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
variations in:
 Salt concentration
 Oxygen regime
 Organic substrate, electron acceptors
 identification of isolates
Oxic media
Summary of previously presented results:
Number of isolates on agar plates
Samples
Medium
DI
DB
DS
AI
AB
AS
UI
UB
US
BI
BB
BS
total
SW
0
0
13
0
0
19
0
0
10
14
0
19
75
12% salt
1
0
3
0
0
10
0
2
4
11
0
11
39
24% salt
0
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
Isolates originating from liquid oxic enrichment cultures
AI
Vibrio sp.
Halomonas sp.
Pseudoalteromonas sp.
Alcanivorax sp.
Marinobacter sp.
Chromohalobacter sp.
Aeromarinobacter sp.
Thiobacillus sp.
Pseudomonas sp.
-Proteobacterium
Bacillus sp.
3
total
6
BI
1
2
DI
AS
BS
DS
US
DB
1
1
1
1
4
1
1
1
1
1
1
3
2
1
2
6
2
1
3
3
3
6
8
1
1
6
1
total
4
2
9
1
2
1
4
2
3
1
9
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
47083
RNA-group
AS6
BS6
NRRLB14851 (Marine)
HTA437 (Mariana Trench)
BS12
G550K (glacial Ice, 500,000 yr old)
MK03
KASA34 (alkalaphilic, cold desert)
B.macroides
1
DS9
PL16 (marine sediments)
DS5
MB-5 (marine sediments)
YKJ-10 (fermented seafood)
US7
BS29
1
LMG20241 (mural painting)
AS28
B. pumilus
BS3
1
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
Halobacillus, Gracilibacillus
Virgibacillus, Salibacillus
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
6
1
US4
Jukes-Cantor
Fitch-Margoliash
B. licheniformis
DSK25 (deep-sea)
US1
G200-T16 (Glacial ice)
KSM-KP43 (alkaliphilic)
G200-N5 (glacial ice, 200 yr old)
0.1
DS10
1
2
Anoxic media
Summary of previuosly presented results:
Number of isolates from anoxic enrichments
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
Affiliation of anaerobic isolates
AI
Halobacteroides lacunaris (99%)
BI
UI
5
Halanaerobium sp. (2 types)
AS
BS
DS
5
2
total
10
3
5
Orenia sp. (94%)
1
1
Sulfospirillum sp. (89%)
1
1
CFB-group (89%)
2
2
Clostridium sp. (6 types)
12
Methanohalophilus mahii (99%)
4
total
6
5
3
21
4
16
4
4
1
Clostridium barkeri
Clostridium propionicum DSM 1682T
Clostridium oroticum
Clostridium aerotolerans
Clostridium indolis
(XV)
(XIVb)
(XIVa)
(XIVa)
(XIVa)
AN-AS6C
AN-US3
AN-AS8
Peptostreptococcus micros
Sporoanaerobacter acetigenes
Clostridium acidiurici
(XIII)
(XII)
(XII)
AN-AS6E
AN-BS1C
AN-AS3B
Thermohalobacter berrensis
Clostridium paradoxum
‘Tepidibacter thalassicus’
Clostridium sp. FL4
(XII)
(XI)
AN-AS4C
AN-AS17
AN-AS4B
Clostridium oceanicum
Clostridium proteolyticum
Clostridium pfennigii
Clostridium cellulolyticum
Clostridium sporosphaeroides
Clostridium thermoautotrophicum
Thermoanaerobacterium thermosulfurigenes
Thermoanaerobacter ethanolicus
Sporomusa sphaeroides
Bacillus subtilis
Clostridium spiroforme
Actinomyces bovis
0.1
Maximum likelihood
(I)
(II)
(III)
(IV)
(VI)
(VII)
(V)
(IX)
(XVIII)
Sporocytophaga myxococcoides
Porphyromoas asaccharolytica
Bacteroides fragilis
Flexibacter aggregans ssp. catalaticus
Cytophaga marinoflava
Flavobacterium aquatile
Tenacibaculum maritimum
Chryseobacterium gleum
Empedobacter brevis
Rikenella microfusus
Anaerophaga thermohalophila
Marinilabilia salmonicolor
Cytophaga fermentans
CFB-Group
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
Sulfurospirillum sp. strain 18.1
Sulfurospirillum deleyianum
Sulfurospirillum barnesii
Chlorobium limicola
0.1
Maximum likelihood
e-Proteobacteria
A
B
10 mm
10 mm
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
Haloferax volcanii
Halorubrum sodomense
Halorubrum vacuolatum
Halorubrum saccharovorum
54R
Methanospirillum hungatei
Atalante-A
Halogeometricum borinquense
Natronobacterium gregoryi
Natrialba asiatica
Natrialba magadii
Natronorubrum bangense
Haloterrigena thermotolerans
Natrinema pellirubrum
Halobiforma haloterrestris
Natronococcus occultus
Halobacterium salinarum
Halococcus salifodinae
Halococcus morrhuae
Natronomonas pharaonis
Halobaculum gomorrense
Halomicrobium mukohataei
Haloarchaea have been
isolated from
L’Atalante Basin
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
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
Isolates with reference to affiliation and environment sampled
Halobacteriaceae (Archaea)
Methanosarcinaceae (Archaea)
Halanaerobiaceae (Low G+C Gram-positives)
Clostridiaceae (Low G+C Gram-positives)
Bacillaceae (Low G+C Gram-positives)
Lactobacilleae (Low G+C Gram-positives)
Actinomycetales (High G+C Gram-positives)
Alteromonadaceae-Proteobacteria)
Vibrionaceae-Proteobacteria)
Halomonadaceae-Proteobacteria)
Pseudomonaceae-Proteobacteria)
Chromatiaceae (-Proteobacteria)
Enterobacteriaceae-Proteobacteria)
Rhodobacteraceae-Proteobacteria)
Campylobacteraceae (e-Proteobacteria)
Rikenellaceae (Bacteroides)
Interfaces
Sediments
1
10
3
44
5
2
1
3
2
2
1
2
1
4
6
16
100
1
4
11
1
5
6
-
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
Aerobic isolates from anoxic enrichments:
 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
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