Biodegradation of saturate fraction of crude oil and production of signature carboxylic acids.

Bacteria degrading large portion of saturated hydrocarbons are important for crude oil bioremediation. This study investigates Novosphingobium sp. S1, Gordonia amicalis S2 and Gordonia terrae S5 capability of degrading wide range of saturated hydrocarbons from Congo Bilondo crude oil and discusses the degradation pathway. A parallel analytical approach combining GC-MS and LC-HRMS enabled characterization of saturated hydrocarbons and comprehensive determination of carboxylic acid metabolites produced during biodegradation, respectively. Results showed that the three strains could efficiently degrade the n-alkanes (C10-C28) as well as methyl-substituted alkanes (C11-C26). The series of mono-, hydroxy- and dicarboxylic acids identified in this study confirmed the active biodegradation of the saturate fraction and suggest their degradation was via the bi-terminal oxidation pathway. This is the first study linking these bacterial species to bi-terminal oxidation of the saturated hydrocarbons. The study highlights the potential application of the bacterial strains in the bioremediation of crude oil contaminated sites. Additionally, while carboxylic acids is indicated as a suitable and valuable metabolic biomarker, its application is considered feasible and cost effective for rapid monitoring and evaluation of hydrocarbon biodegradation.

Quorum sensing in biofilms: a key mechanism to target in ecotoxicological studies.

Our environment is heavily contaminated by anthropogenic compounds, and this issue constitutes a significant threat to all life forms, including biofilm-forming microorganisms. Cell-cell interactions shape microbial community structures and functions, and pollutants that affect intercellular communications impact biofilm functions and ecological roles. There is a growing interest in environmental science fields for evaluating how anthropogenic pollutants impact cell-cell interactions. In this review, we synthesize existing literature that evaluates the impacts of quorum sensing (QS), which is a widespread density-dependent communication system occurring within many bacterial groups forming biofilms. First, we examine the perturbating effects of environmental contaminants on QS circuits; and our findings reveal that QS is an essential yet underexplored mechanism affected by pollutants. Second, our work highlights that QS is an unsuspected and key resistance mechanism that assists bacteria in dealing with environmental contamination (caused by metals or organic pollutants) and that favors bacterial growth in unfavourable environments. We emphasize the value of considering QS a critical mechanism for monitoring microbial responses in ecotoxicology. Ultimately, we determine that QS circuits constitute promising targets for innovative biotechnological approaches with major perspectives for applications in the field of environmental science.

Features of the Opportunistic Behaviour of the Marine Bacterium Marinobacter algicola in the Microalga Ostreococcus tauri Phycosphere.

Although interactions between microalgae and bacteria are observed in both natural environment and the laboratory, the modalities of coexistence of bacteria inside microalgae phycospheres in laboratory cultures are mostly unknown. Here, we focused on well-controlled cultures of the model green picoalga Ostreococcus tauri and the most abundant member of its phycosphere, Marinobacter algicola. The prevalence of M. algicola in O. tauri cultures raises questions about how this bacterium maintains itself under laboratory conditions in the microalga culture. The results showed that M. algicola did not promote O. tauri growth in the absence of vitamin B12 while M. algicola depended on O. tauri to grow in synthetic medium, most likely to obtain organic carbon sources provided by the microalgae. M. algicola grew on a range of lipids, including triacylglycerols that are known to be produced by O. tauri in culture during abiotic stress. Genomic screening revealed the absence of genes of two particular modes of quorum-sensing in Marinobacter genomes which refutes the idea that these bacterial communication systems operate in this genus. To date, the 'opportunistic' behaviour of M. algicola in the laboratory is limited to several phytoplanktonic species including Chlorophyta such as O. tauri. This would indicate a preferential occurrence of M. algicola in association with these specific microalgae under optimum laboratory conditions.

AES and ToF-SIMS combination for single cell chemical imaging of gold nanoparticle-labeled Escherichia coli.

A chemical fingerprint of the Escherichia coli cell surface labeled by gelatin coated gold nanoparticles was obtained by combining Auger Electron Spectroscopy (AES) for single cell level chemical images, and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) Tandem MS for unambiguous molecular identification of co-localized species.

Description of Palleronia rufa sp. nov., a biofilm-forming and AHL-producing Rhodobacteraceae, reclassification of Hwanghaeicola aestuarii as Palleronia aestuarii comb. nov., Maribius pontilimi as Palleronia pontilimi comb. nov., Maribius salinus as Palleronia salina comb. nov., Maribius pelagius as Palleronia pelagia comb. nov. and emended description of the genus Palleronia.

Strain MOLA 401T was isolated from marine waters in the southwest lagoon of New Caledonia and was shown previously to produce an unusual diversity of quorum sensing signaling molecules. This strain was Gram-negative, formed non-motile cocci and colonies were caramel. Optimum growth conditions were 30°C, pH 8 and 3% NaCl (w/v). Based on 16S rRNA gene sequence analysis, this strain was found to be closely related to Pseudomaribius aestuariivivens NBRC 113039T (96.9% of similarity), Maribius pontilimi DSM 104950T (96.4% of similarity) and Palleronia marisminoris LMG 22959T (96.3% of similarity), belonging to the Roseobacter group within the family Rhodobacteraceae. As its closest relatives, strain MOLA 401T is able to form a biofilm on polystyrene, supporting the view of Roseobacter group strains as prolific surface colonizers. An in-depth genomic study allowed us to affiliate strain MOLA 401T as a new species of genus Palleronia and to reaffiliate some of its closest relatives in this genus. Consequently, we describe strain MOLA 401T (DSM 106827T=CIP 111607T=BBCC 401T) for which we propose the name Palleronia rufa sp. nov. We also propose to emend the description of the genus Palleronia and to reclassify Maribius and Hwanghaeicola species as Palleronia species.

Biofilm formation as a microbial strategy to assimilate particulate substrates.

In most ecosystems, a large part of the organic carbon is not solubilized in the water phase. Rather, it occurs as particles made of aggregated hydrophobic and/or polymeric natural or man-made organic compounds. These particulate substrates are degraded by extracellular digestion/solubilization implemented by heterotrophic bacteria that form biofilms on them. Organic particle-degrading biofilms are widespread and have been observed in aquatic and terrestrial natural ecosystems, in polluted and man-driven environments and in the digestive tracts of animals. They have central ecological functions as they are major players in carbon recycling and pollution removal. The aim of this review is to highlight bacterial adhesion and biofilm formation as central mechanisms to exploit the nutritive potential of organic particles. It focuses on the mechanisms that allow access and assimilation of non-dissolved organic carbon, and considers the advantage provided by biofilms for gaining a net benefit from feeding on particulate substrates. Cooperative and competitive interactions taking place in biofilms feeding on particulate substrates are also discussed.

Pleomorphochaeta naphthae sp. nov., a new anaerobic fermentative bacterium isolated from an oil field.

A novel anaerobic fermentative bacterium, strain SEBR 4209T, was isolated from a water sample of a Congolese oil field. Strain SEBR 4209T is phylogenetically related to the genus Pleomorphochaeta, in the family Spirochaetaceae. Its closest relatives are Pleomorphochaeta caudata SEBR 4223T (94.5 % 16S rRNA gene sequence similarity) and Pleomorphochaeta multiformis MO-SPC2T (94.3 % similarity). Like the other members of this genus, cells have a pleomorphic morphology, in particular an annular shape and long stalks. Optimal growth was observed at 37 °C, at pH between 6.8 and 7.0, and with 40 g l-1 NaCl. This strain was only able to grow by fermentation of carbohydrates. The fermentation products from glucose utilization were acetate, ethanol, CO2 and H2. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c. The major polar lipids were phosphoglycolipids, phospholipids and glycolipids. The G+C content of the DNA was 29.6 mol%. Based on phenotypic characteristics and phylogenetic traits, strain SEBR 4209T is considered to represent a novel species of the genus Pleomorphochaeta, for which the name Pleomorphochaetanaphthae sp. nov. is proposed. The type strain is SEBR 4209T (=DSM 104684T=JCM 31871T).

AupA and AupB Are Outer and Inner Membrane Proteins Involved in Alkane Uptake in Marinobacter hydrocarbonoclasticus SP17.

This study describes the functional characterization of two proteins, AupA and AupB, which are required for growth on alkanes in the marine hydrocarbonoclastic bacterium Marinobacter hydrocarbonoclasticus The aupA and aupB genes form an operon whose expression was increased upon adhesion to and biofilm formation on n-hexadecane. AupA and AupB are outer and inner membrane proteins, respectively, which are able to interact physically. Mutations in aupA or/and aupB reduced growth on solid paraffin and liquid n-hexadecane, while growth on nonalkane substrates was not affected. In contrast, growth of aup mutants on n-hexadecane solubilized in Brij 58 micelles was completely abolished. Mutant cells had also lost the ability to bind to n-hexadecane solubilized in Brij 58 micelles. These results support the involvement of AupA and AupB in the uptake of micelle-solubilized alkanes and provide the first evidence for a cellular process involved in the micellar uptake pathway. The phylogenetic distribution of the aupAB operon revealed that it is widespread in marine hydrocarbonoclastic bacteria of the orders Oceanospirillales and Alteromonadales and that it is present in high copy number (up to six) in some Alcanivorax strains. These features suggest that Aup proteins probably confer a selective advantage in alkane-contaminated seawater.IMPORTANCE Bacteria are the main actors of the biological removal of hydrocarbons in seawater, and so, it is important to understand how they degrade hydrocarbons and thereby mitigate marine environmental damage. Despite a considerable amount of literature about the dynamic of microbial communities subjected to hydrocarbon exposure and the isolation of strains that degrade hydrocarbons, most of the genetic determinants and molecular mechanisms of bacterial hydrocarbon uptake remain unknown. This study identifies two genes, aupA and aupB, in the hydrocarbonoclastic bacterium Marinobacter hydrocarbonoclasticus that are present frequently in multiple copies in most of the marine hydrocarbon-degrading bacteria for which the genomic sequence is available. AupA and AupB are two novel membrane proteins interacting together that are involved in the uptake of alkanes dissolved in surfactant micelles. The function and the phylogenetic distribution of aupA and aupB suggest that they might be one attribute of the remarkable adaptation of marine hydrocarbonoclastic bacteria that allow them to take advantage of hydrocarbons.

Impact of temperature on Marinobacter hydrocarbonoclasticus SP17 morphology and biofilm structure during growth on alkanes.

Alkanes are widespread pollutants found in soil, freshwater and marine environments. Marinobacter hydrocarbonoclasticus (Mh) strain SP17 is a marine bacterium able to use many hydrophobic organic compounds, including alkanes, through the production of biofilms that allow their poor solubility to be overcome. This study pointed out that temperature is an environmental factor that strongly affects the biofilm formation and morphology of Mh on the model alkanes, hexadecane and paraffin. We showed that Mh biofilm formation and accumulation of intracytoplasmic inclusions are higher on solid alkanes (hexadecane at 10 °C and paraffin at 10 °C and 30 °C) than on liquid alkane (hexadecane at 30 °C) or soluble substrate (lactate at both temperatures). We also found that Mh produces more extracellular polymeric substances at 30 °C than at 10 °C on alkanes and none on lactate. We observed that bacterial length is significantly higher at 10 °C than at 30 °C on lactate and hexadecane. On paraffin, at 30 °C, the cell morphology is markedly altered by large rounded or irregularly shaped cytoplasmic inclusions. Altogether, the results showed that Mh is able to adapt and use alkanes as a carbon source, even at low temperature.

Pleomorphochaeta caudata gen. nov., sp. nov., an anaerobic bacterium isolated from an offshore oil well, reclassification of Sphaerochaeta multiformis MO-SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov. as the type strain of this novel genus and emended description of the genus Sphaerochaeta.

A strictly anaerobic Gram-stain-negative bacterium, designated strain SEBR 4223T, was isolated from the production water of an offshore Congolese oil field. Cells were non-motile, pleomorphic and had spherical, annular or budding shapes, often exhibiting long stalks. Strain SEBR 4223T grew on a range of carbohydrates, optimally at 37 °C and pH 7, in a medium containing 40 g l-1 NaCl. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c and the major polar lipids were phosphoglycolipids, phospholipids, glycolipids and diphosphatidylglycerol. The G+C content of the DNA was 28.7 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain SEBR 4223T and Sphaerochaeta multiformis MO-SPC2T formed a cluster with similarity to other species of the genus Sphaerochaeta of of less than 86 %. On the basis of the phenotypic characteristics and taxonomic analyses, we propose a novel genus, Pleomorphochaeta gen. nov., to accommodate the novel species Pleomorphochaeta caudata sp. nov., with SEBR 4223T (=DSM 103077T=JCM 31 475T) as the type strain. We also propose the reclassification of Sphaerochaeta multiformis MO SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov., the type strain of this novel genus and emend description of the genus Sphaerochaeta.

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates.

Substrates specialization in lipid compounds and hydrocarbons of Marinobacter genus.

The impact of petroleum contamination and of burrowing macrofauna on abundances of Marinobacter and denitrifiers was tested in marine sediment mesocoms after 3 months incubation. Quantification of this genus by qPCR with a new primer set showed that the main factor favoring Marinobacter abundance was hydrocarbon amendment followed by macrofauna presence. In parallel, proportion of nosZ-harboring bacteria increased in the presence of marcrofauna. Quantitative finding were explained by physiological data from a set of 34 strains and by genomic analysis of 16 genomes spanning 15 different Marinobacter-validated species (Marinobacter hydrocarbonoclasticus, Marinobacter daeopensis, Marinobacter santoriniensis, Marinobacter pelagius, Marinobacter flavimaris, Marinobacter adhaerens, Marinobacter xestospongiae, Marinobacter algicola, Marinobacter vinifirmus, Marinobacter maritimus, Marinobacter psychrophilus, Marinobacter lipoliticus, Marinobacter manganoxydans, Marinobacter excellens, Marinobacter nanhaiticus) and 4 potential novel ones. Among the 105 organic electron donors tested in physiological analysis, Marinobacter pattern appeared narrow for almost all kinds of organic compounds except lipid ones. Strains of this set could oxidize a very large spectrum of lipids belonging to glycerolipids, branched, fatty acyls, and aromatic hydrocarbon classes. Physiological data were comforted by genomic analysis, and genes of alkane 1-monooxygenase, haloalkane dehalogenase, and flavin-binding monooxygenase were detected in most genomes. Denitrification was assessed for several strains belonging to M. hydrocarbonoclasticus, M. vinifirmus, Marinobacter maritinus, and M. pelagius species indicating the possibility to use nitrate as alternative electron acceptor. Higher occurrence of Marinobacter in the presence of petroleum appeared to be the result of a broader physiological trait allowing this genus to use lipids including hydrocarbon as principal electron donors.

The marine bacterium Marinobacter hydrocarbonoclasticus SP17 degrades a wide range of lipids and hydrocarbons through the formation of oleolytic biofilms with distinct gene expression profiles.

Hydrophobic organic compounds (mainly lipids and hydrocarbons) represent a significant part of the organic matter in marine waters, and their degradation has an important impact in the carbon fluxes within oceans. However, because they are nearly insoluble in the water phase, their degradation by microorganisms occurs at the interface with water and thus requires specific adaptations such as biofilm formation. We show that Marinobacter hydrocarbonoclasticus SP17 develops biofilms, referred to as oleolytic biofilms, on a large variety of hydrophobic substrates, including hydrocarbons, fatty alcohols, fatty acids, triglycerides, and wax esters. Microarray analysis revealed that biofilm growth on n-hexadecane or triolein involved distinct genetic responses, together with a core of common genes that might concern general mechanisms of biofilm formation. Biofilm growth on triolein modulated the expression of hundreds of genes in comparison with n-hexadecane. The processes related to primary metabolism and genetic information processing were downregulated. Most of the genes that were overexpressed on triolein had unknown functions. Surprisingly, their genome localization was restricted to a few regions identified as putative genomic islands or mobile elements. These results are discussed with regard to the adaptive responses triggered by M. hydrocarbonoclasticus SP17 to occupy a specific niche in marine ecosystems.

Genome sequence of the marine bacterium Marinobacter hydrocarbonoclasticus SP17, which forms biofilms on hydrophobic organic compounds.

Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration.

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface.

Biofilm formation by marine hydrocarbonoclastic bacteria is commonly observed and has been recognized as an important mechanism for the biodegradation of hydrocarbons. In order to colonize new oil-water interfaces, surface-attached communities of hydrocarbonoclastic bacteria must release cells into the environment. Here we explored the physiology of cells freshly dispersed from a biofilm of Marinobacter hydrocarbonoclasticus developing at the hexadecane-water interface, by combining proteomic and physiological approaches. The comparison of the dispersed cells' proteome with those of biofilm, logarithmic- and stationary-phase planktonic cells indicated that dispersed cells had lost most of the biofilm phenotype and expressed a specific proteome. Two proteins involved in cell envelope maturation, DsbA and CtpA, were exclusively detected in dispersed cells, suggesting a reshaping of the cell envelopes during biofilm dispersal. Furthermore, dispersed cells exhibited a higher affinity for hexadecane and initiated more rapidly biofilm formation on hexadecane than the reference planktonic cells. Interestingly, storage wax esters were rapidly degraded in dispersed cells, suggesting that their observed physiological properties may rely on reserve mobilization. Thus, by promoting oil surface colonization, cells emigrating from the biofilm could contribute to the success of marine hydrocarbonoclastic bacteria in polluted environments.

Behavior of Marinobacter hydrocarbonoclasticus SP17 cells during initiation of biofilm formation at the alkane-water interface.

Hexadecane assimilation by Marinobacter hydrocarbonoclasticus SP17 occurs through the formation of a biofilm at the alkane-water interface. In this study we focused on the interactions of cells with the alkane-water interface occurring during initiation of biofilm development. The behavior of cells at the interface was apprehended by investigating alterations of the mechanical properties of the interface during cell adsorption, using dynamic drop tensiometry measurements. It was found that after having reached the hexadecane-water interface, by a purely thermal diffusion process, cells released surface-active compounds (SACs) resulting in the formation of an interfacial visco-elastic film. Release of SACs was an active process requiring protein synthesis. This initial interaction occurred on metabolizable as well as non-metabolizable alkanes, indicating that at this stage cells are not affected by the nature of the alkane forming the interface. In contrast, at a later stage, the nature of the interface turned out to exert control over the behavior of the cells. The availability of a metabolizable alkane at the interface influenced cell activity, as revealed by cell cluster formation and differences in the interfacial elasticity.

Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the alkane-water interface reveals novel proteins and cellular processes involved in hexadecane assimilation.

Many hydrocarbon-degrading bacteria form biofilms at the hydrocarbon-water interface to overcome the weak accessibility of these poorly water-soluble substrates. In order to gain insight into the cellular functions involved, we undertook a proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm developing at the hexadecane-water interface. Biofilm formation on hexadecane led to a global change in cell physiology involving modulation of the expression of 576 out of 1144 detected proteins when compared with planktonic cells growing on acetate. Biofilm cells overproduced a protein encoded by MARHY0478 that contains a conserved domain belonging to the family of the outer membrane transporters of hydrophobic compounds. Homologs of MARHY0478 were exclusively found in marine bacteria degrading alkanes or possessing alkane degradation genes, and hence presumably constitute a family of alkane transporters specific to marine bacteria. Interestingly, we also found that sessile cells growing on hexadecane overexpressed type VI secretion system components. This secretion system has been identified as a key factor in virulence and in symbiotic interaction with host organisms. This observation is the first experimental evidence of the contribution of a type VI secretion system to environmental adaptation, and raises the intriguing question about the role of this secretion machine in alkane assimilation.

Cytoplasmic wax ester accumulation during biofilm-driven substrate assimilation at the alkane--water interface by Marinobacter hydrocarbonoclasticus SP17.

During growth on n-alkanes, the marine bacterium Marinobacter hydrocarbonoclasticus SP17 formed a biofilm at the alkane-water interface. We showed that hexadecane degradation was correlated with biofilm development and that alkane uptake is localized in the biofilm but not in the bulk medium. Biofilms were observed in cultures on metabolizable n-alkanes (C8-C28) and n-alcohols (C12 and C16), but were formed neither on non-metabolizable alkanes (pristane, heptamethylnonane and n-C32) nor on inert substrata (glass, polystyrene and Permanox). This substratum specificity indicates that biofilm formation is determined by the presence of an interface between an insoluble substrate and the aqueous phase. Simultaneously with biofilm growth, planktonic cells were released from the biofilm. Detached cells were in a non-growing state, implying that the growing population was exclusively located within the biofilm. Planktonic and sessile cells exhibited differences in their ultrastructure and lipid content. Biofilm cells contained a large amount of wax esters (0.47mg/mg protein) in rounded or irregularly shaped cytoplasmic inclusions, whereas detached cells displayed rod-shaped inclusions and contained 5 times fewer wax esters (0.10mg/mg protein) than their sessile counterparts. This study points out the inter-relationship between biofilm formation, insoluble substrate uptake and lipid storage.