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1. Sampling: Sampl es Retrieval

Culturing the DHABs bacteria: the most suitable techniques for DHABs samples retrieval and treatment. 1. Sampling: Sampl es Retrieval. High Pressure (HP). vs. Atmospheric pressure (AP). HPSS. High pressure maintenance system.

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1. Sampling: Sampl es Retrieval

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  1. Culturing the DHABs bacteria: the most suitable techniques for DHABs samples retrieval and treatment

  2. 1. Sampling: SamplesRetrieval High Pressure (HP) vs Atmospheric pressure (AP) HPSS High pressure maintenance system • The bacterial HP metabolic rates were often higherthan their counterpart measured at the atmospheric pressure conditions • The samples incubation at HP conditions could yield more representative DHABs bacterial communities

  3. 2. Sampling: samples transferring Direct isolation on solid media From Niskin to: Aerobic culture Enrichment AEROBIOSIS

  4. 2. Sampling: samples transferring Widdel anaerobic tranfer of water on board 2001 From Niskin to: Under Nitrogen flux Hungate tubes with different liquid media (on board) Glove box for solid media (on board) ANAEROBIOSIS

  5. 2. Sampling: samples transferring From Niskin to: 2002 Under Nitrogen flux Hungate tubes with different liquid media (on board) Glove box for solid media (on board) ANAEROBIOSIS

  6. To Widdel or not to Widdel?…. Samples from body brines have yieldedstrictanaerobes both with (2001) and without (2002) the use of Widdel: NO NECESSARYfor Body brines (highly reduced) No attempt have been made to isolate from Interface without the use of Widdel, but with the Widdel Interfaces Have also yielded strict anaerobes: POSSIBLY USEFUL for Interface (less reduced layer) Strict anaerobes Widdel NO Widdel ?

  7. PhyloGroup vs Media Media Phylog.

  8. Sampling in Oxic/Anoxic Conditions

  9. 3. Sample treatment: Medium efficiency 2001 - Interface

  10. T1 (Archaea) T2,T4(Haloanaerobiales) e-T17 g-T7(Halomonadaceae) g-T8-(Pseudomonadaceae) a-T14 • CPS-SW a-T14 • DSMZ-193 • P1 • HS-GT T11 (CFB) g-T7(Halomonadaceae) g-T8-(Pseudomonadaceae) Interface - Anoxic Media

  11. 3. Sample treatment: Medium efficiency 2001 - Interface

  12. Interface - Oxic Media T10 (Halothiobacillus) a-T14 g-T8-(Pseudomonadaceae) T22-Staphylococcus T25-Bacilli • B • CPS-SW g-T7(Halomonadaceae) g-T8-(Pseudomonadaceae)

  13. 3. Sample treatment: Medium efficiency BodyBrine

  14. BodyBrine - Anoxic Media DSMZ.193 Bacilli g-T7 Haloanaerobiales

  15. BodyBrine - Oxic Media Bacilli g-T8 g-T8 g-T7

  16. 3. Sample treatment: Medium efficiency Sediment

  17. Sediment - Anoxic Media T2 Haloanaerobiaceae T15,T26-Clostridium T19,T20,T21,T25,T8-Firmicutes T8-g (Pseudomonadaceae) T12,T15,T16,T17 Firmicutes 18 Sulfurospirillum sp. T3 Orenia marismortui T21,T25 Firmicutes T12,T25 Firmicutes Archaea T21,T25 Firmicutes DSMZ.193 T5 T12 Firmicutes

  18. 3. Sample treatment: Medium efficiency Sediment

  19. Sediment - Oxic Media T1 Archaea – Metanosarcinales Equivalent T13 a-Rhodobacteriales • -Proteobacteria Firmicutes

  20. Conclusions: Sampling & preliminary samples treatment • The High Pressure should be maintained from samples retrieval to the strains culturing steps (up to the end of the incubation of bacterial cultures) • 2) The use of Widdel is not necessary for maintaining anoxia in Body brine samples during their transferring onboard. It could be essential for Interface samples (less reduced source samples) • 3) The first enrichment-step must be made in anoxia, the presence of O2 causing blooms of Firmicutes (spore forming), and a loss of the DHABs-adapted bacteria

  21. Conclusions: Selection of culture media • According to the nMDS comparison of the used media (variables: diversity and dominance of the selected bacterial populations) several media are equivalent (Eq) • Thebest Eq medium yields the wider range of phylogroups (higher diversity indexes, lower dominance) • Some specific media for targeted phylogroups (Archaea, e-Proteobacteria) can not be substituted and must complement the Eq optimum one. • The detected best Eq medium and specific media are universal for all DHABs layers

  22. Conclusions: Suggested media ? What is the most important parameter that influences the selectivity of the media (substrate, NaCl conc…) ¿

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