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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
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
2. Characterization of isolates
3. Evaluation of the relevance of obtained isolates in situ
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
Isolation under oxic conditions
Isolates
DNA extraction
Amplificatioon of 16S rRNAgene
RFLP
Sequencing
t-RFLP
Physiological
characterization of
representative strains
University of Essex
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
0
0
3
0
0
6
0
0
1
8
0
7
25
multi-Bh
0
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
0
0
16
0
0
29
0
0
14
25
0
30
University of Essex
Preliminary results from partial
sequencing of eight isolates
Strain Closest relatives
Identity Strain
Closest relatives
Identity
AS1
Bacillus clausii
Bacillus subtilis
Bacillus halodurans
95.8 %
93.8 %
92.4 %
BS5
Bacillus niacini
Bacillus aestuarii
Bacillus macroides
98.7 %
98.0 %
96.7 %
AS2
Bacillus subtilis
Bacillus pumilus
Bacillus halodurans
92.2 %
92.2 %
89.0 %
BS6
100 %
Alkaliphilic Bacillus sp.
Bacillus horikoshii
Bacillus halodurans
99.1 %
97.8 %
91.9 %
BS12
Marine Bacillus sp.
Bacillus horikoshii
Bacillus megaterium
99.7 %
95.5 %
94.8 %
BS13
Bacillus sp. from
bacterioplankton
Bacillus niacini
Bacillus megaterium
Bacillus sp. from wall
painting
Uncultured soil bacterium
Bacillus macroides
Bacillus licheniformis
Bacillus subtilis
Bacillus halodurans
AS9
AS11
96.0 %
95.0 %
98.0 %
98.0 %
96.7 %
98.7 %
98.1 %
97.7 %
University of Essex
Physiological properties of aerobic strains
The appearance of isolates from the sediments is different
from those of the interface:
most of the strains from sediment samples were sporeforming (probably Bacillus-related)
only a few strains of the Bannock interface were sporeforming
Strains further characterized (Bacillus-like bacteria from
l’Atalante and Bannock basin)
 were capable facultatively anaerobic growth on artificial
seawater with fermentable substrates
 could grow in liquid media to salt concentrations of up to
15%
University of Essex
Relevance of aerobic isolates 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
Isolated organisms unlikely to be very active in situ
University of Essex
Future work:
Further attempts to isolate extremely halophilic aerobic
microorganisms:
To obtain Halobacteria or other true aerobic halophiles the media
constituents (buffer, major salts, organic compounds) and growth
conditions (pH, temperature) can be altered
Further treatment of isolates already obtained:
 Screening by RFLP fingerprinting
 Partial sequencing of isolates representative of a fingerprint
 Physiological characterization of isolates only remotely related to
known organisms
University of Essex
Anoxic enrichments
Media for on board
inoculation
Subsample for t-RFLP
analysis
Direct
amendment of
brine and
interface
samples
Media
inoculated in
the lab
Positive
enrichments
 Further cultivation
 Isolation by deep-agar
dilution series
 Partial sequencing
 Physiological
characterization of
representative strains
University of Essex
Number of positive enrichments cultures
(with respect to salt regime and sample)
Samples
B
DI
0
DB
0
DS
0
AI
2
AB
0
AS
7
UI
0
UB
0
Bh
0
0
1
0
0
5
0
0
D
0
0
0
—
—
—
—
direct
0
1
—
2
0
—
Hl
—
—
0
1
—
Mp
—
—
0
0
MM
—
—
0
MM10%
—
—
total
0
1
Medium
US
0
total
BI
0
BB
0
BS
0
1
2
0
2
—
—
—
—
—
4
0
—
3
1
—
0
11
2
0
—
1
1
—
0
5
0
0
—
0
0
0
0
1
1
—
—
2
—
—
1
—
—
1
4
0
0
0
1
—
0
0
2
0
1
4
1
5
0
17
4
0
3
8
1
5
9
11
amendment
University of Essex
Number of positive enrichments cultures
(with respect to organic substrate)
Organic substrate
Electron acceptor
Rumen extract
Polymers
Organic acids
W-C broth
H.l.
Fatty acids
Ethanol, lactate
Sugars
Lactate
TMA, formate
Formate, acetate
Fatty acids
Sulfate
Sulfate
Sulfate
Sulfate
-Sulfate
Sulfate
Sulfate
Sulfate
--Sulfate
Positive
enrichments
4
3
2
10
5
2
3
4
4
2
4
2
University of Essex
Physiological properties of anerobic cultures
 Positive enrichments predominantly on high-salt
media and on substrates that can be fermented
 Most cultures grow relatively fast on media with a
high salt concentration
University of Essex
Relevance of anaerobic cultures for the
DHAB microbial community
Enrichments grow readily under conditions
similar to those in the DHAB
Possibly active in the DHAB
University of Essex
Future work:
Further isolation attempts:
 for SRB
 enrichment experiments with brines
concentrated through filtration
Further treatment of enrichments already obtained:
 complete isolation procedure
 partial sequencing of 16S rRNA gene
 physiological characterization of representative phylotypes