Isolamento e caracterização de bactérias resistentes a surfactantes

Mestrado em Biologia MarinhaAs estratégias de biorremediação de hidrocarbonetos envolvem frequentemente a aplicação de surfactantes de modo a aumentar biodisponibilidade de compostos pouco solúveis em água. Todavia, os surfactantes sintéticos são tóxicos para muitas bactérias degradadoras de hidrocarbonetos, reduzindo a taxa de mineralização do poluente. Uma estratégia interessante seria o recurso a bactérias com ambas as capacidades: degradar poluentes hidrofóbicos e resistir aos surfactantes. A microcamada superficial marinha (sea surface microlayer - SML) constitui a interface entre a hidrosfera e a atmosfera, sendo geralmente definida como o milímetro superior da coluna de água. Este compartimento natural é simultaneamente rico em poluentes hidrofóbicos e surfactantes. Neste trabalho pesquisou-se a SML quanto à presença de bactérias resistentes a surfactantes através de culturas de enriquecimento com dodecil sulfato de sódio (SDS – soduim dodecyl sulfate) e brometo de cetil trimetilamónio (CTAB – cetyl trimethylammonium bromide). Sobre um subconjunto dos isolados obtidos, realizou-se PCR (polymerase chain reaction) para detecção do gene gacA - um marcador específico de Pseudomonas – e do gene ndo – codificante para a naftaleno dioxigenase, uma enzima envolvida na degradação de hidrocarbonetos aromáticos policíclicos (PAH - polycyclic aromatic hydrocarbons -). Os isolados foram ainda testados quanto à produção de biossurfactantes. Os resultados obtidos revelam uma elevada abundância de bactérias resistentes a surfactantes na SML. O PCR do gene gacA revelou a predominância de Pseudomonas no subconjunto de isolados (44%). Contudo, não foi obtida amplificação para o gene ndo. A produção de biossurfactantes foi detectada por cultura em meio sólido em 5 isolados, todos pertencentes ao género Pseudomonas. A sequenciação do gene 16S do RNA ribossomal evidencia similaridade filogenética entre os isolados identificados e estirpes produtoras de biossurfactantes e degradadoras de poluentes hidrofóbicos isoladas em diferentes ambientes naturais.Bioremediation efforts sometimes rely on surfactants to enhance hydrocarbon bioavailability. However, most synthetic surfactants employed can be sometimes toxic to degrading microorganisms, reducing the clearance rate of the pollutant. An interesting strategy is to employ bacteria with both the desired attributes: biodegradative capability and tolerance to surfactants. The sea surface microlayer (SML) is the uppermost layer of the water column and represents an interface between the hydrosphere and atmosphere. It is simultaneously rich in hydrophobic pollutants and surfactants. In this study we screened a natural surfactant-rich compartment, the sea surface microlayer, for surfactant-resistant bacteria, using enrichment cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). A random set of isolates obtained from enrichment cultures were tested by PCR for the Pseudomonas genus marker gacA gene and for the ndo gene, encoding for polycyclic aromatic hydrocarbon (PAH) degrading enzyme naphthalene dioxygenase. Also, isolates were screened for biosurfactant production in solid medium. Results show a high relative abundance of surfactant resistant bacteria in the SML. gacA targeted PCR revealed that 44% of the surfactantresistant isolates are Pseudomonads. However, PCR of ndo gene involved in PAH-degrading pathways was not successful with current primers. Biosurfactant production in solid medium was detected in 5 isolates, all belonging to the genus Pseudomonas. Sequencing of 16S rRNA revealed high phylogenetic similarity between the isolates obtained in this study and hydrocarbon-degrading and biosurfactant-producing bacteria from other natural environments

Descontaminação de petróleo por comunidades microbianas bênticas

Doutoramento em BiologiaOs sedimentos marinhos são um reservatório de hidrocarbonetos petrogénicos libertados naturalmente ou acidentalmente para o ambiente marinho. Nos sedimentos marinhos, os hidrocarbonetos são usados como fonte de carbono e energia por comunidades bacterianas complexas. Contudo, a eficiência de biodegradação poderá ser limitada por fatores ambientais. Este trabalho aborda o previsível impacto das condições particulares do mar profundo, da acidificação dos oceanos e da adição de dispersantes químicos nos processos de biodegradação de hidrocarbonetos em ambientes marinhos. Numa primeira fase, a função de destoxificação primária das bactérias degradadoras de hidrocarbonetos aromáticos policíclicos (HAP) nos sedimentos do mar profundo foi avaliado através de uma compilação de informação disponível na literatura científica e também através de uma análise dependente do cultivo envolvendo culturas de enriquecimento de sedimentos de vulcões de lama do mar profundo. Posteriormente, o impacto interativo da acidificação do oceano e da contaminação por hidrocarbonetos petrogénicos em comunidades bacterianas bênticas foi avaliado, em experiências de simulação multifatorial em sistema de microcosmo previamente executadas, com sedimentos subsuperficiais estuarinos. Finalmente, foi executado uma experiência multifatorial em sistema de microcosmos para avaliar o impacto da aplicação de dispersantes químicos em situações simuladas de derrame de hidrocarbonetos em sedimentos estuarinos portuários. Os resultados obtidos, através da análise da fração cultivável, indicam que nos sedimentos do mar profundo a comunidade bacteriana degradadora de HAP é distinta da encontrada noutros sedimentos marinhos devido à predominância de bactérias relacionadas com o género Bacillus. Nos ensaios de microcosmos, apesar das diferenças entre os cenários testados, as comunidades bacterianas revelaram-se em geral, estáveis. Nos sedimentos subsuperficiais estuarinos, as alterações abióticas impostas foram provavelmente atenuadas pela barreira sedimentar sobrejacente e a comunidade bacteriana pareceu ser estável em termos de estrutura e atividade. Do mesmo modo, a dispersão química de hidrocarbonetos petrogénicos, apesar de aumentar a biodisponibilidade de PAH, não alterou significativamente a composição das comunidades bacteriana de sedimentos superficiais estuarinos. Possivelmente, a exposição prévia do sedimento portuário a poluição por hidrocarbonetos poderá ter condicionado a resposta da comunidade bêntica bacteriana à contaminação por petróleo. Em conclusão, a degradação bacteriana de hidrocarbonetos é um processo ubíquo em sedimentos marinhos e as comunidades bacterianas degradadoras revelam elevada estabilidade relativamente à variação de fatores ambientais.The marine sediment compartment is a key sink for naturally and accidentally released oil hydrocarbons in the marine environment. Here, complex communities of interacting bacterial species will efficiently use oil hydrocarbons as sources of carbon and energy. However, the efficiency of the biodegradation process can be limited by some near-future scenarios. This work addresses different environmental scenarios regarding oil hydrocarbon biodegradation in marine sediments. First, the role of bacteria as primary detoxifiers of polycyclic aromatic hydrocarbons in deep-sea sediments was evaluated through the compilation of available data and through a culture-dependent analysis of enrichment cultures derived mud volcano sediments. Next, the impact of the interactive effects of ocean acidification and oil hydrocarbon contamination was further analyzed in subsurface estuarine sediments. Finally, the impact of chemically dispersed oil in estuarine port sediments is evaluated through a multi-factorial microcosm simulation. Results show that , in deep sea mud volcano sediments, the culturable fraction of the PAH-degrading bacterial community seems distinct from other environments, with a predominance of Bacillus-like bacteria. In the microcosmbased assays, despite the differences between them, the overall bacterial community exhibit a reliable stability. In subsurface sediments, abiotic changes tested were possibly attenuated by the superficial sediment barrier and bacterial seem stable to environmental changes. Also, the chemical dispersion of oil, despite enhancing PAH concentration, did not impose significant alterations to the bacterial community composition at the marine sediment surface. The potential pre-exposure of the port sediment to oil hydrocarbon pollution may have preconditioned the response of the benthic bacterial communities to oil contamination. In conclusion, oil-hydrocarbon biodegradation is ubiquitous and communities exhibit a structural stability to environmental changes

Isolation of Surfactant-Resistant Pseudomonads from the Estuarine Surface Microlayer

Bioremediation efforts often rely on the application of surfactants to enhance hydrocarbon bioavailability. However, synthetic surfactants can sometimes be toxic to degrading microorganisms, thus reducing the clearance rate of the pollutant. Therefore, surfactant-resistant bacteria can be an important tool for bioremediation efforts of hydrophobic pollutants, circumventing the toxicity of synthetic surfactants that often delay microbial bioremediation of these contaminants. In this study, we screened a natural surfactant-rich compartment, the estuarine surface microlayer (SML), for cultivable surfactant-resistant bacteria using selective cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Resistance to surfactants was evaluated by colony counts in solid media amended with critical micelle concentrations (CMC) of either surfactants, in comparison with non-amended controls. Selective cultures for surfactant-resistant bacteria were prepared in mineral medium also containing CMC concentrations of either CTAB or SDS. The surfactantresistant isolates obtained were tested by PCR for the Pseudomonas genus marker gacA gene and for the naphthalene-dioxygenase-encoding gene ndo. Isolates were also screened for biosurfactant production by the atomized oil assay. A high proportion of culturable bacterioneuston was tolerant to CMC concentrations of SDS or CTAB. The gacA-targeted PCR revealed that 64% of the isolates were Pseudomonads. Biosurfactant production in solid medium was detected in 9.4% of tested isolates, all affiliated with genus Pseudomonas. This study shows that the SML is a potential source of surfactant-resistant and biosurfactant-producing bacteria in which Pseudomonads emerge as a relevant group

Brevibacterium EB3 inoculation enhances rhizobacterial community interactions leading to improved growth of Salicornia europaea

Plant growth-promoting bacteria (PGPB) can revolutionize sustainable agriculture by improving crop yields and resilience in the face of climate change and soil degradation. However, one of the challenges of using PGPB is identifying strains that can colonize and establish beneficial relationships with plant hosts and microbiomes. This study examined the effects of single and co-inoculations with three PGPB strains (Brevibacterium casei EB3, Pseudomonas oryzihabitans RL18, and Bacillus aryabhattai SP20) on the rhizosphere microbiome of the halophyte crop Salicornia europaea. 16S rRNA gene amplicon sequencing was used to analyze the rhizosphere bacterial communities' diversity, structure, and composition. PGPB inoculations significantly changed the diversity and structure of the bacterial communities in the rhizosphere, accounting for 74 % of the total variability. The strain B. casei EB3 was the most effective at colonizing the rhizosphere and establishing interactions with other beneficial community members. Notably, the treatments associated with higher plant yield, consistently featured the presence of B. casei EB3 and higher connectivity between this strain and taxa known to promote growth and alleviate salt stress in plants such as Marinobacterium, Pseudomonas and Vibrio. These findings are consistent with bacterial inoculants' direct and indirect effect in boosting bacteria-plant cooperation within the rhizosphere, ultimately leading to a shift towards an optimized rhizosphere and beneficial traits for plants

Bacteriophages with Potential for Inactivation of Fish Pathogenic Bacteria: Survival, Host Specificity and Effect on Bacterial Community Structure

Phage therapy may represent a viable alternative to antibiotics to inactivate fish pathogenic bacteria. Its use, however, requires the awareness of novel kinetics phenomena not applied to conventional drug treatments. The main objective of this work was to isolate bacteriophages with potential to inactivate fish pathogenic bacteria, without major effects on the structure of natural bacterial communities of aquaculture waters. The survival was determined in marine water, through quantification by the soft agar overlay technique. The host specificity was evaluated by cross infection. The ecological impact of phage addition on the structure of the bacterial community was evaluated by DGGE of PCR amplified 16S rRNA gene fragments. The survival period varied between 12 and 91 days, with a higher viability for Aeromonas salmonicida phages. The phages of Vibrio parahaemolyticus and of A. salmonicida infected bacteria of different families with a high efficacy of plating. The specific phages of pathogenic bacteria had no detectable impact on the structure of the bacterial community. In conclusion, V. parahaemolyticus and A. salmonicida phages show good survival time in marine water, have only a moderated impact on the overall bacterial community structure and the desired specificity for host pathogenic bacteria, being potential candidates for therapy of fish infectious diseases in marine aquaculture systems

Phage therapy as an approach to prevent Vibrio anguillarum infections in fish larvae production

Fish larvae in aquaculture have high mortality rates due to pathogenic bacteria, especially the Vibrio species, and ineffective prophylactic strategies. Vaccination is not feasible in larvae and antibiotics have reduced efficacy against multidrug resistant bacteria. A novel approach to controlling Vibrio infections in aquaculture is needed. The potential of phage therapy to combat vibriosis in fish larvae production has not yet been examined. We describe the isolation and characterization of two bacteriophages capable of infecting pathogenic Vibrio and their application to prevent bacterial infection in fish larvae. Two groups of zebrafish larvae were infected with V. anguillarum (∼106 CFU mL-1) and one was later treated with a phage lysate (∼108 PFU mL-1). A third group was only added with phages. A fourth group received neither bacteria nor phages (fish control). Larvae mortality, after 72 h, in the infected and treated group was similar to normal levels and significantly lower than that of the infected but not treated group, indicating that phage treatment was effective. Thus, directly supplying phages to the culture water could be an effective and inexpensive approach toward reducing the negative impact of vibriosis in larviculture

Integrated analysis of bacterial and microeukaryotic communities from differentially active mud volcanoes in the Gulf of Cadiz

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults

Assessing the Effects of Rotifer Feed Enrichments on Turbot (Scophthalmus maximus) Larvae and Post-Larvae Gut-Associated Bacterial Communities

Live feed enrichments are often used in fish larvicultures as an optimized source of essential nutrients to improve larval growth and survival. In addition to this, they may also play an important role in structuring larval-associated microbial communities and may help improve their resistance to diseases. However, there is limited information available on how larval microbial communities and larviculture water are influenced by different live feed enrichments. In the present study, we investigated the effects of two commercial rotifer enrichments (ER) on turbot (Scophthalmus maximus) larval and post-larval gut-associated bacterial communities during larviculture production. We evaluated their effects on bacterial populations related to known pathogens and beneficial bacteria and their potential influence on the composition of bacterioplankton communities during larval rearing. High-throughput 16S rRNA gene sequencing was used to assess the effects of different rotifer enrichments (ER1 and ER2) on the structural diversity of bacterial communities of the whole turbot larvae 10 days after hatching (DAH), the post-larval gut 30 DAH, and the larviculture water. Our results showed that different rotifer feed enrichments were associated with significant differences in bacterial composition of turbot larvae 10 DAH, but not with the composition of larval gut communities 30 DAH or bacterioplankton communities 10 and 30 DAH. However, a more in-depth taxonomic analysis showed that there were significant differences in the abundance of Vibrionales in both 10 DAH larvae and in the 30 DAH post-larval gut fed different RE diets. Interestingly, the ER1 diet had a higher relative abundance of specific amplicon sequence variants (ASVs) related to potential Vibrio-antagonists belonging to the Roseobacter clade (e.g., Phaeobacter and Ruegeria at 10 DAH and Sulfitobacter at 30 DAH). In line with this, the diet was also associated with a lower relative abundance of Vibrio and a lower mortality. These results suggest that rotifer diets can affect colonization by Vibrio members in the guts of post-larval turbot. Overall, this study indicates that live feed enrichments can have modulatory effects on fish bacterial communities during the early stages of development, which includes the relative abundances of pathogenic and antagonist taxa in larviculture systems

Polycyclic aromatic hydrocarbons in deep sea sediments: Microbe–pollutant interactions in a remote environment

Recalcitrant polycyclic aromatic hydrocarbons (PAHs) released into seawater end up in the deep sea sediments (DSSs). However, their fate here is often oversimplified by theoretical models. Biodegradation of PAHs in DSSs, is assumed to be similar to biodegradation in surface habitats, despite high hydrostatic pressures and low temperatures that should significantly limit PAH biodegradation. Bacteria residing in the DSSs (related mainly to α- and γ-Proteobacteria) have been shown to or predicted to possess distinct genes, enzymes and metabolic pathways, indicating an adaptation of these bacterial communities to the psychro-peizophilic conditions of the DSSs. This work summarizes some of the most recent research on DSS hydrocarbonoclastic populations and mechanisms of PAH degradation and discusses the challenges posed by future high CO2 and UV climate scenarios on biodegradation of PAHs in DSSs