Objectives: Proof for the Existence of Bacterial Life in Environments Characterized By

BIODEEP (EVK3-2000-00042) First year Scientific Report

Workpackage number 5: Microbial metabolic activities

Lead contractor: CNRS DR 12

Participants: CNRS DR 12, CoNISMa, UESSEX, TUBS

Starting date: month 9

3.1. Objectives

Determination of the distribution, taxonomy and diversity of microorganisms from DHABs, and isolation of strains with biotechnological potential

3.2. Methodology and scientific achievements

Partner CNRS DR 12. Objectives were to proof, by cultivation independent approaches, the existence of bacterial life in environments characterized by extreme conditions.

Cultivation independent approaches were applied to analyze the rates of microbial activities characterizing the oxic water and the brine at their in situ chemical and physical conditions. Water and brine samples were collected using Niskin bottles and 2 different pressure-retaining samplers. Sediments were obtained by using a multicorer. Suspended particulate matter was collected by filtration on 142-mm diameter filter Nuclepore, 1-µm mesh. Six parameters were analyzed: 1) aminopeptidase rate, 2) phosphatase rate (using AMC-Leu and MUF-P analogs, respectively), 3) 14C-glutamic acid assimilation and respiration, 4) bacterial production (using 3H-Leucine), 5) methane production (with or without different methane precursors), 6) sulfate reduction (using 35S-sulphate).

Results: Bacterial activities in the oxic water column give reference data vs their counterparts in the DHABs. At 1000-m, hydrolytic activities were one order of magnitude lower than the maximal in the surface water. Activities at the interface brine/seawater are very high in comparison to the adjacent upper layer, at least similar to those in the surficial layers. Maximal enzymatic and assimilation rates are at the interface of L'Atalante basin. Bacterial activities were detected within the brines of all the basins. In all brine samples enzymatic activities were stronger than in deep oxic water (maximum in L'Atalante). Bacterial production and respiration rates are similar to those in the deep oxic water for all basins, with highest values in Bannock. At the interface brine/sediment, and then in the sediment, activities were detected, but at lower rates than in their oxic counterparts.

Methane production was evidenced in all samples, but those from the oxic zone out of the DHABs. Potential production was higher than natural production, showing that in situ availability of nutrients limits this activity. In the 4 DHABs, CH4 production was minimum in water, then increased, from upper to lower interface, and was maximum in the body brines, where natural concentration of methane were higher. Maximal methane production was in sediment. Sulfate reduction was evidenced in the majority of the samples. In contrast with methanogenesis, the maximal sulfate-reduction was not in sediment but in the brines or at the seawater/brine interface. Maximal SR rate was in the lower interface of L’Atalante.

Partner UEssex objectives: Optimisation of culture Media to cultivate bacteria adapted to DHAB conditions

Methods: Prior to the cruise, many different types of media were tested for a variety of features, for example: ability to support growth of different types of known halophilic microorganisms; maintenance of appropriate pH; no precipitation. About 70 permutations of media were used to enrich for a wide diversity of microorganisms. (Table 1, and the main substrates and terminal electron acceptors are shown in the footnotes to Table 2 (aerobic enrichments) and Table 4 (anaerobic enrichments).

Results: Aerobic enrichments: Isolates were obtained using media with seawater (SW, 35gl-1) or moderate salt concentration (Bh, 119gl-1), but not with media of high salt concentration (medium B, 236gl-1). In addition, aerobes were isolated only from sediment samples or Bannock basin interface samples (Table 2). No isolates were derived from brines or L’Atalante, Urania and Discovery basin interface samples. Most of the isolates from sediment samples were spore-forming microorganisms, whereas only a few of the isolates from the Bannock basin interface formed spores. Growth tests on 24 isolates from L’Atalante and Bannock basin sediment on media with salt concentrations ranging from 2 to 20% w/v showed that growth was faster at lower salt concentration; only 13 strains grew on media with more than 15% w/v salt. Tests in anoxic medium with a seawater salt concentration revealed that 23 out of 24 isolates could grow anaerobically.

Anoxic enrichments: Numerous positive anoxic enrichment cultures were obtained, most of which were from L’Atalante basin sediment on medium B, a medium with a high salt content of 230gl-1 (Table 3). Most of the positive enrichments were obtained with organic compounds that can be fermented by microorganisms, e.g. sugars, lactate and pyruvate (Table 4). The positive enrichments are in isolation; some pure cultures have nearly been established.

Partner TUBS objectives: 1) preliminary analysis of oil-degrading community from the interfacial layer of Urania basin to determine the main players in crude oil enriched culture, 2) construction of »archive” and expression libraries from »oil”- community DNA for the purpose of further analysis and exploitation of enzymatic potential.

Methods:1) Isolation of culturable microorganisms from communities amended with crude oil, The Urania basin interphase was aliquoted (50 ml) in closed vials, spiked with crude weathered oil and incubated under aerobic conditions for six weeks. Total DNA was extracted using G’nomeTM DNA Extraction procedure (Bio101, CA).. The DNA was PCR-amplified using F27 and R1492 oligonucleotides under standard conditions, the amplicons were cloned into pCR2.1 vector (Invitrogen). Resulting E. coli clones were transferred in two 96-well-plates (each, for enrichment and control), and re-amplified with plasmid-located primers M13 and rM13. The amplicons were analysed for RFLP – patterns, and representative clones have been selected for the sequencing.

In parallel, extinction dilution cultures have been obtained for strain isolation. Those were plated from a number of dilutions to the solid medium based on authentic autoclaved seawater sample supplemented with 1.8 % agar and vapour of the crude oil. Visible colonies have been detected after 5-7 days. Colony-PCR with the same primers as for 16S rRNA community clone library was performed which was followed by RFLP-analysis of separate amplicons to find the differences. After that, sequencing analysis was performed for the selected isolates.

2) Making 16S rRNA gene libraries to analyse the same microbial communities. Metagenomic libraries have been constructed using 2 techniques: Firstly, a SuperCosTM system (Stratagene, CA), to »archive” the total DNA, and, secondly, an expression library in Lambda-ZAPTM (Stratagene, CA) to evaluate the enzymatic activities, have been created. The libraries have been established, (Fig 1 and 2 in Annex)

Results. The isolates obtained belonged to three major groups: A) 50 % are related to Marinobacter CAB, sequence identities varied between 98 and 100 %. B) 25 % to Pseudomonas stutzerii-P. balearica group, 98—100% sequence identities., C) 25 % to Proteobacteria, group of Ruegeria, 98 % sequence identity. In control no isolates were obtained after immediate plating of the sample to the oil-containing solid medium.

Library. PCR-amplification uncovered the dominance of Marinobacter CAB with presence of the sequences in 95 % of the clones. Pseudomonad sequences have been found only in 5 clones and Ruegeria in two, out of 192 clones.

About 8000 clones in SuperCos with an average insert size of 42 kbp were obtained.

Expression library: Eight ml with a phage titre of 2x 104 phage units per l were obtained (average insert size of 8 kbp). The latter will be channelled to WP7 for further analysis on the diverse enzymes (for enzymes and detection systems see Table 3 in Annex). Being applied to the phage lawn, these substrates/indicators made possible to identify and clean-up a number of phage clones harbouring a number of above enzymatic activities.

Partner CoNISMa (Dto di Biologia Animale ed Ecologia Microbica, University of Messina). Objectives:Data collection about optimal growth conditions of isolates for further enrichment cultures and strain isolations (D24). Preliminary steps included selection of the most suitable media for enriching and, possibly, isolating DHABs bacterial populations (see WP4).

Methods: DHABs samples were anaerobically transferred into several Hungate tubes containing 100-fold concentrated liquid minimal media supplied with different C sources. Some aliquots of the original samples were also transferred to aerobic media and incubated in presence of oxygen (table 1). The enriched cultures were microscopically screened for growth after 15, 30, 45, 60, 80, 120, 180 d. Increase in bacterial density was considered significant when a 0.5 variance was recorded. Every positive cultures were transferred to a fresh liquid medium and inoculated to a corresponding agarised medium. Transferring under anoxic conditions was done within a glove-box filled with an oxygen-free gas mixture (N2:CO2:H2 90:5:5 % v/v/v). Screening was by using a magnifying lent and a colony counter system.

Results: Few of the 15 tested media (P1 and P2) appeared to give a relatively fast growth of bacterial populations, that were screened as positive 15 days after seeding(Bannock, Urania, L’Atalante interface layers). Both media were strongly enriched in organic C and the P2 was also amended with trimethylamine (2g/l), that favours the growth of methanogenic bacteria. Table 2 refers about the media where the different DHABs samples showed growth after a due incubation time. According to the experimental results, the Lower Interface from Urania basin was containing bacteria able to grow anaerobically by using crude oil as sole source of carbon. Since the main objective of our experimental contribution was to focus on numerically representative bacteria that are inhabiting the DHABs and that are actively growing within the basins, the aerobic enriched cultures have not been screened for isolation, yet. No anaerobic growth on agar media was recorded up to date, except than for the Bannock interface P2 enriched sample, after transferring to the PA16 agar medium. Due to the difficulties in separating two phylogenetic populations in order to obtain pure cultures, proceedings for the CFU physiological characterisation are still impeded.

3.4 Discussion and conclusion

Evidence for diverse microbial activities in all the studied samples of brine clearly show that viewed cells are living organisms. The metabolic rates are higher in samples respecting all the most characteristic conditions of DHABs than in their decompressed counterparts, this data is the proof that DHAB populations are adapted such extreme conditions for live and are actually active in all the studied DHABs. Results evidenced presently active methanogenic archaea and sulfate reducing bacteria in the DHABs, with preferential development of methanogens in the body brines and sediment, whereas sulfate reducing bacteria were rather associated with interface seawater/brine and body brine (CNRS DR 12).Preliminary data suggest that isolates obtained in oxic media are moderately halophilic or halotolerant microorganisms. Extremely halophilic aerobes could not be enriched. The aerobic isolates from the sediments are probably mostly derived from spores deposited during sediment formation and may not be active in the anoxic brines and sediments. In addition, although these isolates are capable of facultative anaerobic growth, they may be outcompeted by halophilic anaerobic organisms in situ (UEssex). The studied oil-induced community is dominated by a common hydrocarbonoclastic bacterium, Marinobacter sp. CAB. The enrichment culture served as a starting point to generate bacteriophage expression library for further analysis of enzymatic activities. The major limitation in constructions of libraries is the shortage of material in control samples: the library making requires high DNA quantities (at the levels of few micrograms), which can be easily achieved in enrichment cultures. Enrichments, however, drastically reduce the diversity of microbial communities and mask their genetic/metabolic potential. In order to generate a comprehensive metagenomic expression library, extended sample volumes are required (> 100 l per sample) (TUBS). According to the results of the different enrichments, we suppose that DHABs are inhabited by populations well adapted to the environmental conditions (EABs). EABs do not necessarily exhibit long duplication times since their growth is strongly stimulated by easily accessible C sources (P1, P2). In presence of recalcitrant matter (P10), growth was delayed. Difficulties in growing the enriched populations on agar may be due to their physiological limits confirmed by their need of easily accessible organic compounds. Due to the difficulties in separating the 2 associated taxa forming colonies on the PA16 medium, we hypothesize that DHABs populations are actively interacting for organic matter recycling (CoNISMa).

3.5 Plan and Objectives for the Next Period

Partners will carry on with the studies on the genetic material and growing strains obtained from the first Biodeep cruise samples. The 16SrRNA gene of all aerobic isolates will be amplified and their diversity assessed using RFLP analysis. The 16SrRNA gene of representative isolates with different RFLP fingerprints will then be partially sequenced.The physiology of organisms with sequences only distantly related to known species will be studied. The isolation of anaerobes will continue. Pure cultures will be related to known microorganisms by partial sequencing of their 16SrRNA gene, and organisms only distantly related to known species will be characterised (UEssex). Estimation of oil degradation rates and analysis of oil-induced communities will be using FISH. Subcloning, sequencing, expression and product analysis of the selected phage clones (TUBS).All the WP5 partners will be implicated in the second Biodeep cruise planned June 2002. Data got during the first cruise will be used to improve the methodology mainly to obtain adapted growing cells.