Microorganisms in seafloor basalts - Biogeosciences

Biogeosciences Discuss., 3, 273–307, 2006 www.biogeosciences-discuss.net/3/273/2006/ © Author(s) 2006. This work is licensed under a Creative Commons License.

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Microorganisms in seafloor basalts J. Einen et al.

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Microbial colonization and alteration of basaltic glass 1

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˚ , I. H. Thorseth , and T. Torsvik J. Einen , C. Kruber , L. Øvreas 1 2

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Department of Biology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway Department of Earth Science, University of Bergen, Allegt. 41, N-5007 Bergen, Norway

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Received: 20 December 2005 – Accepted: 5 January 2006 – Published: 7 March 2006 Correspondence to: J. Einen ([email protected])

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Microorganisms have been reported to be associated with the alteration of the glassy margin of seafloor pillow basalts (Thorseth et al., 2001, 2003; Lysnes et al., 2004). The amount of iron and other biological important elements present in basalts and the vast abundance of basaltic glass in the earth’s crust, make glass alteration an important process in global element cycling. To gain further insight into microbial communities associated with glass alteration, five microcosm experiments mimicking seafloor conditions were inoculated with seafloor basalt and incubated for one year. Mineral precipitations, microbial attachment to the glass and glass alteration were visualized by scanning electron microscopy (SEM), and the bacterial community composition was fingerprinted by PCR and denaturing gradient gel electrophoresis (DGGE) in combination with sequencing. SEM analysis revealed a microbial community with low morphological diversity of mainly biofilm associated and prosthecate microorganisms. Approximately 30 nm thick alteration rims developed on the glass in all microcosms after one year of incubation; this however was also seen in non inoculated controls. Calcium carbonate precipitates showed parallel, columnar and filamentous crystallization habits in the microcosms as well as in the sterile controls. DGGE analysis showed an alteration in bacterial community profiles in the five different microcosms, as a response to the different energy and redox regimes and time. In all microcosms a reduction in number of DGGE bands, in combination with an increase in cell abundance were recorded during the experiment. Sequence analysis showed that the microcosms were dominated by four groups of organisms with phylogenetic affiliation to four taxa: The Rhodospirillaceae, a family containing phototrophic marine organisms, in which some members are capable of heterotrophic growth in darkness and N2 fixation; the family Hyphomicrobiaceae, a group of prosthecate oligotrophic organisms; the genus Rhizobium, N2 fixating heterotrophs; and the genus Sphingomonas, which are known as bio-film producing oligotrophs. Although no bioalteration of glass could be confirmed from our experiments, oligotrophic surface adhering bacteria such as the Sphingomonas sp. and 274

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Hyphomicrobium sp. may nevertheless be important for bioalteration in nature, due to their firm attachment to glass surfaces, and their potential for biofilm production.

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1 Introduction

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When the basaltic magma is extruded into cold seawater at the mid-ocean spreading ridges, the surface layer is cooled so rapidly that its constituent ions do not have enough time to crystallize, but arrests into an amorphous glass rim. As the glass rim is more brittle than the crystalline interior, the density of fractures and cracks are highest in the glassy margins, giving a large surface to mass ratio for water-rock interactions and microbial attachment and growth. The lack of systematic atomic arrangements and the high numbers of cracks in the glass results in a higher dissolution rate for glass then for crystalline basalt, regardless of identical element composition. Subsequent eruptions bury the lava flows, and the pillow lava layer can have substantial depth of the ridge axis. The basalt continues to alter after burial, due to seawater percolations through the pillow lava layer. Oxidized compounds which can be used as electron acceptors and carbon sources are supplied to microorganisms deep within the basaltic layer with the seawater (D’Hondt et al., 2004), and dissolution of glass provides electron donors such as reduced iron and manganese. Microorganisms have been proved by DNA specific staining and chemical analysis to be associated with the altered glass in seafloor pillow lavas (Thorseth et al., 1995). Furthermore, cells and etch marks formed by microorganisms have been directly observed to be associated with altered glass surfaces in seafloor basalt using SEM (Thorseth et al., 1991, 2003). Culture independent molecular phylogenetic techniques have also proved that basaltic glass is colonized by a diverse and unique microflora (Thorseth et al., 2001; Lysnes et al., 2004). One can expect two types of microbial dissolution of glass, passive glass alteration induced by changes in the surrounding environment caused by organisms affecting pH, Eh, or concentration of dissolved ions. Organisms can also actively dissolve glass in order to use the elements either in an assimilatoric 275


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way to obtain trace elements or in a dissimilatoric way to obtain energy. In basaltic glass reduced forms of Fe and Mn, are potential electron donors. Gallionella ferruginea and Leptothrix discophora, are considered classical organisms capable of lithoautotrophic Fe oxidation at neutral pH. Recently it has been shown that groups of microorganisms distantly related with the heterotrophic organisms Marinobacter sp. and Hyphomonas sp. are also capable of lithoautotrophic growth at circum neutral pH on a range of reduced Fe components (Edwards et al., 2003). Basaltic glass compromises a significant component of the oceanic crust, is easily altered and important in the global cycling of elements (Staudigel and Hart, 1983). As microorganisms play a major role in element cycling in other habitats, it’s important to increase our knowledge in how microorganisms colonize and alter pillow lava basalt. Bacterial alteration of basaltic glass has even been reported after such short incubation time as one year in laboratory experiments, at room temperature and with high nutrition levels (Staudigel et al., 1995, 1998; Thorseth et al., 1995). The intension of this study was to observe and measure microbial colonization and bioalteration of basaltic glass under controlled conditions, near to the natural environment of seafloor basalts. Five microcosm experiments mimicking seafloor conditions were set up with different nutrient and redox regimes. Synthetic glass was added artificial seawater and inoculated with seafloor basalt. The microbial colonisation processes were visualized using SEM and followed genetically using PCR-DGGE. 2 Materials and methods

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2.1 Inoculum Printer-friendly Version

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Glassy basalt was collected by dredging the seafloor at 16 different locations in the ˚ neovulcanic zone of the Mohns ridge during the SUBMAR 2001 cruise with R/V Hakon Mosby in the Norwegian/Greenland Sea. The samples displayed from none to highly oxidized surfaces. The samples were handled aseptically and the glassy margin was 276

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chipped of with hammer and chisel and crushed in a pestle. Two sets of inoculums were initiated; one that was prepared and held under aerobic conditions and one that was prepared and held anaerobicly. The crushed glass was transferred to 100 mL serum bottles completely filled with either aged oxic or anoxic sterile seawater. All equipment used was sterilized. Samples for anoxic inoculum was prepared inside a disposable glove box (Model S30-20, Instrument for Research and Industry Inc.) filled with N2 . The samples were stored at 10◦ C in the dark for one year prior its use as inoculum.

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2.2 Synthetic glass

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Synthetic glass was made by melting powdered unaltered seafloor basalt from the Mohns ridge in a platinum crucible, 10 mm diameter and 60 mm high in size at 1250◦ C for 3 h, before it was quenched in distilled water. The oxygen fugacity was controlled in a H2 -CO2 mixture and held at nickel-nickel oxide buffer oxygen fugacity (Deines et al., 1974). The synthetic glass was subsequent crushed to grains of

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Microorganisms in seafloor basalts

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Fig. 6. DGGE gels showing a genetic fingerprint of the bacterial communities in the two aerobic microcosms 1B (with CH4 ), 2B (with acetate) and the three anaerobic microcosms 3B (with H2 ), 4B (unamended) and 5B (with acetate) during the twelve months of incubation. The arrows indicate sequenced bands. The numbers below each lane shows the band/sequence number of each band in that particular lane, numbered from top to bottom. The numbering given here corresponds to the numbers in the phylogenetic tree (Fig. 9). M = marker.

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Microorganisms in seafloor basalts

Anoxic incubation

J. Einen et al.

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Oxic incubation

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Fig. 7. Clustering of samples based band patterns from the DGGE gel in Fig. 6. The sample names are constructed with microcosm name followed by sampling number. i.e. 1B2 means second sampling (6 months of incubation) of microcosms 1B.

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Figure 8: Attached bacterial on glass surfaces during experiment. Glass Fig. 8. Attached bacterial cellscells on glass surfaces during the the experiment. (A)(A) andand (B)(B) Glass grains from microcosm 1B (aerobe with CH4 ) showing typical attached bacteria at 3 months grains from microcosm 1B (aerobe with CH4) showing typical attached bacteria at 3 months (A) and 12 months (B). (C) Growth of bacteria on glass surface in microcosms 2B (aerobe with 12 (B). months. (D) Cells glass surface after 9 month (A)acetate) and 12 after months (C) Growth ofattached bacteria to on the glass surface in microcosms 2Bunder (aerobe anaerobe conditions with H2 from microcosm 3B. (E) Exopolymer producing cells from microwith4Bacetate) afterwith 12 no months. (D) Cells to the surfacebacterial after 9 month under cosm (anaerobic amendment) afterattached 12 months. (F)glass Prostechate cells from microcosm (Anaerobic withHacetate) after 12 month. anaerobe 5B conditions with from microcosm 3B. (E) Exopolymer producing cells from

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microcosm 4B (anaerobic with no amendment) after 12 months. (F) Prostechate bacterial cells from microcosm 5B (Anaerobic with304 acetate) after 12 month

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[X05567] Archaeoglobus fulgidus [M83548] Aquifex pyrophilus [AY349389] Peptococcus sp.

3B0-23* 3B0-25*

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1. Clostridiales group [U41562] Pelobacter venetianus 3B0-22* [AF099062] Desulfotalea psychrophilus

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[AF038843] Sulfurospirillum barnesii 4B2-48 [AB080645] Sulfuricurvum kujiense [L40808] Thiomicrospira denitrificans [AY616755] Halomonas variabilis 3B1-35 99 2. Halomonas group 5B1-67 [U85907] Shewanella gelidimarina [AB003191] Photobacterium profundum 4B1-45 5B1-62 3. Alteromonadaceae group 99 1B1-8 [U85844] Colwellia demingiae [AJ298748] Nitrosococcus halophilus [AF016981] Methylobacter sp. 5B1-65 4. Gamma proteobacteria group 5B2-68 95

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[AJ227759] Caulobacter fusiformis [AB017203] Prosthecomicrobium pneumaticum 4B1-46 4B2-51 4B3-54 5B1-63 [AB110414] Blastochloris sulfoviridis 1B4-15 4B4-57 29 78 3B4-41 2B3-19 78 99 [AJ582227] Loktanella vestfoldensis 51 [AJ534225] Jannaschia helgolansis 4B4-58 4B4-60 80 [AF513476] Rhodospirillaceae bacterium PH30 99 1B0-2* 89 34 1B2-12 32 1B1-11 32 1B0-3* 1B0-6* 32 1B0-5* 32 1B1-9 32 2B2-18 32 32 1B0-7* 32 1B0-4* 1B0-1* 46 1B4-16 [D30791] Rhodobium marinum [AB087720] Rhodopseudomonas julia 4B4-56 5B4-71 5B4-72 36 3B1-28 4B2-49 44 4B4-59 3B1-32 3B4-40 [D49423] Pseudomonas riboflavina 21 [AJ227814] Hyphomonas jannaschia 99 2B2-17 2B4-20 [X94198] Xanthobacter agilis 50 [AJ132378] Defluvibacter lusdtiae 3B4-42 29 14 [D12789] Phyllobacterium myrsinacearum 59 [Y15403] Aquamicrobium defluvium 51 3B1-34 5B1-66 39 504B4-61 5B3-70 4B2-52 46 40 2B4-21 99 3B1-31 54 3B0-26* 5B1-64 22 5B3-69

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[D13722] Sphingomonas adhaesiva 4B2-50 3B3-37 4B1-43 97 4B1-44 98 92 3B3-38 3B1-29 83 3B4-39 52 1B4-13 98 4B4-55 97 [U63956] Sphingomonas sp. 97 4B3-53

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5. Hyphomicrobiaceae group 1

6. Rhodobacteraceae group

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7. Rhodospirillaceae group

8. Hyphomicrobiaceae group 2

9. Rhizobiales group

10. Hyphomonas group

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11. Phyllobacteriaceae group

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12. Sphingomonas group

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Fig. 9.

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Fig. 9. Neighbour Joining tree showing the sequenced DGGE band (bold) and reference sequences from GenBank. The phylogenetic taxa indicated on the right hand side of the tree are based on the phylogenetic affiliation of the reference sequences. The tree was bootstrapped a 100 times, and values are shown on the nodes (100% values are not shown). The bar represents 0.1 substitutions per nucleotide position. Archaeoglobus fulgidus was used as outgroup. Sequences marked with asterisks are retrieved from the inoculums.

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Microorganisms in seafloor basalts Group 1B0 1B1 1B2 1B3 1B4 2B0 2B1 2B2 2B3 2B4 3B0 3B1 3B2 3B3 3B4 4B0 4B1 4B2 4B3 4B4 5B0 5B1 5B2 5B3 5B4 1. Clostridiales 2. Halomonas 3. Alteromonadaceae 4. Gamma proteobacteria group 5. Hyphomicrobiaceae group 1 6. Rhodobacteraceae 7. Rhodospirillaceae 8. Hyphomicrobiaceae group 2 1 9. Rhizobiales group 10. Hyphomonas 11. Phyllobacteriaceae 1 12. Sphingomonas

Fig. 10. Presence of phylogenetic groups during the experiment in the five different microcosms. Black squares means that at least one sequence from that sample group into the specified phylogenetic group as established in Fig. 9. The sample names are constructed with microcosm name followed by sampling number. i.e. 1B2 means second sampling (6 months of incubation) of microcosms 1B.

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