Mecanismos de resposta a mercúrio em plantas de sapal

Mestrado em Métodos Biomoleculares AvançadosOs sapais encontram-se entre os ecossistemas mais produtivos do mundo, suportando um número elevado de espécies vegetais e animais. O mercúrio é considerado um dos metais mais tóxicos, em que os seus efeitos adversos têm repercussões ao longo da cadeia alimentar de todo o ecossistema. A recuperação do mercúrio de lamas e esgotos industriais é obrigatória por lei, contudo, a descontaminação de zonas estuarinas e cursos de água que já se encontram poluídos tem sido negligenciada, dado que as técnicas correntes de remediação de mercúrio utilizam processos físico-químicos, dispendiosos, perturbadores do ambiente e frequentemente ineficazes. Um sistema de fitorremediação, em que as plantas extraem, sequestram ou desintoxicam o mercúrio dos sedimentos de sistemas aquáticos poderá ser uma solução mais atraente. No entanto, esta técnica encontra-se ainda numa fase inicial de desenvolvimento, sendo necessária uma maior investigação sobre os mecanismos moleculares subjacentes à acumulação de metais de modo a optimizar os processos inerentes a esta técnica de remediação. Neste contexto, o presente trabalho incidiu sobre o estudo do papel que as espécies vegetais A. patula, H. portulacoides, J. maritimus e P. australis possuem na descontaminação de uma área específica do sapal da Ria de Aveiro, o Esteiro de Estarreja, sujeito a uma elevada contaminação por mercúrio. Os principais objectivos deste trabalho incluíram, numa primeira fase, a análise dos níveis de contaminação dos rizosedimentos das várias espécies de plantas bem como da sua caracterização físico-química, o que permitiu avaliar os níveis de toxicidade experimentados pelas espécies. A análise da acumulação e distribuição de Hg nos diferentes órgãos das várias espécies vegetais, bem como a quantificação da biomassa aérea permitiram determinar o potencial de fitorremediação de cada uma das espécies da comunidade do Esteiro de Estarreja. O trabalho apresentado engloba também a análise de mecanismos moleculares responsáveis pela tolerância a Hg nestas espécies, com particular incidência nos seus efeitos a nível oxidativo através da quantificação de espécies reactivas de oxigénio e de danos oxidativos e na subsequente resposta antioxidante através da quantificação de defesas antioxidantes enzimáticas. A quantificação dos níveis de tióis (glutationa e fitoquelatinas), conhecidos mecanismos na sequestração intracelular de metais em plantas, foi igualmente determinada, tendo sido avaliado o seu contributo na tolerância a Hg nestas espécies. Os resultados obtidos demonstraram que as diferentes espécies apresentaram diferenças importantes na acumulação e distribuição de Hg. As espécies dicotiledóneas A. patula e H. portulacoides apresentaram níveis semelhantes de Hg nos seus órgãos e as espécies monocotiledóneas J. maritimus e P. australis acumularam mais metal nos seus órgãos subterrâneos. Foi igualmente determinada a distribuição de Hg nas diferentes fracções celulares, uma vez que a partição metálica é fundamental para inferir o grau de toxicidade que as plantas experienciam e a mobilidade de Hg no sapal. Embora se tenham verificado diferenças importantes na acumulação e distribuição de Hg entre as várias espécies estudadas, a estratégia geral que adoptaram para lidar com exposições ambientais de Hg foi a imobilização do metal na fracção insolúvel, o que sugere que esta estratégia é o principal mecanismo de tolerância a Hg nestas espécies. A acumulação e distribuição de Hg, em conjunto com o trabalho subsequente, determinaram a capacidade de fitorremediação das espécies estudadas. Assim, J. maritimus e P. australis apresentaram-se como as melhores candidatas para processos de fitoestabilização e todas as espécies, com excepção de A. patula, apresentam capacidades semelhantes de fitoextracção. A avaliação de parâmetros relacionados com o stresse oxidativo permitiu inferir os níveis de stresse em que cada planta se encontrava. Observou-se que todas as espécies apresentaram sintomas de stresse oxidativo associados à acumulação de Hg. Contudo, A. patula foi a espécie que apresentou níveis mais elevados de stresse oxidativo, ao contrário de J. maritimus e P. australis que se apresentaram com menos danos oxidativos. Estas diferenças entre as espécies deveram-se, possivelmente, à acção das enzimas antioxidantes. As dicotiledóneas apresentaram níveis mais baixos de todas as enzimas nas folhas, enquanto que as monocotiledóneas apresentaram níveis mais elevados de SOD e GPx, particularmente nos órgãos subterrãneos. A determinação de tióis (glutationa e fitoquelatinas) permitiu estimar a sua importância na tolerância ao mercúrio. Os resultados obtidos trouxeram novos dados sobre o papel das fitoquelatinas em exposições ambientais a Hg. Observou-se que em todas as espécies houve síntese de fitoquelatinas, tendo-se observado diferenças entre elas. Nas dicotiledóneas e nos órgãos aéreos das monocotiledóneas a síntese de PCs foi, de facto, a principal resposta à presença de Hg. Contudo, e contrariamente ao que se observa na maioria dos trabalhos de laboratório, nas espécies monocotiledóneas, o órgão com maior acumulação de mercúrio foi aquele que apresentou menor conteúdo em fitoquelatinas, sugerindo que a síntese destes agentes quelantes não parece ser o principal mecanismo de tolerância a mercúrio, podendo entrar em acção outros mecanismos de tolerância complementares. Os resultados obtidos neste trabalho permitiram uma melhor compreensão sobre as estratégias que estas plantas de sapal utilizaram para lidar com uma elevada e crónica contaminação ambiental por Hg, mas também criaram novas perspectivas sobre os seus mecanismos e respostas moleculares à presença deste metal no ambiente. Assim, este trabalho poderá ser o ponto de partida para o desenvolvimento de competências com capacidade de intervenção na fitorremediação de áreas de sapal contaminadas, bem como para estudos de gestão ambiental e do impacto da contaminação por metais em ecossistemas estuarinos. ABSTRACT: Salt marshes rank among the most productive ecosystems on earth. Among heavy metals, mercury is considered one of the most toxic metals and its effects have repercussions throughout the food chain of the entire ecosystem. The recovery of mercury from industrial sludge and liquid wastes is now required by governments, but the cleanup of previously polluted estuarine areas and waterways has been neglected. This is due partly to the fact that the physical and chemical remediation techniques currently used to extract or immobilize mercury are extremely expensive, environmentally disruptive and sometimes ineffective. A phytoremediation system, in which plants extract, sequester and/or detoxify mercury pollutants, may be a more attractive solution. However, this technique is still in a developmental stage and requires further research on the molecular mechanisms underlying the accumulation of metals in order to optimize the processes inherent to this remediation technique. Under this context, the present work focused on the study of the role that the salt marsh species A. patula, H. portulacoides, J. maritimus and P. australis have on the decontamination of a specific salt marsh area of the Ria de Aveiro, the Estarreja Channel, subject to a high mercury contamination. Thus, the main objectives of this work included, first, the analysis of the contamination levels in the species rhizosediments and their physic-chemical characterization, which allowed inferring the toxicity levels experienced by organisms colonizing these sediments ad their role in metal availability. The accumulation and distribution of Hg in the several plants as well as the aerial biomass quantification determined the phytoremediation potential of each species colonizing the Estarreja channel. The work presented also includes the analysis of molecular mechanisms responsible for plant Hg tolerance, with particular focus on the oxidative stress effects, through the quantification of reactive oxygen species and oxidative damage and on the subsequent antioxidant response through the quantification of enzymatic antioxidant defences. The levels of thiols (glutathione and phytochelatins), known mechanisms in intracellular sequestration of metals in plants, was also determined and its contribution to the Hg tolerance in these species was assessed. The results showed that the several species presented significant differences in Hg accumulation and distribution. The dicotyledons A. patula and H. portulacoides revealed similar Hg levels in all their organs and monocotyledonous J. maritimus and P. australis accumulated more metal in their belowground organs. It was also determined the distribution of Hg in different cell fractions, since the metal partition is essential to infer the degree of toxicity that plants experience and to assess the Hg mobility in the salt marsh system. Although there were important differences in the accumulation and distribution of Hg between the studied species, the overall strategy they have adopted to deal with Hg environmental exposure was the metal immobilization in cell walls, suggesting that this strategy is the main Hg tolerance mechanism in these species. The Hg accumulation and distribution, together with the subsequent work, determined the phytoremediation capacity of these species. Thus, J. maritimus and P. australis presented itself as the best candidates for Hg phytostabilization and with A. patula exception, all species presented similar phytoextraction capabilities. The evaluation of parameters related to oxidative stress allowed to infer the levels of stress that each plant experienced. It was observed that all species showed oxidative stress symptoms associated with Hg accumulation. However, A. patula was the species with higher oxidative stress levels, unlike J. maritimus and P. australis that had less oxidative damage. These differences between species are due to the action of antioxidant enzymes. The dicotyledons showed lower levels of enzyme activities in the leaves, while the monocots had higher levels of SOD and GPx, particularly in the belowground organs. The thiol quantification (glutathione and phytochelatins) allowed to estimate its importance in the plants Hg tolerance. The results have brought new data on the role of phytochelatin in Hg environmental exposures. It was noticed that PC synthesis occurred in all species, however important differences were observed between them. In dicotyledons and in aerial organs of monocots the synthesis of PCs was, in fact, the main response to the Hg accumulation. However, in opposition to most laboratory works, in the monocots, the organs with higher Hg accumulation were the ones that showed a lower phytochelatin content, suggesting that the synthesis of these chelating agents is not the main mechanism behind Hg tolerance, and that other complementary tolerance mechanisms may come to action. The results obtained in this work provided a better understanding of the strategies used by salt marsh plants to cope with a strong and chronic environmental Hg contamination, but also created new perspectives on their molecular mechanisms and responses to the presence of metal in the environment. Thus, this work may be the starting point for the development of skills with intervention capacity in the phytoremediation of salt marsh contaminated areas as well as for environmental management studies and impact of metal disposal in estuarine ecosystems

Hydrocarbonoclastic bacteria: from bioremediation to bioenergy feedstock

Tese de Doutoramento em Engenharia Química e Biológica.Bacterial storage lipids are being considered as viable alternative feedstocks for industrial and biotechnological applications, compared to conventional ones. The production of these bacterial compounds can be obtained from different carbon sources, including inexpensive and recalcitrant wastes. This thesis explores the potential of using hydrocarbonoclastic bacteria to obtain lipid reserve substances from hydrocarbon-based wastes, promoting a more economic and environmentally sustainable biotechnological process combining pollution reduction and production of added value compounds. The production of storage lipids by a hydrocarbon degrading bacterium, Rhodococcus opacus B4, from glucose, acetate and hexadecane, was reported for the first time and compared to Rhodococcus opacus PD630, the best known triacylglycerols (TAG) accumulating bacterium. TAG was the main storage compound produced by both strains. R. opacus B4 presented 2 to 3 fold higher TAG volumetric productions than R. opacus PD630 when cultivated in hexadecane. Subsequently, the ability of R. opacus B4 to decontaminate hexadecane-contaminated cork sorbents while producing lipid storage compounds was shown. Two types of cork sorbents, natural and thermally treated cork, were used. R. opacus B4 was able to degrade practically all hexadecane impregnated in both cork sorbents and TAG was the main neutral lipid compound produced. The potential of the obtained lipid-rich biomass for biomethane production was assessed and an efficent conversion to methane was observed. An indigenous microbial community enriched in carbon storage producing hydrocarbon degrading bacteria was developed from a hydrocarbon-based wastewater (containing lubricant and engine oil waste), using feast and famine conditions in the form of alternating periods of presence of the carbon substrate followed by its absence. The most dominant bacteria identified belongs to hydrocarbon degrading Rhodococcus, Acinetobacter and Pseudomonas genera, known for producing mainly triacylglycerols (TAG), wax esters (WE) and polyhydroxyalkanoates (PHA), respectively. The enriched community was able to accumulate small amounts of TAG and PHA, and higher levels of a non-identified lipid as well as to efficiently degrade the wastewater hydrocarbons. The effect of different cultivation parameters on storage compound production by the developed enriched community was evaluated, using central composite circumscribed design based on surface response methodology. The interaction between carbon and nitrogen concentrations positively influenced PHA accumulation whereas interaction between carbon and nitrogen concentrations with cultivation time affected PHA in a negative way. Regarding neutral lipids production, nitrogen concentration and the interaction between carbon and nitrogen concentrations were the significant parameters. Neutral lipids produced were essentially TAG, presenting a highly diversity of chemical structures composed by a narrow range of fatty acids. Finally, a genome-based comparative analysis of genes and metabolic reactions responsible for TAG, WE and polyhydroxybutyrate (PHB) biosynthesis in members of Rhodococcus, Acinetobacter, Alcanivorax and Pseudomonas genera was performed to get deeper knowledge on carbon storage compounds metabolism in hydrocarbonoclastic bacteria. The presence of genes coding for complete metabolic pathways for TAG and PHB biosynthesis was detected in all species. Rhodococcus strains are highly enriched in genes involved in TAG and PHB metabolism, whereas Alcanivorax, Acinetobacter and Pseudomonas have a high number of genes coding for enzymes involved in PHB production.Os lípidos de reserva bacterianos têm sido considerados como matéria-prima alternativa aos lípidos convencionais para aplicações biotecnológicas e industriais. A produção destes compostos pode ser obtida a partir de diferentes fontes de carbono, incluindo resíduos recalcitrantes e economicamente acessíveis. Esta tese teve como principal objetivo explorar o potencial de bactérias hidrocarbonoclásticas para a produção de lípidos de reserva a partir de resíduos contendo hidrocarbonetos. Esta capacidade pode contribuir para o desenvolvimento de um processo biotecnológico enconomicamente e ambientalmente sustentável, envolvendo a descontaminação biológica de resíduos e a produção de compostos de valor acrescentado. A produção de lipidos de reserva em Rhodococcus opacus B4, uma bactéria degradadora de hidrocarbonetos, a partir de glucose, acetato e hexadecano, foi descrita pela primeira vez e comparada com a bactéria Rhodococcus opacus PD630, considerada como a melhor produtora de triacilgliceróis (TAG). Os triacilgliceróis foram os principais compostos produzidos por ambas as estirpes. R. opacus B4 apresentou uma produção volumétrica de TAG duas a três vezes superior à de R. opacus PD630 quando cultivadas em hexadecano. A capacidade de R. opacus B4 para degradar hexadecano adorvido a cortiça e produzir lípidos de reserva foi avaliada. Foram utilizados dois tipos de adsorventes de cortiça, cortiça natural e cortiça termicamente tratada. R. opacus B4 degradou eficazmente o hexadecano impregnado em ambas as cortiças e os triacilgliceróis foram os principais lípidos de reserva produzidos. O potencial de produção de metano a partir da biomassa rica em lípidos obtida foi analisado, tendo-se observado uma eficiente conversão a metano. O desenvolvimento de uma comunidade microbiana enriquecida em bactérias hidrocarbonoclásticas produtoras de compostos de reserva foi obtido a partir de águas residuais contendo lubrificantes e óleo de motor, aplicando períodos alternados de presença e ausência de fonte de carbono. As bactérias mais dominantes identificadas pertencem a géneros hidrocarbonoclásticos, nomeadamente Rhodococcus, Acinetobacter e Pseudomonas, conhecidas como produtoras de TAG, ésteres de cera (WE) e polihidroxialcanoatos (PHA), respetivamente. A comunidade enriquecida acumulou baixas quantidades de TAG e PHA e quantidades superiores de um lípido desconhecido, tendo degradado eficientemente os hidrocarbonetos presentes na água residual. O efeito de diferentes parâmetros de cultivo na produção de compostos de reserva pela comunidade enriquecida foi avaliado através de um desenho fatorial de experiências baseado na metodologia de superfície de resposta (RSM). A interação entre as concentrações de carbono e azoto influenciou positivamente a acumulação de PHA enquanto que as interações entre o período de cultivo e as concentrações de carbono e azoto e o tempo de cultivo afetaram negativamente a produção de PHA. A produção de lípidos neutros foi significativamente influenciada pela concentração de azoto e pela interação entre as concentrações de carbono e azoto. Os lípidos neutros produzidos foram essencialmente TAG, apresentando uma elevada diversidade de estruturas químicas compostas por uma ampla gama de ácidos gordos. Foi efetuada uma análise comparativa de genes e reações metabólicas responsáveis pela síntese de TAG, WE e polihidroxibutiratos (PHB) em espécies pertencentes aos géneros Rhodococcus, Acinetobacter, Alcanivorax e Pseudomonas. A presença de genes que codificam para enzimas pertencentes a todas as vias metabólicas relativas à produção de TAG e PHB foi detetada em todas as espécies estudadas. As estirpes de Rhodococcus opacus apresentam um número elevado de genes envolvidos no metabolismo de TAG e PHB, enquanto que Alcanivorax borkumensis SK2, Acinetobacter baylyi ADP1 e Pseudomonas putida KT2440 possuem uma grande diversidade de genes que codificam para enzimas envolvidas na produção de PHB.Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/64500/2009.Fundação para a Ciência e a Tecnologia (FCT) Strategic Project of UID/BIO/04469/2013 unit.The project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462).The Project BioEnv - Biotechnology and Bioengineering for a sustainable world, ref Norte - 07-0124-FEDER-000048, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER

Sequencing batch airlift reactors (SBAR): a suitable technology for treatment and valorization of mineral oil wastewaters towards lipids production

Produced water (PW) and spent oil-based wastewaters are some of the largest mineral oil wastewaters produced. Due to the high toxicity of hydrocarbons, several countries set stringent discharge limits and its treatment is compulsory before discharge. In this work, biological treatment of mineral oil wastewaters coupled with the production of bacterial lipids is demonstrated in sequential batch airlift reactors (SBAR). Two SBAR (2 L working volume) were used for treatment of PW and lubricant-based wastewater (LW), inoculated with Alcanivorax borkumensis SK2 (SBARAb+PW) and Rhodococcus opacus B4 (SBARR.o+LW), respectively. A total petroleum hydrocarbon removal (TPH) efficiency up to 96% and 80% were achieved for SBARAb+PW and SBARR.o+LW, respectively. Intracellular lipids production in SBARAb+PW increased when lower TPH/N ratios and higher feast stage duration were applied (up to 0.74 g g-1 cell dry weight (CDW)), whereas in SBARR.o+LW higher lipids production was observed for higher TPH/N ratios (0.94 g g-1 in CDW). Triacylglycerols (TAG) were the main intracellular lipid accumulated in both SBARAb+PW and SBARR.o+LW operations, while wax ester (WE) production was only observed extracellularly in the SBARAb+PW.This work was supported by the Portuguese Foundation for Science and Technology (FCT) and European Regional Development Fund (FEDER) under the scope of project SaltOil+ (POCI-01-0145-FEDER030180); the strategic programmes UIDB/04469/2020, UID/BIA/4050/ 2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019; and by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Research of Rita M. Silva was supported by PhD grant SFRH/BD/116154/2016, funded by FCT.info:eu-repo/semantics/publishedVersio

Biological saline produced water treatment, a sustainable process towards lipids production

Oil and gas industry is responsible for the generation of large volumes of oil-contaminated wastewaters, such as saline produced water (PW), that without proper treatment can cause environmental contamination. Alcanivorax borkumensis SK2 is a biosurfactant producer capable of degrading and converting hydrocarbons into bacterial lipids under nutrient limiting conditions (e.g. nitrogen and/or oxygen). Recently, the industrial interest in biosurfactants and bacterial lipids for biotechnological applications (e.g. bioremediation or biofuels production) is increasing. Pursuing the interest of treating and simultaneously valorizing PW, a sequencing batch airlift reactor (SBAR) strategy consisting of sequential cycles of feast and famine stages was developed. A. borkumensis SK2 was used as bioreactor inoculum and the effect of cycle duration, total petroleum hydrocarbon to nitrogen ratio (TPH/N) and dissolved oxygen (DO) concentration (7-8 mg L-1 and 2-3 mg L-1 (famine and feast stage); and 7-8/1-2 mg L-1 (famine/feast stage)) were investigated. The system provided an efficient PW treatment, achieving TPH removal efficiencies in a narrow range from 90 ± 2.1 to 96 ± 1.8 %. Intracellular lipid production increased from 0.48 to 0.74 g g-1 of cellular dry weight (CDW) with the application of higher feast stage duration and lower TPH/N ratios suggesting that nitrogen availability is the most relevant factor to promote accumulation. Under 2-3 mg L-1 and 7-8/1-2 mg L-1 of oxygen, lipid accumulation dropped to 0.50 g g-1 of CDW. Intracellular lipid profile changed according to the DO concentration. Triacylglycerols (TAG) and wax esters (WE) were accumulated under maximum and limiting DO concentrations, while polyhydroxyalkanoates (PHA) accumulation was triggered by the application of alternated DO conditions, indicating that the type of intracellular compound can be selected by manipulating the oxygen concentration in the SBAR. Extracellular lipid production (TAG and WE) was not significantly affected by none of the operational conditions applied (0.06 g L-1). Moreover, the production of a cell-bound and an extracellular glycolipid biosurfactant capable of reducing the medium surface tension from 65 to approximately 41 mN m-1 was observed. The proposed strategy showed that biological PW treatment in a SBAR can be a sustainable process through the production of added-value compounds contributing to a circular economy model.This research was supported by the Portuguese Foundation for Science and Technology (FCT I.P.) and European Regional Development Fund (ERDF) under the scope of project SaltOil+ (POCI-01- 0145- FEDER-030180) (Portugal 2020, COMPETE 2020); Rita M. Silva PhD grant (SFRH/BD/116154/2016) was funded by FCT.info:eu-repo/semantics/publishedVersio

Iron compounds in anaerobic degradation of petroleum hydrocarbons: a review

Waste and wastewater containing hydrocarbons are produced worldwide by various oil-based industries, whose activities also contribute to the occurrence of oil spills throughout the globe, causing severe environmental contamination. Anaerobic microorganisms with the ability to biodegrade petroleum hydrocarbons are important in the treatment of contaminated matrices, both in situ in deep subsurfaces, or ex situ in bioreactors. In the latter, part of the energetic value of these compounds can be recovered in the form of biogas. Anaerobic degradation of petroleum hydrocarbons can be improved by various iron compounds, but different iron species exert distinct effects. For example, Fe(III) can be used as an electron acceptor in microbial hydrocarbon degradation, zero-valent iron can donate electrons for enhanced methanogenesis, and conductive iron oxides may facilitate electron transfers in methanogenic processes. Iron compounds can also act as hydrocarbon adsorbents, or be involved in secondary abiotic reactions, overall promoting hydrocarbon biodegradation. These multiple roles of iron are comprehensively reviewed in this paper and linked to key functional microorganisms involved in these processes, to the underlying mechanisms, and to the main influential factors. Recent research progress, future perspectives, and remaining challenges on the application of iron-assisted anaerobic hydrocarbon degradation are highlighted.This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (POCI-01-0145-FEDER-016575) and of the strategic funding of UIDB/04469/2020 unit. It was also funded by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, and by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte—BioEcoNorte project (NORTE-01-0145-FEDER-000070).info:eu-repo/semantics/publishedVersio

A mathematical model for bioremediation of hydrocarbon-contaminated soils

The AQUASIM executable file is available in DataRepositoriUM (https://doi.org/10.34622/datarepositorium/2BUM2J, accessed on 15 September 2022).Bioremediation of hydrocarbons in soil is a highly complex process, involving a multiplicity of physical, chemical and biological phenomena. Therefore, it is extremely difficult to control and boost the bioremediation of these systems after an oil spill. A mathematical model was developed to assist in the prediction and decision-making regarding the in situ bioremediation of hydrocarbon-contaminated soils. The model considered the most relevant processes involved in the mass transfer and biodegradation of alkanes over time and along the depth of a flooded soil column. Aliphatic hydrocarbons were chosen since they are less water soluble than aromatics and account for 50–90% of the hydrocarbon fraction in several petroleum products. The effect of adding oxygen, nitrate, iron (III) or sulfate as electron acceptors was then simulated (bioremediation scenarios). Additionally, and to feed the model, batch assays were performed to obtain experimental data on hydrocarbon adsorption to soil particles (more than 60% of hydrocarbons tends to be adsorbed to soil particles), as well as hydrocarbon biodegradation rates in the presence of nitrate (0.114 d−1) and oxygen (0.587 d−1). The model indicates that saturated hydrocarbon removal occurs mainly with adsorption/desorption and transport processes in the upper layers of soil due to methanogenic biodegradation in deeper layers, since the other microbial processes are soon limited by the lack of electron acceptors. Simulation results show that higher initial electron acceptor concentrations led to higher hydrocarbon removal, confirming that the model is performing in accordance with the expected. Close to the surface (at 0.1 m depth), all scenarios predicted more than 83% hydrocarbon removal after two years of simulation. Soil re-aeration results in faster hydrocarbon removal (more than 20% after one year) and surfactants addition (around 15% after one year) may also accelerate soil bioremediation. With this model, the simultaneous contributions of the various physicochemical and biological processes are integrated, facilitating the simulation and comparison of different bioremediation scenarios. Therefore, it represents a useful support tool for the management of contaminated sites.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (PTDC/AAG-TEC/3500/2014; POCI-01-0145-FEDER-016575), the strategic funding of UIDB/04469/2020 unit and by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Toxicity of the 3,4-methylenedioxymethamphetamine and its enantiomers to Daphnia magna after isolation by semipreparative chromatography

MDMA (3,4-methylenedioxymethamphetamine) is a chiral psychoactive recreational drug sold in illicit markets as racemate. Studies on the impact of MDMA on aquatic organisms are scarce. While enantioselectivity in toxicity in animals and humans has been reported, none is reported on aquatic organisms. This study aimed to investigate the ecotoxicological effects of MDMA and its enantiomers in Daphnia magna. For that, enantiomers (enantiomeric purity > 97%) were separated by liquid chromatography using a homemade semipreparative chiral column. Daphnids were exposed to three concentrations of (R,S)-MDMA (0.1, 1.0 and 10.0 µg L−1) and two concentrations of (R)- and (S)-enantiomers (0.1 and 1.0 µg L−1) over the course of 8 days. Morphophysiological responses were dependent on the substance form and daphnia development stage, and they were overall not affected by the (R)-enantiomer. Changes in swimming behaviour were observed for both the racemate and its enantiomers, but enantioselective effects were not observed. Reproductive or biochemical changes were not observed for enantiomers whereas a significant decrease in acetylcholinesterase and catalase activity was noted at the highest concentration of (R,S)-MDMA (10 µg L−1). Overall, this study showed that sub-chronic exposure to MDMA racemate and its enantiomers can interfere with morphophysiological and swimming behaviour of D. magna. In general, the (R)-enantiomer demonstrated less toxicity than the (S)-enantiomer.This work was financially supported by national funds through the FCT/MCTES (PIDDAC), under the project PTDC/CTA-AMB/6686/2020, and partially supported through the projects UIDB/04423/2020 and UIDP/04423/2020 (Group of Natural Products and Medicinal ChemistryCIIMAR)

Vaginal discharge in pregnant women: comparison between syndromic approach and examination of clinical nursing practice

This is a study for assessment of a quantitative approach in pregnant women (N=104), in which findings of vaginal infection were compared. The findings were obtained by two means, flowchart of vaginal discharge, and typical examinations in the clinical nursing practice. Data were collected from January to July 2011 through interviews and gynecological examinations. The flowchart showed no efficacy to identify candidiasis and trichomoniasis. Furthermore, it showed low sensitivity (0.0%; 50%) and positive predictive value (0.0%; 3.6%) for both infections, and low specificity for trichomoniasis (46%). The flowchart was shown to be satisfactory for bacterial vaginosis, with high sensitivity (100%), negative predictive value (100%), and accuracy (74%). We conclude that use of the flowchart should be reassessed, as it was not able to identify important infections in pregnant women. A continuous effort must be directed for development of effective tests in order to prevent the spread of infection and reduce the number of unnecessary treatments.Estudio evaluativo con enfoque cuantitativo, con una muestra de 104 gestantes, cuyo objetivo fue comparar los resultados de infecciones vaginales en las gestantes, obtenidos a partir del diagrama de flujo vaginal y las pruebas presentes en la práctica clínica de enfermería. Los datos fueron recolectados por entrevista y examen ginecológico, realizados de enero a julio del 2011. El diagrama de flujo no fue eficaz en la identificación de candidiasis y tricomoniasis, presentando baja sensibilidad (0,0%; 50%) y valor predictivo positivo (0,0%; 3,6%), para las dos infecciones y baja especificidad para tricomoniasis (46%). Para vaginosis bacteriana, se mostró satisfactoria, con alta sensibilidad (100%), valor predictivo negativo (100%) y precisión (74%). Se concluye que el uso del diagrama necesita ser revisado, ya que no fue eficaz en la identificación de infecciones importantes en las gestantes. Los esfuerzos para desarrollar pruebas efectivas deben ser continuos, con el objetivo de prevenir la propagación de infecciones y reducir tratamientos innecesarios.Estudo avaliativo de abordagem quantitativa, com amostra de 104 gestantes, com o objetivo de comparar os achados de infecções vaginais em gestantes obtidos por meio do fluxograma de corrimento vaginal com exames presentes na prática clínica da Enfermagem. Os dados foram coletados por meio de entrevista e exame ginecológico realizados de janeiro a julho de 2011. O fluxograma não se mostrou eficaz na identificação de candidíase e tricomoníase, apresentou baixa sensibilidade (0,0%; 50%) e valor preditivo positivo (0,0%; 3,6%) para as duas infecções e baixa especificidade para tricomoníase (46%). Mostrou-se satisfatório para vaginose bacteriana, com alta sensibilidade (100%), valor preditivo negativo (100%) e acurácia (74%). Conclui-se que o emprego do fluxograma precisa ser reavaliado, visto que não foi eficaz em identificar infecções importantes em gestantes. Os esforços para o desenvolvimento de testes eficazes devem ser contínuos, com intuito de prevenir a disseminação de infecções e reduzir tratamentos desnecessários

Long-chain fatty acids degradation by desulfomonile species and proposal of candidatus Desulfomonile palmitatoxidans

The datasets generated for this study can be found in the European Nucleotide Archive (ENA)–LS453291 (https://www.ebi.ac.uk/ena/browser/view/LS453291), PRJEB26656 (https://www.ebi.ac.uk/ena/browser/view/PRJEB26656), PRJEB35900 (https://www.ebi.ac.uk/ena/browser/view/PRJEB35900).Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16:0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of the Desulfomonile genus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong to Desulfomonile genus [ca. 92% 16S rRNA gene sequences closely related to Desulfomonile spp.; and ca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between the Desulfomonile sp. in HP culture and the type strain Desulfomonile tiedjei strain DCB-1T, we propose a novel species designated as Candidatus Desulfomonile palmitatoxidans. This bacterium shares 94.3 and 93.6% 16S rRNA gene identity with Desulfomonile limimaris strain DCB-MT and D. tiedjei strain DCB-1T, respectively. Based on sequence abundance of Desulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species Ca. Desulfomonile palmitatoxidans most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture and D. tiedjei strain DCB-1T with short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, nor D. tiedjei strain DCB-1T degraded palmitate when incubated with Methanobacterium formicicum as a possible methanogenic syntrophic partner. Unlike D. tiedjei strain DCB-1T, Ca. Desulfomonile palmitatoxidans lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genus Desulfomonile is proposed. Our study reveals an unrecognized LCFA degradation feature of the Desulfomonile genus.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (PTDC/AAG-TEC/3500/2014; POCI-01-0145-FEDER016575), and of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio