BIODEEP (EVK3-2000-00042) Second year Scientific Report
(April 1st 2002 – March 31st 2003)
WP 5: Microbial metabolic activities
Lead contractor: CNRS DR 12
Participants: CNRS DR12 (2), CoNISMa (1), UESSEX (6), TUBS (7)
3.1. Objectives
Understanding the ecological relations between the microbial communities and the functioning of
DHABs environments by (1) the study of the microbial activity of natural bacterial consortia and
(2) the physiological characterization and the metabolic activities among microbial isolates.
3.2. Methodology and scientific achievements
3.2.1. Study of the microbial activity of natural bacterial consortia
Partner CNRS DR 12. Objectives were to evidence bacterial life in environments characterized by
extreme conditions.
Cultivation independent approaches were applied to analyze the rates of microbial activities
characterizing the seawater-brine interface, the brine, the brine-sediment interface, and the
sediments; in addition in Urania Basin, samples were collected at the brine-heavy brine interface, in
the heavy brine, and in the mud pit. Both in Urania Basin and Bannock Basin, methanogenic
activity was studied in samples collected in the sediment-trap.
Different parameters were analyzed: (1) 14C-glutamic acid assimilation and respiration, (2) bacterial
production (using 3H-Leucine), (3) 14C-glucose assimilation and respiration [Biodeep-3 cruise], (4)
methane production (natural and potential methane production - and natural methane production
rate during Biodeep-3 cruise), (5) concentration of dissolved methane, (6) sulfate reduction (using
35
S-sulphate).
Results: The results of the second cruise (Biodeep-3, June 2002) confirm data we previously
obtained during the first cruise (Biodeep-2, August-September 2001): despite extreme conditions
for life microbial populations are able to exert metabolic activities within all the studied samples
(interface, brine and sediments) of the 4 studied basins.
Microbial heterotrophic activity: Globally, heterotrophic microbial activities appeared to be optimal
in the Bannock basin and minimal in the Discovery basin. Within the data set, microbial
communities of the Bannock brine appears the most active for biomass production and glutamate
uptake, at the opposite the less active consortia should be those from the Discovery brines. The
metabolic activity drastically increases in the near bottom brine, and as usually observed, the
sediment harbored heterotrophic activities are clearly higher than in the immediately overlying
liquid phase.
Anaerobic bacterial activities: Methane production activity was detected in all the studied samples
(84 different samples were studied for methanogenesis). In general, methane production increased
along the interface, with maximal values observed in the body brine, decreased in the bottom brine
overlying the sediment, and increased again in the sediment, where values were similar or slightly
higher than those observed in the body brine. Globally, methane production is maximal in Urania
Basin, decreases in L'Atalante and Bannock Basins, and becomes minimal Discovery Basin; the
concentration of dissolved methane decreases in the same way.
The sulfate reducing activity was more aleatory, (among the 43 different samples studied for sulfate
reduction, 6 harbored nil activity). In general, the rate of sulfate reduction increased along interface,
being maximal in the lower interface layer (Bannock, Discovery) or in the body brine (L'Atalante,
Urania); after a decrease in the bottom brine, sulfate reduction attained maximal rates in the
sediment. Globally, sulfate reduction rate is maximal in L'Atalante Basin, and decreases to
Bannock, Urania and Discovery Basins.
BIODEEP (EVK3-2000-00042) Second year Scientific Report
(April 1st 2002 – March 31st 2003)
For all the studied microbial metabolic activities, Discovery exhibited the lowest values, suggesting
that this 'younger' brine contains the less active bacterial consortia or the less numerous bacterial
populations,
3.2.2. Physiological characterization and metabolic activities among microbial isolates
Partner CNRS DR 12. Objective was the physiological characterization of 41 isolated strains
regarding NaCl concentration, temperature, and pressure.
Results
NaCl concentration: Most of the anaerobic facultative isolated (32/41) are moderate halophiles with
an optimum of growth in 50 or 100‰ NaCl. A majority (30/32) is able to grow at high salt
concentration 200 or 250‰ NaCl; 3 isolates exhibit optimal growth rate at 100‰ NaCl, 9 are
halotolerant (optimum 10‰ NaCl - isolated from interface); one, isolated from the upper interface
of Urania, is moderate halotolerant; and 5 are extremely halotolerant (up to 200‰).
Temperature: Most of the strains show optimal growth at 35°C, except 5 isolated, which did not
grow at 15°C.
Pressure: No significant growth could be observed under pressure conditions (300 bars) as
compared to atmospheric pressure, indicating the absence of barophilic bacteria among the isolates,
although 2 of them could be were barotolerant.
Partner UEssex. Objectives were to understand the physiology of the isolates from WP4 and
determine their importance in the DHABs.
Most methods involve using media with different salt concentrations, pH etc., or adding compounds
as growth substrates or to indicate the presence of extracellular polymer-degrading enzymes.
Results:
Aerobic isolates: Among the aerobic isolates obtained, most are either related to Proteobacteria or
the low G+C Gram positive organisms. They thrive on seawater medium and are most probably
moderately halophilic (marine) organisms. Most of the Proteobacteria are derived from the
interfaces. They are related to organisms commonly found in marine bacterioplankton and in marine
deep-sea environments. Some of the obtained strains can be referred to as extremely halotolerant.
Most of the low G+C Gram positive organisms are derived from sediments. They are spore formers,
most are related to Bacillus-like organisms. Most of the strains were only moderately halophilic
These observations lead to the assumption that the cultivated aerobic sporeformers are derived from
dormant spores and are not active in situ. However, there are a few exceptions, six strains (4 strains
from L’Atalante sediments, and 2 from Urania sediments) were found to be halophilic, and could
possibly be active in situ.
Anaerobic isolates: The strictly anaerobic isolates and enrichments consisted of extremely
halophilic, (from L'Atalante sediment, and L'Atalante, Urania, and Bannock interfaces) moderately
halophilic (from Bannock interface, L'Atalante, Bannock, Discovery, and Urania sediments).
The extremely halophilic methanogenic Archaea (from L'Atalante sediment) and Halanaerobiaceae
(from L'Atalante, Urania, and Bannock interfaces and L'Atalante sediment), the moderately
halophilic anaerobes (Bannock interfaces) and another extremely halophilic facultatively anaerobe
(from interface and sediment of L'Atalante) could be active in situ.
Partner CONISMA DISTAM. Objectives were to study (1) the distribution of metabolic activities
among strains isolated in the 4 DHABs, and (2) the anaerobic methane oxidation (AMO) in Urania
brine and mud pit
Results:
Distribution of metabolic activities among strains isolated in the 4 DHABs: We characterized some
metabolic activities on strains isolated from samples inoculated during the 2001 cruise.
BIODEEP (EVK3-2000-00042) Second year Scientific Report
(April 1st 2002 – March 31st 2003)
- Oil utilisation as unique carbon source: Screening results reveal that the activity in oil
degradation is higher in Bannock respect to the other basins, and is prevalent in the sediments rather
than in the water/brine interface.
- Lipase-esterase activity: The lipase-esterase activity on saturated long chain fatty acids is
widespread among the isolates of water/brine interface, and L’Atalante showed the lowest activity.
The hydrolytic activity on unsaturated fatty acids prevailed in the water/brine interface too, but it
was much lower as compared with the saturated substrate. The activities on saturated fatty acids
were relatively more represented in higher saline conditions, at 5% NaCl.
- Production of polysaccharides Around 50% of the tested strains produced exopolysaccharides,
except for the Discovery isolates which showed lower percentage. Water/brine interface (W/B
interface), brine/sediment interface and sediments showed similar percentages of strains producing
exopoysaccharides, around 35-40%. A global view of these results showed that in the Bannock
basin a relatively large amount of the cultivable microflora has interesting metabolic capabilities,
while Discovery showed the lowest activity. Except that for oil degradation activity, the higher
percentage of active strains was found in the water/brine interface.
- Salinity tolerance: All the strains grow at seawater salinity, while only few strains do not tolerate
absence of NaCl in the growing medium. Few strains isolated mostly from Urania and Discovery
tolerate >15% NaCl. L’Atalante isolates have the lowest values of tolerance.
Anaerobic methane oxidation(AMO) in Urania brine and mud pit: After 30 days of incubation at in
situ temperature, no differences in methane partial pressure have been evaluated between the
samples added with bromodeoxiuridine and methane, and the control without BrdU, without
methane, or sterilized. These data show that in no one of the samples AMO occurred, and for this
reason we let the incubation to prosecute.
Partner CONISMA DBAEM Messina. Objective was the physiological characterization of
isolated strains regarding (1) salinity tolerance and (2) metabolic potentialities.
Results:
On different isolates from DHABs (Alteromonas, Marinobacter, Idiomarina, Halomonas,
Alcanivorax and Pseudomonas), the growth curves were made in aerobic and microaerophilic
condition using Bioscreen technologies. The metabolic versatility of isolates were analyzed by
testing their capability to metabolize of Phenantrene, Napthalene, Hexadecane, Catechol, Gentisic
acid and Protocatechuic acid.
Partner TUBS. Objectives were to analyze the microbial colonization of rheolite/basalt surfaces
exposed for approximately 12 months to oxic seawater (Urania basin) and anoxic brines (Urania
and Bannock basins).
Results
Urania seawater: The highest colonization was observed on the surfaces from the samples exposed
to the oxic seawater over Urania basin on basalt matrices. The presence of cells, some of them
obviously dividing, has been recorded; exopolysaccharide matrices covering both, basalt and
rheolite surfaces were also detected.
Urania brine: Poor presence of both, organogenic material and microorganism cells. Seldom events
of finding of cell-like structures on rheolite surfaces were contrasting the absolute lack of any
presence of life on the basalt granules.
Bannock brine: Similar pattern than observed in Urania brine. The microbial colonization was
obviously extremely poor.
The environmental conditions in the anoxic hypersaline brines strongly inhibited the microbial
colonization. In the overlying seawater, in contrast to anoxic hypersaline counterpart, the visible
development of microorganisms (dividing cells, development of the EPS matrices) occurred on the
both minerals tested; the stronger colonization being detected on the basalt surface.
BIODEEP (EVK3-2000-00042) Second year Scientific Report
(April 1st 2002 – March 31st 2003)
3.3. Socio-Economic Relevance and Policy Implications
We evidence active microorganisms able to develop within extreme conditions encountered within
the DHABs. The isolation of bacterial strains originating from these biotopes and their
physiological characterization suggests that some new biotechnological products could be obtained.
3.4. Discussion and Conclusion
Experiments performed during the second cruise (Biodeep-3, June 2002) confirm the results
obtained during the previous year (Biodeep-2 cruise, August-September 2001): microorganisms are
able to develop in extreme conditions occurring in the 4 studied DHABs.
Isolation and physiological characterization of anaerobic and halophilic isolates indicate that these
bacteria can be active in situ, which agrees with the detection of diverse microbial activities in
numerous and various samples collected within the 4 studied DHABs. In the same way, isolation of
extremely halophilic methanogenic Archaea and Halanaerobiaceae corroborates the detection of
methanogenic activity in the brine and sediment.
Process of Anaerobic Methane Oxidation
Two experiment series were performed simultaneously on samples collected in Urania Basin (brine,
mud pit and sediment) to evidence anaerobic methane oxidation. Theoretically, this process is
performed by a bacterial consortium including methanogenic Archaea and sulfate reducing bacteria.
(1) One experiment series, performed by CNRS DR 12, implicated the comparison of (i) the rate of
methane production in the presence and absence of inhibitor of sulfate reduction (molybdate), and
(ii) the rate of sulfate reduction in the presence and absence of inhibitor methane production (BES).
(2) The second experiment series, performed by CoNiSMA-DISTAM and UESSEX, implicated the
incorporation of BrdU (Bromodeoxiuridine) in the DNA of sulfate reducing bacteria susceptible to
carry out anaerobic methane oxidation.
The molybdate addition results in an increase of the methane production rate (x17 in the brine; x1.5
in the Mud Pit and x5.7 in the sediment). Two explanations are possible for this increase: (1) the
methanogenic Archaea produce more methane as more substrates are available (these substrates
being previously used by sulfate reducers, when they are not inhibited by molybdate). (2) The
methanogenic Archaea produce the same quantity of methane, but as methane is not used by sulfate
reducers, which are inhibited by molybdate, the final concentration of methane produced is higher
than in the absence of molybdate.
The other experiments involving the consumption of methane by sulfate reducers did give
significant results, which could suggest that, in the studied samples, sulfate reducers are not
involved in the anaerobic methane oxidation (further experiments must be performed to corroborate
or not this hypothesis).
3.5. Plan and objectives for the next period
The objectives of the next year are still related to Project management, in particular regarding the
following aspects:
Previous cruises: Exploitation of results obtained during the previous cruises (Biodeep-2 and
Biodeep-3 cruises), performing some additional analyses on the collected and studied samples.
Preparation of the next and last cruise (Biodeep-5 planed for November 2003). Determination of the
samples to collect and the studies to perform, in view to confirm and complete the previous results
* Sampling along interface and within the sediment of the various DHABs to complete our sample
collection
* Addition of new experiments in view to confirm the previous results obtained during the firsts
cruises and to make complete our understanding about bacterial activities and physiological
characterization of bacterial strains implicated in these activities.
* Analyses of the collected samples (on board and at the laboratory)
Exploitation of the results and writing of scientific papers and reports.