Transcript Nessun titolo diapositiva - ULR CoNISMa di Milano

Partner 1c
CoNISMa, Consorzio Nazionale
Interuniversitario Scienze del Mare
DISTAM
Università degli Studi di Milano
Daniele Daffonchio
Tullio Brusa
Alessandro Favini
Sara Borin
Lorenzo Brusetti
Enrica Canzi
Claudia Sorlini
WP4
“Determination of the distribution, taxonomy and diversity of microorganisms from DHABs, and isolation of strains with biotechnological
potential”
1st year objectives:
 optimisation of cultivation protocols
 selection and isolation of microbial pure cultures from DHABs samples
 molecular characterization of isolated strains
 strains phylogenetic characterization
Culture media
set up
Heterotrophic
bacteria
Oligotrophic
bacteria
Halophiles or
halotolerant
Bacteria
Oil-degrading
bacteria
Medium
Sea Water medium (246 DSM)
%NaCl Main substrates
3
Beef extract 10g
Peptone 10g
Marine Broth (Difco 2216)
3
Peptone 1g
yeast extract 1g
Sea Water medium (246 DSM) 1:10
3
Beef extract 5g
diluted
Peptone 1g
Marine Broth (Difco 2216) 1:10 diluted
3
Peptone 0.5g
yeast extract 0.1g
Akagi medium
3
Polypepton 10mg
proteose pepton 5mg
soyton 5 mg
yeast extract 5mg
Akagi medium 1:10 diluted
3
Polypepton 1mg
proteose pepton 0.5mg
soyton 0.5mg
yeast extract 0.5mg
Halobacteria Medium (372 DSM)
20
Casaminoacids 5g
yeast extract 5g
Halobacterium Medium (97 DSM)
25
Casaminoacids 7.5g
yeast extract 10g
Mixed aged brines with yeast
Yeast extract 1g
Aged sea water with crude oil
3
Crude oil 1g
Crude oil alone
Crude oil 1g
Mixed cultures obtained from DHABs inoculation on board
OXIC Growth
Brine
diluted
Marine Broth
Marine Broth
diluted
Halobacteria medium
DSM 97
Halobacterium medium
Sea water + oil
Brine + oil
Brine + yeast
Total
A
A
U
U
B
B
D
D
A
A
U
U
B
B
D
D
Sediment
A U B
A U B
A
A
D
B
U
D
D
U
B
D
D
B
D
D
A
U
U
D
8
U
U
oligotrophic
Akagi
Akagi diluted
DSM372
D
D
Brine-Sediment
D A U B
D A U B
U
B
U
U
4
5
6
2
6
5
7
Heterotrophic
oxic
Medium
Water-Brine
Sea Water medium D A U B
Sea Water medium D A U B
Oil degraders
5
2
4
7
3
ANOXIC Growth
diluted
Marine Broth
Marine Broth
diluted
D
D
U
Brine
Brine-Sediment
D A U B
D A U B
D
D
A
A
U
U
B
B
Sediment
A U B
A U B
A
A
U
U
B
B
oligotrophic
Akagi
Akagi diluted
DSM372
Halobacteria medium
DSM 97
Halobacterium medium
Sea water + oil
Brine + oil
Brine + yeast
Total
Heterotrophic
anoxic
Medium
Water-Brine
Sea Water medium D A
B
Sea Water medium D
B
Oil degraders
4
1
1
2
4
4
4
4
4
4
4
Pure strains isolated from mixed cultures
OXIC Growth
diluted
Marine Broth
Marine Broth
diluted
Halobacteria medium
DSM 97
Halobacterium medium
Sea water + oil
Brine + oil
Brine + yeast
Total
2
3
2
2
3
2
Brine
5
4
1
1
Sediment
2 4 2
3 4 2
3
2
2
5
1
1
Brine-Sediment
3 1 6 2
1
1
3
1
2
1
1
1
Oil degraders
14
9
9
11
8
5
10
5
3
5
9
ANOXIC Growth
Water-Brine
2
Brine
Brine-Sediment
2 1
Sediment
2
diluted
Marine Broth
Marine Broth
1
diluted
Akagi
Akagi diluted
DSM372
anoxic
Total
Ox Anox
D Discovery 25 5
Halobacteria medium
DSM 97
Halobacterium medium
Sea water + oil
Brine + oil
Brine + yeast
Total
6
Heterotrophic
Medium
Sea Water medium
Sea Water medium
oligotrophic
Akagi
Akagi diluted
DSM372
Water-Brine
3
3 3
5
1
oxic
Heterotrophic
Medium
Sea Water medium
Sea Water medium
3
2
1
2
A Atalante
19 3
U Urania
28
B Bannok
22
Pure strains isolated from mixed cultures
Oxic Isolates
16
14
oxic
interfaces
brines
sediments
12
Discovery
Atalante
8
Urania
Bannok
6
4
Anoxic Isolates
2
3
interfaces
0
WB
B
BS
S
brines
sites
sediments
2
Discovery
Atalante
number
number
10
Urania
Bannok
1
anoxic
0
WB
B
BS
S
sites
Cell morphology typing
16A L’Atalante
13B Bannock
1D Discovery
18A L’Atalante
24A L’Atalante
1D Discovery
10D Discovery
Molecular typing of isolated strains
Isolated
strains













ITS-PCR
species/subspecies
specific
16S rDNA sequencing
identification
Group 1
Halobacillus
Group 2
Halomonas
Group 3
Alteromonas
Group 4
nnn
Group 5
nnn
Group n
nnn
Rep-PCR
interspecies
diversity
characterisation
Molecular typing of isolates based on
16S-23S rDNA Intergenic Transcribed Spacer PCR
tDNA
23S rDNA
16S rDNA
ITS-HHP
Polymorphisms in
Number
length
sequence
Heteroduplex bands
Homoduplex bands
5S
Molecular typing of isolates based on
16S-23S rDNA Intergenic Transcribed Spacer PCR
L’Atalante
Bannock
Discovery
Urania
n° strains
7
17
20
25
ITS groups
1, 3, 4, 5, 6
1, 2, 14, 15, 22, 23, 24, 38, 39, 40, 41, 42
4, 18, 19, 20, 21, 25, 33, 34, 35, 36, 37
1, 4, 7, 9, 10, 11, 12, 13, 14, 16, 17, 26, 27,
28, 29, 30, 31, 32
42 ITS groups
3 groups found in different basins
23 Groups with only 1 isolate
Strains identification
1 strain for each ITS group will be identified by comparing its 16S rDNA
sequence with sequences databases
Strain
6A
12A
13D
11D
6D
17D
4B
Basin
L’Atalante
L’Atalante
Discovery
Discovery
Discovery
Discovery
Bannock
Sample
WB
S
BS
BS
WB
WB
WB
ITS group
Closest relative
6
Alteromonas macleodii
3
Bacillus cohnii
4
Halobacillus trueperi
20
Bacillus firmus
25
Halomonas cupida
n.d.
Chromohalobacter israelensis
n.d.
Pseudoalteromonas ruthenica
N° bases % of homology
397
91%
401
98%
385
99%
450
99%
430
96%
484
98%
414
93%
6D Halomonas cupida
11D Bacillus firmus
12A Bacillus cohnii
13D Halobacillus trueperi
4B Pseudoalteromonas ruthenica