1. BIODEEP-WP4 BIODEEP-WP5 Andrea Sass , Terry McGenity
University of Essex
BIODEEP-WP4
Determination of the distribution, taxonomy and diversity of micro-organisms 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

2. 1. Isolation of bacteria from different physiological groups
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

3. Major properties of media
University of Essex
Major properties of media

4. Physiological characterization of representative strains
University of Essex
Isolation under oxic conditions
DNA extraction
Amplificatioon of 16S rRNAgene
RFLP
Sequencing
t-RFLP
Isolates
Physiological characterization of representative strains

5. Number of isolates on agar plates
University of Essex
Number of isolates on agar plates
total 39 36 25 14

6. Preliminary results from partial sequencing of eight isolates
University of Essex
Preliminary results from partial
sequencing of eight isolates

7. Physiological properties of aerobic strains
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 spore-forming (probably Bacillus-related)
only a few strains of the Bannock interface were spore-forming
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%

8. Relevance of aerobic isolates for the DHAB microbial community
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

9. 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

10. Subsample for t-RFLP analysis
University of Essex
Anoxic enrichments
Media for on board inoculation
Subsample for t-RFLP analysis
Direct amendment of brine and interface samples
Positive enrichments
Further cultivation
Isolation by deep-agar dilution series
Partial sequencing
Physiological characterization of representative strains
Media inoculated in the lab

11. Number of positive enrichments cultures
University of Essex
Number of positive enrichments cultures
(with respect to salt regime and sample) 9 5 1 4 11
total

12. Number of positive enrichments cultures
University of Essex
Number of positive enrichments cultures
(with respect to organic substrate)

13. Physiological properties of anerobic cultures
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

14. Relevance of anaerobic cultures for the DHAB microbial community
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

15. Future work: Further isolation attempts: for SRB
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