CNR
IAMC - Istituto per l’Ambiente Marino
Costiero
Messina, Italy
Università degli Studi di Messina
WP4-WP5
State of the Art April 2003
Isolates analysis
Anaerobic chamber
0
SOLID Brine based media
O2
Reisolation by
liquid and solid cultures
O2
ONR Saline Medium
(3%Salinity)
Strain collection
Phylogentical and physiological
charachterisation
No Isolates from BodyBrine
Isolates analysis WP4-WP5
Phylogenetical Analysis
Taxonomic
Diversity
REP Fingerprinting
Aerobic/Anaerobic
growth curves
Methabolic analysis “Bottle Neck”
and others
Conclusions
Physiological
Application
Isolates analysis
Isolated strains
Bulleidia e.
Methanococcus jannacii
Desulfococcus biaticus
Arcobacter butzleril
Alteromonas macleodi
Idiomarina loihensis
Halomonas variabilis
339-27-530
364 365
367-27
369-27
3691492 370
Marinobacter aqueoli
357
351g
348g-530
345
340-27
350
Marinobacter aqueoli
374b
351b
Alteromonas sp.
354
374l
363
Marinobacter sp.
348b
Marinobacter hydrocarbonoclasticus
369
Pseudomonas sp.
Pseudomonas sp.
328
Pseudomonas pseudoalcaligenes
Pseudomonas sp.
353
359
Alcanivorax sp.
358
Alcanivorax Venustit
368
Fundibacter jadensi
Alcanivorax borkumensis
Sulfitobacter pontiacus
0.
Roseobaccter sp.
1
Isolates analysis
Phylogenetical Analysis
Taxonomic
REP Fingerprinting
Aerobic/Anaerobic
growth curves
Methabolic analysis “Bottle Neck”
and others
Conclusions
Diversity
Physiological
Application
Isolates analysis
REP Fingerprinting
Repetitive Extragenic Palyndromic Analysis
M
2
3S
5
1
6
TAACGGCGCTCCACA ………………….TGTGGAGCGCCGTTA
4
GENE
ATTGCCGCGAGGTGT…………………..ACACCTCGCGGCAAT
2
1
6
3
5
4
Isolates analysis
REP Fingerprinting
Isolates analysis
REP Fingerprinting
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacte hydrocarbonoclasticus
Halomonas variabilis
Marinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
Pseudoalteromonas antarctica
Halomonas variabilis
Halomonas variabilis
Halomonas variabilis
Alcanivorax venusti
Alcanivorax sp.
Marinobacter hydrocarbonoclasticus
Pseudomonas pseudoalcaligenes
Marinobacter hydrocarbonoclasticus
Alteromonas sp.
Marinobacter aqueoli
Idiomarina loiensis
Idiomarina loiensis
Marinobacter aqueoli
Isolates analysis
Phylogenetical Analysis
Taxonomic
REP Fingerprinting
Aerobic/Anaerobic
growth curves
Diversity
Physiological
Methabolic analysis “Bottle Neck”
and others
Conclusions
Application
Isolates analysis
Aerobic/Anaerobic growth curves
Bioscreen analysis
Reading at 640nm every 1.30h
For 10days
4 replicate
Aerobic
ONR
+
Pyruvate
Anaerobic
Mineral oil
ONR
+
Pyruvate
Isolates analysis
Aerobic/Anaerobic growth curves
Aerobic
340
345
134h
114h
178h
142h
Anaerobic
363
348
42h
102h
Alteromonas sp.
Marinobacter h.
Idiomarina l.
Marinobacter a.
351
350
354
356
122h
138h
34h
82h
62h
64h
88h
128h
Marinobacter h.
Marinobacter h.
Marinobacter a.
Ten days, four replicate
Idiomarina l.
Isolates analysis
Aerobic/Anaerobic growth curves
Aerobic
358
359
Anaerobic
353
339
108h
50h
116h
85h
Alcanivorax v.
Alcanivorax v.
365
367
Alteromonas sp.
Halomonas v.
364
369
88h
92h
72h
72h
104h
94h
92h
78h
Halomonas v.
Halomonas V.
Halomonas v.
Ten days, four replicate
Halomonas v.
Isolates analysis
Aerobic/Anaerobic growth curves
Aerobic
320
323
Anaerobic
326
328
184h
200h
54h
80h
Idiomarina l.
Marinobacter a.
327
330
124h
116h
182h
166h
Marinobacter h.
Marinobacter a.
Marinobacter h.
333
Pseudomonas p.
Ten days, four replicate
Pseudomonas p.
Isolates analysis
REP Fingerprinting
Marinobacter aqueoli
Marinobacter
339
327 aqueoli
Marinobacter aqueoli
124h hydrocarbonoclasticus
Marinobacte
Halomonas
182hvariabilis
Marinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
Pseudoalteromonas antarctica
50h
Halomonas variabilis
Halomonas variabilis 85h
Halomonas
variabilis v.h.
Marinobacter
Halomonas
Alcanivorax venusti
Alcanivorax sp.
369
345 hydrocarbonoclasticus
Marinobacter
Pseudomonas pseudoalcaligenes
Marinobacter hydrocarbonoclasticus
114h sp.
Alteromonas
Marinobacter
142h aqueoli
Idiomarina loiensis
72h
Idiomarina loiensis
92h
Marinobacter
Halomonas
v. h.
Marinobacter aqueoli
Isolates analysis
Aerobic/Anaerobic growth curves
Problems:
• Preliminary data (not shown) seem to suggest that the
isolates are not able to grow on pure brine.
Future:
•
•
To try the same experiment in aerobic and anaerobic
condition using a salt gradient from SeaWater to pure Brine
To try different substrates
Isolates analysis
Phylogenetical Analysis
Taxonomic
REP Fingerprinting
Aerobic/Anaerobic
growth curves
Diversity
Physiological
Methabolic analysis “Bottle Neck”
and others
Conclusions
Application
Isolates analysis
Methabolic analysis
Why:
• The study of the degradation of aromatic compounds has a very
applicative meaning in order to evaluate the possible employ in
bioremediation
• The major part of dangerous or difficoult to remove compounds are
aromatics
• A great number of polluted areas is associated with hypersaline
environment
Methabolic analysis (Catechol,
Gentisate, Protocatechuic acid)
Isolates analysis
Multiple culture on ONR
+
Aromatic compound
Tested by turbidimetry of growing cells after 4 days
The samples were incubated in anaerobic chamber to
avoid spontaneous oxydation of substrates
Catechol
Oxygen
Gentisate
Oxygen
Isolates analysis
Catechol
Gentisate
Methabolic analysis (Catechol,
Gentisate, Protocatechuic acid)
Isolates analysis
Catechol
Gentisate
Methabolic analysis (Catechol,
Gentisate, Protocatechuic acid)
Isolates analysis
Catechol
Gentisate
Methabolic analysis (Catechol,
Gentisate, Protocatechuic acid)
Isolates analysis
Methabolic analysis (Naphtalene,
Fenantrene, Hexadecane)
Direct cultivation on ONR Agar
Blank in ONR without carbon source
Hexadecane
Methabolic analysis (Catechol,
Gentisate, Protocatechuic acid)
Isolates analysis
#
Catechol Gentisic ac. Protocatech.ac.
368
++
NS
NS
359
++
+++
+
340
+++
NS
+
353
+++
+
NS
339
NS
+
+++
364
+++
NS
NS
365
NS
NS
++
367
+
NS
NS
369
++
NS
+++
320
++
NS
+
348b
NS
++
+
351
+
+++
NS
357
+++
+++
++
323
++
NS
NS
330
++
+
NS
354
++
NS
NS
363
NS
NS
+
370
+
+
NS
326
+++
+
+
327
+++
NS
+++
345
+++
NS
NS
350
+++
+
NS
374
+++
NS
NS
328
+
+
NS
333
++
NS
NS
Species
Alcanivorax sp.
Alcanivorax venusti
Alteromonas sp.
Alteromonas sp.
Halomonas variabilis
Halomonas variabilis
Halomonas variabilis
Halomonas variabilis
Halomonas variabilis
Idiomarina l
Idiomarina l
M arinobacter aqueoli
M arinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
M arinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
Pseudomonas pseudoalcaligenes
Pseudomonas pseudoalcaligenes
Preliminar
data
Isolates analysis
368
358
359
369
340
353
339
364
365
367
320
348b
323
330
354
363
370
357
351b
351g
326
327
350
374
328
333
Phe
+
Naph
+
+
+
+
++
+
+
Hex
++
++
++
+
++
++
+
+
+
++
+
++
+
+
+
++
+
+
+
++
++
++
++
++
++
++
+
++
++
++
+
++
Control
+
Alcanivorax sp.
Alcanivorax venusti
Alcanivorax venusti
+
Halomonas variabilis
Alteromonas sp.
Alteromonas sp.
+
Halomonas variabilis
+
Halomonas variabilis
+
Halomonas variabilis
+
Halomonas variabilis
Idiomarina l
Idiomarina l
+
Marinobacter aqueoli
+
Marinobacter aqueoli
+
Marinobacter aqueoli
Marinobacter aqueoli
+
Marinobacter aqueoli
+
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
+
Marinobacter hydrocarbonoclasticus
+
Marinobacter hydrocarbonoclasticus
+
Marinobacter hydrocarbonoclasticus
+
Marinobacter hydrocarbonoclasticus
+
Pseudomonas pseudoalcaligenes
Pseudomonas pseudoalcaligenes
357
368
358
359
369
340
353
339
364
365
367
320
348b
323
330
354
363
370
348g
351b
351g
326
327
345
350
374
328
333
Cat
+++
++
Gen
+++
NS
Protoc
++
NS
++
++
+++
+++
NS
+++
NS
+
++
NS
++
++
++
NS
+
+++
NS
NS
+
+
NS
NS
NS
NS
++
NS
+
NS
NS
+
+
+++
+
NS
+++
NS
++
NS
+
+
NS
NS
NS
+
NS
+
+++
NS
+++
+++
+++
+++
+++
+
++
+
NS
NS
+
NS
+
NS
+
+++
NS
NS
NS
NS
NS
Phe
+
+
Naph
+
+
+
+
+
++
+
+
Hex
++
++
++
++
+
++
++
+
+
+
++
+
++
+
+
+
++
++
++
++
++
+
++
++
+
++
++
++
+
++
Blank
Species
+
Alcanivorax Sp
+
Alcanivorax sp.
Alcanivorax venusti
Alcanivorax venusti
+
Halomonas variabilis
Alteromonas sp.
Alteromonas sp.
+
Halomonas variabilis
+
Halomonas variabilis
+
Halomonas variabilis
+
Halomonas variabilis
Idiomarina l
Idiomarina l
+
Marinobacter aqueoli
+
Marinobacter aqueoli
+
Marinobacter aqueoli
Marinobacter aqueoli
+
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
Marinobacter aqueoli
+
Marinobacter hydrocarbonoclasticus
+
Marinobacter hydrocarbonoclasticus
Marinobacter hydrocarbonoclasticus
+
Marinobacter hydrocarbonoclasticus
+
Marinobacter hydrocarbonoclasticus
+
Pseudomonas pseudoalcaligenes
Pseudomonas pseudoalcaligenes
Isolates analysis
Methabolic analysis
Future:
• To repeat the experiments in salinity gradient
• To check and charachterize the genes coding for the
enzymes involved in the HC pathways by PCR and
sequencing
• To purify and charachterize the obtained enzymes
Isolates analysis
Conclusions
Phylogenetical Analysis
•The cultivatable components of bacterial communities from DHABs are not
corresponding at all with the active components within the basins
(Allochthnous/contaminant bacteria?)
REP Fingerprinting
Aerobic/Anaerobic growth curves
Methabolic analysis (Catechol, Gentisate, Protocatechuic
acid)
Conclusions
Isolates analysis
Conclusions
Phylogenetical Analysis
REP Fingerprinting
•Isolates with identical 16S rDNA sequences show different REP fingerprint.
•The REP analysis shows an higher and variegate diversity in respect to the 16S
rDNA sequence analysis that henances the low efficiency of 16S rDNA analysis.
Aerobic/Anaerobic growth curves
Methabolic analysis (Catechol, Gentisate, Protocatechuic
acid)
Conclusions
Isolates analysis
Conclusions
Phylogenetical Analysis
REP Fingerprinting
Aerobic/Anaerobic growth curves
•Every isolate shows peculiar growth curves and is able to grow both in aerobic
and anaerobic conditions
•Each isolate grows faster in aerobic than in anaerobic conditions but in general
the maximum density for both conditions is comparable
•The clustering of the isolates based on the obtained growth curves seems to
correspond to the REP-16S rDNA grouping
Methabolic analysis (Catechol, Gentisate, Protocatechuic
acid)
Conclusions
Isolates analysis
Conclusions
Phylogenetical Analysis
REP Fingerprinting
Aerobic/Anaerobic growth curves
Methabolic analysis (Catechol, Gentisate, Protocatechuic
acid)
•Some isolates are able to degrade different aromatic compounds at the marine
salt conditions. The question on the range of salinity at which this capability is
maintained is still opened.
•Further analyses on the aromatic compound spectra are planned (included the
halogenated compounds) as well as the molecular study of the bacterial
enzymes involved in the HC pathways.
Conclusions
Isolates analysis
Conclusions
Phylogenetical Analysis
REP Fingerprinting
Aerobic/Anaerobic growth curves
Methabolic analysis (aromatic compounds)
Other substrates
Conclusion
We have a lot of work to do!
Special Thank’s to the CNR staff