Adélia Varela

Impact of ionic liquids on extreme microbial biotypes from soil

This work aims at identifying, amongst extreme soil biotypes at locations of high salinity and high hydrocarbon load, microbial strains able to survive short or long-term exposure to the presence of selected ionic liquids. We have evaluated the impact of ionic liquids on the diversity of the soil microbiota to identify which microbial strains have higher survival rates towards ionic liquids, and consequently those which might possibly play a major role in their biotic fate. To the best of our knowledge, this is the first study of this kind. Soils, from a region in Portugal (Aveiro) were sampled and the bacterial and fungal strains able to survive after exposure to high concentrations of selected ionic liquids were isolated and further characterised. We have mainly focused on two types of cations: imidazolium – the most commonly used; and cholinium – generally perceived as benign. The surviving microbial strains were isolated and taxonomically identified, and the ionic liquid degradation was analysed during their cultivation. The continuing exposure of the microbial strains to petroleum hydrocarbons is likely to be the basis for their acquired resistance to some imidazolium salts; also, the higher capacity of fungi – compared to bacteria – to grow, even during their exposure to these liquid salts, became evident in this study.

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

UNLABELLED Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE). Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (β-glucosidases and endo-1,5-α-l-arabinosidase). Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of β-glucosidase in cork secretome. BIOLOGICAL SIGNIFICANCE Cork degradation by fungi remains largely overlook. Herein we aimed at understanding how A. nidulans colonise cork cell walls and how this relates to wood colonisation. To address this, the protein species consistently present in the secretome were analysed, as well as major alterations occurring in the substrate, including lignin degradation compounds being released. The obtained data demonstrate that this fungus has superficially attacked the cork cell walls apparently by using both enzymatic and Fenton-like reactions. Only a few polysaccharide degrading enzymes could be detected in the secretome which was dominated by protein species associated with autolysis. Lignin degradation was corroborated by the identification of some degradation products, but the suberin barrier in the cell wall remained virtually intact. Comparative proteomics revealed that cork and wood colonisation share a common set of enzymatic mechanisms.

Understanding fungal functional biodiversity during the mitigation of environmentally dispersed pentachlorophenol in cork oak forest soils

Pentachlorophenol (PCP) is globally dispersed and contamination of soil with this biocide adversely affects its functional biodiversity, particularly of fungi - key colonizers. Their functional role as a community is poorly understood, although a few pathways have been already elucidated in pure cultures. This constitutes here our main challenge - elucidate how fungi influence the pollutant mitigation processes in forest soils. Circumstantial evidence exists that cork oak forests in N. W. Tunisia - economically critical managed forests are likely to be contaminated with PCP, but the scientific evidence has previously been lacking. Our data illustrate significant forest contamination through the detection of undefined active sources of PCP. By solving the taxonomic diversity and the PCP-derived metabolomes of both the cultivable fungi and the fungal community, we demonstrate here that most strains (predominantly penicillia) participate in the pollutant biotic degradation. They form an array of degradation intermediates and by-products, including several hydroquinone, resorcinol and catechol derivatives, either chlorinated or not. The degradation pathway of the fungal community includes uncharacterized derivatives, e.g. tetrachloroguaiacol isomers. Our study highlights fungi key role in the mineralization and short lifetime of PCP in forest soils and provide novel tools to monitor its degradation in other fungi dominated food webs.

A three-act play: pentachlorophenol threats to the cork oak forest soils mycobiome

Atmospheric release of persistent organic pollutants (POPs) constitutes a silent threat through chronic contamination of soils at global scale; yet fundamental understanding of their occurrence, sources and fate is still largely lacking. Similar to a three act play, this review comprises Setup, Confrontation and Resolution. The first emphasises the eighty years of the history of pentachlorophenol (PCP) usage, only recently classified as POP. The second focus on active sources of PCP pollution, including inside cork oak forests in N.W. Tunisia; a threat partially neutralised by the soil microbial diversity, especially fungi. As Resolution, the need for improved knowledge on the global distribution and impacts of PCP in soil microbial diversity as means to preserve the multi-functionality of terrestrial ecosystem is emphasised.

Harmonisation of physical and chemical methods for soil management in Cork Oak forests - Lessons from collaborative investigations

As part of a collaborative project to investigate human impacts on Quercus suber L. (cork oak) forests, five research groups from countries in Europe and North Africa undertook a survey of soil quality (physical properties, potentially toxic elements) at sites in NW Tunisia and NW Sardinia. All groups performed the analysis of soil samples after agreeing prescribed methodologies to ensure harmonisation and the production of a robust and reliable data set. The data produced were compared using basic statistical methods and revealed strong positive correlation despite minor operational variation. The data indicates that inter and intra laboratory variability differed from parameter to parameter and that good agreement was obtained where methodology was common. Collaborative research introduced the need for common communication plans and exchange of information not normally supplied in analytical reporting. Key words: Forest soil quality, inter-comparison, Quercus suber L., cork oak, Tunisia, Sardinia, chemical analysis, potentially toxic elements.

Proteomic Insights on the Metabolism of Penicillium janczewskii during the Biotransformation of the Plant Terpenoid Labdanolic Acid

Plant terpenoids compose a natural source of chemodiversity of exceptional value. Many of these compounds own biological/pharmacological activity, others are regarded as unique chemical skeletons for the synthesis of derivatives with improved properties. Functional chemical modification of terpenoids through biotransformation frequently relies on the use of Ascomycota strains, but information on major cellular responses is still largely lacking. Penicillium janczewskii mediates a stereo-selective hydroxylation of labdanolic acid (LA)—terpenoid found abundantly in Cistus ladanifer—producing 3β-hydroxy-labdanolic acid with yields >90%. Herein, combined analyses of mycelial and extracellular differential proteomes demonstrated that the plant terpenoid increased stress responses, especially against oxidative stress (e.g., accumulation of superoxide dismutase) and apparently altered mitochondria functioning. One putative cytochrome P450 monooxygenase differentially accumulated in the secretome and the terpenoid bioconversion was inhibited in vivo in the presence of a P450 inhibitor. The stereo-selective hydroxylation of the plant terpenoid is likely mediated by P450 enzymes, yet its unequivocal identity remains unclear. To the best of our knowledge, this is the first time that proteomics was used to investigate how a plant terpenoid impacts the metabolism of a filamentous fungus during its efficiently biotransformation. Our findings may encourage the development of new strategies for the valorization of plant natural resources through biotechnology.