The link between the microbial ecology, gene expression, and biokinetics of denitrifying polyphosphate-accumulating systems under different electron acceptor combinations.

The emission of the greenhouse gas nitrous oxide (N2O) can occur during biological nutrient removal. Denitrifying enhanced biological phosphorus removal (d-EBPR) systems are an efficient means of removing phosphate and nitrogen, performed by denitrifying polyphosphate-accumulating organisms (d-PAOs). The aim of this work was to study the effect of various combinations of electron acceptors, nitrate (NO3-), nitrite (NO2-), and N2O, on the denitrification pathway of a d-EBPR system. Batch tests were performed with different electron acceptor combinations, to explore the denitrification pathway. Reverse transcriptase-qPCR (RT-qPCR) and high-throughput sequencing, combined with chemical analysis, were used to study gene expression, microbial diversity, and denitrification kinetics. The potential for N2O production was greater than the potential for its reduction in most tests. A strong correlation was observed between the N2O reduction rate and the relative gene expression of nitrous oxide reductase per nitrite reductase (nosZ/(nirS + nirK)), suggesting that the expression of denitrifying marker genes is a strong predictor of the N2O reduction rate. The d-EBPR community maintained a core population with low variations throughout the study. Furthermore, phylogenetic analyses of the studied marker genes revealed that the organisms actively involved in denitrification were closely related to Thauera sp., Candidatus Accumulibacter phosphatis, and Candidatus Competibacter denitrificans. Moreover, Competibacter-related OTUs seem to be important contributors to the N2O reduction capacity of the system, likely scavenging the N2O produced by other organisms. Overall, this study contributes to a better understanding of the microbial biochemistry and the genetics involving biological denitrification removal, important to minimize N2O emissions in wastewater treatment plants.

Plant growth-promoting Burkholderia species isolated from annual ryegrass in Portuguese soils.

AIMS: To search for culturable Burkholderia species associated with annual ryegrass in soils from natural pastures in Portugal, with plant growth-promoting effects. METHODS AND RESULTS: Annual ryegrass seedlings were used to trap Burkholderia from two different soils in laboratory conditions. A combined approach using genomic fingerprinting and sequencing of 16S rRNA and recA genes resulted in the identification of Burkholderia strains belonging to the species Burkholderia graminis, Burkholderia fungorum and the Burkholderia cepacia complex. Most strains were able to solubilize mineral phosphate and to synthesize indole acetic acid; some of them could produce siderophores and antagonize the phytopathogenic oomycete, Phytophthora cinnamomi. A strain (G2Bd5) of B. graminis was selected for gnotobiotic plant inoculation experiments. The main effects were the stimulation of root growth and enhancement of leaf lipid synthesis and turnover. Fluorescence in situ hybridization and confocal laser microscopy evidenced that strain G2Bd5 is a rhizospheric and endophytic colonizer of annual ryegrass. CONCLUSIONS: This work revealed that annual ryegrass can naturally associate with members of the genus Burkholderia. A novel plant growth promoting strain of B. graminis was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel strain belongs to the plant-associated Burkholderia cluster and is a promising candidate for exploitation as plant inoculant in field conditions.

Occurrence and low pressure ultraviolet inactivation of yeasts in real water sources.

Low pressure ultraviolet photolysis proved to be an efficient treatment to achieve inactivation of different yeast species (Candida sp., Cryptococcus carnescens, Metschnikowia viticola/Candida kofuensis, Rhodosporidium babjevae, Rhodotorula minuta, and Rhodotorula mucilaginosa) isolated from water sources with very different compositions. The sensitivity degree of various yeast isolates to UV treatment varied among different genera. Species isolated from surface water gained additional photoprotective resistance as a defence mechanism to be able to survive under constant sunlight conditions compared to the groundwater isolates. Yeasts were found to be more resistant to UV treatment than E. coli, Cryptosporidium, and Giardia.

Quantitative proteomic analysis of ibuprofen-degrading Patulibacter sp. strain I11.

Ibuprofen is the third most consumed pharmaceutical drug in the world. Several isolates have been shown to degrade ibuprofen, but very little is known about the biochemistry of this process. This study investigates the degradation of ibuprofen by Patulibacter sp. strain I11 by quantitative proteomics using a metabolic labelling strategy. The whole-genome of Patulibacter sp. strain I11 was sequenced to provide a species-specific protein platform for optimal protein identification. The bacterial proteomes of actively ibuprofen-degrading cells and cells grown in the absence of ibuprofen was identified and quantified by gel based shotgun-proteomics. In total 251 unique proteins were quantitated using this approach. Biological process and pathway analysis indicated a number of proteins that were up-regulated in response to active degradation of ibuprofen, some of them are known to be involved in the degradation of aromatic compounds. Data analysis revealed that several of these proteins are likely involved in ibuprofen degradation by Patulibacter sp. strain I11.

Transcriptional analysis of virulence-related genes in enterococci from distinct origins.

AIMS: The role of enterococci in food fermentation and as probiotics counteracts with their increasing importance as human pathogens. Over the years, several virulence factors have been described, mainly in clinical strains but also in food isolates. However, differential expression of such traits may modulate the pathogenic potential of the harbouring enterococci. To further unravel such differential response, this study aims to identify environmental cues responsible for triggering the expression of virulence-related genes. METHODS AND RESULTS: The differential expression of eight virulence-related genes (cylMBAI, agg, esp, efaA(fs) and efaAfm) in 16 enterococci from distinct origins, grown in conditions simulating environmental colonization and infection sites, was analysed by reverse transcriptase PCR. The expression profiles were environmental and strain-dependent, because no constant response was observed neither for clinical nor food enterococci. CONCLUSIONS: Virulence expression profiles are strain-specific and unrelated with strain's origin or species allocation. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study constitutes the first approach aimed at the evaluation of the differential expression of enterococcal virulence-related genes combining so many growth environments, enterococcal species and origins. So, with this investigation, we intend to contribute to the clarification of enterococcal pathogenicity potential, especially for food strains.

Small but powerful: Light-emitting diodes for inactivation of Aspergillus species in real water matrices.

This study addressed the effectiveness of light emitting diodes to achieve inactivation of three different Aspergillus species (Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus) in a real water matrix. Three single small ultraviolet-C diodes emitting light at two different wavelengths were tested: 255 nm that is similar to the wavelength emitted by low pressure mercury lamps and 265 nm that is closer to the maximum absorbance wavelength of DNA. The ultraviolet-C diodes emitting light at 265 nm were found to be more effective than the 255 nm, achieving 3-log, 1-log and 5-log inactivations of Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus using less than 20 mJ/cm2 (13,97 mJ/cm2; 7,28 mJ/cm2; 19,74 mJ/cm2). The diodes have also affected the morphology of the fungal spores and increased the percentage of damaged and dead spores.

Bottled water: analysis of mycotoxins by LC-MS/MS.

The presence of mycotoxins in food samples has been widely studied as well as its impact in human health, however, information about its distribution in the environment is scarce. An analytical method comprising a solid phase extraction procedure followed by liquid chromatography tandem mass spectrometry analysis was implemented and validated for the trace analysis of mycotoxins in drinking bottled waters. Limits of quantification achieved for the method were between 0.2ngL(-1) for aflatoxins and ochratoxin, and 2.0ngL(-1) for fumonisins and neosolaniol. The method was applied to real samples. Aflatoxin B2 was the most frequently detected mycotoxin in water samples, with a maximum concentration of 0.48±0.05ngL(-1) followed by aflatoxin B1, aflatoxin G1 and ochratoxin A. The genera Cladosporium, Fusarium and Penicillium were the fungi more frequently detected. These results show that the consumption of these waters does not represent a toxicological risk for an adult.

Biodegradation of pesticides using fungi species found in the aquatic environment.

Relatively limited attention has been given to the presence of fungi in the aquatic environment compared to their occurrence in other matrices. Taking advantage and recognizing the biodegradable capabilities of fungi is important, since these organisms may produce many potent enzymes capable of degrading toxic pollutants. Therefore, the aim of this study was to evaluate the potential ability of some species of filamentous fungi that occur in the aquatic environment to degrade pesticides in untreated surface water. Several laboratory-scale experiments were performed using the natural microbial population present in the aquatic environment as well as spiked fungi isolates that were found to occur in different water matrices, to test the ability of fungi to degrade several pesticides of current concern (atrazine, diuron, isoproturon and chlorfenvinphos). The results obtained in this study showed that, when spiked in sterile natural water, fungi were able to degrade chlorfenvinphos to levels below detection and unable to degrade atrazine, diuron and isoproturon. Penicillium citrinum, Aspergillus fumigatus, Aspergillus terreus and Trichoderma harzianum were found to be able to resist and degrade chlorfenvinphos. These fungi are therefore expected to play an important role in the degradation of this and other pollutants present in the aquatic environment.

Biogenic amines occurrence in wine. Amino acid decarboxylase and proteolytic activities expression by Oenococcus oeni.

This work deals with the study of the proteolytic and amino acid decarboxylase activities of selected Oenococcus oeni isolates and the effect of yeast autolysis on biogenic amines production in wine. A total of 220 isolates of O. oeni were tested for decarboxylase and proteolytic activity. Only six isolates showed both activities, but only after a period of adaptation in a growth medium containing wine. The results reported on this paper show that proteolytic activity was dependent on medium composition and bacterial growth phase. It can be assumed that the ability of O. oeni to use wine peptides and to produce biogenic amines is not a constant characteristic of this species, and enzymatic system expression appears to be closely dependent on nutritional and energetical composition of the medium. It also seems to be strain dependent and not widespread among this bacterial community.

Cork stoppers industry: defining appropriate mould colonization.

AIMS: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. METHODS AND RESULTS: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. CONCLUSIONS: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.

Free chlorine inactivation of fungi in drinking water sources.

The effectiveness of free chlorine for the inactivation of fungi present in settled surface water was tested. In addition, free chlorine inactivation rate constants of Cladosporium tenuissimum, Cladosporium cladosporioides, Phoma glomerata, Aspergillus terreus, Aspergillus fumigatus, Penicillium griseofulvum, and Penicillium citrinum that were found to occur in different source waters were determined in different water matrices (laboratory grade water and settled water). The effect of using different disinfectant concentrations (1 and 3 mg/l), temperatures (21 and 4 °C), and pH levels (6 and 7) was addressed. The sensitivity degree of different fungi isolates to chlorine disinfection varied among different genera with some species showing a higher resistance to disinfection and others expected to be more prone to protection from inactivation by the water matrix components. When the disinfection efficiency measured in terms of the chlorine concentration and contact time (Ct) values needed to achieve 99% inactivation were compared with the Ct values reported as being able to achieve the same degree of inactivation of other microorganisms, fungi were found to be more resistant to chlorine inactivation than bacteria and viruses and less resistant than Cryptosporidium oocysts.

Assessment of the presence and dynamics of fungi in drinking water sources using cultural and molecular methods.

A comparison of different isolation techniques and culture media for detection of filamentous fungi and yeasts in the aquatic environment revealed that the use of membrane filtration with the media dichloran rose bengal chloramphenicol (DRBC) optimized fungi detection in terms of abundance and variety in three untreated water sources with very different characteristics (surface water, spring water, and groundwater). The diversity of the fungi population captured by direct DNA extraction of fungi collected by membrane filtration was compared with the isolates obtained after selective growth using different culture media through amplification of the internal transcribed spacer gene and denaturing gradient gel electrophoresis (DGGE). The Czapek-Dox agar, Sabouraud dextrose agar, and DRBC media showed closer similarities to those obtained by the uncultured biomass for the different water sources. Based on these data and the best enumeration results, DRBC is recommended for the assessment of fungi in water sources using culture-based methods. DGGE was also used to monitor temporal variations in the fungal population structure and showed that each water matrix possessed a distinct population profile as well as that changes in the fungal community can be expected in the different matrices throughout the year.

Registered designation of origin areas of fermented food products defined by microbial phenotypes and artificial neural networks.

Cheese produced from raw ewes' milk and chouriço, a Portuguese dry fermented sausage, are still produced in a traditional way in certain regions of Portugal by relying on colonization by microbial populations associated with the raw materials, equipment, and local environments. For the purpose of describing the product origins and types of these fermented foods, metabolic phenotypes can be used as descriptors of the product as well as to determine the presence of compounds with organoleptic value. The application of artificial neural networks to the metabolic profiles of bacterial isolates was assayed and allowed the separation of products from different regions. This method could then be used for the Registered Designation of Origin certification process of food products. Therefore, besides test panel results for these traditionally produced food products, another tool for validating products for the marketplace is available to the producers. The method can be improved for the detection of counterfeit products.