Transcript Kein Folientitel - ULR CoNISMa di Milano

University of Essex
BIODEEP-WP4
Determination of the distribution, taxonomy and diversity of microorganisms from DHABs, and isolation of strains with
biotechnological potential
BIODEEP-WP5
Understanding of ecological relations between the microbial
communities and the functioning of DHAB environments
Andrea Sass , Terry McGenity, Boyd McKew
University of Essex
Objectives:
1. Isolation of bacteria from different physiological groups
Alterations:
 Salt concentration
 Oxygen regime
 Organic substrate, electron acceptors (“food”)
Halophilic, halotolerant, marine organisms
Aerobic and anaerobic
degraders of different classes of organic compounds
University of Essex
after strain isolation:
 DNA extraction
 Amplification of 16S rRNA gene
 Screening through RFLP
 Sequencing
 t-RFLP fingerprint of representative strains
 Physiological characterization of selected strains
University of Essex
Major properties of media
Ions etc.
B
Bh
D
Hl
Mp
MM
MM
CHM SW
10%
Cl- (mM)
3830
1915
6840
3520
2605
378
1750
3610
590
Na+ (mM)
3300
1665
50
3440
2580
342
1710
3625
518
Mg2+ (mM)
300
150
3470
10
25
15
15
81
49
SO42- (mM)
100
50
71
0
12
10
10
81
28
K+ (mM)
100
50
15
54
5
4
4
27
10
Ca+ (mM)
15
8
2
1
1
1
1
0
10
Salt content (gl-1) 236
119
348
207
155
23
103
212
35
pH
7.0
5.5
7.0
7.0
7.0
7.0
7.6
7.8
7.0
University of Essex
1. Results from isolation on agar plates under oxic conditions
Number of isolates on agar plates
Samples
total
Medium
DI
DB
DS
AI
AB
AS
UI
UB
US
BI
BB
BS
CPS-SW
0
0
8
0
0
9
0
0
6
7
0
9
39
multi-SW
0
0
5
0
0
10
0
0
4
7
0
10
36
CPS-Bh
1
0
3
0
0
6
0
2
1
8
0
7
25
multi-Bh
1
0
0
0
0
4
0
0
3
3
0
4
14
CPS-B
0
0
0
0
0
0
0
0
0
0
0
0
0
multi-B
0
0
0
0
0
0
0
0
0
0
0
0
0
CHM
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
University of Essex
Affiliation of isolates from agar plates:
 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 the Bannock interface are
related to organisms commonly found in marine
plankton
Affiliation of aerobic strains from l‘Atalante sediment
Strain
Examples for clostest relatives
Similarity*
AS1, AS5, AS24, AS25, AS26,
Bacillus sp. from salt marsh sediment
100%
AS2, AS4,
Bacillus subtilis
90%
AS3
Bacillus sp. from intertidal sediment,
88%, 88%
AS29
Halobacillus litoralis
AS6, AS11, AS17, AS21
“Bacillus barbaricus” (isolated from wall
100%
painting)
AS7
marine Bacillus sp., Bacillus pumilus
95%, 95%
AS8A
“Bacillus naphtovorans”, B. aquaemaris”
98%, 98%
AS8B, AS13
Bacillus. sp. from glacial ice, Jeotgalibacillus 98%, 95%
alimentarius
AS9
Bacillus. sp from glacial ice, B. horikoshii
100%, 98%
AS10
Bacillus marinus
97%
AS12
Bacillus subtilis
94%
AS15, AS27
isolate from mural painting, “B. aquaemaris”
100%, 99%
AS16
Bacillus subtilis
99%
AS18
Bacillus sp. from intertidal sediment
99%
AS19
“Bacillus naphtovorans”,
98%
AS20
Bacillus sp. from intertidal sediment
99%
AS22
Bacillus sp. from intertidal sediment
90%
AS23
Halomonas aquamarina
100%
AS28
Bacillus niacini
99%
* based on an approximately 500 bp long partial sequence
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Total number of
isolates
l‘Atalante
Number and affiliation of isolates not related to
Bacillus sp.
29
1 (Halomonas sp.)
30
4 (Pseudomonas sp.)
16
1 (Halomonas sp.)
14
3 (2 Halomonas sp., 1 Pseudoalteromonas sp.)
25
24 (15 Alteromonas sp., 7 Marinobacter sp.,
sediment
Bannock
sediment
Discovery
sediment
Urania
sediment
Bannock
interface
1 Pseudoalteromonas sp., 1 Thiobacillus sp.)
affiliation of Isolates from Urania brine: Staphylococcus sp. from deep-subsurface
from Discovery interface: Bacillus sp.
University of Essex
Relevance of aerobic isolates from agar
plates for the DHAB microbial community
 Isolates from sediments possibly derived from resting cells
 Isolates from interfaces possibly marine bacteria derived from
the oxic water column
 Investigation of possible activity of Bacillus sp. in situ still in
progress
University of Essex
other enrichment approaches under oxic conditions:
enrichment in liquid oxic medium under varying conditions
(salt, organic substrate)
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
 only one isolate from high salt medium
1
1
6
1
total
4
2
9
1
2
1
4
2
3
1
9
University of Essex
approaches with samples from cruise in 2002:
tests with eight aerobic liquid media with high salt concentration
(approx. 20%) and variations in buffer, pH, organic substrate
preliminary results:
four cultures on agar plates
(likely to be extremely halotolerant only)
University of Essex
2. Results from isolation from liquid medium under anoxic conditions
Number of positive anoxic enrichments cultures
(with respect to salt regime and sample)
Samples
DB
0
DS
0
AI
1
AB
0
AS
4
UI
0
UB
0
US
0
BI
1
BB
0
BS
0
total
B
DI
0
Bh
0
0
1
0
0
4
0
0
1
2
0
3
11
D
0
0
0
—
—
—
—
—
—
—
—
—
0
direct
0
0
—
1
0
—
3
0
—
3
0
—
7
Hl
—
—
0
1
—
2
0
—
0
1
—
1
5
Mp
—
—
0
0
0
2
—
0
2
0
0
2
6
MM
—
—
0
—
—
2
—
—
1
—
—
2
5
MM10%
—
—
0
0
0
4
—
0
0
2
0
3
9
total
0
0
1
3
0
18
3
0
5
9
0
11
Medium
6
amendment
University of Essex
Number of positive enrichments cultures
(with respect to organic substrate)
Organic substrate
Rumen extract
Polymers
Organic acids
Broth
H.l.
Fatty acids
Ethanol, lactate
Sugars
Lactate
TMA, formate
Formate, acetate
Monomers
Electron acceptor
Sulfate
Sulfate
Sulfate
Sulfate
-Sulfate
Sulfate
Sulfate
Sulfate
--DMSO
Positive
enrichments
2
4
4
12
6
2
4
2
5
6
2
3
University of Essex
Number of anaerobic and extremely halophilic isolates
AI
BI
UI
AS
BS
US
DS
Total
6
25
3
31
15
2
1
Strictly anaerobic
6
5
3
24
4
1
1
Extremely halophilic
6
2
3
9
0
0
0
Marine
0
23
0
22
15
2
1
University of Essex
Affiliation of anaerobic isolates
AI
Halobacteroides lacunaris (99%)
BI
UI
AS
BS
DS
10
5
5
total
Halanaerobium sp. KT-2/3-3 (Kebris
deep (99%), Halanaerobium
2
2
saccharolytica (97%)
Halanaerobium saccharolytica (97%)
3
3
Orenia sp. (94%)
1
1
Sulfospirillum sp. (89%)
1
1
CFB-group (89%)
2
2
Clostridium sp. (6 types)
12
Methanohalophilus mahii (99%)
4
Halorhabdus utahensis (99%)
1
total
6
16
4
4
1
5
3
21
4
1
40
University of Essex
Summary:
 Strains related to anaerobic extremely
halophiles from every basin
 Different anaerobic halophiles obtained
from every basin
 From sediments predominantly marine
anaerobic sporeformers were obtained
 Most strains from l‘Atalante basin
 Archaebacteria from l‘Atalante interface
and sediment
 No SRB
University of Essex
Physiological properties of anerobic isolates
 Halanaerobiales : all except one (from DS) extremely halophilic
 CFB-group: grow only anaerobically
 Clostridia: all exept one (aerobic) grow in anoxic seawater
 Halorhabdus sp.: grows only (yet) anaerobically
University of Essex
Affiliation of aerobic isolates from anoxic enrichment cultures
AI
Marinobacter hydrocarbonoclasticus
BI
UI
AS
BS
DS
total
13
13
Vibrio sp.
2
2
Idiomarina sp.
1
1
Alteromonas sp.
1
1
-Proteobacterium
1
1
Enterobacteriaceae
2
2
(99%)
Halobacillus trueperi (95%)
5
Bacillus sp.
Actinomyces
1
Lactobacilliales
total
0
20
0
6
1
6
7
7
2
3
1
1
11
0
37
Summary:
 Aerobic marine organisms (out of anoxic
enrichments) were isolated from BI, AS and BS
 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
University of Essex
Relevance of anaerobic cultures for the
DHAB microbial community
Many isolates (Halanaerobiaceae,
Methanohalophilus sp., Halorhabdus sp.,
Halobacillus sp.) grow readily under conditions
similar to those in the DHAB
Possibly active in the DHAB
(t-RFLP signature of Halanaerobiaceae
could not be found in profiles from brines)
University of Essex
approaches with samples from cruise in 2002:
more enrichments for SRB:
 Medium with two different salt
concentrations (24%, 15%)
 Electron donors: alcohols, fatty acids,
benzoic acid
 Higher volume and more inoculum
 In liquid culture and in „gradient tubes“
preliminary results: no growth in enrichments
media or gradient tubes yet
Acknowledgements:
Boyd McKew