Cristiana Cravo-Laureau

Anaerobic n-alkane metabolism by a sulfate-reducing bacterium, Desulfatibacillum aliphaticivorans strain CV2803T.

ABSTRACT The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.

Impact of oil on bacterial community structure in bioturbated sediments

Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions – with tidal cycles and natural seawater – was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g−1 wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities.

Marine coastal sediments microbial hydrocarbon degradation processes: contribution of experimental ecology in the omics’era

Coastal marine sediments, where important biological processes take place, supply essential ecosystem services. By their location, such ecosystems are particularly exposed to human activities as evidenced by the recent Deepwater Horizon disaster. This catastrophe revealed the importance to better understand the microbial processes involved on hydrocarbon degradation in marine sediments raising strong interests of the scientific community. During the last decade, several studies have shown the key role played by microorganisms in determining the fate of hydrocarbons in oil-polluted sediments but only few have taken into consideration the whole sediment’s complexity. Marine coastal sediment ecosystems are characterized by remarkable heterogeneity, owning high biodiversity and are subjected to fluctuations in environmental conditions, especially to important oxygen oscillations due to tides. Thus, for understanding the fate of hydrocarbons in such environments, it is crucial to study microbial activities, taking into account sediment characteristics, physical-chemical factors (electron acceptors, temperature), nutrients, co-metabolites availability as well as sediment’s reworking due to bioturbation activities. Key information could be collected from in situ studies, which provide an overview of microbial processes, but it is difficult to integrate all parameters involved. Microcosm experiments allow to dissect in-depth some mechanisms involved in hydrocarbon degradation but exclude environmental complexity. To overcome these lacks, strategies have been developed, by creating experiments as close as possible to environmental conditions, for studying natural microbial communities subjected to oil pollution. We present here a review of these approaches, their results and limitation, as well as the promising future of applying “omics” approaches to characterize in-depth microbial communities and metabolic networks involved in hydrocarbon degradation. In addition, we present the main conclusions of our studies in this field.

Role of environmental fluctuations and microbial diversity in degradation of hydrocarbons in contaminated sludge

Little is known about microbial communities involved in hydrocarbon degradation, whether it be their structural and functional diversity or their response to environmental constraints such as oxygen fluctuation. Here, current knowledge of the impact of diversity and redox oscillations upon ecosystem processes is reviewed. In addition, we present the main conclusions of our studies in this field. Oxic/anoxic oscillations had a strong impact upon bacterial community structures, influencing their ability to degrade hydrocarbons and their capacity to reduce hydrocarbon toxicity. Furthermore, a decrease in functional diversity has a strong impact on pollutant degradation.

Response of Archaeal Communities to Oil Spill in Bioturbated Mudflat Sediments

The response of archaeal community to oil spill with the combined effect of the bioturbation activity of the polychaetes Hediste diversicolor was determined in mudflat sediments from the Aber-Benoît basin (Brittany, French Atlantic coast), maintained in microcosms. The dynamics of the archaeal community was monitored by combining comparative terminal restriction fragment length polymorphism (T-RFLP) fingerprints and sequence library analyses based on 16S rRNA genes and 16S cDNA. Methanogens were also followed by targeting the mcrA gene. Crenarchaeota were always detected in all communities irrespective of the addition of H. diversicolor and/or oil. In the presence of oil, modifications of archaeal community structures were observed. These modifications were more pronounced when H. diversicolor was added resulting in a more diverse community especially for the Euryarchaeota and Thaumarchaeota. The analysis of mcrA transcripts showed a specific structure for each condition since the beginning of the experiment. Overall, oiled microcosms showed different communities irrespective of H. diversicolor addition, while similar hydrocarbon removal capacities were observed.

Role of environmental factors and microorganisms in determining the fate of polycyclic aromatic hydrocarbons in the marine environment

Polycyclic aromatic hydrocarbons (PAHs) are widespread in marine ecosystems and originate from natural sources and anthropogenic activities. PAHs enter the marine environment in two main ways, corresponding to chronic pollution or acute pollution by oil spills. The global PAH fluxes in marine environments are controlled by the microbial degradation and the biological pump, which plays a role in particle settling and in sequestration through bioaccumulation. Due to their low water solubility and hydrophobic nature, PAHs tightly adhere to sediments leading to accumulation in coastal and deep sediments. Microbial assemblages play an important role in determining the fate of PAHs in water and sediments, supporting the functioning of biogeochemical cycles and the microbial loop. This review summarises the knowledge recently acquired in terms of both chronic and acute PAH pollution. The importance of the microbial ecology in PAH-polluted marine ecosystems is highlighted as well as the importance of gaining further in-depth knowledge of the environmental services provided by microorganisms.

Anaerobic 1-Alkene Metabolism by the Alkane- and Alkene-Degrading Sulfate Reducer Desulfatibacillum aliphaticivorans Strain CV2803T

ABSTRACT The alkane- and alkene-degrading, marine sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, known to oxidize n-alkanes anaerobically by fumarate addition at C-2, was investigated for its 1-alkene metabolism. The total cellular fatty acids of this strain were predominantly C-(even number) (C-even) when it was grown on C-even 1-alkenes and predominantly C-(odd number) (C-odd) when it was grown on C-odd 1-alkenes. Detailed analyses of those fatty acids by gas chromatography-mass spectrometry after 6- to 10-week incubations allowed the identification of saturated 2- and 4-ethyl-, 2- and 4-methyl-, and monounsaturated 4-methyl-branched fatty acids with chain lengths that correlated with those of the 1-alkene. The growth of D. aliphaticivorans on (per)deuterated 1-alkenes provided direct evidence of the anaerobic transformation of these alkenes into the corresponding 1-alcohols and into linear as well as 10- and 4-methyl-branched fatty acids. Experiments performed with [13C]bicarbonate indicated that the initial activation of 1-alkene by the addition of inorganic carbon does not occur. These results demonstrate that D. aliphaticivorans metabolizes 1-alkene by the oxidation of the double bond at C-1 and by the subterminal addition of organic carbon at both ends of the molecule [C-2 and C-(ω-1)]. The detection of ethyl-branched fatty acids from unlabeled 1-alkenes further suggests that carbon addition also occurs at C-3. Alkylsuccinates were not observed as potential initial intermediates in alkene metabolism. Based on our observations, the first pathways for anaerobic 1-alkene metabolism in an anaerobic bacterium are proposed. Those pathways indicate that diverse initial reactions of 1-alkene activation can occur simultaneously in the same strain of sulfate-reducing bacterium.

The anaerobic hydrocarbon biodegrading bacteria: An overview

Abstract Hydrocarbons are widespread in our environment. The number of bacteria known to oxidize hydrocarbons in the absence of oxygen has considerably increased during the last ten years. Anaerobic bacteria have been shown capable of utilizing hydrocarbons not only in consortia but also in pure cultures. The results obtained in the framework of MATBIOPOL project on anaerobic hydrocarbon degradation by denitrifying bacteria and by enrichment cultures maintained under methanogenic conditions are exposed together with the present knowledge on hydrocarbon biodegradation.

Impact of microbial diversity depletion on xenobiotic degradation by sewage-activated sludge.

Microbial diversity is generally considered as having no effect on the major processes of the ecosystem such as respiration or nutrient assimilation. However, information about the impact of diversity on minor functions such as xenobiotic degradation is scant. We studied the role of diversity on the capacity of an activated-sludge microbial community to eliminate phenanthrene, a polycyclic aromatic hydrocarbon. We also assessed the impact of diversity erosion on the ability of activated sludge to oxidize a wide range of organic compounds. The diversity of activated sludge was artificially modified by dilution to extinction followed by regrowth stage which led to communities with similar biomass but displaying a diversity gradient. The capacity of activated-sludge community to degrade phenanthrene was greatly modified: at high levels of diversity, the community was able to mineralize phenanthrene whereas at medium levels it first of all partially lost its ability to mineralize this pollutant and at the lowest diversity, the activated sludge completely lost its capacity to transform phenanthrene. Diversity depletion also reduced the metabolic diversity and biomass productivity of sewage-activated sludge. This study demonstrates that diversity erosion can greatly affect major ecosystem services such as pollutant removal.

Anaerobic oxidation of n-alkenes by sulphate-reducing bacteria from the genus Desulfatiferula: n-ketones as potential metabolites.

Two alkene-degrading sulphate-reducing bacteria from the genus Desulfatiferula (Desulfatiferula olefinivorans strain LM2801(T) and Desulfatiferula sp. strain BE2801) were investigated for their 1-alkene metabolism. Their total cellular fatty acids were predominantly C-even when they were grown on C-even 1-alkene (1-hexadecene), whereas a mixture of fatty acids with C-odd or C-even carbon chains predominated when cells were grown on C-odd 1-alkene (1-pentadecene). This is consistent with the fatty acid composition of other sulphate-reducing strains previously reported to grow on n-alkenes. Linear and 3-OH-fatty acids appear to be the main fatty acids produced by the two Desulfatiferula strains. The analysis of their neutral lipids led to identifying several n-alkanols and n-ketones with the same number of carbon atoms as the alkene growth substrate and with functionality located between C-1 and C-5. Growth of strains LM2801(T) and BE2801 on (per) deuterated 1-alkenes provided direct evidence of their anaerobic transformation to corresponding 1-alkanols, n-ketones and linear (3-OH-) fatty acids. These results demonstrate that Desulfatiferula strains oxidize a 1-alkene by oxidation of the double bond at C-1, but also at C-2 to C-5 (after eventual isomerization of the double bond) yielding the corresponding C-2 to C-5 n-ketones (via the corresponding n-alkanols). The formation of specific 3-OH-fatty acids by elongation of shorter chain fatty acids was also demonstrated. Based on our observations, pathways for anaerobic 1-alkene metabolism in sulphate-reducing bacteria from the genus Desulfatiferula are proposed. They indicate that n-ketones can constitute new metabolites of the biodegradation of n-alkenes in anaerobic environments.