Nitrogen and sulfur for phosphorus: Lipidome adaptation of anaerobic sulfate-reducing bacteria in phosphorus-deprived conditions.

Understanding how microbial lipidomes adapt to environmental and nutrient stress is crucial for comprehending microbial survival and functionality. Certain anaerobic bacteria can synthesize glycerolipids with ether/ester bonds, yet the complexities of their lipidome remodeling under varying physicochemical and nutritional conditions remain largely unexplored. In this study, we thoroughly examined the lipidome adaptations of Desulfatibacillum alkenivorans strain PF2803T, a mesophilic anaerobic sulfate-reducing bacterium known for its high proportions of alkylglycerol ether lipids in its membrane, under various cultivation conditions including temperature, pH, salinity, and ammonium and phosphorous concentrations. Employing an extensive analytical and computational lipidomic methodology, we identified an assemblage of nearly 400 distinct lipids, including a range of glycerol ether/ester lipids with various polar head groups. Information theory-based analysis revealed that temperature fluctuations and phosphate scarcity profoundly influenced the lipidome's composition, leading to an enhanced diversity and specificity of novel lipids. Notably, phosphorous limitation led to the biosynthesis of novel glucuronosylglycerols and sulfur-containing aminolipids, termed butyramide cysteine glycerols, featuring various ether/ester bonds. This suggests a novel adaptive strategy for anaerobic heterotrophs to thrive under phosphorus-depleted conditions, characterized by a diverse array of nitrogen- and sulfur-containing polar head groups, moving beyond a reliance on conventional nonphospholipid types.

Legacy and dispersant influence microbial community dynamics in cold seawater contaminated by crude oil water accommodated fractions.

Dispersants, used for combating oil spills, increase hydrocarbon bioavailability promoting their biodegradation. Oil weathering process introduces harmful soluble hydrocarbons, such as polycyclic aromatic hydrocarbons (PAHs), into the water column, resulting in water-accommodated fraction (WAF). The presence of dispersants can influence the weathering process by increasing PAHs solubility, toxicity and biodegradability. However, little is known on how dispersants affect microbial communities and their degradation capacities, especially in cold environment where low temperature decreases microbial activity and thus hydrocarbon degradation. Here, we investigated the microbial community dynamics in cold water contaminated by WAF prepared from crude oil with or without a commercial dispersant (Finasol OSR52). The WAFs, prepared with Naphthenic North Atlantic crude oil, were used to contaminate seawater from Norwegian cold sites, one oil-contaminated and the other pristine. The WAF-contaminated seawaters were maintained in microcosms at 4 °C for 21 days. The content of PAHs and microbial compositions (16S rRNA gene sequencing) were determined at days 0, 7, 14 and 21. In addition, the 96 h toxicity assay with adult Acartia tonsa revealed WAFs toxicity at days 0 and 21. The toxicity of WAF mixtures, with and without dispersant, against Acartia tonsa was reduced during the experiment, but PAHs removal was not increased. The water from the oil-contaminated site showed the highest PAHs removal revealing legacy effect (presence of microorganisms adapted to PAHs). Additionally, our results reveal: i) microbial community plasticity allowing the adaptation to the presence of PAHs and dispersant, ii) specific bacteria taxa probably involved in PAHs degradation, and iii) dispersants shape the microbial communities dynamics by stimulating potential dispersant-degrading taxa, such as Fusibacter. Thus, our results provide valuable insights on the role of microbial community in determining the fate of water-solubilized hydrocarbon in cold environment while questioning the role of dispersant used for fighting oil spill.

Mono- and dialkyl glycerol ether lipids in anaerobic bacteria: biosynthetic insights from the mesophilic sulfate reducer Desulfatibacillum alkenivorans PF2803T.

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1'-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1'-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.

Desulfatiferula berrensis sp. nov., a n-alkene-degrading sulfate-reducing bacterium isolated from estuarine sediments.

A novel sulfate-reducing bacterium designated strain BE2801(T) was isolated from oil-polluted estuarine sediments (Berre Lagoon, France). Cells were Gram-stain-negative, motile, slightly curved or vibrioid rods. Optimal growth of strain BE2801(T) occurred at 30-32 °C, 0.5-1.5% NaCl (w/v) and pH 7.2-7.4. Strain BE2801(T) grew with C4 to C20 fatty acids or C12 to C20 n-alkenes as electron donors. Acetate and carbon dioxide were the oxidation products. The major cellular fatty acids were C16 : 0, C(16 : 1)ω7c and C(18 : 1)ω7. The DNA G+C content was 50.2 mol%. 16S rRNA and dsrAB gene sequence analysis indicated that strain BE2801(T) was a member of the family Desulfobacteraceae within the class Deltaproteobacteria. DNA-DNA hybridization with the most closely related taxon demonstrated 14.8 % relatedness. Based on phenotypic and phylogenetic evidence, strain BE2801(T) ( = DSM 25524(T) = JCM 18157(T)) is proposed to be a representative of a novel species of the genus Desulfatiferula, for which the name Desulfatiferula berrensis sp. nov. is suggested.

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.

Impacts of bioremediation schemes for the mitigation of a low-dose anthracene contamination on free-living marine benthic nematodes.

A microcosm experiment was used to examine (1) the effects of different bioremediation schemes on degradation of anthracene and the structure of free-living marine nematodes in a lightly contaminated (4.5 µg g(-1)) sediment from Bizerte lagoon and (2) the responses of the nematode community upon an artificial spiking of a low dose anthracene (1 µg g(-1)). For that purpose sediment microcosms were incubated in laboratory for 40 days. Bioremediation techniques decreased the anthracene contamination, and interestingly, biodegradation were more efficient when anthracene was artificial supplied into the sediment suggesting that the addition of bioavailable anthracene stimulated the bacterial community to adjust towards a PAH-degrading community. Spiking with this low dose of anthracene provoked significant changes in the nematode community structure and abundance, with the elimination of specific species such as Mesacanthion diplechma, the decrease of the dominant species Oncholaimus campylocercoides and the increase in abundance of opportunistic species such as Spirinia parasitifera. This would suggest a low tolerance of the nematode community despite the presence of a weak anthracene contamination in the sediment that could have allow dominance of an anthracene tolerant nematode species. Anthracene toxicity was alleviated in biostimulation treatments, leading to a strong increase in nematode abundance, concomitantly with changes in the nematode community structure; Prochromadorella neapolitana became the most abundant species.

Greenhouse gas emission potential of tropical coastal sediments in densely urbanized area inferred from metabarcoding microbial community data.

Although benthic microbial community offers crucial insights into ecosystem services, they are underestimated for coastal sediment monitoring. Sepetiba Bay (SB) in Rio de Janeiro, Brazil, holds long-term metal pollution. Currently, SB pollution is majorly driven by domestic effluents discharge. Here, functional prediction analysis inferred from 16S rRNA gene metabarcoding data reveals the energy metabolism profiles of benthic microbial assemblages along the metal pollution gradient. Methanogenesis, denitrification, and N2 fixation emerge as dominant pathways in the eutrophic/polluted internal sector (Spearman; p < 0.05). These metabolisms act in the natural attenuation of sedimentary pollutants. The methane (CH4) emission (mcr genes) potential was found more abundant in the internal sector, while the external sector exhibited higher CH4 consumption (pmo + mmo genes) potential. Methanofastidiosales and Exiguobacterium, possibly involved in CH4 emission and associated with CH4 consumers respectively, are the main taxa detected in SB. Furthermore, SB exhibits higher nitrous oxide (N2O) emission potential since the norB/C gene proportions surpass nosZ up to 4 times. Blastopirellula was identified as the main responsible for N2O emissions. This study reveals fundamental contributions of the prokaryotic community to functions involved in greenhouse gas emissions, unveiling their possible use as sentinels for ecosystem monitoring.

Mono- to tetra-alkyl ether cardiolipins in a mesophilic, sulfate-reducing bacterium identified by UHPLC-HRMSn: a novel class of membrane lipids.

The composition of membrane lipids varies in a number of ways as adjustment to growth conditions. Variations in head group composition and carbon skeleton and degree of unsaturation of glycerol-bound acyl or alkyl chains results in a high structural complexity of the lipidome of bacterial cells. We studied the lipidome of the mesophilic, sulfate-reducing bacterium, Desulfatibacillum alkenivorans strain PF2803T by ultra-high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMSn). This anaerobic bacterium has been previously shown to produce high amounts of mono-and di-alkyl glycerol ethers as core membrane lipids. Our analyses revealed that these core lipids occur with phosphatidylethanomamine (PE) and phosphatidylglycerol (PG) head groups, representing each approximately one third of the phospholipids. The third class was a novel group of phospholipids, i.e., cardiolipins (CDLs) containing one (monoether/triester) to four (tetraether) ether-linked saturated straight-chain or methyl-branched alkyl chains. Tetraether CDLs have been shown to occur in archaea (with isoprenoid alkyl chains) but have not been previously reported in the bacterial Domain. Structurally related CDLs with one or two alkyl/acyl chains missing, so-called monolyso-and dilyso-CDLs, were also observed. The potential biosynthetic pathway of these novel CDLs was investigated by examining the genome of D. alkenivorans. Three CDL synthases were identified; one catalyzes the condensation of two PGs, the other two are probably involved in the condensation of a PE with a PG. A heterologous gene expression experiment showed the in vivo production of dialkylglycerols upon anaerobic expression of the glycerol ester reductase enzyme of D. alkenivorans in E. coli. Reduction of the ester bonds probably occurs first at the sn-1 and subsequently at the sn-2 position after the formation of PEs and PGs.

Cold sediment microbial community shifts in response to crude oil water-accommodated fraction with or without dispersant: a microcosm study.

In cold environments, the low temperature slows down microbial metabolisms, such as the biodegradation processes of hydrocarbons, which are often stimulated by the addition of dispersants in oil spill disasters. In this study, we investigated the effects of hydrocarbon water-accommodated fraction (WAF) prepared with and without dispersant on benthic microbial communities in a microcosm experiment in which hydrocarbon removal was observed. Both WAFs contained similar polycyclic aromatic hydrocarbon (PAH) content. The microcosm experiment, set up with either pristine or contaminated sediments, was conducted for 21 days at 4 °C under WAF and WAF + dispersant conditions. The behavior of bacterial communities in response to WAF and WAF + dispersant was examined at both DNA and RNA levels, revealing the effect of WAF and WAF + dispersant on the resident and active communities respectively. The contaminated sediment showed less taxa responsive to the addition of both WAF and WAF + dispersant than the pristine sediment, indicating the legacy effect by the presence hydrocarbon-degrading and dispersant-resistant taxa inhabiting the contaminated sediment.

Marine Fungi Select and Transport Aerobic and Anaerobic Bacterial Populations from Polycyclic Aromatic Hydrocarbon-Contaminated Sediments.

The organization of microbial communities in marine sediment relies on complex biotic and abiotic interactions. Among them, the interaction between fungi and bacteria plays a crucial role building specific microbial assemblages, resulting in metabolic networks adapted to environmental conditions. The fungal-bacterial interaction (FBI) includes bacterial translocation via fungal mycelia, allowing bacterial dispersion, and ecological niche colonization. In order to demonstrate that the translocation of bacteria through fungal mycelia involves bacterial selection, the mycelia of two fungi isolated from marine coastal sediment, Alternaria destruens F10.81 and Fusarium pseudonygamai F5.76, showing different strategies for uptake of polycyclic aromatic hydrocarbon (PAH), homogenous internalization and vacuole forming respectively, were used to translocate bacteria through hydrophobic hydrocarbon contaminated sediments. A. destruens F10.81 selected four specific bacteria, while bacterial selection by F. pseudonygamai F5.76 was not evident. Among the bacteria selected by A. destruens F10.81, Spirochaeta litoralis, known as strictly anaerobic bacterium, was identified, indicating that A. destruens F10.81 selects and transports both aerobic and anaerobic bacteria. Such a result is consistent with the observed formation of anoxic micro-niches in areas surrounding and affected by fungal hyphae. Our findings provide new insights on the selection and dispersion of bacterial communities by fungi, which are crucial for the organization of microbial communities and their functioning in coastal PAH-contaminated sediments. IMPORTANCE The study provides advances for understanding fungal-bacterial relationships, particularly on the selection and dispersion of bacterial communities by fungi, which are crucial for the organization of microbial communities and their functioning in coastal PAH-contaminated sediments. The transportation of bacteria via fungal hyphae (fungal highway) results in bacterial selection; in particular, fungal hyphae offer adequate conditions for the transport of both aerobic and anaerobic bacteria through hydrophobic patches for the colonization of novel niches.

New insights into microbial community coalescence in the land-sea continuum.

The land-sea continuum constitutes a mixing zone where soil microbial communities encounter, via runoff, those inhabiting marine coastal sediment resulting in community coalescence. Here, we propose an experimental approach, mimicking the land-sea continuum, to study the microbial community coalescence events in different situations, by 16S and 18S rRNA genes metabarcoding. The microbial community structure of sediment diverged with the soil inputs. For prokaryotes, phylogenetic enrichment and amplicon sequence variants (ASVs) replacements characterized the community changes in sediment receiving soil inputs. For fungi, despite phylogenetic enrichment was not observed, the fungal ASVs richness was maintained by soil inputs. Comparison of microbial communities revealed ASVs specific to sediment receiving soil inputs, and also ASVs shared with soil and/or runoff. Among these specific ASVs, four bacterial and one fungal ASVs were identified as indicators of coalescence. Our study provides evidences that coalescence involves the mixing of microorganisms and of the environment.

Vertical organization of microbial communities in Salineta hypersaline wetland, Spain.

Microbial communities inhabiting hypersaline wetlands, well adapted to the environmental fluctuations due to flooding and desiccation events, play a key role in the biogeochemical cycles, ensuring ecosystem service. To better understand the ecosystem functioning, we studied soil microbial communities of Salineta wetland (NE Spain) in dry and wet seasons in three different landscape stations representing situations characteristic of ephemeral saline lakes: S1 soil usually submerged, S2 soil intermittently flooded, and S3 soil with halophytes. Microbial community composition was determined according to different redox layers by 16S rRNA gene barcoding. We observed reversed redox gradient, negative at the surface and positive in depth, which was identified by PERMANOVA as the main factor explaining microbial distribution. The Pseudomonadota, Gemmatimonadota, Bacteroidota, Desulfobacterota, and Halobacteriota phyla were dominant in all stations. Linear discriminant analysis effect size (LEfSe) revealed that the upper soil surface layer was characterized by the predominance of operational taxonomic units (OTUs) affiliated to strictly or facultative anaerobic halophilic bacteria and archaea while the subsurface soil layer was dominated by an OTU affiliated to Roseibaca, an aerobic alkali-tolerant bacterium. In addition, the potential functional capabilities, inferred by PICRUSt2 analysis, involved in carbon, nitrogen, and sulfur cycles were similar in all samples, irrespective of the redox stratification, suggesting functional redundancy. Our findings show microbial community changes according to water flooding conditions, which represent useful information for biomonitoring and management of these wetlands whose extreme aridity and salinity conditions are exposed to irreversible changes due to human activities.

Functional redundancy in response to runoff input upholds microbial community in hydrocarbon-contaminated land-sea continuum.

An experimental approach mimicking the land-sea continuum in microcosms was developed in order to determine the effect of the terrigenous inputs by soil runoff on the microbial functional potential in hydrocarbon (HC) contaminated marine coastal sediment. We hypothesized that the coalescent event increases the functional potential of microbial communities in marine coastal sediments, influencing the fate of HC in marine coastal ecosystems. The microbial functional potential including the HC degradation ability was assessed by DNA-array to compare the sediment receiving or not terrigenous inputs. The removal of HC and the functional gene richness in sediment was unchanged with the terrigenous inputs. However, the gene variants (GVs) composition was modified indicating functional redundancy. In addition, functional indicators including GVs related to sulfite reduction, denitrification and polyaromatic degradation were identified in higher proportion in sediment receiving terrigenous inputs. The terrigenous inputs modified the functional co-occurrence networks, showing a reorganization of the GVs associations with an increase of the network complexity. Different keystone GVs ensuring similar functions were identified in networks with or without terrigenous inputs, further confirming functional redundancy. We argue that functional redundancy maintains the structure of microbial community in hydrocarbon-contaminated land-sea continuum mixing zone. Our results provide helpful functional information for the monitoring and management of coastal environment affected by human land-based activities.

Microbial indicators along a metallic contamination gradient in tropical coastal sediments.

The structure and diversity of microbial community inhabiting coastal sediments reflect the exposition to contaminants. Aiming to assess the changes in the microbiota from Sepetiba Bay (SB, Brazil) sediments, correlations between the 16S rRNA gene data (V4-V5 region), metal contamination factors (CF), and the ecological risk classification provided by the Quality Ratio (QR) index were considered. The results show that microbial diversity differs significantly between the less (SB external sector) and the most (SB internal sector) polluted sectors. Also, differences in the microbial community structure regarding the ecological risk classifications validated the QR index as a reliable tool to report the SB chronic contamination. Microbial indicator genera resistant to metals (Desulfatiglans, SEEP-SRB1, Spirochaeta 2, among others) presented mainly anaerobic metabolisms. These genera are related to the sulfate reducing and methanogenic metabolisms probably participating in the natural attenuation processes but also associated with greenhouse gas emissions. In contrast, microbial indicator genera sensitive to metals (Rubripirellula, Blastopirellula, Aquibacter, among others) presented mainly aerobic metabolisms. It is suggested that future works should investigate the metabolic functions to evaluate the influence of metallic contaminants on microbial community inhabiting SB sediment.

Methanogenic octadecene degradation by syntrophic enrichment culture from brackish sediments.

A microbial enrichment culture from brackish sediments was able to grow on octadec-1-ene (an unsaturated aliphatic hydrocarbon) as sole source of carbon and energy, under methanogenic conditions. Octadecene degradation is stopped either when bromoethanesulfonic acid, a selective inhibitor of methanogenesis is introduced, or when hydrogen is introduced. In the presence of bromoethanesulfonic acid, the degradation is restored by the addition of a hydrogenotrophic sulfate-reducing microorganism with sulfate. Results of molecular biodiversity, which revealed the presence of bacteria as well as of acetoclastic and hydrogenotrophic methanogens, are consistent with a syntrophic degradation involving Bacteria and Archaea. This is the first demonstration of syntrophic alkene degradation by microbial communities, showing that syntrophy is more widespread than we could have thought so far. These results highlight the need for a better understanding of microbial interactions and their role in the organic-matter degradation in polluted environments.

Fungal-bacterial network in PAH-contaminated coastal marine sediment.

Fungal microbiome interacts with the other biotic components in coastal sediment playing a key role in the overall coordination of the whole microbial community. These interactions are affected by human activities, such as the constant affluence of polycyclic aromatic hydrocarbons (PAHs). Although fungi and bacteria interactions have been found to play a key role in PAH bioremediation in soil, the effect of PAHs on fungal diversity and their specific interactions with bacteria in coastal sediments are yet to be investigated. The understanding of fungal bacterial interactions under PAH contamination is critical for further bioremediation regarding the important fungal diversity observed in coastal sediment. Here, we investigated the fungal bacterial co-occurrence in PAH-contaminated sediments. The co-occurrence network, constructed with sequencing data (bacterial 16S and fungal 18S rRNA genes barcoding) from 51 PAH-contaminated samples, revealed modules dominated by either fungi or bacteria, reflecting probably the different types of interaction possible between fungi and bacteria. Then, a network constructed from non-contaminated sample data was compared with a network built from the corresponding PAH-contaminated samples issued from a mesocosm experiment. The comparison revealed the effect of PAHs in fungi and bacteria interactions, characterized by a PAH-contaminated network exhibiting less abundant and diverse fungal and bacterial ASVs than the non-contaminated network. However, the links between the remaining ASVs in the PAH-contaminated network showed stronger correlations. Noteworthy, an ASV affiliated to Chrytridiomycota phylum was identified as a keystone fungal ASV forming a module in association with facultative anaerobic and anaerobic bacteria affiliated to the families Prolixibacteraceae, Fusobacteriaceae, and Desulfobulbaceae. These results suggest that fungi promote bacterial anaerobic metabolisms, which are important to cope with the presence of PAHs in sediments. Our study reveals the importance of fungal bacterial interactions in coastal sediments paving the way for future studies to fully understand fungal role in coastal sediment.

Microbial diversity alteration reveals biomarkers of contamination in soil-river-lake continuum.

Microbial communities inhabiting soil-water-sediment continuum in coastal areas provide important ecosystem services. Their adaptation in response to environmental stressors, particularly mitigating the impact of pollutants discharged from human activities, has been considered for the development of microbial biomonitoring tools, but their use is still in the infancy. Here, chemical and molecular (16S rRNA gene metabarcoding) approaches were combined in order to determine the impact of pollutants on microbial assemblages inhabiting the aquatic network of a soil-water-sediment continuum around the Ichkeul Lake (Tunisia), an area highly impacted by human activities. Samples were collected within the soil-river-lake continuum at three stations in dry (summer) and wet (winter) seasons. The contaminant pressure index (PI), which integrates Polycyclic aromatic hydrocarbons (PAHs), alkanes, Organochlorine pesticides (OCPs) and metal contents, and the microbial pressure index microgAMBI, based on bacterial community structure, showed significant correlation with contamination level and differences between seasons. The comparison of prokaryotic communities further revealed specific assemblages for soil, river and lake sediments. Correlation analyses identified potential "specialist" genera for the different compartments, whose abundances were correlated with the pollutant type found. Additionally, PICRUSt analysis revealed the metabolic potential for pollutant transformation or degradation of the identified "specialist" species, providing information to estimate the recovery capacity of the ecosystem. Such findings offer the possibility to define a relevant set of microbial indicators for assessing the effects of human activities on aquatic ecosystems. Microbial indicators, including the detection of "specialist" and sensitive taxa, and their functional capacity, might be useful, in combination with integrative microbial indices, to constitute accurate biomonitoring tools for the management and restoration of complex coastal aquatic systems.

Microbial community metabolic alterations and resistance to metals and antibiotics driven by chronic exposition to multiple pollutants in a highly impacted tropical coastal bay.

Microbial communities from Sepetiba Bay (SB, Rio de Janeiro, Brazil), characterized by 16S rRNA gene (V4-V5 region) sequencing analysis, were found to be correlated with the metallic contamination factor and the Quality Ratio (QR) index. Consistently, the predicted function of microbial communities, obtained with Tax4Fun2, showed that the functional patterns in SB internal sector under the highest anthropogenic pressure were different from that observed in the external sector with the lowest contamination level. Signal transduction, cellular community, membrane transport, and energy metabolism were among the KEGG pathways favored by metallic contamination in the SB internal sector, while lipid metabolism, transcription, and translation were among the pathways favored in the SB external sector. Noteworthy, the relative proportions of KEGG pathways and genes associated with metallic homeostasis showed significant differences according to the SB sectors, consistently with the ecological risk classification (QR index) of sediments. The functional prediction approach is an economically viable alternative and presents an overview of the main pathways/genes favored in the SB microbiota exposed to long-term pollution. In contrast, the microgAMBI, ecological status index based on bacterial community composition, was not consistent with the metallic contamination of SB, suggesting that this index requires improvements to be applied in tropical areas. Our study also revealed a strong correlation between metal resistance genes (MRG) and antibiotic resistance genes (ARG), indicating that MRG and ARG are co-selected by the metallic contamination prevailing in SB.

Low-cost gel-filled microwell array device for screening marine microbial consortium.

In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 µm × 600 µm × 700 µm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.

Development of molecular driven screening for desulfurizing microorganisms targeting the dszB desulfinase gene.

COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) were developed for the detection of the dszB desulfinase gene (2'-hydroxybiphenyl-2-sulfinate desulfinase; EC 3.13.1.3) by polymerase chain reaction (PCR), which allow to reveal larger diversity than traditional primers. The new developed primers were used as molecular monitoring tool to drive a procedure for the isolation of desulfurizing microorganisms. The primers revealed a large dszB gene diversity in environmental samples, particularly in diesel-contaminated soil that served as inoculum for enrichment cultures. The isolation procedure using the dibenzothiophene sulfone (DBTO2) as sole sulfur source reduced drastically the dszB gene diversity. A dszB gene closely related to that carried by Gordonia species was selected. The desulfurization activity was confirmed by the production of desulfurized 2-hydroxybiphenyl (2-HBP). Metagenomic 16S rRNA gene sequencing showed that the Gordonia genus was represented at low abundance in the initial bacterial community. Such observation highlighted that the culture medium and conditions represent the bottleneck for isolating novel desulfurizing microorganisms. The new developed primers constitute useful tool for the development of appropriate cultural-dependent procedures, including medium and culture conditions, to access novel desulfurizing microorganisms useful for the petroleum industry.