Abstract Dyes are recalcitrant compounds that resist conventional biological treatments. The degradation of three textile dyes (Indigo, RBBR and Sulphur Black), and the dye-containing liquid effluent and solid waste from the Municipal Treatment Station, Americana, São Paulo, Brazil, by the cyanobacteria Anabaena flos-aquae UTCC64, Phormidium autumnale UTEX1580 and Synechococcus sp. PCC7942 was evaluated. The dye degradation efficiency of the cyanobacteria was compared with anaerobic and anaerobic-aerobic systems in terms of discolouration and toxicity evaluations. The discoloration was evaluated by absorption spectroscopy. Toxicity was measured using the organisms Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. The three cyanobacteria showed the potential to remediate textile effluent by removing the colour and reducing the toxicity. However, the growth of cyanobacteria on sludge was slow and discoloration was not efficient. The cyanobacteria P. autumnale UTEX1580 was the only strain that completely degraded the indigo dye. An evaluation of the mutagenicity potential was performed by use of the micronucleus assay using Allium sp. No mutagenicity was observed after the treatment. Two metabolites were produced during the degradation, anthranilic acid and isatin, but toxicity did not increase after the treatment. The cyanobacteria showed the ability to degrade the dyes present in a textile effluent; therefore, they can be used in a tertiary treatment of effluents with recalcitrant compounds.

The halogenated compounds aeruginosin and cyanopeptolin (protease inhibitors) are well documented in several cyanobacteria. In this work, the presence of genetic coding for aeruginosin and cyanopeptolin in Sphaerocavum genus and their characterization by mass spectrometry and potential toxic effects were investigated. Three strains of Sphaerocavum brasiliense (CCIBt 3094, CCIBt 3096 and CCIBt 3316) isolated from different reservoirs in São Paulo State, and one (CCIBt 3179) from Rio Grande do Norte State were selected for this study. Fragments of aerA-aerB and mcnC-mcnE gene regions (aeruginosin and cyanopeptolin, respectively) were amplified by polymerase chain reaction (PCR) and sequenced. Compounds were characterized by mass spectrometry. All the studied strains presented aeruginosin and cyanopeptolin synthetase genes. Mass spectrometry showed the presence of several aeruginosin and cyanopeptolin variants. The Allium test showed that the ethanolic extract promoted toxic and cytotoxic actions in acute and chronic treatments. These naturally occurring protease inhibitors can play an important role in the physiopathology of human diseases.

Cianobactérias estão amplamente distribuídas no ambiente e podem ser uma alternativa eficaz e econômica para a remoção de corantes de efluentes de indústrias têxteis. O presente trabalho investigou o potencial de seis linhagens de cianobactérias de descolorir onze tipos de corantes têxteis. A absorção máxima de cada corante foi verificada usando um espectrofotômetro. Espectrometria de massas foi utilizada na verificação da degradação e possível remoção de corantes pelas cianobactérias. Os resultados mostraram que todas as linhagens avaliadas foram capazes de remover indigo, amarelo palanil, amarelo indantreno, azul indantreno, azul dispersol, vermelho indantreno e vermelho dispersol em mais de 50%. O isolado brasileiro Phormidium sp. CENA135 foi capaz de descolorir e remover completamente o índigo azul BANN 30. Este estudo confirmou a capacidade das cianobactérias de descolorir e, possivelmente, degradar corantes têxteis estruturalmente diferentes, sugerindo a possibilidade de sua aplicação em estudos de biorremediação.

Cyanobacteria are widely distributed in the environment and may be an effective and economic alternative for removing dyes from textile industry effluents. The present work investigated the potential of six cyanobacterial strains in decolorizing eleven types of textile dyes. The maximum absorbance of each dye was verified using a spectrophotometer. Mass spectrometry was used to verify the removal and possible degradation of dyes by the cyanobacteria. The results showed that all of the evaluated cyanobacteria were able to remove indigo, palanil yellow, indanthrene yellow, indanthrene blue, dispersol blue, indanthrene red and dispersol red by more than 50%. The Brazilian isolate Phormidium sp. CENA135 was able to decolorize and completely remove indigo blue BANN 30. This study confirmed the capacity of cyanobacteria to decolorize and possibly to structurally degrade different textile dyes, suggesting the possibility of their application in bioremediation studies.

Os sideróforos provenientes de Xylella fastidiosa de videiras foram investigados. Tais metabólitos seqüestram ferro, um elemento essencial, do hospedeiro, o que os torna um potencial fator de patogenicidade. Em um meio de cultura em placa com limitação de ferro, tais sideróforos foram detectados pela reação com o complexo cromoazurol S (CAS). Diferentes métodos de análise instrumentais foram utilizados para caracterização dos sideróforos, como: cromatografia de afinidade por metal imobilizado (IMAC), cromatografia micelar eletrocinética capilar (MEKC) e espectrometria de massas com ionização por electrospray (ESI-MS). Os resultados obtidos confirmaram a produção de sideróforos. A extração do(s) composto(s) por IMAC com Fe3+ imobilizado foi uma etapa importante. O(s) sideróforo(s) não foi separado por eletroforese capilar de zona, indicando sua neutralidade sob os pHs investigados. As análises por MEKC apresentaram um pico diferente (quando comparadas à análise do controle), com caráter levemente hidrofóbico. A espectrometria de massas mostrou que os compostos alvos podem ter uma massa molecular relativa dentro da esperada para sideróforos, como: 875, 1004 e 1092 Da.

Siderophore molecules from grapevine Xylella fastidiosa were investigated. Such metabolites sequester iron, an essential element, from the host, making them a potential factor of pathogenicty. In an iron-limited medium, siderophores were detected in culture plates of X. fastidiosa containing the complex Chromeazurol S (CAS). A combination of different instrumental analyses was used for siderophore(s) characterization, such as: immobilized metal affinity chromatography (IMAC), micelar electrokinetic capillary chromatography (MEKC) and electrospray-mass spectrometry (ESI-MS). The results show that grapevine X. fastidiosa produced siderophore(s), as confirmed by the CAS plate assay. The extraction of the compound(s) using IMAC with immobilized Fe3+ was important for analyte purification. The chelator was not separated by capillary zone electrophoresis, indicating the possibility of a neutral compound under the investigated pHs. MEKC runs presented a different peak (when compared to the control analysis) which represented a slightly hydrophobic compound. Mass spectrometry showed that the compound(s) may have a relative molecular mass within the expected range for siderophore molecules, such as 875, 1004 and 1092 Da.

Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.

In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy.

Vários gêneros de cianobactérias produzem metabólitos secundários tóxicos, entre eles as hepatotoxinas microcistinas. A análise de microcistinas em águas para abastecimento humano é uma exigência do Ministério da Saúde (Portaria 518/2004), mas essa portaria ainda não estabelece o método de extração e análise a serem usados e a quantificação da toxina é comumente realizada por ELISA ("enzyme-linked immunosorbent assay") ou HPLC (cromatografia líquida de alta eficiência), cuja eficiência depende do método de extração utilizado. Neste trabalho foi desenvolvido um método simples, rápido e barato de extração para o isolamento e identificação de microcistinas. Para isso, selecionou-se a linhagem Microcystis aeruginosa NPLJ-4 descrita como produtora de microcistina-LR. Oito diferentes tratamentos foram testados para determinar a melhor extração da toxina. As amostras foram analisadas por LC-MS (cromatografia líquida acoplada a espectrometria de massas), ELISA e Q-TOF ("quadrupole time-of-flight"). Os resultados mostraram que a melhor extração foi a que usou sonicação das amostras diluídas em água. O método proposto permite o processamento rápido das amostras e estabelece um método de extração para análise e identificação de microcistina-LR e outras variantes.

Several cyanobacterial genera produce toxic secondary metabolites, the most well-known of which are the hepatotoxic microcystins (MCYSTs). Microcystin analyses in drinking water are a requirement of the Health Ministry (Regulation 518/2004) in Brazil, but this regulation does not establish which extraction and analytical method should be used; toxin quantification is usually carried out by ELISA (enzyme-linked immunosorbent assay) or HPLC (high performance liquid chromatography), the efficiency of which depends on the extraction method used. In this work a simple, fast and cheap method of extraction was developed for the isolation and identification of MCYSTs. For this, the strain Microcystis aeruginosa NPLJ-4, reported to be a MCYST-LR producer, was selected. Eight different treatments were tested to determine the best MCYST extraction. Samples were applied in LC-MS (liquid chromatography-mass spectrometry), ELISA and Q-TOF (quadrupole time-of-flight). The most efficient extraction was achieved by sonicating samples diluted in water. The proposed method permits rapid sample processing, and establishes an extraction method for both the analysis and identification of MCYST-LR and other variants.

Este trabalho teve como objetivo avaliar o conteúdo de antocianinas no suco e as características físicas e químicas dos frutos de oito variedades de laranjas sanguíneas e de laranja Valência, e, também, verificar os efeitos do armazenamento dos frutos a 10ºC, durante um período de até 60 dias, nos parâmetros avaliados. Os teores de antocianina foram determinados utilizando-se de um método espectrofotométrico, assim como dez características físicas e químicas dos frutos e dos sucos foram avaliadas antes e durante o armazenamento. Todas as variedades de laranja avaliadas apresentaram naturalmente baixos teores ou nenhum teor de antocianina no suco. O armazenamento durante um período de até 60 dias, em baixa temperatura, possibilitou acúmulo significativo de antocianina no suco, porém de maneira desigual nas variedades de laranjas sanguíneas testadas. As variedades Moro foram as que apresentaram suco contendo os maiores teores de antocianina no final do armazenamento. À exceção de duas variedades, Sanguinelli (Marrocos e Polidari), as demais variedades sanguíneas avaliadas podem ser consideradas como semelhantes entre si e adequadas ao consumo. O armazenamento dos frutos a 10ºC, durante o período máximo de 60 dias, alterou significativamente somente as variáveis: largura de frutos, teor de SS, acidez e o rendimento de suco.

The aim of this study was to assess the content of anthocyanins in the juice and the physical and chemical characteristics of the fruit of eight blood orange varieties and of orange Valencia, and also verify the effects of storage of fruits at 10ºC, for a period of up to 60 days, in the same parameters. The anthocyanin content was determined using a spectrofotometric method and ten physical and chemical characteristics of the fruits and juice were also evaluated before and during the storage. Under natural conditions all varieties showed fruits with low anthocyanin content in the juice. Cold storage during a period of up to 60 days enables a significant accumulation of anthocyanin in the juice, but in a different level in the evaluated blood orange varieties. Moro is the variety that showed the highest anthocyanin content in the end of the storage period. Except for two Sanguinelli varieties (Morocco and Polidari), the other varieties can be considered as similar as others and suitable for consumption. The storage of fruits to 10ºC during the maximum period of 60 days significantly modifies only fruit width, juice content in fruits, soluble solids content and acidity of the juice.

O objetivo deste trabalho foi estudar a produção de sideróforos pelas bactérias endofíticas Methylobacterium spp., que ocupam o mesmo nicho ecológico que Xylella fastidiosa subsp. pauca (Xfp), em plantas cítricas. A produção de sideróforos, pelas linhagens de Methylobacterium, foi testada por meio do ensaio de cromoazarol-ágar (chromeazurol agar assay-CAS), teste de Csáky (tipo hidroxamato) e do teste de Arnow (tipo catecol). Além disso, a habilidade de Xfp em utilizar sideróforos produzidos por bactérias endofíticas, como fonte de ferro, in vitro, foi avaliada. Todas as 37 linhagens de Methylobacterium spp. testadas foram positivas para a produção de sideróforos, pelo teste CAS-ágar. Methylobacterium spp. foram capazes de produzir sideróforos do tipo hidroxamato, mas não do tipo catecol. O crescimento in vitro de Xfp foi estimulado pela presença de sideróforos no sobrenadante de Methylobacterium mesophilicum endofítica.

The objective of this work was to study the production of siderophores by endophytic bacteria Methylobacterium spp., which occupy the same ecological niche as Xylella fastidiosa subsp. pauca (Xfp) in citrus plants. The siderophore production of Methylobacterium strains was tested according to chromeazurol agar assay test (CAS), Csáky test (hydroxamate-type) and Arnow test (catechol-type). In addition, the ability of Xfp to use siderophores, in vitro, produced by endophytic bacteria as source of iron, was evaluated. All 37 strains of Methylobacterium spp. tested were CAS-positive for siderophore production. Methylobacterium spp. produced hydroxamate-type, but not catechol-type siderophores. In vitro growth of Xfp was stimulated by the presence of supernatant siderophores of endophytic Methylobacterium mesophilicum.

Pleiotropic drug resistance (PDR) proteins, a subfamily of the ATP-binding cassette (ABC) transporters, have been recently shown to play a role in plant defense against biotic and abiotic stresses. However, nothing is known about their expression in citrus. To investigate the occurrence of PDR homologues in citrus species, we have surveyed EST sequences from different tissues and conditions of the Citrus Expressed Sequence Tags (CitEST) database, through sequence similarity search analyses and inspections for characteristic PDR domains. Multiple sequence alignments, prediction of transmembrane topology and phylogenetic analysis of PDR-like proteins were additionally performed. This study allowed the identification of nine putative proteins showing characteristic PDR features in citrus species under various conditions, which may indicate a potential correlation between PDRs and stress and metabolism of citrus plants. Moreover, a tissue-specific putative PDR-like protein was found in sweet orange fruits. To our knowledge, this is the first report regarding the identification of citrus ESTs encoding PDR-like proteins as well as the first to identify a putative full ABC transporter with specific expression in fruits.

Bacterial production of siderophores may involve specific genes related to nonribosomal peptide and polyketide biosynthesis, which have not been fully identified in the genome of Xylella fastidiosa strain 9a5c. However, a search for siderophore-related genes in strain 9a5c indicated five membrane receptors, including siderophore, ferrichrome-iron and hemin receptors. All these biomolecules are thought to be associated with iron transport and utilization. Eighty isolates obtained from citrus orchards containing trees that developed citrus variegated chlorosis (CVC) were screened for siderophore production. The results demonstrated that only 10 of the isolates did not produce siderophores. Additional strains obtained from coffee, almond, mulberry, elm, ragweed, periwinkle and grape also infected by X. fastidiosa were also shown by the chromeazurol bioassay to produce siderophores. In order to correlate siderophore production with the presence of siderophore-related genes, a polymerase chain reaction (PCR) was developed using specific primers for the catechol-type ferric enterobactin receptor (pfeA) and the hydroxamate-type ferrisiderophore receptor (fiuA) genes of strain 9a5c. The PCR results confirmed our hypothesis by demonstrating that amplification products were detected in all strains except for those isolates that did not produce siderophores.