeneity and an estimated proportion Nitrospira clones EPR3968-O8a-Bc32 and EPR3970- of invariable sites. Maximum likelihood bootstrapping was MO1A-Bc27 are 96% similar to the chemolithotrophic carried out with 1000 replicates for the non-Proteobacteria nitrite-oxidizing Nitrospira marina bacterium (Teske et al., tree, and 500 replicates for the other trees. Bootstrap propor- 1994). Denitrification is broadly distributed phylogeneti- tions were obtained by reading all trees into PAUP* to obtain a majority rule consensus. Phylogenetic trees were visualized cally and many possible candidates were also observed. with TREEVIEW (Page, 1996). Specifically, numerous clones cluster with Pseudomonas (g-Proteobacteria, Fig. 1), of which many species reduce Rarefaction analysis and diversity comparisons nitrate, and clone EPR3967-O2-Bc51 is 99% similar to a denitrifying Acidovorax sp. (Khan et al., 2002). Interest- A distance matrix generated in ARB (using the neighbour- ingly, a number of nitrate reducing bacteria also have Fe joining method with the Jukes-Cantor correction and a bac- oxidizing capabilities (Straub et al., 2004). teria filter) from the aligned sequence data was used as the input for rarefaction analysis and richness estimates using In this study, we show evidence to support the inference the computer program DOTUR (Schloss and Handelsman, that alteration reactions occurring on basalt may be fuel- 2005). A distance level of 0.03 was selected to compare ling this biosphere. We demonstrate a generally positive sequences for both rarefaction and richness indicators. Using correlation between basalt alteration and both the abun- the ‘list’ output file from these DOTUR analyses, the computer dance and structure of the bacterial communities existing program SONS (Schloss and Handelsman, 2006) was used to in this bare-rock environment. Additionally, as alteration compare bacterial community structure and OTU overlap proceeds, redox stratification and chemical niche creation among the different seafloor lavas also at an OTU definition of 0.03. on basalts support a wide diversity of possible metabolic pathways in this habitat. Further examination, larger data sets and cultivation studies targeting the metabolisms Acknowledgements discussed herein are required to reveal the foundations The authors thank Dan Fornari, Maurice Tivey and Hans influencing the microbial populations colonizing subma- Schouten (WHOI) for accommodating C.M.S and W.B. on

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 86–98 96 C. M. Santelli, V. P. Edgcomb, W. Bach and K. J. Edwards research cruise AT11-7. Everyone at the Josephine Bay Paul basalt weathering on the global carbon cycle. Chem Geol Center at the Marine Biological Laboratories, especially 202: 257–273. Hilary Morrison and David Mark Welch, have been tremen- Eberhard, C., Wirsen, C.O., and Jannasch, H.W. (1995) Oxi- dously helpful and resourceful with sequencing and phyloge- dation of polymetal sulfides by chemolithoautotrophic bac- netic analyses. Eric Webb and Stefan Sievert (WHOI) shared teria from deep-sea hydrothermal vents. Geomicrobiol J their lab space and equipment for sequencing. Patrick 13: 145–164. Schloss (U. Wisconsin-Madison/U. 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