Sandra Chaves

Diversity and impact of prokaryotic toxins on aquatic environments: a review.

Microorganisms are ubiquitous in all habitats and are recognized by their metabolic versatility and ability to produce many bioactive compounds, including toxins. Some of the most common toxins present in water are produced by several cyanobacterial species. As a result, their blooms create major threats to animal and human health, tourism, recreation and aquaculture. Quite a few cyanobacterial toxins have been described, including hepatotoxins, neurotoxins, cytotoxins and dermatotoxins. These toxins are secondary metabolites, presenting a vast diversity of structures and variants. Most of cyanobacterial secondary metabolites are peptides or have peptidic substructures and are assumed to be synthesized by non-ribosomal peptide synthesis (NRPS), involving peptide synthetases, or NRPS/PKS, involving peptide synthetases and polyketide synthases hybrid pathways. Besides cyanobacteria, other bacteria associated with aquatic environments are recognized as significant toxin producers, representing important issues in food safety, public health, and human and animal well being. Vibrio species are one of the most representative groups of aquatic toxin producers, commonly associated with seafood-born infections. Some enterotoxins and hemolysins have been identified as fundamental for V. cholerae and V. vulnificus pathogenesis, but there is evidence for the existence of other potential toxins. Campylobacter spp. and Escherichia coli are also water contaminants and are able to produce important toxins after infecting their hosts. Other bacteria associated with aquatic environments are emerging as toxin producers, namely Legionella pneumophila and Aeromonas hydrophila, described as responsible for the synthesis of several exotoxins, enterotoxins and cytotoxins. Furthermore, several Clostridium species can produce potent neurotoxins. Although not considered aquatic microorganisms, they are ubiquitous in the environment and can easily contaminate drinking and irrigation water. Clostridium members are also spore-forming bacteria and can persist in hostile environmental conditions for long periods of time, contributing to their hazard grade. Similarly, Pseudomonas species are widespread in the environment. Since P. aeruginosa is an emergent opportunistic pathogen, its toxins may represent new hazards for humans and animals. This review presents an overview of the diversity of toxins produced by prokaryotic microorganisms associated with aquatic habitats and their impact on environment, life and health of humans and other animals. Moreover, important issues like the availability of these toxins in the environment, contamination sources and pathways, genes involved in their biosynthesis and molecular mechanisms of some representative toxins are also discussed.

Anaerobic bio-removal of uranium (VI) and chromium (VI): Comparison of microbial community structure

Several microbial communities, obtained from uranium contaminated and non-contaminated samples, were investigated for their ability to remove uranium (VI) and the cultures capable for this removal were further assessed on their efficiency for chromium (VI) removal. The highest efficiency for removal of both metals was observed on a consortium from a non-contaminated soil collected in Monchique thermal place, which was capable to remove 91% of 22 mg L(-1) U(VI) and 99% of 13 mg L(-1) Cr(VI). This study revealed that uranium (VI) removing communities have also ability to remove chromium (VI), but when uranium (VI) was replaced by chromium (VI) several differences in the structure of all bacterial communities were observed. TGGE and phylogenetic analysis of 16S rRNA gene showed that the uranium (VI) removing bacterial consortia are mainly composed by members of Rhodocyclaceae family and Clostridium genus. On the other hand, bacteria from Enterobacteriaceae family were detected in the community with ability for chromium (VI) removal. The existence of members of Enterobacteriaceae and Rhodocyclaceae families never reported as chromium or uranium removing bacteria, respectively, is also a relevant finding, encouraging the exploitation of microorganisms with new abilities that can be useful for bioremediation.

Mechanism of uranium (VI) removal by two anaerobic bacterial communities.

The mechanism of uranium (VI) removal by two anaerobic bacterial consortia, recovered from an uncontaminated site (consortium A) and other from an uranium mine (consortium U), was investigated. The highest efficiency of U (VI) removal by both consortia (97%) occurred at room temperature and at pH 7.2. Furthermore, it was found that U (VI) removal by consortium A occurred by enzymatic reduction and bioaccumulation, while the enzymatic process was the only mechanism involved in metal removal by consortium U. FTIR analysis suggested that after U (VI) reduction, U (IV) could be bound to carboxyl, phosphate and amide groups of bacterial cells. Phylogenetic analysis of 16S rRNA showed that community A was mainly composed by bacteria closely related to Sporotalea genus and Rhodocyclaceae family, while community U was mainly composed by bacteria related to Clostridium genus and Rhodocyclaceae family.

Effect of uranium (VI) on two sulphate-reducing bacteria cultures from a uranium mine site

This work was conducted to assess the impact of uranium (VI) on sulphate-reducing bacteria (SRB) communities obtained from environmental samples collected on the Portuguese uranium mining area of Urgeiriça. Culture U was obtained from a sediment, while culture W was obtained from sludge from the wetland of that mine. Temperature gradient gel electrophoresis (TGGE) was used to monitor community changes under uranium stress conditions. TGGE profiles of dsrB gene fragment demonstrated that the initial cultures were composed of SRB species affiliated with Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Desulfomicrobium spp. (sample U), and by species related to D. desulfuricans (sample W). A drastic change in SRB communities was observed as a result of uranium (VI) exposure. Surprisingly, SRB were not detected in the uranium removal communities. Such findings emphasize the need of monitoring the dominant populations during bio-removal studies. TGGE and phylogenetic analysis of the 16S rRNA gene fragment revealed that the uranium removal consortia are composed by strains affiliated to Clostridium genus, Caulobacteraceae and Rhodocyclaceae families. Therefore, these communities can be attractive candidates for environmental biotechnological applications associated to uranium removal.

Insect-symbiont systems: From complex relationships to biotechnological applications

Microbial symbiosis is a ubiquitous aspect of life and was a major element in the ability of insects to explore several adverse environments. To date, the study of symbiosis in insects has been impaired by the unculturability of most symbionts. However, some molecular methods represent powerful tools to help understand insect‐microorganism associations and to disclose new symbiont‐host systems. Beyond playing an essential role in nutrition and development of the insects, symbionts can produce bioactive compounds that protect the host against adverse environmental conditions, predators and/or direct competitors. Since the search for natural bioactive products and new enzymes is a developing area, understanding the diversity and nature of symbiont‐host relationships paves the way for the exploitation of new resources in biotechnology. Furthermore, genetic transformation of the symbionts with genes that code for compounds that are toxic for pathogenic and phytopathogenic agents is also a promising area of application of the insect‐symbiont relationships. The search for new bioactive compounds, the use of symbionts for pest and disease control and the molecular strategies applied for these purposes are issues of particular interest for innovative biotechnological applications and are addressed in the present review.

Photoprotective Bioactivity Present in a Unique Marine Bacteria Collection from Portuguese Deep Sea Hydrothermal Vents

Interesting biological activities have been found for numerous marine compounds. In fact, screening of phylogenetically diverse marine microorganisms from extreme environments revealed to be a rational approach for the discovery of novel molecules with relevant bioactivities for industries such as pharmaceutical and cosmeceutical. Nevertheless, marine sources deliverables are still far from the expectations and new extreme sources of microbes should be explored. In this work, a marine prokaryotic collection from four Mid-Atlantic Ridge (MAR) deep sea hydrothermal vents near the Azores Islands, Portugal, was created, characterized and tested for its photoprotective capacity. Within 246 isolates, a polyphasic approach, using chemotaxonomic and molecular typing methods, identified 23-related clusters of phenetically similar isolates with high indexes of diversity. Interestingly, 16S rRNA gene sequencing suggested the presence of 43% new prokaryotic species. A sub-set of 139 isolates of the prokaryotic collection was selected for biotechnological exploitation with 484 bacterial extracts prepared in a sustainable upscalling manner. 22% of the extracts showed an industrially relevant photoprotective activity, with two extracts, belonging to new strains of the species Shewanella algae and Vibrio fluvialis, uniquely showing UV-A, UV-B and UV-C protective capacity. This clearly demonstrates the high potential of the bacteria MAR vents collection in natural product synthesis with market applications.

Evaluation of growth, biochemical and bioaccumulation parameters in Pelophylax perezi tadpoles, following an in-situ acute exposure to three different effluent ponds from a uranium mine.

Mining activities invariably produce metal contaminated effluents. Depending on factors such as pH and metal concentration the toxicity of the effluent may vary. To assess the effects of three characteristically different effluent ponds from a deactivated uranium mine, with toxicologically relevant data, an in situ exposure with Pelophylax perezi tadpoles, was conducted. Tadpoles were exposed to the three effluent ponds, ranked by increasing order of metals concentrations (REF, M1, M2). Survival, growth, metal accumulation, antioxidant enzymes (catalase, glutathione peroxidase and glutathione reductase) and lipid peroxidation (LPO) were determined in tadpoles. As well, physical and chemical variables of the effluents were measured. Death percentage in the effluents was 3.17 (REF), 9.84 (M1) and 42.86% (M2) and was not coincident with metal accumulation which was highest in tadpoles exposed to M1, while metal contents in M2 tadpoles were quite similar to those recorded in REF tadpoles. However, high mortality in M2 was attributed to the extremely low pH (≈3.77). From the three effluents M2 tadpoles had the lowest growth and the antioxidant enzymatic activity was only affected in the case glutathione peroxidase (GPx) with significantly higher activity in M1, being in accordance with the highest accumulation of metals. LPO, usually associated with metal accumulation, had the following pattern M1>REF>M2. Overall, effluent toxicity in tadpoles exposed to M2 effluent seems to be primarily an effect of pH while in M1 toxicity is mainly owed to high metal concentrations. The effluent acidity seems to reduce metal accumulation probably due to damage in the integument, affecting ion uptake. The results obtained bring a better understanding of the toxicological processes that local P. perezi population is subjected to, mainly in the early life stages. Furthermore this study highlights the influence of pH in the toxicity of metal rich effluents.

Molecular assessment of microbiota structure and dynamics along mixed olive oil and winery wastewaters biotreatment

The major parcel of the degradation occurring along wastewater biotreatments is performed either by the native microbiota or by added microbial inocula. The main aim of this study was to apply two fingerprinting methods, temperature gradient gel electrophoresis (TGGE) and length heterogeneity-PCR (LH-PCR) analysis of 16S rRNA gene fragments, in order to assess the microbiota structure and dynamics during mixed olive oil and winery wastewaters aerobic biotreatment performed in a jet-loop reactor (JLR). Sequence homology analysis showed the presence of bacterial genera Gluconacetobacter, Klebsiella, Lactobacillus, Novosphingobium, Pseudomonas, Prevotella, Ralstonia, Sphingobium and Sphingomonas affiliated with five main phylogenetic groups: alpha-, beta- and gamma-Proteobacteria, Firmicutes and Bacteroidetes. LH-PCR analysis distinguished eight predominant DNA fragments correlated with the samples showing highest performance (COD removal rates of 67 up to 75%). Cluster analysis of both TGGE and LH-PCR fingerprinting profiles established five main clusters, with similarity coefficients higher than 79% (TGGE) and 62% (LH-PCR), and related with hydraulic retention time, indicating that this was the main factor responsible for the shifts in the microbiota structure. Canonical correspondence analysis revealed that changes observed on temperature and O2 level were also responsible for shifts in microbiota composition. Community level metabolic profile analysis was used to test metabolic activities in samples. Integrated data revealed that the microbiota structure corresponds to bacterial groups with high degradative potential and good suitability for this type of effluents biotreatments.

Effects of Increased Nitrogen Availability in Mediterranean Ecosystems: A Case Study in a Natura 2000 Site in Portugal

Nitrogen (N) enrichment has been pinpointed as a main driver for biodiversity change. Most of our knowledge of effects of increased N availability on ecosystems comes from northern Europe and America. Most other ecosystem types have been neglected. Although Mediterranean ecosystems are N-limited biodiversity hotspots, very little is known about the effects of N enrichment in these systems. In contribution to filling this gap, our study examined the short-term effects of N enrichment in a N-manipulation (doses and forms) field study of a severely nutrient-limited Mediterranean ecosystem located in a Natura 2000 site in Portugal. N availability (dose and forms) was modified by the addition of 40 and 80 kg N ha−1 year−1 as NH4NO3 or 40 kg N as NH4 + ha−1 year−1 (control plots are not fertilized) since January 2007. The studied ecosystem was highly N responsive, i.e., visible changes were seen within one year of N additions: vascular plant and soil microbial diversity (soil bacteria and arbuscular mycorrhizal fungal spores) increased. Also the N concentration in leaves and litter increased, while the carbon-to-nitrogen (C/N) ratio of leaves and litter decreased.

Differential gene expression in Iberian green frogs (Pelophylax perezi) inhabiting a deactivated uranium mine.

Iberian green frogs (Pelophylax perezi) were found inhabiting a deactivated uranium mine, especially an effluent pond, seriously contaminated with metals and radionuclides. These animals were previously assessed for oxidative stress parameters and did not revealed significant alterations. In order to better understand which mechanisms may be involved in the ability to withstand permanent contamination gene expression analysis was performed in the liver, through suppression subtractive hybridization (SSH). The SSH outcome in the liver revealed the up-regulation of genes coding for the ribosomal protein L7a and for several proteins typical from blood plasma: fibrinogen, hemoglobin and albumin. Besides their normal function, some of these proteins can play an important role as protective agents against oxidative stress. This work provides new insights on possible basal protection mechanisms that may act in organisms exposed chronically to contamination.