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

BIODEEP-WP4

University of EssexUniversity of Essex

Andrea Sass , Terry McGenity, Boyd McKew

cultivation experiments with a suite of oxic and anoxic media

variations in: Salt concentration Oxygen regime Organic substrate, electron acceptors

identification of isolates

Samples

Medium DI DB DS AI AB AS UI UB US BI BB BS

SW 0 0 13 0 0 19 0 0 10 14 0 19

12% salt 1 0 3 0 0 10 0 2 4 11 0 11

24% salt 0 0 0 0 0 0 0 0 0 0 0 0

total 1 0 16 0 0 29 0 2 14 25 0 30

total

75

39

0

Number of isolates on agar plates

Summary of previously presented results:

Oxic media

AI BI DI AS BS DS US DB total

Vibrio sp. 3 1 4 Halomonas sp. 1 1 2 Pseudoalteromonas sp. 2 1 1 4 1 9 Alcanivorax sp. 1 1 Marinobacter sp. 1 1 2 Chromohalobacter sp. 1 1 Aeromarinobacter sp. 1 3 4 Thiobacillus sp. 2 2 Pseudomonas sp. 1 2 3 -Proteobacterium 1 1 Bacillus sp. 2 3 3 1 9

total 6 6 3 6 8 1 6 1

Isolates originating from liquid oxic enrichment cultures

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)

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

0.1

47083AS6

BS6NRRLB14851 (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)US7BS29LMG20241 (mural painting)AS28

B. pumilusBS3

YY (Paper mill)AS7

MB9 (marine sediments)US13AS2AS3

BS18Virgib. pantothenticus2-9-3 (salt crystal, 293– 250 million yr old)

Gracilib. halotoleransMarinococcus halophilus

BS17Halob. salinus (salt lake)

Halob. litoralisHalob. trueperiUS16

B. halophilusMarinococcus albus

Halob. halophilusHalob. (salt lake)

OS-5 (coastal marsh)AS5

KSM-S237 (alkaliphilic)DS16BS25

DSM 8724 (alkaliphilic)B. alcalophilus

MN-003 (oil contaminated marine sediments)DS1

AS12US2B. licheniformisUS4B. licheniformis

DSK25 (deep-sea)US1G200-T16 (Glacial ice)KSM-KP43 (alkaliphilic)

G200-N5 (glacial ice, 200 yr old)DS10 2

1

1

6

1

1

1

Jukes-CantorFitch-Margoliash

Halobacillus, GracilibacillusVirgibacillus, Salibacillus

RNA-group

AI BI UI AS BS US DS

Total 6 25 3 31 15 2 1

Strictly anaerobic 5 5 3 24 4 1 1

Extremely halophilic 6 2 3 9 0 0 0

Marine 0 23 0 22 15 2 1

Anoxic media

Number of isolates from anoxic enrichments

Summary of previuosly presented results:

AI BI UI AS BS DS total

Halobacteroides lacunaris (99%) 5 5 10

Halanaerobium sp. (2 types) 2 3 5

Orenia sp. (94%) 1 1

Sulfospirillum sp. (89%) 1 1

CFB-group (89%) 2 2

Clostridium sp. (6 types) 12 4 16

Methanohalophilus mahii (99%) 4 4

total 6 5 3 21 4 1

Affiliation of anaerobic isolates

Clostridium barkeri (XV)Clostridium propionicum DSM 1682T (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-AS17AN-AS4C

AN-AS3BAN-BS1CAN-AS6E

AN-AS6C

AN-AS8AN-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

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 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

10 m

10 m

A B

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

0.1Maximum likelihood

54RAtalante-A

Atalante B2 - 12

Atalante B2 - 10

T36T31

clMT17T42AN-AI3

Methanospirillum hungatei

Halogeometricum borinquense

Halobaculum gomorrense

Halococcus salifodinaeHalococcus 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 AHalosimplex 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

L’Atalante Basin

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

Interfaces Sediments

Halobacteriaceae (Archaea) 1 1

Methanosarcinaceae (Archaea) - 4

Halanaerobiaceae (Low G+C Gram-positives) 10 6

Clostridiaceae (Low G+C Gram-positives) - 16

Bacillaceae (Low G+C Gram-positives) 3 100

Lactobacilleae (Low G+C Gram-positives) - 1

Actinomycetales (High G+C Gram-positives) - 4

Alteromonadaceae-Proteobacteria) 44 11

Vibrionaceae-Proteobacteria) 5 1

Halomonadaceae-Proteobacteria) 2 5

Pseudomonaceae-Proteobacteria) 1 6

Chromatiaceae (-Proteobacteria) 3 -

Enterobacteriaceae-Proteobacteria) 2 -

Rhodobacteraceae-Proteobacteria) 2 -

Campylobacteraceae (-Proteobacteria) 1 -

Rikenellaceae (Bacteroides) 2 -

Isolates with reference to affiliation and environment sampled

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

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:

Bannock interface:High number of isolates, mostly aerobic

L’Atalante 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?