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

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

Major properties of media University of Essex Major properties of media

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

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

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

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%

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

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

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

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

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

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

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

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