Characterization of brackish anaerobic bacteria involved in hydrocarbon degradation: A combination of molecular and culture-based approaches

Cravo-Laureau C., Matheron R., Cayol J., Joulian C., Hirschler-Réa A.

A novel marine sulfate-reducing bacterium, strain CV2803T, which is able to oxidize aliphatic hydrocarbons, was isolated from a hydrocarbon-polluted marine sediment (Gulf of Fos, France). The cells were rod-shaped and slightly curved, measuring 0·6×2·2–5·5 μm. Strain CV2803Tstained Gram-negative and was non-motile and non-spore-forming. Optimum growth occurred in the presence of 24 g NaCl l−1, at pH 7·5 and at a temperature between 28 and 35 °C. Strain CV2803Toxidized alkanes (from C13to C18) and alkenes (from C7to C23). The DNA G+C content was 41·4 mol%. Comparative sequence analyses of the 16S rRNA gene and dissimilatory sulfite reductase (dsrAB) gene and those of other sulfate-reducing bacteria, together with its phenotypic properties, indicated that strain CV2803Twas a member of a distinct cluster that contained unnamed species. Therefore, strain CV2803T(=DSM 15576T=ATCC BAA-743T) is proposed as the type strain of a novel species in a new genus,Desulfatibacillum aliphaticivoransgen. nov., sp. nov.

Wieland A., K�hl M., McGowan L., Four�ans A., Duran R., Caumette P., Garc�a de Oteyza T., Grimalt J.O., Sol� A., Diestra E., Esteve I., Herbert R.A.

The microenvironment and community composition of microbial mats developing on beaches in Scapa Flow (Orkney Islands) were investigated. Analysis of characteristic biomarkers (major fatty acids, hydrocarbons, alcohols, and alkenones) revealed the presence of different groups of bacteria and microalgae in mats from Waulkmill and Swanbister beach, including diatoms, Haptophyceae, cyanobacteria, and sulfate-reducing bacteria. These analyses also indicated the presence of methanogens, especially in Swanbister beach mats, and therefore a possible role of methanogenesis for the carbon cycle of these sediments. High amounts of algal lipids and slightly higher numbers (genera, abundances) of cyanobacteria were found in Waulkmill Bay mats. However, overall only a few genera and low numbers of unicellular and filamentous cyanobacteria were present in mats from Waulkmill and Swanbister beach, as deduced from CLSM (confocal laser scanning microscopy) analysis. Spectral scalar irradiance measurements with fiber-optic microprobes indicated a pronounced heterogeneity concerning zonation and density of mainly anoxygenic phototrophs in Swanbister Bay mats. By microsensor and T-RFLP (terminal restriction fragment length polymorphism) analysis in Swanbister beach mats, the depth distribution of different populations of purple and sulfate-reducing bacteria could be related to the microenvironmental conditions. Oxygen, but also sulfide and other (inorganic and organic) sulfur compounds, seems to play an important role in the stratification and diversity of these two major bacterial groups involved in sulfur cycling in Swanbister beach mats.

Kniemeyer O., Fischer T., Wilkes H., Glöckner F.O., Widdel F.

ABSTRACT Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m -xylene, respectively. However, strain EbS7 did not oxidize naphthalene, m -xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n -hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobic n -alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.

Cervantes F.J., Dijksma W., Duong-Dac T., Ivanova A., Lettinga G., Field J.A.

ABSTRACT The anaerobic microbial oxidation of toluene to CO 2 coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [ 13 C]toluene were recovered as 13 CO 2 in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [ 13 C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.

Widdel F., Rabus R.

Saturated and aromatic hydrocarbons are wide-spread in our environment. These compounds exhibit low chemical reactivity and for many decades were thought to undergo biodegradation only in the presence of free oxygen. During the past decade, however, an increasing number of microorganisms have been detected that degrade hydrocarbons under strictly anoxic conditions.

Maidak B.L.

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.

Ehrenreich P., Behrends A., Harder J., Widdel F.

The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C6H14). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C8H18). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C16H34). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen.

Herbert R.A.

It is generally considered that nitrogen availability is one of the major factors regulating primary production in temperate coastal marine environments. Coastal regions often receive large anthropogenic inputs of nitrogen that cause eutrophication. The impact of these nitrogen additions has a profound effect in estuaries and coastal lagoons where water exchange is limited. Such increased nutrient loading promotes the growth of phytoplankton and fast growing pelagic macroalgae while rooted plants (sea-grasses) and benthic are suppressed due to reduced light availability. This shift from benthic to pelagic primary production introduces large diurnal variations in oxygen concentrations in the water column. In addition oxygen consumption in the surface sediments increases due to the deposition of readily degradable biomass. In this review the physico-chemical and biological factors regulating nitrogen cycling in coastal marine ecosystems are considered in relation to developing effective management programmes to rehabilitate seagrass communities in lagoons currently dominated by pelagic macroalgae and/or cyanobacteria.

Galushko, Minz, Schink, Widdel

Incubation of marine sediment in anoxic, sulphate-rich medium in the presence of naphthalene resulted in the enrichment of sulphate-reducing bacteria. Pure cultures with short, oval cells (1.3 by 1.3-1.9 microm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulphate reduction to sulphide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulphate-reducing bacteria of the delta-subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were used in addition to naphthalene. Quantification of substrate consumption, sulphide formation and formed cell mass revealed that naphthalene was completely oxidized with sulphate as the electron acceptor.

So C.M., Young L.Y.

ABSTRACT An alkane-degrading, sulfate-reducing bacterial strain, AK-01, was isolated from an estuarine sediment with a history of chronic petroleum contamination. The bacterium is a short, nonmotile, non-spore-forming, gram-negative rod. It is mesophilic and grows optimally at pH 6.9 to 7.0 and at an NaCl concentration of 1%. Formate, fatty acids (C 4 to C 16 ) and hydrogen were readily utilized as electron donors. Sulfate, sulfite, and thiosulfate were used as electron acceptors, but sulfur, nitrite, and nitrate were not. Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequence indicate that AK-01 is most closely related to the genera Desulfosarcina , Desulfonema , and Desulfococcus in the delta subdivision of the class Proteobacteria . It is phenotypically and phylogenetically different from strains Hxd3 and TD3, two previously reported isolates of alkane-degrading, sulfate-reducing bacteria. The alkanes tested to support growth of AK-01 had chain lengths of C 13 to C 18 . 1-Alkenes (C 15 and C 16 ) and 1-alkanols (C 15 and C 16 ) also supported growth. The doubling time for growth on hexadecane was 3 days, about four times longer than that for growth on hexadecanoate. Mineralization of hexadecane was indicated by the recovery of 14 CO 2 from cultures grown on [1- 14 C]hexadecane. Degradation of hexadecane was dependent on sulfate reduction. The stoichiometric ratio (as moles of sulfate reduced per mole of hexadecane degraded) was 10.6, which is very close to the theoretical ratio of 12.25, assuming a complete oxidation to CO 2 . Anaerobic alkane degradation by sulfate reducers may be a more widespread phenomenon than was previously thought.

Rabus, Wilkes, Schramm, Harms, Behrends, Amann, Widdel

Denitrifying bacteria were enriched from freshwater sediment with added nitrate as electron acceptor and crude oil as the only source of organic substrates. The enrichment cultures were used as laboratory model systems for studying the degradative potential of denitrifying bacteria with respect to crude oil constituents, and the phylogenetic affiliation of denitrifiers that are selectively enriched with crude oil. The enrichment culture exhibited two distinct growth phases. During the first phase, bacteria grew homogeneously in the aqueous phase, while various C1-C3 alkylbenzenes, but no alkanes, were utilized from the crude oil. During the second phase, bacteria also grew that formed aggregates, adhered to the crude oil layer and emulsified the oil, while utilization of n-alkanes (C5 to C12) from the crude oil was observed. During growth, several alkylbenzoates accumulated in the aqueous phase, which were presumably formed from alkylbenzenes. Application of a newly designed, fluorescently labelled 16S rRNA-targeted oligonucleotide probe specific for the Azoarcus/Thauera group within the beta-subclass of Proteobacteria revealed that the majority of the enriched denitrifiers affiliated with this phylogenetic group.

Patricia B., Jean-Claude B.

A microbial surfactant was investigated for its potential to enhance the biodegradation of heptadecane. The bioemulsifier used in this study was extracted from culture supernatants of Pseudomonas nautica after growth on heptadecane. The heptadecane uptake rate-could be increased 15-fold by the addition of 1.5 mg protein of bioemulsifier per 100 mg of heptadecane. Scanning electron microscopy showed that bioemulsification was the main mode allowing the transfer of hydrocarbon in the presence of the isolated compound.

Ward D.M., Ferris M.J., Nold S.C., Bateson M.M.

SUMMARY This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

Heider J., Spormann A.M., Beller H.R., Widdel F.

The capacity of some bacteria to metabolize hydrocarbons in the absence of molecular oxygen was first recognized only about ten years ago. Since then, the number of hydrocarbon compounds shown to be catabolized anaerobically by pure bacterial cultures has been steadily increasing. This review summarises the current knowledge of the bacterial isolates capable of anaerobic mineralization of hydrocarbons, and of the biochemistry and molecular biology of enzymes involved in the catabolic pathways of some of these substrates. Several alkylbenzenes, alkanes or alkenes are anaerobically utilized as substrates by several species of denitrifying, ferric iron-reducing and sulfate-reducing bacteria. Another group of anaerobic hydrocarbon degrading bacteria are ‘proton reducers’ that depend on syntrophic associations with methanogens. For two alkylbenzenes, toluene and ethylbenzene, details of the biochemical pathways involved in anaerobic mineralization are known. These hydrocarbons are initially attacked by novel, formerly unknown reactions and oxidized further to benzoyl-CoA, a common intermediate in anaerobic catabolism of many aromatic compounds. Toluene degradation is initiated by an unusual addition reaction of the toluene methyl group to the double bond of fumarate to form benzylsuccinate. The enzyme catalyzing this first step has been characterized at both the biochemical and molecular level. It is a unique type of glycyl-radical enzyme, an enzyme family previously represented only by pyruvate-formate lyases and anaerobic ribonucleotide reductases. Based on the nature of benzylsuccinate synthase as a radical enzyme, a hypothetical reaction mechanism for the addition of toluene to fumarate is proposed. The further catabolism of benzylsuccinate to benzoyl-CoA and succinyl-CoA appears to occur via reactions of a modified β-oxidation pathway. Ethylbenzene is first oxidized at the methylene carbon to 1-phenylethanol and subsequently to acetophenone, which is then carboxylated to 3-oxophenylpropionate and converted to benzoyl-CoA and acetyl-CoA. Anaerobic mineralization of alkanes involves an oxygen-independent oxidation to fatty acids, followed by β-oxidation. In one strain of an alkane-mineralizing sulfate-reducing bacterium, the activation appears to proceed via a chain-elongation, possibly by addition of a C1-group at the terminal methyl group of the alkane. Finally, aspects concerned with the regulation and ecological significance of anaerobic hydrocarbon catabolic pathways are discussed.

Phelps C.D., Kerkhof L.J., Young L.Y.

A stable and sediment-free, benzene mineralizing, sulfate-reducing culture that resisted repeated attempts at isolation was examined using molecular approaches such as traditional cloning and sequencing and a direct PCR fingerprinting method for 16S rRNA genes. Despite the culture's long exposure to benzene as the only carbon and energy source (over 3 years) and repeated dilutions of the original enrichment, this consortium has remained relatively complex. Cloning and sequence analysis identified 12 unique small subunit rRNA genes. The 16S rRNA genes belong to different eubacterial phyla, including Proteobacteria, Cytophagales and Gram-positives. There is one deeply branching clone which is not closely related to any known, sequenced, bacterium. A different clone, however, is closely related to a known sulfidogenic, aromatic hydrocarbon degrader. To assess 16S rRNA gene cloning efficiency, a fingerprinting method based on fluorescent, end-labeling of PCR product (16S rRNA genes) and screening by restriction length polymorphism analysis (RFLP) was employed. The data obtained indicated that we had cloned and characterized nearly all of the eubacterial 16S rRNA genes amplified from the consortia.

Barrouilhet S., Monperrus M., Tessier E., Khalfaoui-Hassani B., Guyoneaud R., Isaure M., Goñi-Urriza M.

Mercury (Hg) is a global pollutant of environmental and health concern; its methylated form, methylmercury (MeHg), is a potent neurotoxin. Sulfur-containing molecules play a role in MeHg production by microorganisms. While sulfides are considered to limit Hg methylation, sulfate and cysteine were shown to favor this process. However, these two forms can be endogenously converted by microorganisms into sulfide. Here, we explore the effect of sulfide (produced by the cell or supplied exogenously) on Hg methylation. For this purpose, Pseudodesulfovibrio hydrargyri BerOc1 was cultivated in non-sulfidogenic conditions with addition of cysteine and sulfide as well as in sulfidogenic conditions. We report that Hg methylation depends on sulfide concentration in the culture and the sulfides produced by cysteine degradation or sulfate reduction could affect the Hg methylation pattern. Hg methylation was independent of hgcA expression. Interestingly, MeHg production was maximal at 0.1–0.5 mM of sulfides. Besides, a strong positive correlation between MeHg in the extracellular medium and the increase of sulfide concentrations was observed, suggesting a facilitated MeHg export with sulfide and/or higher desorption from the cell. We suggest that sulfides (exogenous or endogenous) play a key role in controlling mercury methylation and should be considered when investigating the impact of Hg in natural environments.

Goñi-Urriza M., Duran R.

Microbial mats are well-structured bacterial communities where the different functional groups strongly interact. They develop on shorelines contaminated after oil spills and have the capacity to grow on the spilled oil. Their crude oil degradation capacity is now well established in laboratory studies, demonstrating efficient degradation of model hydrocarbon compounds. However, their in situ degradation capacities are more controversial. In many cases, it is likely that the mats are trapping oil in deeper anoxic zones, protecting the oil from abiotic transformations. In the last decade, high-throughput sequencing approaches have improved our knowledge of the biodiversity of microbial mats allowing us to describe the changes of microbial communities in response to the presence of crude oil. Many strains, belonging to all of the main functional groups found in microbial mats, have been described for their capacity to degrade oil compounds under both oxic and anoxic conditions. Their abundance significantly increases in the mats after an oil input, highlighting the potential of microbial mats for oil degradation. But microbial mats appear, in many cases, ineffective for in situ bioremediation. Nevertheless, ex situ use of mats communities for oil degradation is promising for bioremediation.

Bridou R., Rodriguez-Gonzalez P., Stoichev T., Amouroux D., Monperrus M., Navarro P., Tessier E., Guyoneaud R.

In this study, axenic cultures of sulfate-reducing (SRB) and nitrate-reducing (NRB) bacteria were examined for their ability to methylate inorganic tin and to methylate or dealkylate butyltin compounds. Environmentally relevant concentrations of natural abundance tributyltin (TBT) and 116Sn-enriched inorganic tin were added to bacterial cultures to identify bacterial-mediated methylation and dealkylation reactions. The results show that none of the Desulfovibrio strains tested was able to induce any transformation process. In contrast, Desulfobulbus propionicus strain DSM-6523 degraded TBT either under sulfidogenic or non-sulfidogenic conditions. In addition, it was able to alkykate 116Sn-enriched inorganic tin leading to the formation of more toxic dimethyltin and trimethyltin. A similar capacity was observed for incubations of Pseudomonas but with a much greater dealkykation of TBT. As such, Pseudomonas sp. ADR42 degraded 61% of the initial TBT under aerobic conditions and 35% under nitrate-reducing conditions. This is the first work reporting a simultaneous TBT degradation and a methylation of both inorganic tin species and TBT dealkykation products by SRB and NRB under anoxic conditions. These reactions are environmentally relevant as they can control the mobility of these compounds in aquatic ecosystems; as well as their toxicity toward resident organisms.

Goñi-Urriza M., Duran R.

Microbial mats are well-structured bacterial communities where the different functional groups strongly interact. They develop on shorelines contaminated after oil spills and have the capacity to grow on the spilled oil. Their crude oil degradation capacity is now well established in laboratory studies, demonstrating efficient degradation of model hydrocarbon compounds. However, their in situ degradation capacities are more controversial. In many cases, it is likely that the mats are trapping oil in deeper anoxic zones, protecting the oil from abiotic transformations. In the last decade, high-throughput sequencing approaches have improved our knowledge of the biodiversity of microbial mats allowing us to describe the changes of microbial communities in response to the presence of crude oil. Many strains, belonging to all of the main functional groups found in microbial mats, have been described for their capacity to degrade oil compounds under both oxic and anoxic conditions. Their abundance significantly increases in the mats after an oil input, highlighting the potential of microbial mats for oil degradation. But microbial mats appear, in many cases, ineffective for in situ bioremediation. Nevertheless, ex situ use of mats communities for oil degradation is promising for bioremediation.

Ranchou-Peyruse M., Goñi-Urriza M., Guignard M., Goas M., Ranchou-Peyruse A., Guyoneaud R.

The strain BerOc1T was isolated from brackish sediments contaminated with hydrocarbons and heavy metals. This strain has been used as a model strain of sulfate-reducer to study the biomethylation of mercury. The cells are vibrio-shaped, motile and not sporulated. Phylogeny and physiological traits placed this strain within the genus Pseudodesulfovibrio . Optimal growth was obtained at 30 °C, 1.5 % NaCl and pH 6.0–7.4. The estimated G+C content of the genomic DNA was 62.6 mol%. BerOc1T used lactate, pyruvate, fumarate, ethanol and hydrogen. Terminal electron acceptors used were sulfate, sulfite, thiosulfate and DMSO. Only pyruvate could be used without a terminal electron acceptor. The major fatty acids were C18 : 0, anteiso-C15 : 0, C16 : 0 and C18 : 1ω7. The name Pseudodesulfovibrio hydrargyri sp. nov. is proposed for the type strain BerOc1T (DSM 10384T=JCM 31820T).

Stal L.J., Bolhuis H., Cretoiu M.S.

Microbial mats are structured, small-scale microbial ecosystems, and similar as biofilms cover a substratum like a tissue. A general characteristic of a microbial mat is the steep physicochemical gradients that are the result of the metabolic activities of the mat microorganisms. Virtually every microbial mat is formed through autotrophic metabolism and through the fixation of atmospheric dinitrogen. Chemoautotrophic organisms fuel these processes in the absence of light. In illuminated environments photoautotrophic organisms are the driving force and these mats are subject of this chapter. In the vast majority of cases, primary production by the oxygenic phototrophic cyanobacteria is the basis of a diverse community that forms a living entity with a macroscopic habitus. This entity has its own physiology that is the result of interaction, communication, cooperation, and competition of the individual functional groups of microorganisms. Organic matter is remineralized and in sulfur-dominated environments sulfate-reducing bacteria are responsible for end-oxidation that leads to the production of sulfide, which is used by anoxygenic photoautotrophic bacteria. Aerobic and anaerobic anoxygenic phototrophic bacteria and proteorhodopsin-containing bacteria are important as secondary producers and take care of the decomposition of organic matter in a process that is aided by light.

Goñi Urriza M., Gassie C., Bouchez O., Klopp C., Guyoneaud R.

ABSTRACT Desulfovibrio BerOc1 is a sulfate-reducing bacterium isolated from the Berre lagoon (French Mediterranean coast). BerOc1 is able to methylate and demethylate mercury. The genome size is 4,081,579 bp assembled into five contigs. We identified the hgcA and hgcB genes involved in mercury methylation, but not those responsible for mercury demethylation.

Horel A., Mortazavi B., Sobecky P.A.

We investigated different types of biostimulation practices to enhance degradation of weathered conventional diesel fuel in sandy beach sediments from coastal Alabama. Biodegradation rates were measured following the addition of either inorganic nutrients, or organic matter derived from either plant material (Spartina alterniflora) or fish tissue (Chloroscombrus chrysurus) both common to the region. The greatest hydrocarbon degradation rates were observed in the C. chrysurus amended treatments (k=0.0119 d(-1)). Treatment with fish-derived organic matter increased the degradation rates by 104% as compared to control treatments, while inorganic nutrient addition increased the degradation rates by 57%. The addition of plant derived organic matter, however, only marginally enhanced the degradation rates (~7%) during the course of the study. Bacterial 16S rRNA analyses revealed that most sediment microorganisms belonged to the classes; Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Bacteroidetes. The most frequently abundant hydrocarbon degraders were mostly similar to Achromobater sp., Microbulbifer sp., Ruegeria sp., and Pseudomonas sp.

Duran R., Bonin P., Jezequel R., Dubosc K., Gassie C., Terrisse F., Abella J., Cagnon C., Militon C., Michotey V., Gilbert F., Cuny P., Cravo-Laureau C.

The present study aimed to examine whether the physical reworking of sediments by harrowing would be suitable for favouring the hydrocarbon degradation in coastal marine sediments. Mudflat sediments were maintained in mesocosms under conditions as closer as possible to those prevailing in natural environments with tidal cycles. Sediments were contaminated with Ural blend crude oil, and in half of them, harrowing treatment was applied in order to mimic physical reworking of surface sediments. Hydrocarbon distribution within the sediment and its removal was followed during 286 days. The harrowing treatment allowed hydrocarbon compounds to penetrate the first 6 cm of the sediments, and biodegradation indexes (such as n-C18/phytane) indicated that biodegradation started 90 days before that observed in untreated control mesocosms. However, the harrowing treatment had a severe impact on benthic organisms reducing drastically the macrofaunal abundance and diversity. In the harrowing-treated mesocosms, the bacterial abundance, determined by 16S rRNA gene Q-PCR, was slightly increased; and terminal restriction fragment length polymorphism (T-RFLP) analyses of 16S rRNA genes showed distinct and specific bacterial community structure. Co-occurrence network and canonical correspondence analyses (CCA) based on T-RFLP data indicated the main correlations between bacterial operational taxonomic units (OTUs) as well as the associations between OTUs and hydrocarbon compound contents further supported by clustered correlation (ClusCor) analysis. The analyses highlighted the OTUs constituting the network structural bases involved in hydrocarbon degradation. Negative correlations indicated the possible shifts in bacterial communities that occurred during the ecological succession.

Goñi-Urriza M., Corsellis Y., Lanceleur L., Tessier E., Gury J., Monperrus M., Guyoneaud R.

The proteins encoded by the hgcA and hgcB genes are currently the only ones known to be involved in the mercury methylation by anaerobic microorganisms. However, no studies have been published to determine the relationships between their expression level and the net/gross methylmercury production. This study aimed to decipher the effect of growth conditions on methylmercury production and the relationships between hgcA and hgcB expression levels and net methylation. Desulfovibrio dechloroacetivorans strain BerOc1 was grown under sulfidogenic conditions with different carbon sources and electron donors as well as under fumarate respiration. A good correlation was found between the biomass production and the methylmercury production when the strain was grown under sulfate-reducing conditions. Methylmercury production was much higher under fumarate respiration when no sulfide was produced. During exponential growth, hgcA and hgcB gene expression levels were only slightly higher in the presence of inorganic mercury, and it was difficult to conclude whether there was a significant induction of hgcA and hgcB genes by inorganic mercury. Besides, no relationships between hgcA and hgcB expression levels and net mercury methylation could be observed when the strain was grown either under sulfate reduction or fumarate respiration, indicating that environmental factors had more influence than expression levels.

Duran R., Goňi-Urriza M.S.

Microbial mats are structured bacterial communities in which interactions between the different functional groups are enhanced. Such mats developed on shore lines contaminated after oil spills and showed the capacity to grow on the spilled oil. Their capacity to degrade crude oil is nevertheless subject to controversy. While many laboratory studies demonstrated degradation of model hydrocarbon compounds, other suggested that, in situ, the mats protected the oil from abiotic transformations. Many strains, belonging to all of the main functional groups found in microbial mats, have been described for their capacity to degrade oil compounds under either oxic and anoxic conditions. Thus the potential for oil degradation exists in the microbial mats but in situ bioremediation by using mats seems ineffective. Nevertheless, ex situ use of mats communities for oil degradation is a promising way of biodegradation.

Païssé S., Goñi-Urriza M.S., Fahy A., Duran R.

Bacterial community fingerprinting approaches are widely used to characterize the structure of bacterial communities and assess their dynamics upon changing environmental conditions. We describe here a protocol for terminal restriction fragment length polymorphism (T-RFLP) that offers the possibility to obtain good resolution community profiles. In addition, we describe a number of statistical approaches for analysing T-RFLP data. T-RFLP can be carried out in high-throughput and semi-automated, which makes it possible to rapidly compare bacterial communities from a large number of samples. We will present examples of community adaptation in response to oil exposure.

Ranchou-Peyruse M., Monperrus M., Bridou R., Duran R., Amouroux D., Salvado J.C., Guyoneaud R.

Mercury methylation has been extensively reported in the literature among “Firmicutes” and “Proteobacteria.” Nevertheless, results are hardly comparable because of differences in initial inorganic mercury concentrations used. The use of stable isotopic tracers now permits to study mercury transformations at concentrations close to environmental levels. Here, several strains, including strict fermentative and sulphate-reducing bacteria, were tested for their mercury methylation capacities and the results were compared with data available to date. Under such conditions, mercury methylation only occurs among the delta-Proteobacteria. The absence of relation between taxonomic/phylogenetic affiliation and mercury methylation capacities was pointed out and discussed for environmental studies.

Bordenave S., Goñi-urriza M., Vilette C., Blanchard S., Caumette P., Duran R.

The aim of this work was to characterize bacterial ring-hydroxylating dioxygenase (RHD) diversity in a pristine microbial mat and follow their diversity changes in response to heavy fuel oil contamination. In order to describe the RHDs diversity, new degenerate primers were designed and a nested-PCR approach was developed to gain sensitivity and wider diversity. RHD diversity in artificially contaminated mats maintained in microcosms and in chronically contaminated mats was analysed by clone libraries and terminal restriction fragment length polymorphism (T-RFLP) at genomic and transcriptomic levels. The RHD diversity in the pristine microbial mat was represented by Pseudomonas putida nahAc-like genes and no increase of diversity was detected after 1 year of oil contamination. The diversity observed in a 30 year chronically polluted microbial mat was represented by four main RHD clusters and two new genes revealing higher polyaromatic hydrocarbon (PAH) degradation capacity. This study illustrates that a single petroleum contamination (such as oil spill) is not enough to involve a detectable modification of RHD diversity. The new degenerate primers described here allowed RHD gene amplification from pristine and contaminated samples thereby showing their diversity. The proposed approach solves one of the main problems of functional gene analysis providing effective amplification of the environmental diversity of the targeted genes.

Ranchou-Peyruse A., Herbert R., Caumette P., Guyoneaud R.

Phototrophic anoxygenic purple bacteria play a key role in many aquatic ecosystems by oxidizing sulfur compounds and low-molecular-weight organic compounds using light as energy source. In this study, molecular methods based upon pufM gene (photosynthetic unit forming gene) were compared with culture-dependent methods to investigate anoxygenic purple phototrophic communities in sediments of an eutrophic brackish lagoon. Thirteen strains, belonging to eight different genera of purple phototrophic bacteria were isolated with a large dominance of the metabolically versatile purple non-sulfur bacteria (eight strains), some purple sulfur bacteria (three strains) and two strains belonging to the Roseobacter clade (aerobic phototrophs). The pufM genes amplified from the isolated strains were not detected by the molecular methods [terminal-restriction fragment length polymorphism (T-RFLP)] applied on in situ communities. An environmental clone library of the pufM gene was thus constructed from sediment samples. The results showed that most of the clones probably corresponded to aerobic phototrophic bacteria. Our results demonstrate that the culture-dependent techniques remain the best experimental approach for determining the diversity of phototrophic purple non-sulfur bacteria whereas the molecular approach clearly illustrated the abundance of organisms related to the Roseobacter clade in these eutrophic sediments.