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Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with biotechnological potential BIODEEP-WP4.

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Presentation on theme: "Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with biotechnological potential BIODEEP-WP4."— Presentation transcript:

1 Determination of the distribution, taxonomy and diversity of micro-organisms from DHABs, and isolation of strains with biotechnological potential BIODEEP-WP4 University of Essex 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 Number of isolates on agar plates Summary of previously presented results: Oxic media

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) -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 0.1 47083 AS6 BS6 NRRLB14851 (Marine) HTA437 (Mariana Trench) BS12 G550K (glacial Ice, 500,000 yr old) MK03 KASA34 (alkalaphilic, cold desert) B.macroides DS9 PL16 (marine sediments) DS5 MB-5 (marine sediments) YKJ-10 (fermented seafood) US7 BS29 LMG20241 (mural painting) AS28 B. pumilus BS3 YY (Paper mill) AS7 MB9 (marine sediments) US13 AS2 AS3 BS18 Virgib. pantothenticus 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 BS25 DSM 8724 (alkaliphilic) B. alcalophilus MN-003 (oil contaminated marine sediments) DS1 AS12 US2 B. licheniformis US4 B. licheniformis DSK25 (deep-sea) US1 G200-T16 (Glacial ice) KSM-KP43 (alkaliphilic) G200-N5 (glacial ice, 200 yr old) DS10 2 1 1 6 1 1 1 Jukes-Cantor Fitch-Margoliash Halobacillus, Gracilibacillus Virgibacillus, Salibacillus RNA-group

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

9 Affiliation of anaerobic isolates

10 Clostridium barkeri (XV) Clostridium propionicum DSM 1682 T (XIVb) Clostridium oroticum (XIVa) Clostridium aerotolerans (XIVa) Clostridium indolis (XIVa) Peptostreptococcus micros (XIII) Sporoanaerobacter acetigenes (XII) Clostridium acidiurici (XII) Clostridium paradoxum (XI) Clostridium sp. FL4 Clostridium oceanicum (I) AN-AS4B AN-AS17 AN-AS4C AN-AS3B AN-BS1C AN-AS6E AN-AS6C AN-AS8 AN-US3 Clostridium proteolyticum (II) Clostridium pfennigii Clostridium cellulolyticum (III) Clostridium sporosphaeroides (IV) Clostridium thermoautotrophicum (VI) Thermoanaerobacterium thermosulfurigenes (VII) Sporomusa sphaeroides (IX) Thermoanaerobacter ethanolicus (V) Bacillus subtilis Clostridium spiroforme (XVIII) Actinomyces bovis 0.1 Thermohalobacter berrensis (XII) Tepidibacter thalassicus Maximum likelihood

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

12 10 m A B

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

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

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 lAtalante basin Archaebacteria from lAtalante interface and sediment No SRB

18 from BI, AS and BS all moderately halophilic predominantly Gram negative -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 Aerobic isolates from anoxic enrichments:

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

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