Degradation of selected PAHs by laccase-mediator system in soil

This study investigated the degradation of selected PAHs using laccase produced through cocultivation of Trichoderma viride (EXF8977) and Penicillium chrysogenum (EXF1818) on solid state fermentation and its mediator system. The main objective of this work was to evaluate the ability of the laccase-mediator systems to degrade PAHs in soil, as well as the effects of natural and synthetic mediators on the laccase activity during the process of PAHs degradation. The laccase production was optimized in packed-bed bioreactor, by testing different airflow (0.05 L min-1 , 0.1 L min-1 and 0.2 L min-1 ), temperature (RT, 30 and 40 °C) and time (2 and 3 weeks) on kiwi peels as substrate using single fungi species and cocultivation. Extracellular laccase activity was measured spectrophotometrically using 0.5 mM 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate on 420 nm and enzyme activity was expressed in units per millilitre (U/mL). Batch degradation experiments were carried out in 100 mL Erlenmeyer flask on soil pH 5, containing PAH mixture (fluorene, anthracene, pyrene, chrysene and benzo(a)pyrene) from 50 till 600 ppm in dark at 20, 30 and 40 °C. In order to obtain optimum laccase degradation conditions different concentration (0.1 mM, 0.5 mM and 1 mM) of synthetic (ABTS) and natural mediators (p-coumaric acid and ferulic acid) were studied. Samples were collected after 0, 3 and 5 incubation days and the compounds were measured by UHPLC. All the assays including controls were performed in triplicate. Thereafter column packed bed reactor was set up to evaluate the degradation with continue flow (0.015 mL/min) of crude laccase enzyme with 1 mM ABTS, p-coumaric acid and ferulic acid for 25 days, by taking samples each 5 days and evaluating degradation ratio and the remaining concentration of laccase in samples. The selected fungi were able to produce 2.95 U/mL of laccase during SSF in Erlenmeyer flask, however this amount increased with continuous airflow. Highest values were obtained at aeration rates 0.2 L min-1 , with a production of 6.32 U/mL, followed by 0.1 and 0.05 L min -1 with productions of 5.45 and 3.79 U/mL, respectively. Data showed laccase extracted from cocultivation of Penicillium chrysogenum and Trichoderma viride on kiwi peels with an appropriate mediator promoted the degradation of fluorene, anthracene, pyrene, chrysene and benzo(a)pyrene in concentration from 50 ppm till 600 ppm. Reaction system reached highest degradation at 3 days in case of fluorene, chrysene and benzo(a)pyrene, but with anthracene and pyrene maximum degradation rate was observed between 3 and 5 days. The optimizing mediator for fluorene and pyrene was p-coumaric acid, with removal of 81% and 98%, respectively. For anthracene, the best mediator was ABTS with 42% degradation, and laccase with ferulic acid achieved highest degradation rate with chrysene, 61%, and benzo(a)pyrene, 5.4%. However, in packet bed reactor with continuous flow of laccase with mediator at 0.015 mL/min, for 25 days at RT (26- 28 °C), ABTS was found to be the optimum mediator for fluorene, anthracene, chrysene and benzo(a)pyrene, with degradation reaching 74.8 %, 71.9%, 81.8% and 96%, respectively. Around 72.2 % of phenanthrene was degraded with ferulic acid as mediator, and 99.9% degradation of pyrene was reached with p-coumaric acid. Less than 60% degradation rate in 25 days was observed for phenanthrene and anthracene with p-coumaric acid as mediator. The results achieved in the present research demonstrated the utility of laccase from SSF production and its use in bioremediation in soil, especially to reduce the concentration of PAH. However, more research must be done in real conditions.(undefined)info:eu-repo/semantics/publishedVersio

Ligninolytic enzymes production during polycyclic aromatic hydrocarbons degradation: effect of soil pH, soil amendments and fungal co-cultivation

Soil microorganisms play an important role in the degradation of PAHs and use various metabolic pathways for this process. The effect of soil pH, different soil amendments and the co-cultivation of fungi on the degradation of PAHs in soil and on the activity of ligninolytic enzymes was evaluated. For that purpose, three fungi were studied: Trichoderma viride, Penicillium chrysogenum and Agrocybe aegerita. Biodegradation assays with a mixture of 200 ppm PAHs (fluorene, pyrene, chrysene, and benzo[a]pyrene---50 ppm each) were set up at room temperature for 8 weeks. The maximum laccase activity by solid state fermentation---SSF (7.43 U/g) was obtained by A. aegerita on kiwi peels with 2 weeks and the highest manganese peroxidase activity (7.21 U/g) was reached in 4 weeks, both at pH 7. Fluorene, pyrene, and benzo[a]pyrene achieved higher degradation rates in soil at pH 5, while chrysene was more degradable at pH 7. About 85--90\% of the PAHs were degraded by fungal remediation. The highest degradation of fluorene was achieved by co-cultivation of A. aegerita and P. chrysogenum, remaining 14\% undegradable. Around 13\% of pyrene stay undegradable by A. aegerita and T. viride and by A. aegerita and P. chrysogenum, both systems supported in kiwi peels, while 11\% of chrysene remained in soil by the co-cultivation of these fungi, supported by peanut shells. Regarding benzo[a]pyrene, 13\\% remained in soil after treatment with A. aegerita. Despite the increase in degradation of some PAHs with co-cultivation, higher enzyme production during degradation was observed when fungi were cultivated alone.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of UID/BIO/04469/2013 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. Ziva Vipotnik is a recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) - Doctoral Program in Applied and Environmental Microbiology (DP_AEM); operation NORTE-08-5369-FSE000060; co-financed by North 2020 through the European Social Fund (ESF).info:eu-repo/semantics/publishedVersio

Biodegradation of chrysene and benzo[a]pyrene and removal of metals from naturally contaminated soil by isolated Trametes versicolor strain and laccase produced thereof

"Available online 11 June 2022"The objective of this study was to assess the degradation rates of chrysene and benzo[a]pyrene, as well as the removal of aluminium and iron from contaminated soil collected in the upper layer (030 cm) in Lagos, Southwest Nigeria. Trametes versicolor was isolated from this soil and used in degradation experiments, with plantain peels as support. After 8 weeks, 82.0% of chrysene degradation was achieved by T. versicolor, and by adding support this increased to 91.0%. Benzo[a]pyrene was less degradable, with 38.0% and 49.1% of degradation, respectively. Trametes versicolor was also capable of accumulate 46.1% of aluminium and 57.2% of iron. By adding plantain peels, these amounts increased to 48.2% and 61.8%, respectively. At the same time, laccase was produced by Trametes versicolor on plantain peels, achieving 37.8 U/g of crude laccase during SSF at 30 °C for 3 weeks. Laccase degradation experiments were set up in packed-bed reactor (PBR), with a constant feed of 21.6 mL/day of laccase, with and without mediators. In 35 days, 75.9% degradation of chrysene was achieved by laccase. The highest degradation was observed with ABTS (2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) as mediator, 87.9%. Benzo[a]pyrene degradation with laccase reached 35.6%, raising to 38.8 % with ferulic acid as mediator. In addition, 99.2% of iron and 99.6% of aluminium was removed by laccase, being the treatment for this last mediated with ABTS.This study was supported by the Portuguese Foundation for Science and Technology (FCT), Portugal under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of UID/BIO/04469/2013 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. Ziva Vipotnik is a recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) - Doctoral Program in Applied and Environmental Microbiology (DP_AEM); operation NORTE-08-5369-FSE-000060; co-financed by North 2020 through the European Social Fund (ESF).info:eu-repo/semantics/publishedVersio

Recovery of rare earth elements from wastewater towards a circular economy

The use of rare earth elements is a growing trend in diverse industrial activities, leading to the need for eco-friendly approaches to their efficient recovery and reuse. The aim of this work is the development of an environmentally friendly and competitive technology for the recovery of those elements from wastewater. Kinetic and equilibria batch assays were performed with zeolite, with and without bacterial biofilm, to entrap rare earth ions from aqueous solution. Continuous assays were also performed in column setups. Over 90% removal of lanthanum and cerium was achieved using zeolite as sorbent, with and without biofilm, decreasing to 70% and 80%, respectively, when suspended Bacillus cereus was used. Desorption from the zeolite reached over 60%, regardless of the tested conditions. When in continuous flow in columns, the removal yield was similar for all of the rare earth elements tested. Lanthanum and cerium were the elements most easily removed by all tested sorbents when tested in single- or multi-solute solutions, in batch and column assays. Rare earth removal from wastewater in open setups is possible, as well as their recovery by desorption processes, allowing a continuous mode of operation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of UID/BIO/04469/2013 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, Portugal.info:eu-repo/semantics/publishedVersio

Biorremediação e biodegradação de selecionados hidrocarbonetos poliaromáticos por fungos

Tese de doutoramento em Engenharia Química e BiológicaDespite the amount of pollutants in the ecosphere has been increasing for a long time, soil contaminated with polyaromatic hydrocarbons (PAH) still remains one of the most important environmental problems worldwide. PAH are universal pollutants with a big variation in their concentration levels in different regions. PAH tend to bind to clays as these have high surface area, which allows more binding and adsorption of PAH on finer soil fractions. However, clay particles reduce in size as time goes by, diminishing porosity and transferable species in soil, and with aging of PAH also lose bioavailability, becoming persistent. Despite the different reported bioremediation techniques, this work focus on the biological degradation of PAH with fungi and their enzymes, mainly laccase and laccase-mediator systems. Laccases are receiving attention from different researchers due to their specific nature. They may be used in different industrial applications as they present the ability to oxidize a broad spectrum of phenolic and nonphenolic compounds. Moreover, processes with laccases are quite competitive with conventional chemical processes in different industries. However, the main limiting factor to their application still remains – large-scale production of laccase, mainly of highly active and stable laccase, is still to be attained. The work is globally divided into two parts, depending on the soil used: artificially contaminated or naturally contaminated soils. In the first part of this work the effect of soil pH and of different supports was evaluated, and the enzyme activities during the PAH degradation in soil were measured. Moreover, laccase degradation was set up, produced by fungal co- cultivation, using kiwi peels as substrate. The produced laccase was applied to PAH contaminated soil to evaluate its efficiency on enzymatic bioremediation. In a second part, heavily PAH and metals polluted soil obtained from Lagos, Nigeria, was used. Trametes versicolor was isolated from this soil and used in degradation experiments, with plantain peels as support. Laccase degradation assays were also set up, in batch and in PBR, with and without mediators. The mediators used were ferulic acid, coumaric acid and ABTS. It was observed that mediators as ABTS and ferulic acid were more successful enhancing PAH degradation.Apesar da crescente poluição da ecosfera que se verifica desde há muito tempo, a contaminação dos solos com hidrocarbonetos poliaromáticos (PAH) mantem-se como um importante problema ambiental a nível mundial. Os PAH são poluentes universais com uma enorme variação dos seus níveis de concentração em diferentes regiões. Os PAH tendem a ligar-se a argilas graças à elevada área superficial destas últimas, favorecendo a sua ligação e adsorção em frações mais finas de solo. No entanto, com a passagem do tempo e os efeitos de erosão, as partículas de argila vão diminuindo de tamanho, reduzindo a porosidade e a transferência de espécies no solo, assim como os PAH vão perdendo biodisponibilidade, tornando-se mais persistentes. Apesar de diferentes técnicas de biorremediação já terem sido publicadas e exploradas, este trabalho foca-se na degradação biológica de PAH com fungos e suas enzimas, em especial com lacases e sistemas lacases-mediadores. As lacases têm recebido especial atenção por parte dos investigadores graças à sua natureza específica. Elas podem ser usadas em diferentes aplicações industriais já que têm a capacidade de oxidar um largo espetro de compostos fenólicos e não fenólicos. Além disso, processos que usam lacases são bastante competitivos com processos químicos convencionais usados em indústrias distintas. No entanto, o principal fator limitante da sua aplicação ainda permanece – a produção de lacase em larga escala, sobretudo de lacase de elevadas atividade e estabilidade, ainda não foi conseguida Este trabalho está dividido em duas partes gerais, dependendo do solo usado: solo artificialmente contaminado ou solo naturalmente contaminado. Na primeira parte deste manuscrito, o efeito do pH do solo e de diferentes suportes foi avaliado, assim como foram medidas as atividades enzimáticas durante a degradação de PAH no solo. A degradação por lacase foi avaliada por co-cultivo de fungos em substrato de cascas de kiwi. A lacase produzida foi aplicada em solo contaminado por PAH para avaliação da eficiência da biorremediação enzimática. Na segunda parte do trabalho usou-se um solo muito poluído em PAH e metais, obtido em Lagos, na Nigéria. A partir desse solo, isolou-se o Trametes versicolor que foi usado nos ensaios de degradação, com cascas de banana como suporte. Realizaram-se ensaios em batch e em contínuo de degradação de lacase, sem e com mediadores. Os mediadores usados foram o ácido ferúlico, o ácido cumárico e o ABTS. Observou-se que os mediadores ABTS e ácido ferúlico implementam com mais sucesso a degradação dos PAH.This thesis was financially supported by grant NORTE-69- 2015-15, by Doctoral Program in Applied and Environmental Microbiology (DP_AEM); operation NORTE-08-5369-FSE- 000060; co-financed by North 2020 through the European Social Fund (ESF)

Rehabilitation of a historically contaminated soil by different laccases and laccase-mediator system

In this study, the degradation of polyaromatic hydrocarbons (PAH) present in naturally contaminated soil by laccases was evaluated. For that purpose, laccase was produced by solid-state fermentation (SSF) on agroindustrial residues by Trametes versicolor and Agrocybe aegerita. At the same time, commercial laccases from T. versicolor and Aspergillus sp. (Novozym 51,003) were used.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of UID/BIO/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. Ziva Vipotnik is a recipient of a fellowship supported by a doctoral advanced training (call NORTE-69–2015-15) — Doctoral Program in Applied and Environmental Microbiology (DP_AEM); operation NORTE-08–5369-FSE-000060; co-fnanced by North 2020 through the European Social Fund (ESF).info:eu-repo/semantics/publishedVersio

Production of ochratoxin A in dry-cured ham based-media by Aspergillus westerdijkiae

Aspergillus westerdijkiae é um dos mais importantes fungos produtores de Ocratoxina A (OTA). Apesar de ser maioritariamente associado a alimentos de origem vegetal, pode também ser encontrado em produtos cárneos curados, onde Penicillium nordicum é considerado o mais importante produtor de OTA. O objetivo deste estudo foi avaliar as condições ecofisiológicas que influenciam o crescimento de A. westerdijkiae e produção de OTA, em comparação com P. nordicum. Para esse efeito, foi testada a inteação entre os fatores temperatura (15, 20, 25 and 30 °C) e atividade de água (aw) (0.99, 0.97, 0.93, 0.90 and 0.85) obtida pela combinação de solutos iónicos (NaCl) e não-iónicos (glicerol), usando meio de cultura à base de presunto como modelo. Os dois fungos em estudo apresentaram perfis de produção de OTA significativamente diferentes. As condições ótimas de produção de OTA por A. westerdijkiae foram 0,94 – 0,97 aw e 20 – 25 °C, e por P. nordicum foram 0,95 – 0,97 aw e 18 – 22 °C, mas A. westerdijkiae produziu quantidades de OTA significativamente superiores. Nenhuma das espécies produziu a micotoxina a 0,85 aw. O crescimento de ambos os fungos não mostrou ser um bom indicador da produção de OTA. Os resultados obtidos mostram que A. westerdijkiae pode representar um elevado risco de contaminação com OTA em presunto devido à produção de elevadas quantidades da micotoxina numa gama alargada de condições ambientais habitualmente encontadas durante o processo de cura dos produtos cárneos.Aspergillus westerdijkiae is one of the most relevant Ochratoxin A (OTA) producing species of the genus Aspergillus. Although it is usually considered a major risk in carbon- -rich food matrices of plant origin, it has also been isolated from cured meat products, where Penicillium nordicum is considered the major OTA producer. The objective of this work was to evaluate the ecophysiological conditions governing growth and OTA production of A. westerdijkiae in comparison with P. nordicum. For that purpose, the interaction between temperature (15, 20, 25 and 30 °C) and water activity (aw) (0.99, 0.97, 0.93, 0.90 and 0.85), achieved with a combination of ionic (NaCl) and non-ionic (glycerol) solutes, was studied by using dry-cured ham-based medium as a model system. Different OTA production profiles were found between the two fungi. The optimal OTA production conditions for A. westerdijkiae were at 0.94 – 0.97 aw and 20 – 25 °C, and for P. nordicum at 0.95 – 0.97 aw between 18 – 22 °C, but A. westerdijkiae produced significantly higher amouts of OTA. None of the strains was able to produce detectable amounts of OTA at 0.85 aw under all temperatures tested. Growth was not a good indicator of OTA production by A. westerdijkiae or P. nordicum. The obtained results show that A. westerdijkiae may represent a great potential risk of OTA contamination in dry-cured ham due to high OTA production under a wide range of conditions usually encountered during ripening of dry-cured ham.info:eu-repo/semantics/publishedVersio

Chapter 14 - Retention of organic micro-pollutants by sorption processes

This chapter is a review of the impacts of organic micro-pollutants on the environment and quality of life in general, and of the sorption processes that these molecules go through when released into water, air, or soils. Potential sorbents for these compounds are listed and discussed, with particular focus on low-cost green sorbents, that otherwise would constitute another waste to be treated. The economic and environmental advantages of the usage of such wastes is defended. Special attention is given to sorption on activated sludges, mechanisms, and implications, with an indication of the tertiary hybrid treatment to be implemented in wastewater treatment plants or in water treatment plants for the removal of these emergent molecules. This treatment includes biological mineralization and immobilization by adsorption. A critical assessment of the approach to the resolution of the overall problem of emergent contaminants is given, with the introduction of industrial symbiosis as a circular economy approach.This study was supported by the Portuguese Foundation for Science and Technology under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of UID/BIO/04469/2013 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

A review on biological processes for pharmaceuticals wastes abatement: a growing threat to modern society

Over the last decades, the production and consumption of pharmaceuticals and health care products grew manifold, allowing an increase in life expectancy and a better life quality for humans and animals, in general. However, the growth in pharmaceuticals production and consumption comes with an increase in waste production, which creates a number of challenges as well as opportunities for the waste management industries. The conventional current technologies used to treat effluents have shown to be inefficient to remove or just to reduce the concentrations of these types of pollutants to the legal limits. The present review provides a thorough state-of-the-art overview on the use of biological processes in the rehabilitation of ecosystems contaminated with the pharmaceutical compounds most commonly detected in the environment and eventually more studied by the scientific community. Among the different biological processes, special attention is given to biosorption and biodegradation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of the UID/BIO/04469/2013 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