The role of the restriction-modification system of Clostridium pasteurianum on its electro-transformation

Dissertação de mestrado em BioengenhariaClostridium pasteurianum is a Gram-positive and anaerobic bacterium with a great biotechnological potential. It is one of the few microorganisms capable of hydrolyzing glycerol to produce solvents as ethanol and butanol, which have a wide applicability in the market as biofuels. The development of a genetic system for this microorganism would increase its application opportunities since gene overexpression or inactivation could improve their solventogenic characteristics. Its genetic information is already known but this organism has a particular resistance to transformation. This resistance can be explained by a very efficient restriction system that does not allow the entrance of non-methylated DNA or DNA with a methylation pattern different from it. Therefore, foreign DNA must be correctly methylated prior to transformation. For this purpose, a specific methyltransferase is needed to transfer methyl groups to a certain nucleotide of a specific sequence. The goal of this thesis was to create a genetic system in C. pasteurianum that allows genome modification and foreign protein expression, ultimately improving C. pasteurianum DSM 525 transformation. Preliminary simple electro-transformations in which the parameters to make competent cells and the electroporation conditions were altered, did not result in positive results. Being aware of the possibility of a restriction system presence in this organism, experiments with M.MspI methylated DNA were performed, however they demonstrated the inability of this methyltransferase to improve the microorganism transformation. The presence of restriction enzymes was confirmed when a characterization of the restriction system of C. pasteurianum was performed using MspI methylated and non-methylated DNA. The presence of a discrete digestion pattern was detected, and M.MspI methylation could not protect the foreign DNA from C. pasteurianum restriction action. The polyamine spermidine, with known affinity for negatively charged DNA, showed to be efficient against C. pasteurianum crude extract digestion action, however not sufficiently to facilitate this microorganism electrotransformation. By accessing the genome information, the Restriction/Modification (R/M) systems of this microorganism were analyzed. The GATC type IIP R/M system was chosen in order to verify the restriction and methylation enzymes activity with the same target sequence. Three genes, one REase (DpnII) and two MTases (Dam and MdpnII) were cloned in pETduet-1, followed by overproduction in BL21 (DE3). The codon usage of the host and original organism were not compatible, and the protein production in tRNAs provider strains was tested. Protein production was detected, however was not possible to re-confirm their presence. The common protein folding problems were analyzed using a disulfide bond enhancer strain. Nevertheless, the production problem may not be related to this, since no different protein over-production was detected. Restriction reactions with the REase BstUI and C. pasteurianum crude extract, using DNA methylated by M.SssI (m5CG), were developed and showed that the REase responsible for hindering foreign DNA entering C. pasteurianum recognizes the sequence 5'-CGCG- 3'. In a second analysis of the C. pasteurianum genome a methyltransferase-encoding gene was identified that may be involved in methylating the sequence 5'-CGCG- 3'. The in silico analysis was performed and its codon usage was also improved to be compatible with E. coli. In this work, the reasons for C. pasteurianum’s recalcitrance to transformation were identified, the knowledge about its R/M systems was extended, and a proposal to efficiently transform this bacterium was provided.Clostridium pasteurianum DSM 525 é uma bactéria Gram-positiva anaeróbia com um elevado potencial biotecnológico. Este é um dos poucos microrganismos capaz de hidrolisar glicerol para produzir solventes como etanol e butanol, que têm uma grande aplicabilidade no mercado. O desenvolvimento de um sistema genético para este organismo permitiria aumentar as suas oportunidades de aplicação sendo que a sobre-expressão ou inativação de um determinado gene pode melhorar as suas características solventogénicas. A sua informação genética já é conhecida, mas este microrganismo apresenta uma particular resistência à transformação. Esta resistência pode ser explicada pela presença de um eficiente sistema de restrição que não permite a entrada de DNA não metilado ou DNA metilado de forma diferente da própria bactéria. Desta forma, o DNA estranho deve ser corretamente metilado antes da transformação. Para que isto seja possível é necessária a presença de uma metilase específica para transferir grupos metilo para um determinado nucleótido de uma sequência específica. O objetivo desta tese foi criar um sistema genético em C. pasteurianum que permitisse modificações no genoma e a expressão de proteínas heterólogas, ou seja, que permitisse melhorar a transformação de C. pasteurianum DSM 525. Foram realizadas transformações preliminares simples com parâmetros que diferem na forma de obter células competentes e nas condições de eletroporação, contudo os resultados obtidos não foram positivos. Tendo conhecimento da possibilidade da presença de um sistema de restrição neste organismo, foram realizadas experiências com DNA metilado pela enzima M.MspI, sendo que estas demonstraram a incapacidade da metiltransferase para melhorar a transformação deste microrganismo. Foi confirmada a presença de enzimas de restrição aquando da caracterização do sistema de restrição de C. pasteurianum usando DNA não metilado ou metilado pela enzima M.MspI. Foi detetada a presença de um padrão de digestão distinto, verificando-se que a enzima M.MspI não consegue proteger o DNA estranho da ação de restrição de C. pasteurianum. A poliamina espermidina, com conhecida afinidade por DNA negativamente carregado, mostrou ser eficiente contra a ação de digestão do extrato cru de C. pasteurianum, contudo não o suficiente para facilitar a electrotransformação deste microrganismo. Tendo acesso ao genoma, foi então analisado o sistema de Restrição e Modificação (R/M) deste microrganismo. Foi escolhido o sistema R/M tipo IIP GATC para verificar a atividade de enzimas de restrição e metilação com a mesma sequência de reconhecimento. Foram clonados três genes no vetor pETduet-1, uma enzima de restrição (REase – DpnII) e duas metiltransferases (MTases – Dam and Mdpn), seguindo-se a produção em BL21 (DE3). O conjunto de codões usados pelo hospedeiro e pelo organismo de origem não eram compatíveis, foi então testada a produção proteica em estirpes fornecedoras de tRNAs. Foi observada produção proteica contudo não foi possível re-avaliar a sua presença. Foram analisados problemas de enrolamento (do inglês folding), usando uma estirpe que facilita a formação de pontes dissulfito. No entanto, o problema na produção não deve estar associado ao enrolamento proteico sendo que não foi detectada produção proteica nestas condições. Foram desenvolvidas reações de restrição com a REase BstUI e extrato cru de C. pasteurianum usando DNA metilado pela enzima M.SssI (m5CG) e foi mostrado que a REase responsável pelo impedimento da entrada de DNA em C. pasteurianum reconhece a sequência 5'-CGCG- 3'. Numa segunda análise ao genoma de C. pasteurianum DSM 525 foi identificado um gene que codifica uma metiltransferase que pode estar envolvida na metilação da sequência 5'-CGCG- 3'. Foi feita a análise in silico e o tipo de codões usados foi melhorado para ser compatível com E. coli. Neste trabalho, foram identificadas as razões para a resistência deste microrganismo à transformação, foi consolidado o conhecimento sobre o seu sistema de R/M e foi proposta uma metodologia para transformar de forma eficiente esta bactéria

Exploring the diversity of anaerobic sludge towards glycerol valorization

The large amounts of glycerol produced by the biodiesel industry (10 % of the total biodiesel production) can create environmental and economic losses if sustainable strategies are not applied to utilize the surplus of this compound. The production of valuable chemical compounds by anaerobic microorganisms can be a sustainable treatment strategy to add value to waste-glycerol and to the biodiesel industry. The objective of this work was to study the diversity and physiology of anaerobic microorganisms involved in glycerol consumption and valorization. Mesophilic enrichments were developed under sulfate-reducing and methanogenic conditions, using as inoculum anaerobic granular sludge from a brewery wastewater treatment plant. After several transfers, three different stable cultures were obtained, with the capacity to grow with glycerol under different culture conditions. One enrichment, ferments glycerol mainly to propionate, with a yield of 0.88 mM propionate per mM glycerol consumed. This culture is dominated by a bacterium closely affiliated with Propionivibrio pelophilus strain asp 66, 98.5% ID based on 16S rRNA genes. The P. pelophilus strain asp 66 was reported to be unable to grow with glycerol. A second enriched culture was obtained which is constituted of Desulfovibrio alcoholivorans, 99.5% ID, and the methanogen Methanofollis liminatans strain GKZOZ, 98.8% ID. Incubations with 2-bromoethanesulfonate confirmed that this is a syntrophic co-culture. Desulfovibrio alcoholivorans converts the glycerol to acetate and H2 and the methanogenic partner consumes the H2, making glycerol degradation thermodynamically viable. In the third enriched culture, Desulfovibrio sulfodismutans strain ThAcO1, 97.5 % ID, reduces glycerol to acetate, but only in the presence of sulfate or a methanogenic partner. In conclusion, starting from the same inoculum (anaerobic sludge), glycerol could be metabolized through different pathways by the enrichment cultures obtained. Fermentative, syntrophic and sulfate-reducing cultures were enriched forming valuable products that can be used in industrial applications or as energy carriers. Thus, anaerobic microbial communities are an asset to surpass the bottleneck of biodiesel production caused by the surplus of glycerol, allowing it to be sustainably treated and valorized.info:eu-repo/semantics/publishedVersio

Pseudomonas empower syntrophic fatty acids degradation in the presence of oxygen

Anaerobic digestion (AD) processes specifically directed towards biogas production are currently of great interest worldwide, due to the urgent need of more sustainable energy sources. Although not consensual, oxygen has been shown as an ally of AD processes, resulting in more efficient biogas production when added to the systems in vestigial doses. It has been suggested that it stimulates facultative anaerobic bacteria (FAB), which are generally present in the anaerobic communities. These bacteria are involved in several steps of AD (fermentation and acidogenesis) but have also been referred to protect the strict anaerobes from oxidative stress [1]. In this work, the influence of FAB in the degradation of short-, medium- and long-chain fatty acids (C4, butyrate; C8, octanoate; C16, hexadecanoate) by two syntrophic co-cultures was investigated. Syntrophomonas wolfei (Sw)/ Methanospirillum hungatei (Mh) and S. zehnderi (Sz)/ Methanobacterium formicicum (Mf) were pre-grown and Pseudomonas sp. (FAB) were further added, along with each substrate over a range of O2 concentrations (0-2 % v/v). In a second transfer, each culture was exposed to the same O2 concentration range. Both syntrophic co-cultures (Sw+Mh and Sz+Mf) had their activity highly reduced, or even completely inhibited, in the presence of O2. Interestingly, the theoretical CH4 production expected from C8 was reached by consortium Sw+Mh+Ps at days 3, 8 and 28 under 0%, 1% and 2% O2, respectively. The same trend was observed for consortium Sz+Mf+Ps. C4 and C16 degradation occurred similarly to C8 degradation, presenting similar results and the same tendency for both tested consortia. This data suggests a positive interaction and network establishment between these organisms. Apparently, Pseudomonas consumed the oxygen allowing the creation of a reduced environment, a requirement for an effective development of the strict anaerobic syntrophic co-cultures. In the 2nd transfer, the protective support of Pseudomonas was maintained. Moreover, it was verified that the cultures (Sw+Mh+Ps and Sz+Mf+Ps) previously exposed to O2 preserved their activity either under anoxic or microaerophilic conditions. These results show the essential role of Pseudomonas in the protection of syntrophic co-cultures activity, empowering fatty acids degradation under microaerophilic conditions. Furthermore, it highlights the FAB/Pseudomonas importance in real AD systems (where vestigial amounts of O2 can be detected) for the stability and resilience of the system maintaining syntrophic communities functionality and biogas production.info:eu-repo/semantics/publishedVersio

Facultative anaerobic bacteria: key players in syntrophic fatty acids degradation under microaerophilic conditions

Anaerobic digestion (AD) is a mature technology that contributes to the sustainable development through the production of energy and products, using microbes as key players. Oxygen, formerly thought as the nemesis of AD, has been shown to benefit the AD processes when provided in vestigial doses [1]. The beneficial effects of micro-aeration have been attributed to the increased activity of facultative anaerobic bacteria (FAB). Besides being involved in fermentation and acidogenesis, FAB have been referred to act as a protective shield against the damaging effects of oxidative stress to the strict anaerobic communities [2]. However, their role in the syntrophic degradation of fatty acids is not clear. In this work, the relationship between syntrophic bacteria (Syntrophomonas wolfei (Sw) and S. zehnderi (Sz)), methanogens (Methanospirillum hungatei (Mh) and Methanobacterium formicicum (Mf)) and FAB (Pseudomonas strains (Ps)) was investigated, during the degradation of short (C4, butyrate), medium (C8, octanoate) and long (C16, hexadecanoate). The syntrophic co-cultures were pre-grown and the Pseudomonas spp. were further added, along with each substrate, over a range of O2 concentrations (0-2 % v/v). A second transfer was performed, exposing each of the cultures to O2 concentrations between 0-2% (v/v). In the presence of O2 (even at the lower concentrations) the activity of the syntrophic cocultures sharply decreased or even disappeared. However, in the presence of Pseudomonas, methane production occurred, reaching the theoretically expected at days 3, 8 and 28 under 0%, 1% and 2% O2, respectively. These results were obtained for C8 degradation in the presence of Sw+Mh+Ps. The same tendency was observed for C4 and C16 degradation with both consortia. Additionally, in the 2nd transfer, the cultures previously exposed to O2, maintained their activity being able of completely convert substrates to methane, under anoxic and microaerophilic conditions. These results show the essential role of Pseudomonas in the protection of syntrophic coculture activity allowing fatty acids degradation under microaerophilic conditions. Therefore, in real AD systems, where vestigial O2 can be detected, the presence of FAB may result in more stable, resilient, and functional syntrophic communities.FCT under UIDB/04469/2020 unit; and SFRH/BD/132845/2017 grantinfo:eu-repo/semantics/publishedVersio

Aerobic and facultative bacteria: working horses at the service of anaerobic digestion

It is clear that aerobic and facultative anaerobic bacteria have an important role in the first steps of the anaerobic digestion (AD) process, especially when complex organic compounds are degraded. However, their diversity, abundance and function, related to the fine control of process variables such as pH and ORP, and the potential establishment of networks with methanogens and acetogens are far from being fully understood. Here we show some examples demonstrating that microaerophilic and aerobic conditions are critical for accelerating the methane production rates from hydrophobic compounds such as lipids and hydrocarbons. In the later case, a combination of aerobic with methanogenic conditions allow to convert hydrocarbons to methane at accelerated rates, with bacterial lipids as main intermediates. We believe that hybrid fine-controlled microaerophilic AD processes will emerge as the next generation of applications of AD technology to boost the methane production rate from a myriad of anaerobically slow degraded complex substratesinfo:eu-repo/semantics/publishedVersio

Co-cultivation of Thermoanaerobacter strains with a methanogenic partner enhances glycerol conversion

Glycerolrich waste streams produced by the biodiesel, bioethanol and oleochemical industries can be treated and valorized by anaerobic microbial communities to produce methane. As current knowledge of the microorganisms involved in thermophilic glycerol conversion to methane is scarce, thermophilic glyceroldegrading methanogenic communities were enriched. A coculture of Thermoanaerobacter and Methanothermobacter species was obtained, pointing to a nonobligately syntrophic glycerol degradation. This hypothesis was further studied by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelii with glycerol (10 mM) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mM day1, corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic cocultures than in the pure bacterial cultures. The catabolic pathways of glycerol conversion were identified by genome analysis of the two Thermoanaerobacter strains. NADH and reduced ferredoxin formed in the pathway are linked to proton reduction, which becomes thermodynamically favourable when the hydrogen partial pressure is kept low by the hydrogenotrophic methanogenic partner.The authors thank Ruben Gonçalves for preparing the thermophilic biomass and Andreia Salvador for the sup port with the microbial communities’ analysis. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit, Project SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403) and BioTecNorte operation (NORTE-01-0145-FEDER 000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Opera cional Regional do Norte. The authors also acknowledge the financial support of FCT and European Social Fund through the grants attributed to C.P. Magalhaes (SFRH/BD/132845/2017) and A.L. Arantes (PD/BD/128030/2016).info:eu-repo/semantics/publishedVersio

Enhanced glycerol conversion by Thermoanaerobacter strains

Glycerol-rich waste streams produced as a surplus by the biodiesel industry can be treated and valorized by anaerobic microbial communities to produce biogas. Glycerol is a highly reduced compound. Its complete degradation to methane and carbon dioxide requires a syntrophic cooperation of anaerobic bacteria and archaea, either directly or through propionate, lactate or ethanol as intermediates. The aim of this work was to study glycerol valorization to methane by thermophilic microbial communities. Glycerol-degrading methanogenic communities were enriched at 55 ºC. A co-culture of Thermoanaerobacter and Methanothermobacter was obtained pointing to facultatively syntrophic glycerol degradation. This hypothesis was further tested by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelli type strains with glycerol (10 mmol L-1) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mmol L-1 day-1, corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic co-cultures than in pure bacterial cultures. The methanogen acted as a biological electron acceptor, which enhanced glycerol conversion by Thermoanaerobacter species, since it facilitates the redox balance and contributes to a higher energy gain of these bacteria. Therefore, syntrophic glycerol fermentation promotes faster anaerobic treatment of glycerol rich waste streams coupled to methane production.info:eu-repo/semantics/publishedVersio

Livro Vermelho dos Peixes Dulciaquícolas e Diádromos de Portugal Continental

info:eu-repo/semantics/publishedVersio

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Micro-aeration as a strategy to enhance bioconversion of long-chain fatty acids to methane

Tese de doutoramento em Engenharia Química e BiológicaWaste lipids have high energy value, but the amount of lipids effectively converted into methane is still limited, because high lipid loads lead to long-chain fatty acids (LCFA) accumulation, hindering bioconversion. Micro-aeration can improve methane production in AD, and facultative anaerobic bacteria (FAB) or aerobic bacteria (AB) seem to be key microorganisms in the process. In this work, micro-aeration was applied, targeting the intensification of lipids/LCFA biodegradation to methane. Two experiments were performed in batch assays, to study micro-aeration's impact on syntrophic communities' activity. In the first experiment, anaerobic sludge was incubated with ethanol, in the presence of oxygen concentrations up to 5 % (Chapter 3). In the second experiment, two synthetic consortia composed by syntrophic fatty acid degrading bacteria and hydrogenotrophic methanogenic partners were constructed in the laboratory and supplemented with two Pseudomonas strains (Chapter 4). The cultures were subjected to 0-10% O2, and the conversion of palmitate (C16:0), caprylate (C8:0), and butyrate (C4:0) to methane was monitored. The results from the two experiments show that the activity of FAB/AB in general, and Pseudomonas spp. in particular, were crucial for maintaining the syntrophic relationships under micro-aerobic conditions, providing a shielding effect towards the syntrophic communities and limiting the inhibitory effect of oxygen. Indirect micro-aeration was then studied in the degradation of glucose (Chapter 5) or oleate (Chapter 6). Anaerobic bioreactors were compared with bioreactors that received aerated feeding. Organic loading rates of 2 g L-1 d-1 (in COD) were applied in both experiments. The results from the study with glucose indicate that aerated feeding enhances methane production, without interfering with the microbial community composition. However, with oleate, aeration in the feeding acted as a pre-treatment, contributing to oleate to palmitate conversion by AB/FAB even before entering the bioreactor. The reactors that received aerated feeding were able to operate in continuous for longer periods, with higher stability than the anaerobic reactors, and required shorter batch periods for degradation of the palmitate and acetate that accumulated during the continuous phases. In conclusion, this thesis shows that FAB/AB/Pseudomonas spp. have a crucial role in protecting and maintaining functional and resilient syntrophic communities. Moreover, in LCFA biodegradation, micro-aeration can trigger a first aerobic treatment that converts oleate to palmitate, and may facilitate further conversion to methane, relatively to strict anaerobic process.Os lípidos residuais possuem um alto valor energético, mas a quantidade de lípidos convertidos efetivamente a metano ainda é limitada, pois cargas elevadas de lípidos resultam na acumulação de ácidos gordos de cadeia longa (AGCL), dificultando a sua bioconversão. O microarejamento pode melhorar a produção de metano em digestores anaeróbios (DA), e as bactérias anaeróbias facultativas (BAF) ou aeróbias (BA) parecem ser microrganismos com um papel-chave no processo. Neste trabalho, foi aplicado microarejamento com o objetivo de intensificar a biodegradação de lípidos/AGCL a metano. Foram realizados dois ensaios em batch, para estudar o impacto do microarejamento na atividade de comunidades sintróficas. No primeiro ensaio, uma lama anaeróbia foi incubada com etanol, na presença de concentrações de oxigénio até 5 % (Capítulo 3). No segundo ensaio, foram construídos dois consórcios sintéticos compostos por bactérias sintróficas que degradam ácidos gordos e os parceiros metanogénicos hidrogenotróficos, suplementados com duas estirpes de Pseudomonas (Capítulo 4). As culturas foram submetidas a 0-10 % de O2, e a conversão de palmitato (C16:0), caprilato (C8:0) e butirato (C4:0) a metano foi monitorizada. Os resultados dos dois ensaios mostram que em geral a atividade das BAF/BA, e em particular das Pseudomonas spp., foi crucial para a manutenção das relações sintróficas em condições microaeróbias, proporcionando um efeito protetor para as comunidades sintróficas e limitando o efeito inibidor do oxigénio. O microarejamento indireto foi então aplicado na degradação de glucose (Capítulo 5) e oleato (Capítulo 6). Nestes trabalhos, reatores anaeróbios foram comparados com reatores que receberam alimentação arejada. Foi aplicada aos dois ensaios, uma carga orgânica de 2 g L-1 d-1 (em CQO). Os resultados do ensaio com glucose indicam que a alimentação arejada aumenta a produção de metano, sem interferir na composição da comunidade microbiana. No entanto, com o oleato, o arejamento da alimentação atuou como um pré-tratamento, contribuindo para a conversão do oleato a palmitato por BA/BAF antes da entrada no reator. Os reatores que receberam alimentação arejada foram capazes de operar em contínuo por períodos mais longos, com maior estabilidade do que os reatores anaeróbios, e necessitaram de períodos em batch mais curtos para a degradação do palmitato e acetato que acumularam durante as fases em contínuo. Em conclusão, esta tese demonstra que BAF/BA/Pseudomonas spp. têm um papel crucial na proteção e manutenção de comunidades sintróficas funcionais e resilientes. Além disso, na biodegradação de AGCL, o microarejamento pode estimular um primeiro tratamento aeróbio em que se converte o oleato em palmitato e pode facilitar a posterior conversão deste a metano, em comparação com o processo estritamente anaeróbio.I acknowledge the support of Fundação para Ciência e Tecnologia (FCT) through the PhD scholarships SFRH/BD/132845/2017 and COVID/BD/152450/2022, and all the entities supporting this work: FCT under the scope of the strategic funding of UIDB/04469/2020 unit, the European Regional Development Fund under the scope of the Norte2020—Programa Operacional Regional do Norte—BioEcoNorte project (NORTE-01-0145-FEDER-000070)