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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
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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
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Major properties of media
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Isolation under oxic conditions

• DNA extraction
• Amplificatioon of 16S rRNAgene
• RFLP
• Sequencing
• t-RFLP

Isolates
Physiological characterization of representative
strains
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Number of isolates on agar plates
6
Preliminary results from partial sequencing of
eight isolates
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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 lAtalante 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

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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
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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

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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
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Number of positive enrichments cultures (with
respect to salt regime and sample)
12
Number of positive enrichments cultures (with
respect to organic substrate)
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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

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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
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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