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Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with b

University of Essex. BIODEEP-WP4. Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with biotechnological potential. Andrea Sass , Terry McGenity, Boyd McKew . cultivation experiments with a suite of oxic and anoxic media

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Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with b

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  1. University of Essex BIODEEP-WP4 Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with biotechnological potential Andrea Sass , Terry McGenity, Boyd McKew

  2. cultivation experiments with a suite of oxic and anoxic media variations in: • Salt concentration • Oxygen regime • Organic substrate, electron acceptors • identification of isolates

  3. total 75 39 0 Oxic media Summary of previously presented results: Number of isolates on agar plates

  4. Isolates originating from liquid oxic enrichment cultures

  5. new isolates from oxic media (samples cruise 2002): • emphasis on highly saline media, all liquid • growing organisms screened for unique morphology and ability to grow on sea water four strains isolated • Idiomarina sp. (UI, extremely halotolerant, aerotactic) • Bacillus halophilus (BS, halotolerant) • a-Proteobacterium (AS, halotolerant) • Halorhabdus utahensis (AS,extremely halophilic)

  6. Almost all isolates from sediments are related to Bacillus sp. found in marine or other saline environmens or e.g. glacial ice • Almost all isolates from interfaces are related to organisms commonly found in marine plankton

  7. RNA-group 47083 AS6 BS6 NRRLB14851 (Marine) HTA437 (Mariana Trench) 1 BS12 G550K (glacial Ice, 500,000 yr old) MK03 KASA34 (alkalaphilic, cold desert) B.macroides DS9 PL16 (marine sediments) DS5 MB-5 (marine sediments) 1 YKJ-10 (fermented seafood) US7 BS29 LMG20241 (mural painting) AS28 B. pumilus 1 BS3 YY (Paper mill) AS7 MB9 (marine sediments) US13 AS2 AS3 BS18 Virgib. pantothenticus Halobacillus, Gracilibacillus Virgibacillus, Salibacillus 2-9-3 (salt crystal, 293– 250 million yr old) Gracilib. halotolerans Marinococcus halophilus BS17 Halob. salinus (salt lake) Halob. litoralis Halob. trueperi US16 B. halophilus Marinococcus albus Halob. halophilus Halob. (salt lake) OS-5 (coastal marsh) AS5 KSM-S237 (alkaliphilic) DS16 6 BS25 DSM 8724 (alkaliphilic) B. alcalophilus MN-003 (oil contaminated marine sediments) DS1 AS12 1 US2 B. licheniformis US4 Jukes-Cantor Fitch-Margoliash B. licheniformis DSK25 (deep-sea) 1 US1 G200-T16 (Glacial ice) KSM-KP43 (alkaliphilic) 2 G200-N5 (glacial ice, 200 yr old) DS10 0.1

  8. Anoxic media Summary of previuosly presented results: Number of isolates from anoxic enrichments

  9. Affiliation of anaerobic isolates

  10. Clostridium barkeri (XV) Clostridium propionicum DSM 1682T(XIVb) Clostridium oroticum(XIVa) Clostridium aerotolerans(XIVa) Clostridium indolis(XIVa) AN-AS6C AN-US3 AN-AS8 Peptostreptococcus micros (XIII) Sporoanaerobacter acetigenes (XII) Clostridium acidiurici (XII) AN-AS6E AN-BS1C AN-AS3B Thermohalobacter berrensis (XII) Clostridium paradoxum (XI) ‘Tepidibacter thalassicus’ Clostridium sp. FL4 AN-AS4C AN-AS17 AN-AS4B Clostridium oceanicum(I) Clostridium proteolyticum(II) Clostridium pfennigii Clostridium cellulolyticum(III) Clostridium sporosphaeroides (IV) Clostridium thermoautotrophicum(VI) Thermoanaerobacterium thermosulfurigenes (VII) Thermoanaerobacter ethanolicus (V) Sporomusa sphaeroides (IX) Bacillus subtilis Clostridium spiroforme (XVIII) Actinomyces bovis Maximum likelihood 0.1

  11. Sporocytophaga myxococcoides Porphyromoas asaccharolytica Bacteroides fragilis Cytophaga marinoflava Flavobacterium aquatile Tenacibaculum maritimum Chryseobacterium gleum Empedobacter brevis Rikenella microfusus Anaerophaga thermohalophila Marinilabilia salmonicolor Cytophaga fermentans AN-BI4 Uncultured hydrothermal vent bacterium Uncultured hydrothermal vent bacterium Desulfovibrio vulgaris Wolinella succinogenes Helicobacter pylori Thiomicrospira denitrificans Arcobacter nitrofrigilis Campylobacter jejuni AN-BI3A Sulfurospirillum arcachonense Sulfurospirillumsp. strain 18.1 Sulfurospirillum deleyianum Sulfurospirillum barnesii Chlorobium limicola 0.1 Flexibacter aggregans ssp. catalaticus CFB-Group e-Proteobacteria Maximum likelihood

  12. B A 10 mm 10 mm

  13. Halothermothrix orenii Halanaerobium saccharolyticum Halanaerobium congolense AN-UI1A Halanaerobium sp. KT-2/3-3 (Kebrit Deep, Red Sea) AN-BI5B Halanaerobium praevalens Halobacteroides halobius Halanaerobacter chitinivorans Halanaerobacter lacunarum AN-AI1A Orenia salinaria Orenia marismortui AN-DS1 Acetohalobium arabaticum Bacillus subtilis Clostridium butyricum Megasphaera elsdenii Actinomyces bovis 0.1 Maximum likelihood

  14. Methanospirillum hungatei Halobacterium salinarum Natronorubrum bangense Natrialba magadii Natrialba asiatica Natrinema pellirubrum Natronobacterium gregoryi Natronococcus occultus Halococcus salifodinae Halococcus morrhuae Halogeometricum borinquense Haloferax volcanii Halobaculum gomorrense Halorubrum vacuolatum Halorubrum sodomense Halorubrum saccharovorum Natronomonas pharaonis Halomicrobium mukohataei 54R Atalante-A Haloarchaea have been isolated from L’Atalante Basin Haloterrigena thermotolerans Halobiforma haloterrestris Jukes-Cantor distance calculation Fitch-Margoliash treeing algorithm 550 nucleotides of 16S rRNA compared Atalante B2 - 12 Haloarcula marismortui rrnA ‘Haloarcula sinaiiensis’ minor ‘Haloarcula sinaiiensis’ major Haloarcula marismortui rrnB Atalante B2 - 10 Halosimplex carlsbadense ATCC BAA-75 gene A Halosimplex carlsbadense ATCC BAA-75 gene B T36 T31 Halosimplex carlsbadense ATCC BAA-75 gene C clMT17 T42 AN-AI3 Halorhabdus utahensis 10 nuc substitutions per 100 bases

  15. new enrichment approaches with media (samples cruise 2002): • emphasis on: • methane-oxidizing bacteria • sulfate-reducing bacteria • no methane consumption detectable • no growth of sulfate-reducing bacteria • no more anaerobic isolates

  16. Isolates with reference to affiliation and environment sampled

  17. Anaerobic bacteria: • Anaerobic extremely halophilic bacteria from every basin, different from each basin • From sediments predominantly marine anaerobic sporeformers were obtained • Most strains from l‘Atalante basin • Archaebacteria from l‘Atalante interface and sediment • No SRB

  18. Aerobic isolates from anoxic enrichments: • from BI, AS and BS • all moderately halophilic • predominantly Gram negative g-Proteobacteria obtained from BI • predominantly Gram-positive Sporeformers obtained from AS and BS (matches results from isolation on agar plates) • Halobacillus sp. were often isolated anaerobically in high-salt media

  19. Bannock interface: • High number of isolates, mostly aerobic sample taken for cultivation with higher proportion of low salinity (oxic) waters? • L’Atalante and Urania interface: • Fewer isolates, mostly anaerobic samples for cultivation with higher proportion of brine? Quality of interface sample critical for cultivation success

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