Diversity and dynamics of the Vibrio community in well water used for drinking in Guinea-Bissau (West Africa).

Bacteria of the genus Vibrio are ubiquitous in aquatic environments and can be found either in culturable or in a viable but nonculturable (VBNC) state. The genus comprises many pathogenic species accountable for water and food-borne diseases that prove to be fatal, especially in developing countries, as in Guinea-Bissau (West Africa), where cholera is endemic. In order to ascertain the abundance and structure of Vibrio spp. community in well waters that serve as the sole source of water for the population, quantitative polymerase chain reaction (qPCR), PCR-denaturant gradient gel electrophoresis (DGGE), and cloning approaches were used. Results suggest that Vibrio spp. were present throughout the year in acidic, freshwater wells with a seasonal community composition shift. Vibrio spp. abundance was in accordance with the abundance found in coastal environments. Sequences closely related to pathogenic Vibrio species were retrieved from well water revealing exposure of the population to such pathogens. pH, ammonium, and turbidity, regulated by the rain pattern, seem to be the variables that contributed mostly to the shaping and selection of the Vibrio spp. community. These results reinforce the evidence for water monitoring with culture-independent methods and the clear need to create/recover water infrastructures and a proper water resources management in West African countries with similar environmental conditions.

Prevalence of antibiotic resistance in bacteria isolated from drinking well water available in Guinea-Bissau (West Africa).

The dissemination of antibiotic-resistant bacteria and the spread of antibiotic resistance genes are a major public health concern worldwide, being even proposed as emerging contaminants. The aquatic environment is a recognized reservoir of antibiotic resistant bacteria, and antibiotic resistance genes have been recently detected in drinking water. In this study, the water quality and the prevalence of antibiotic resistance of heterotrophic culturable bacteria were characterized seasonally in wells that serve the population of Guinea-Bissau (West Africa) as the sole source of water for drinking and other domestic proposes. The results revealed that well water was unfit for human consumption independently of the season, owing to high acidity and heavy fecal contamination. Moreover, potentially pathogenic bacteria, which showed resistance to the most prescribed antibiotics in Guinea-Bissau, were isolated from well water, posing an additional health risk. Our results suggest that well water not only fosters the transmission of potential pathogenic bacteria, but also represents an important reservoir for the proliferation of antibiotic resistant bacteria, that can aggravate the potential to cause disease in a very vulnerable population that has no other alternative but to consume such water.

Methanethiol accumulation exacerbates release of N2 O during denitrification in estuarine sediments and bacterial cultures.

Microbes play critical roles in the biogeochemical cycling of nitrogen and sulfur in aquatic environments. Here we investigated the interaction between the naturally occurring organic sulfur compound methanethiol (MeSH) and the final step of the denitrification pathway, the reduction of nitrous oxide (N2 O) to dinitrogen (N2 ) gas, in sediment slurries from the temperate Douro and Ave estuaries (NW Portugal) and in pure cultures of the marine bacterium Ruegeria pomeroyi. Sediment slurries and cell suspensions were amended with a range of concentrations of either MeSH (0-120 µM) or methionine (0-5 mM), a known precursor of MeSH. MeSH or methionine additions caused N2 O to accumulate and this accumulation was linearly related to MeSH concentrations in both coastal sediments (R(2) = 0.7-0.9, P < 0.05) and R. pomeroyi cell suspensions (R(2) = 0.9, P < 0.01). Our results suggest that MeSH inhibits the final step of denitrification resulting in N2 O accumulation.

Ability of salt marsh plants for TBT remediation in sediments.

INTRODUCTION: The capability of Halimione portulacoides, Spartina maritima, and Sarcocornia fruticosa (halophytes very commonly found in salt marshes from Mediterranean areas) for enhancing remediation of tributyltin (TBT) from estuarine sediments was investigated, using different experimental conditions. METHODS: The influence of H. portulacoides on degradation of the butyltin compounds was assessed in two different ways: (1) a 9-month ex situ study carried out in a site of Sado River estuary, center of Portugal, which used polluted sediments collected at other nonvegetated site from the same estuary; and (2) a 12-month laboratorial study, using both plant and sediment collected at a relatively clean site of Cávado River estuary, north of Portugal, the sediment being doped with TBT, DBT, and MBT at the beginning of the experiment. The role of both S. fruticosa and S. maritima on TBT remediation in sediments was evaluated in situ, in salt marshes from Marim channel of Ria Formosa lagoon, south of Portugal, which has large areas colonized by each one of these two plants. For estimation of microbial abundance, total cell counts of sediment samples were enumerated by the DAPI direct count method. Butyltin analyses in sediment were performed using a method previously validated, which consisted of headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry after in situ ethylation (with tetraethylborate). RESULTS: Sediments colonized both ex situ and at lab by H. portulacoides displayed TBT levels about 30% lower than those for nonvegetated sediments with identical initial composition, after 9-12 months of plant exposure. In addition, H. portulacoides showed to be able of stimulating bacterial growth in the plant rhizosphere, which probably included degraders of TBT. In the in situ study, which compared the levels of TBT, DBT, and MBT in nonvegetated sediment and in sediments colonized by either S. maritima or S. fruticosa from the same area, TBT and DBT were only detected in nonvegetated sediment, whereas MBT was quantified in most samples. DISCUSSION: This work demonstrated that H. portulacoides has potentiality to be used for enhancing TBT remediation in sediments from salted areas. The results observed in situ for S. maritima or S. fruticosa suggested that these two salt marsh plants also favored TBT remediation. CONCLUSION: Therefore, the application of halophytes in technologies for TBT remediation in sediments seems to be efficient both in situ and ex situ, cost effective, and nondestructive, despite the fact that they have been rarely used for this purpose so far.

Influence of surfactants on the Cu phytoremediation potential of a salt marsh plant.

To assess the possible effect that surfactants commonly found in the aquatic environment may have on the remediation potential of the salt marsh plant Halimione portulacoides, a non-ionic (Triton X-100) and an anionic (sodium dodecyl sulfate, SDS) surfactants were used. Experiments were carried out in the laboratory, either in hydroponics (sediment elutriate) or in sediment soaked in elutriate, using sediment and water from an estuarine salt marsh (Cávado River, NW Portugal). Groups of H. portulacoides (grown in a greenhouse) were exposed for 6d to media with 0.16mM added Cu(II) in the absence and in the presence of each one of the two selected surfactants, at concentrations lower than the respective micellar critical concentration. Cu was determined in solutions, sediments and in different plant tissues before and after experiments. Plant photosynthetic efficiency did not indicate deletory effects of the exposure to the added pollutants. The non-ionic surfactant Triton X-100 and, to a lesser extent, the anionic surfactant SDS too, favored Cu accumulation in the plant roots but not Cu translocation, indicating that surfactants may favor Cu adsorption to the roots (phytostabilization). On the other hand, both surfactants favored Cu solubility from the sediment. Therefore, the presence of surfactants, which are frequently found in estuarine areas, as a result of urban and industrial effluent discharges, may condition metal distribution in those environments.

Influence of a salt marsh plant (Halimione portulacoides) on the concentrations and potential mobility of metals in sediments.

Influence of Halimione portulacoides, commonly found in temperate salt marshes, on sediment metal contents, speciation and potential mobility in case of sediment re-suspension was evaluated. Both colonized and non-colonized sediments were studied for total Cd, Cu, Pb and Zn contents and metal fraction exchangeable to water collected in situ. Sediment elutriates, prepared with water collected from each site, were used to simulate a sediment re-suspension phenomenon. As the characteristics and degree of contamination of sediments may influence system behaviour, salt marshes of two Portuguese estuaries, Cavado (NW coast) and Sado (SW coast), were studied. Cu, Pb and Zn contents higher than ERL (quality guideline, effect range-low) were observed, indicating potential risks for living organisms. Strong Cu-complexing organic ligands, also determined in both water and elutriates, were higher in rhizosediment elutriates, at concentrations similar, or even higher, to those of Cu. Such ligands condition metals speciation in the water column and probably also metal bioavailability. From rhizosediment significant amounts of Cu and Zn were transferred to the aqueous phase, concentrations 2-8 times higher than concentrations present in water. In contrast, elutriates of non-colonized sediment removed metals from water, Cu and Zn levels in elutriates being 2-6 times lower than initial ones. Cd and Pb levels in water and elutriates were not measurable in most cases. Results clearly indicate that metals potential solubility in the rhizosphere of plants was markedly higher than that in the surrounding sediment. The obtained results indicated that H. portulacoides presence (and probably other salt marsh plants) may cause a marked increase in metals concentrations in dissolved phase (pore water or even water column if rhizosediment is re-suspended). As salt marsh plants may be abundant in temperate and subtropical estuaries and costal lagoons, this phenomenon should not be disregard in future studies towards the sustainable management of such environments.

Can PAHs influence Cu accumulation by salt marsh plants?

The presence of polycyclic aromatic hydrocarbons (PAHs) may change the mechanisms of metal uptake, thus influencing kinetics and extent of metal phytoextraction. Studies on the subject are scarce, particularly for salt marsh plants. The aim of this work was to investigate the effect of PAHs on the uptake of Cu by Halimione portulacoides, a plant commonly found in salt marshes. Experiments were carried out in the laboratory, either in hydroponics (sediment elutriate) or in sediment soaked in elutriate, which were prepared with sediment and water from a salt marsh of the Cavado river estuary (NW Portugal). Groups of H. portulacoides (grown in a greenhouse) were exposed to those media during six days. Cu2+ (as Cu(NO3)2), 10(2) and 10(4) microg l(-1), was added to the media as well as 1.6 microg l(-1) of the sixteen EPA priority PAHs (0.1 microg l(-1) of each PAHs). Cu was assayed in solutions, sediments and different plant tissues before and after experiments. After exposure, photosynthetic efficiency and levels of chlorophylls were also measured, indicating that plant stress indicators were identical in all plants independently of the media to which the plants were exposed. PAHs influenced both the soluble Cu fraction and Cu uptake by plants. The amounts of metal accumulated in both roots and stems were significantly higher when the 10(4) microg l(-1) of Cu enriched elutriate was amended with PAHs. Thus, results suggest that PAHs may modify Cu solubility, the Cu sorption by plants and/or the passive penetration of Cu into the root cells. Therefore, the combined effects of different types of pollutants should be taken in consideration when studying the remediation potential of plants, namely in terms of phytoextraction.

Dynamics of nitrous oxide reductase genes (nosZ) in intertidal rocky biofilms and sediments of the Douro River estuary (Portugal), and their relation to N-biogeochemistry.

In this study, temporal variability of nosZ genotypes was evaluated in two intertidal rocky biofilms and two intertidal sediment sites of the Douro River estuary, Portugal. The results were compared to rates of key N-cycle processes and environmental variables to examine possible links between denitrifier community dynamics and N biogeochemistry. Genetic heterogeneity of the nosZ gene was evaluated by terminal restriction fragment length polymorphism analysis (T-RFLP) and by sequencing cloned nosZ gene fragments. Phylogenetic analysis showed that the majority of the nosZ genes detected were most similar to nosZ genes from isolates affiliated with alpha-subclass of the class Proteobacteria. Results revealed low nosZ genotype richness, and hierarchical cluster analysis showed significant differences in the composition of denitrifier communities that inhabit different intertidal environments of the Douro River estuary. Monthly surveys of nosZ genotypes from sandy sediments showed that, while the same T-RFLP peaks were present in all samples, shifts in the relative peak areas of the different nosZ genotypes occurred. Canonical correspondence analysis, based on data from the monthly survey, revealed a strong relationship between the relative peak areas of some T-RFLP operational taxonomic units (OTUs) with denitrification rate and NO3- availability. Results suggest that denitrifiers with specific nosZ genotypes (OTUs) have competitive advantage over others when NO3- fluctuates in the system; these fluctuations reflect, in turn, variability in denitrification rates.

Composition and activity of beta-Proteobacteria ammonia-oxidizing communities associated with intertidal rocky biofilms and sediments of the Douro River estuary, Portugal.

AIMS: To characterize the phylogenetic composition of ammonia-oxidizing bacteria (AOB) of the beta-subclass of the class Proteobacteria in intertidal sediment and rocky biofilms of the Douro estuary, and evaluate relationships with environmental variables and N-biogeochemistry. METHODS AND RESULTS: Cluster analysis of denaturing gradient gel electrophoresis profiles showed differences in beta-Proteobacteria AOB assemblage composition between rocky biofilms and sediments. All sequences obtained from intertidal rocky biofilm sites exhibited phylogenetic affinity to Nitrosomonas sp. lineages, whereas a majority of the sequences from the sediment sites were most similar to marine Nitrosospira cluster 1. Hierarchical cluster analysis based on environmental variables identified two main groups of samples. The first contained samples from rocky biofilm sites characterized by high concentrations of NO2- and NH4+, and high organic matter and chlorophyll a content. The second group contained all of the sediment samples; these sites were characterized by lower values for the variables above. In addition, rocky biofilm sites exhibited higher nitrification rates. CONCLUSIONS: Intersite differences in environmental and/or physical conditions led to the selection of different populations of beta-Proteobacteria AOB, supporting different magnitudes of N-cycling regimes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents an important step in establishing the influence of environmental factors on the distribution of beta-Proteobacteria AOB with possible consequences for N-biogeochemistry.

Survival of faecal indicator bacteria in tropical estuarine waters (Bangpakong River, Thailand).

AIMS: To investigate the survival of cultivable bacteria in the tropical Bangpakong estuary (Eastern Thailand) under different salinities and light conditions. METHODS AND RESULTS: Dark and light microcosm experiments using membrane diffusion chambers were carried out under three different experimental conditions, namely (i) low salinity, (ii) progressive mixing with brackish water and (iii) fast mixing with high salinity water spiked with raw urban sewage. Faecal coliforms declined faster than faecal enterococci, as shown by survival T90 values ranging from 82.2 +/- 4.2 to 14.5 +/- 0.8 h and 97.5 +/- 0.4-20.6 +/- 1.2 h, respectively. The survival of freshwater heterotrophic bacteria was higher but variable (121.2 +/- 5.0-30.1 +/- 14.3 h), whereas that of heterotrophic marine bacteria was rather stable (81.5 +/- 4.2-44.6 +/- 2.5 h). CONCLUSIONS: Overall survival was higher in low salinities. Light had a further deleterious effect, since it accelerated the decay of faecal indicators, particularly in high salinities. Faecal enterococci had a higher resistance to environmental conditions compared with faecal coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is relevant to the understanding of the behaviour of different faecal indicator bacteria and the optimization of sewage treatment plants aimed at the reduction and/or elimination of faecal load discharged into estuarine waters submitted to salinity variations.

Water quality and uses of the Bangpakong River (eastern Thailand).

The Bangpakong River is the most important watershed in the Eastern part of Thailand. Water quality parameters were sampled from June 1998 through May 1999 at 11 sites along a 227 km gradient, covering the wet season (June-November) and the dry season (December-May). Surface water was collected at three different stations per site (close to the banks and in the middle of the river), and analyzed for temperature, dissolved oxygen, turbidity, suspended solids, pH. ammonia, fecal coliforms, biochemical oxygen demand and chemical oxygen demand as well as conductivity, phosphate, and heavy metals. The Scottish water quality index (WQI) was adaptated to the tropical environment. The averaged WQI was low (41%) and quality declined significantly during the dry season (ANOVA, p<0.001). Although the quality rose somewhat at middle sites, only 27% of the WQI values during wet season and 2.5% during dry season were higher than 50%, denoting poor environmental quality. Within each season, the main sources of variability were the differences between sites along the gradient (48% during the wet season, 63% during the dry season), whereas monthly variability represented less than 20% of the variability. The seasonal results show that the river is suitable only for tolerant fish and wildlife species and is of doubtful use for potable water supply during the dry season. As quality improves during the wet period, water can be used for the production of potable water, but only with advanced treatment, and for indirect and noncontact recreational activities. In the middle stretches of the river, higher water quality permits multiple uses at moderate cost.

Crenarchaeota and Euryarchaeota in temperate estuarine sediments.

AIMS: Application of molecular techniques to ecological studies has unveiled a wide diversity of micro-organisms in natural communities, previously unknown to microbial ecologists. New lineages of Archaea were retrieved from several non-extreme environments, showing that these micro-organisms are present in a large variety of ecosystems. The aim was therefore to assess the presence and diversity of Archaea in the sediments of the river Douro estuary (Portugal), relating the results obtained to ecological data. METHODS AND RESULTS: Total DNA was extracted from sediment samples obtained from an estuary deprived of vegetation, amplified by PCR and the resulting DNA fragments cloned. The archaeal origin of the cloned inserts was checked by Southern blot, dot blot or colony blot hybridization. Recombinant plasmids were further analysed by restriction with AvaII and selected for sequencing. Phylogenetic analyses of 14 sequences revealed the presence of members of the domain Archaea. Most of the sequences could be assigned to the kingdom Crenarchaeota. CONCLUSION: Most of these sequences were closely related to those obtained from non-extreme Crenarchaeota members previously retrieved from diverse ecosystems, such as freshwater and marine environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of archaeal 16S rDNA sequences in temperate estuarine sediments emerges as a valuable contribution to the understanding of the complexity of the ecosystem.

Isolation and characterization of two new methanesulfonic acid-degrading bacterial isolates from a Portuguese soil sample.

Two novel bacterial strains that can utilize methanesulfonic acid as a source of carbon and energy were isolated from a soil sample collected in northern Portugal. Morphological, physiological, biochemical and molecular biological characterization of the two isolates indicate that strain P1 is a pink-pigmented facultative methylotroph belonging to the genus Methylobacterium, while strain P2 is a restricted methylotroph belonging to the genus Hyphomicrobium. Both strains are strictly aerobic, degrade methanesulfonate, and release small quantities of sulfite into the medium. Growth on methanesulfonate induces a specific polypeptide profile in each strain. This, together with the positive hybridization to a DNA probe that carries the msm genes of Methylosulfonomonas methylovora strain M2, strongly endorses the contention that a methanesulfonic acid monooxygenase related to that found in the previously known methanesulfonate-utilizing bacteria is present in strains P1 and P2. The isolation of bacteria containing conserved msm genes from diverse environments and geographical locations supports the hypothesis that a common enzyme may be globally responsible for the oxidation of methanesulfonate by natural methylotrophic communities.