Inês Baptista

Wavelength dependence of biological damage induced by UV radiation on bacteria

The biological effects of UV radiation of different wavelengths (UVA, UVB and UVC) were assessed in nine bacterial isolates displaying different UV sensitivities. Biological effects (survival and activity) and molecular markers of oxidative stress [DNA strand breakage (DSB), generation of reactive oxygen species (ROS), oxidative damage to proteins and lipids, and the activity of antioxidant enzymes catalase and superoxide dismutase] were quantified and statistically analyzed in order to identify the major determinants of cell inactivation under the different spectral regions. Survival and activity followed a clear wavelength dependence, being highest under UVA and lowest under UVC. The generation of ROS, as well as protein and lipid oxidation, followed the same pattern. DNA damage (DSB) showed the inverse trend. Multiple stepwise regression analysis revealed that survival under UVA, UVB and UVC wavelengths was best explained by DSB, oxidative damage to lipids, and intracellular ROS levels, respectively.

Effects of UV-B Radiation on the Structural and Physiological Diversity of Bacterioneuston and Bacterioplankton

ABSTRACT The effects of UV radiation (UVR) on estuarine bacterioneuston and bacterioplankton were assessed in microcosm experiments. Bacterial abundance and DNA synthesis were more affected in bacterioplankton. Protein synthesis was more inhibited in bacterioneuston. Community analysis indicated that UVR has the potential to select resistant bacteria (e.g., Gammaproteobacteria), particularly abundant in bacterioneuston.

Diversity in UV sensitivity and recovery potential among bacterioneuston and bacterioplankton isolates.

Aims:  To assess the variability in UV‐B (280–320 nm) sensitivity of selected bacterial isolates from the surface microlayer and underlying water of the Ria de Aveiro (Portugal) estuary and their ability to recover from previous UV‐induced stress.

Effect of tributyltin (TBT) in the metabolic activity of TBT-resistant and sensitive estuarine bacteria

The effect of tributyltin (TBT) on growth and metabolic activity of three estuarine bacteria with different TBT resistance profiles was investigated in an organic‐rich culture medium (TSB) and in phosphate buffered saline (PBS) buffer. Exposure to TBT was assessed by determining its effect on growth (OD600 nm measurement), bacterial productivity (leucine incorporation), viability (CFU counts), aggregation and cell size (from Live/Dead analysis), ATP and NADH concentrations. TBT exposure resulted in decrease of bacterial density, cell size, and metabolic activity. In addition, cell aggregates were observed in the TBT‐treated cultures. TBT strongly affected bacterial cell metabolism and seemed to exert an effect on its equilibrium, interfering with cell activity. Also, TBT toxicity was lower when cells were grown in TSB than in PBS, suggesting that a nutrient‐rich growth medium can protect cells from TBT toxicity. This study contributes to our understanding of the TBT‐resistant cell behavior reflected in its physiology and metabolic activity. This information is of utmost importance for further studies of TBT bioremediation.

Can Volatile Organic Metabolites Be Used to Simultaneously Assess Microbial and Mite Contamination Level in Cereal Grains and Coffee Beans

A novel approach based on headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–ToFMS) was developed for the simultaneous screening of microbial and mite contamination level in cereals and coffee beans. The proposed approach emerges as a powerful tool for the rapid assessment of the microbial contamination level (ca. 70 min versus ca. 72 to 120 h for bacteria and fungi, respectively, using conventional plate counts), and mite contamination (ca. 70 min versus ca. 24 h). A full-factorial design was performed for optimization of the SPME experimental parameters. The methodology was applied to three types of rice (rough, brown, and white rice), oat, wheat, and green and roasted coffee beans. Simultaneously, microbiological analysis of the samples (total aerobic microorganisms, moulds, and yeasts) was performed by conventional plate counts. A set of 54 volatile markers was selected among all the compounds detected by GC×GC–ToFMS. Principal Component Analysis (PCA) was applied in order to establish a relationship between potential volatile markers and the level of microbial contamination. Methylbenzene, 3-octanone, 2-nonanone, 2-methyl-3-pentanol, 1-octen-3-ol, and 2-hexanone were associated to samples with higher microbial contamination level, especially in rough rice. Moreover, oat exhibited a high GC peak area of 2-hydroxy-6-methylbenzaldehyde, a sexual and alarm pheromone for adult mites, which in the other matrices appeared as a trace component. The number of mites detected in oat grains was correlated to the GC peak area of the pheromone. The HS-SPME/GC×GC–ToFMS methodology can be regarded as the basis for the development of a rapid and versatile method that can be applied in industry to the simultaneous assessment the level of microbiological contamination and for detection of mites in cereals grains and coffee beans.

A microcosm approach to evaluate the degradation of tributyltin (TBT) by Aeromonas molluscorum Av27 in estuarine sediments

Tributyltin (TBT) is a biocide extremely toxic to a wide range of organisms, which has been used for decades in antifouling paints. Despite its global ban in 2008, TBT is still a problem of great concern due to the high levels trapped in sediments. Aeromonas molluscorum Av27 is a TBT degrading bacterium that was isolated from an estuarine system. We investigated the ability and the role of this bacterium on TBT degradation in this estuarine system, using a microcosm approach in order to mimic environmental conditions. The experiment was established and followed for 150 days. Simultaneously, changes in the indigenous bacterial community structure were also investigated. The results revealed a maximum TBT degradation rate of 28% accompanied by the detection of the degradation products over time. Additionally, it was observed that TBT degradation was significantly enhanced by the presence of Av27. In addition a significantly higher TBT degradation occurred when the concentration of Av27 was higher. TBT degradation affected the bacterial community composition as revealed by the changes in the prevalence of Proteobacteria subdivisions, namely the increase of Deltaproteobacteria and the onset of Epsilonproteobacteria. However, the addition of Av27 strain did not affect the dominant phylotypes. Total bacterial number, bacterial biomass productivity, 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analyses also indicated alterations on the bacterial community structure over time, with bacteria non-tolerant to pollutants increasing their representativeness, as, for instance, the increase of the number of Alphaproteobacteria clones from 6% in the beginning to 12% at the end of the experiment. The work herein presented confirms the potential of Av27 strain to be used in the decontamination of TBT-polluted environments.

Evaluation of resistance development and viability recovery by toxigenic and non-toxigenic Staphylococcus aureus strains after repeated cycles of high hydrostatic pressure.

In this work, the development of resistance and the recovery of growth after several consecutive cycles of high hydrostatic pressure (HPP) were for the first time evaluated in different strains of Staphylococcus aureus. Three strains of this important and highly resilient to HPP foodborne pathogen were used: a non-enterotoxigenic ATCC 6538 strain, treated with 600 MPa for 30 min at 20 °C, and the toxigenic strains 2153 MA (with enterotoxin A) and 2065 MA (with the enterotoxins A, G and I), treated with 600 MPa for 15 min at 20 °C. After the first treatment, surviving colonies were used to produce new bacterial cultures. This procedure was repeated nine times more for each bacterium or until total inactivation occurred. The inactivation profile of non-enterotoxic strain and the two enterotoxic strains did not change after consecutive cycles, but the toxic strain with three enterotoxins was completely inactivated after the fourth cycle. The three strains did not recover their viability after 14 days. The results indicate that HPP effectively inactivates non-toxigenic and toxigenic strains of S. aureus after a single treatment. The surviving bacteria did not develop resistance after 10 cycles of pressurization and did not recover their viability after 14 days of incubation.

Bacterial biomass production in an estuarine system: high variability of leucine conversion factors and changes in bacterial community structure during incubation

Heterotrophic bacterioplankton play a key role in the transfer of organic carbon from the dissolved pool into planktonic trophic webs. The accurate measurement of bacterial biomass pro- duction (BBP) is crucial for the establishment of carbon budgets in aquatic systems. Estimation of BBP from the incorporation of radiolabelled leucine into bacterial proteins requires the use of empirical, semitheoretical or theoretical conversion factors (CFs). In this study, our estimate of the BBP for the estuarine system of Ria de Aveiro was higher when we used empirical and semitheoretical CFs than when we used theoretical ones. Empirical CFs ranged between 9.26 and 29.81 kg C mol -1 in the marine zone and between 4.25 and 16.88 kg C mol -1 in the brackish zone. Semitheoretical CFs ranged from 5.06 to 9.49 kg C mol -1 in the marine zone and from 5.28 to 9.34 kg C mol -1 in the brack- ish zone. During experiments to determine empirical CFs, the structure of the bacterial community was also analyzed. Sample preparation and the addition of leucine altered the structure of the bacte- rial community, and this probably affected the determination of the empirical CFs. The results show that, besides the effect of the use of an inadequate CF, the estimates of BBP can be biased in a sys- tem-specific way by the alteration of the structure of the bacterial community as a result of sample manipulation (i.e. filtration/dilution, incubation, and addition of non-radiolabelled and radiolabelled leucine).

Contribution of chemical water properties to the differential responses of bacterioneuston and bacterioplankton to ultraviolet‐B radiation

The surface microlayer (SML) is characterized by different physicochemical properties from underlying waters (UW). However, whether these differences in abiotic factors underlie the distinct sensitivity of bacterioneuston (i.e. SML bacteria) and bacterioplankton to environmental stressors remains to be addressed. We investigated the contribution of abiotic factors to the UV-B sensitivity of bacterioneuston and bacterioplankton. Nutrients (especially nitrogen and phosphate) emerged as important determinants of bacterial UV-B sensitivity. The role of particles, nutrients, and dissolved organic components on bacterial UV-B sensitivity was further evaluated using dilution cultures. Filtered samples were twofold more UV sensitive than unfiltered samples, suggesting a UV-protective effect of particles. High nutrient concentrations attenuated bacterial UV-B sensitivity (up to 40%), compared with unamended conditions, by influencing bacterial physiology and/or community composition. Suspending cells in natural water, particularly from the SML, also attenuated UV-B sensitivity (up to 23%), compared with suspension in an artificial mineral solution. Bioassays using Pseudomonas sp. strain NT5I1.2B revealed that chemical water properties influence UV-induced oxidative damage. UV-B sensitivity was associated with high cell-specific activities. The chemical environment of the SML and UW influences UV-B effects on the corresponding bacterial communities. Maintaining low cell activities might be advantageous in stressful environments, like the SML.

Influence of incubation conditions on bacterial production estimates in an estuarine system

This study aimed to assess the influence of incubation conditions in the determination of bacterial production (BP). In order to achieve that goal, experimental setups were performed in situ and in the laboratory under both dark and light conditions. To test spatial and seasonal variations and the different natural light exposure of microorganisms, sampling was performed in two distinct zones of the estuary Ria de Aveiro (Portugal), typifying the marine and brackish water zones of the estuarine system. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments was used to monitor possible alterations in bacterial community composition induced by the incubation conditions. The results showed that BP determined in situ conditions significantly differed from in the laboratory. In the marine zone, a defined pattern of variation was detected, with consistent higher values of BP in laboratory dark conditions. This trend was not present in the brackish water zone. The seasonal and spatial variability of BP observed in field incubations was related to the physical–chemical proprieties of the water column, irradiance levels and the original community composition. The metabolic active profiles of bacteria were substantially different in the several incubation conditions, suggesting that methodological procedure influences the bacterial community composition, and the values of BP reported for aquatic ecosystems could be quite different from the real ones. In the light of these results, we suggest that BP determinations should be conducted under in situ conditions. However, due to execution limitations, BP needs to be frequently determined in the laboratory, and in this case, dark incubations provide more approximate values. This is the method routinely used, and although this incubation condition can cause stimulation of BP, the structure of the bacterial community is more similar to the one obtained with the in situ incubations.