BTEX profile and health risk at the largest bulk port in Latin America, Paranaguá Port.

Port-related activities have a detrimental impact on the air quality both at the point of source and for considerable distances beyond. These activities include, but are not limited to, heavy cargo traffic, onboard, and at-berth emissions. Due to differences in construction, operation, location, and policies at ports, the site-specific air pollution cocktail could result in different human health risks. Thus, monitoring and evaluating such emissions are essential to predict the risk to the community. Environmental agencies often monitor key pollutants (PM2.5, PM10, NO2, SO2), but the volatile organic carbons (VOCs) most often are not, due to its analytical challenging. This study intends to fill that gap and evaluate the VOC emissions caused by activities related to the port of Paranaguá - one of the largest bulk ports in Latin America - by characterizing BTEX concentrations at the port and its surroundings. At seven different sites, passive samplers were used to measure the dispersion of BTEX concentrations throughout the port and around the city at weekly intervals from November 2018 to January 2019. The average and uncertainty of BTEX concentrations (µg m-3) were 0.60 ± 0.43, 5.58 ± 3.80, 3.30 ± 2.41, 4.66 ± 3.67, and 2.82 ± 1.95 for benzene, toluene, ethylbenzene, m- and p-xylene, and o-xylene, respectively. Relationships between toluene and benzene and health risk analysis were used to establish the potential effects of BTEX emissions on the population of the city of Paranaguá. Ratio analysis (T/B, B/T, m,p X/Et, and m,p X/B) indicate that the BTEX levels are mainly from fresh emission sources and that photochemical ageing was at minimum. The cancer risk varied across the sampling trajectory, whereas ethylbenzene represented a moderate cancer risk development for the exposed population in some of the locations. This study provided the necessary baseline data to support policymakers on how to change the circumstances of those currently at risk, putting in place a sustainable operation.

The microbiome of a shell mound: ancient anthropogenic waste as a source of Streptomyces degrading recalcitrant polysaccharides.

Metagenome amplicon DNA sequencing and traditional cell culture techniques are helping to uncover the diversity and the biotechnological potential of prokaryotes in different habitats around the world. It has also had a profound impact on microbial taxonomy in the last decades. Here we used metagenome 16S rDNA amplicon sequencing to reveal the microbiome composition of different layers of an anthropogenic soil collected at a shell mound Sambaqui archeological site. The Samabaqui soil microbiome is mainly composed by phyla Acidobacteria, Rokubacteria, Proteobacteria and Thaumarchaeota. Using culture-dependent analysis we obtained few Streptomyces isolates from the Sambaqui soil. One of the isolates, named Streptomyces sp. S3, was able to grow in minimal medium containing recalcitrant polysaccharides including chitin, xylan, carboxymethylcellulose or microcrystalline cellulose as sole carbon sources. The activities of enzymes degrading these compounds were confirmed in cell free supernatants. The genome sequence revealed not only an arsenal of genes related to polysaccharides degradation but also biosynthetic gene clusters which may be involved in the production of biotechnologically interesting secondary metabolites.

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion using Ni2+ magnetic beads.

To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.

Antigen production and development of an indirect ELISA based on the nucleocapsid protein to detect human SARS-CoV-2 seroconversion.

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Influence of seasonality on the aerosol microbiome of the Amazon rainforest.

The Amazon rainforest is the world's largest tropical forest, and this biome may be a significant contributor to primary biological aerosol (PBA) emissions on a global scale. These aerosols also play a pivotal role in modulating ecosystem dynamics, dispersing biological material over geographic barriers and influencing climate through radiation absorption, light scattering, or acting as cloud condensation nuclei. Despite their importance, there are limited studies investigating the effect of environmental variables on the bioaerosol composition in the Amazon rainforest. Here we present a 16S rRNA gene-based amplicon sequencing approach to investigate the bacterial microbiome in aerosols of the Amazon rainforest during distinct seasons and at different heights above the ground. Our data revealed that seasonal changes in temperature, relative humidity, and precipitation are the primary drivers of compositional changes in the Amazon rainforest aerosol microbiome. Interestingly, no significant differences were observed in the bacterial community composition of aerosols collected at ground and canopy levels. The core airborne bacterial families present in Amazon aerosol were Enterobacteriaceae, Beijerinckiaceae, Polyangiaceae, Bacillaceae and Ktedonobacteraceae. By correlating the bacterial taxa identified in the aerosol with literature data, we speculate that the phyllosphere may be one possible source of airborne bacteria in the Amazon rainforest. Results of this study indicate that the aerosol microbiota of the Amazon Rainforest are fairly diverse and principally impacted by seasonal changes in temperature and humidity.

Uncovering prokaryotic biodiversity within aerosols of the pristine Amazon forest.

Biological aerosols (bioaerosol) are atmospheric particles that act as a dispersion unit of living organisms across the globe thereby affecting the biogeographic distribution of organisms. Despite their importance, there is virtually no knowledge about bioaerosols emitted by pristine forests. Here we provide the very first survey of the prokaryotic community of a bioaerosol collected inside pristine Amazon forest at 2 m above ground. Total atmospheric particles were collected at the Amazon Tall Tower Observatory, subjected to metagenomic DNA extraction and the prokaryotic diversity was determined by 16S rRNA gene amplicon sequencing. A total of 271,577 reads of 250 bp of the 16S rRNA gene amplicon were obtained. Only 27% of the reads could be classified using the 16S SILVA database. Most belonged to Proteobacteria, Actinobacteria and Firmicutes which is in good agreement with other bioaerosol studies. Further inspection of the reads using Blast searches and the 18S SILVA database revealed that most of the dataset was composed of Fungi sequences. The identified microbes suggest that the atmosphere may act as an important gateway to interchange bacteria between plants, soil and water ecosystems.

Influence of ancient anthropogenic activities on the mangrove soil microbiome.

Mangroves are highly productive ecosystems located at the transition between the terrestrial and marine environments. Mangroves play an important role in carbon storage, nutrient cycling and support for the marine food web. Mangrove soils are formed by fine particles rich in organic carbon and are subject to constant fluctuations in oxygen, salinity and nutrient availability due to fresh water flux and tidal variations. Microbes play an important role in nutrient cycling in mangrove soils; however, studies on the mangrove soil microbiome are scarce. Here we compare the microbiome of pristine mangrove soil located in an environmentally protected area in Guaratuba, Southern Brazil, with the microbiome of mangrove soil affected by the presence of carbonaceaous debris eroding from an archeological site known as Sambaqui. We show that although the Sambaqui site has a major effect on soil chemistry, increasing the soil pH by 2.6 units, only minor changes in the soil microbiome were detected indicating resilience of the microbial community to pH variations. The high alpha diversity indexes and predicted metabolic potential suggest that the mangrove soil microbiome not only provides important ecological services but also may host a broad range of microbes and genes of biotechnological interest.

Polysaccharides present in cultivated Teloschistes flavicans symbiosis: comparison with those of the thallus.

The chemical structures of polysaccharides present in aposymbiotically cultured myco- and photobionts of the lichen Teloschistes flavicans were determined, in order to compare them with those previously found in the intact thallus. The mycobiont was cultured on a solid Lilly and Barnett medium and the resulting colonies were freeze dried, defatted, and their polysaccharides were extracted successively with 2%, 10% and 30% aq. KOH, each at 100 degrees C. The extracts were neutralized (HOAc) and fractionated, giving rise to three homogeneous fractions, PFSK2 from 2% KOH, which contained a (1-->4),(1-->6)-linked alpha-glucan (1:1 ratio, pullulan), fraction PK10 from 10% KOH extraction, which was a linear (1-->3)-linked linear beta-glucan (laminaran), and fraction PK30 from 30% KOH extraction, being a branched (1-->3),(1-->6)-linked beta-glucan. The photobiont (Trebouxia sp. de Puymaly) was cultured in liquid nutrient medium, and after purification, a linear (1-->5)-linked beta-galactofuranan was characterized. The galactofuranan and the laminaran were not present in the symbiotic thallus, in contrast to the glucans, showing that the mycobiont alone produces them without participation of the photobiont.

Molecular studies of photobionts of selected lichens from the coastal vegetation of Brazil.

A light microscopic and molecular analysis of photobionts in Ramalina and Cladonia from coastal habitats of Brazil is presented. A Bayesian phylogenetic analysis of ITS rDNA sequences suggests a Trebouxia lineage which is preferentially tropical in geographic distribution. This highly diverse clade also includes the morphological similar species Trebouxia higginsiae and galapagensis. Within the predominantly tropical clade of Trebouxia we distinguish several subclades, three of which are represented in our samples of Ramalina species. Since sexuality has not been recognized in coccal lichenised photobionts until recently, we cannot apply a biological species concept, but when compared with the sequence diversity between known species we conclude that several new species need to be described in this clade. The mutually exclusive presence of other Trebouxia lineages in temperate samples of Ramalina suggests an evolution towards higher selectivity in this genus. A strictly tropical lineage is not conspicuous in the photobionts of the genus Asterochloris sampled from Cladonia so far.

Carbohydrate, glycolipid, and lipid components from the photobiont (Scytonema sp.) of the lichen, Dictyomema glabratum.

The photobiont of the lichen, Dictyonema glabratum (Scytonema sp.), was isolated and cultivated in a soil-extract medium and submitted to chemical analysis. Successive extractions with CHCl3-MeOH, aqueous MeOH, and H2O gave rise to solutions of lipids (25%), low-molecular-weight carbohydrates (22%), and polysaccharides (4%), respectively. TLC of the lipid extract showed the presence of glycolipids, which were further purified and examined by NMR spectroscopy and GC-MS. Monogalactosyldiacylglycerol (1%), digalactosyldiacylglycerol (0.8%), trigalactosyldiacylglycerol (0.4%), and sulfoquinovosyldiacylglycerol (0.5%) were identified. The most abundant fatty acid ester in each fraction was palmitic (C16:0), but a great variation of the ester composition from one to another was found. Others present were those of C12:0, C14:0, C15:0, C16:1, C17:0, C18:0, C18:1, C18:2, C18:3, C22:0, C22:2, and C24:0. The lipid extract was also subjected to acid methanolysis, which gave rise to dodecane, 2-Me-heptadecane, 2,6-Me2-octadecane, and 8-Me-octadecane, methyl esters of C14:0, C15:0, C16:0, C16:1, C17:0, C18:0, C18:1, C18:2, C20:0, and C24:0 fatty acids, and the dimethyl ester of decanedioic acid. The polysaccharide had mainly Glc, Gal, and Man, with small amounts of 3-O-methylrhamnose and 2-O-methylxylose, both found in plants, and unexpectedly, some of the units were beta-galactofuranose, typical of fungal, but not cyanobacterial polysaccharides. The low-molecular-weight carbohydrates showed mannose as the main free reducing sugar, which differs from Nostoc sp. and Trebouxia sp. photobionts.

A fungus-type beta-galactofuranan in the cultivated Trebouxia photobiont of the lichen Ramalina gracilis.

A structural characterization of polysaccharides extracted from the aposymbiotically cultured photobiont of the lichen Ramalina gracilis was carried out in order to compare them with those previously found in the symbiotic thallus. The photobiont was isolated from thallus fragments, following the method of Yamamoto, and cultivated in a liquid nutrient medium. Freeze-dried cells were defatted, and the polysaccharides extracted successively with water and aq. 10% KOH, each at 100 degrees C. After purification, the soluble fractions provided a polysaccharide containing a (1-->5)-linked beta-galactofuranosyl backbone, substituted in a small proportion at O-6 by beta-Galf units. Amylose was also found, as insoluble material obtained on freeze-thawing of the alkaline extract. These polysaccharides have not been found in the symbiotic thallus of Ramalina gracilis, which contained only water-soluble (isolichenan) and insoluble glucans (nigeran and laminaran), and galactomannan. Surprisingly, the galactofuranan has similarities with those found in some fungal cell walls.

Linear beta-mannose-containing polysaccharide, beta-xylan, and amylose from the cultured photobiont Trebouxia sp. of the ascolichen Ramalina celastri.

The cultured photobiont Trebouxia sp. of Ramalina celastri was successively extracted at 100 degrees C with hot water, 2% aqueous KOH, and 10% aqueous KOH to give polysaccharide-containing fractions A (2.9%), B (3.9%), and C (0.9% yield) respectively. The intact biont contained 3.8% amylose, which was present in each fraction, and was identified by a blue color formed with iodine solution. In fraction A, and following retrogradation from aqueous solution, it was characterized by (13)C-NMR spectroscopy. Fraction B was treated with alpha-amylase to give a water-soluble fraction consisting mainly of beta-mannose-containing polysaccharides (1.5% yield), whose main component had dn/dc 0.162 and M(r) 17 kDa. Fraction C was subjected to freeze-thawing and the precipitate was treated with alpha-amylase to give a resistant, linear, low molecular mass (1-->4)-linked beta-xylan. The beta-D-mannopyranan preparation contained mainly of 3-O- (28%), 4-O- (11%), and 6-O-substituted Manp units (35%), with 3-O-substituted Rhap units (11%). A controlled Smith degradation provided a beta-mannan with nonreducing end- (8%), 3-O- (85%) and 6-O-substituted units, showing (1-->3)- and (1-->6)-linked structures in the original polysaccharide. These could be present as block-type structures.

A new pullulan and a branched (1-->3)-, (1-->6)-linked beta-glucan from the lichenised ascomycete Teloschistes flavicans.

The polysaccharides formed on hot alkaline extraction of the ascomycetous lichen Teloschistes flavicans were fractionated to give two glucans, which were characterised by methylation analysis and 1D and 2D NMR spectroscopy. One was a branched beta-glucan containing (1-->3) and (1-->6) linkages, a structure which is more typical of basidiomycetes rather than ascomycetes, which have linear glucans. The other was an alpha-glucan with alternating (1-->4) and (1-->6) linkages, found for the first time in Nature. This structure can be classified as a pullulan, which has been isolated from the fungi Aureobasidium pullulans, Tremella mesenterica, and Cyttaria harioti, but has different ratios of the component glycosidic linkages. The significance of the presence of the isolated alpha- and beta-glucans is discussed.