Caracterização do património geomorfológico do Parque Natural do Douro Internacional (NE de Portugal) com vista à sua valorização

O Parque Natural do Douro Internacional (PNDI) é uma área protegida pertencente à Rede Nacional de Áreas Protegidas, sob a alçada do Instituto da Conservação da Natureza e da Biodiversidade. O Parque localiza-se no nordeste transmontano, numa área de 851 km2, que acompanha longitudinalmente os rios Douro e Águeda, através de um troço fronteiriço e ao longo de 130 km. Os vales do tipo canhão fluvial com as sua vertentes abruptas, as arribas, destacam-se entre outras geoformas no PNDI. O presente trabalho visa caracterizar e quanti!car a relevância do Património Geomorfológico, inserido no riquíssimo Património Geológico existente na área, bem como a apresentação de propostas de valorização. Foi feita a caracterização dos geomorfossítios inventariados no âmbito dum projecto anterior, tendo-se concluído que os aspectos de maior importância da paisagem do PNDI são o Planalto Mirandês, os relevos residuais, as geoformas graníticas e os vales profundos do rio Douro e afluentes. De seguida, procedeu-se à quantificação da relevância, utilizando uma adaptação dos métodos propostos por Cendrero (2000) e Brilha (2005) para o Património Geológico, obtendo-se uma seriação dos geomorfossítios quanti!cados, o que permite concluir quais os locais com maior potencial para valorização e divulgação. Para a valorização destes geomorfossítios propõem-se várias estratégias como a implementação de painéis interpretativos temáticos, inseridos num percurso rodoviário com o tema “Rota das Arribas”, passando pelos miradouros mais emblemáticos dos rios Douro e Águeda. O Património Geomorfológico do PNDI é um dos ex-libris do Parque, pelo que deverá ser valorizado, constituindo uma importante valência para o impulso do geoturismo na região.The International Douro Natural Park (IDNP) is a protected area that belongs to the Protected Areas National Network, managed by the Nature and Biodiversity Conservation Institute. It is located in northeastern Portugal, with an area of 851 km2. The Park follows the Douro and Águeda rivers, through the border with Spain, along 130 km. The fluvial canyons and cliffs associated with these rivers are important landscape elements in the IDNP. The present work intends to characterize and quantify the relevance of Geomorphological Heritage, inserted in the rich Geological Heritage of this area, as also to present valorization strategies. A characterization of potential geomorphosites identified in a previous project was developed, which highlights the most important elements of the IDNP landscape, namely the Miranda Plain, residual reliefs, granitic landforms and deep valleys of the Douro and the Águeda rivers. A quantitative assessment was also applied, based on a modified version of the models proposed by Cendrero (2000) and Brilha (2005) for the Geological Heritage. A final ranking of the geomorphosites was proposed which establishes the valuable sites that must be included in conservation strategies or selected for geotourism and educational programs. Several strategies were proposed to value these geomorphosites, such as thematic interpretative panels and a car route, the “Arribas Route”, joining the most important viewpoints of the Douro and the Águeda rivers. The Geomorphological Heritage of the IDNP is an ex-libris of this Natural Park. It must be recognized, valued and considered as a major contribution to the geotourism in the region

CH4 production at moderate H2/CO2 pressures insights on the use of anaerobic granular sludge as biocatalyst

Introduction: The continuous increase in energy consumption and the intensive use of fossil fuels, lead to the emission of greenhouse gases (GHG) and, in particular, to an increase in the concentration of CO2 in the atmosphere. In this context, the improvement in global awareness and the demand for sustainable technologies and products strongly contribute to laid plans to combat climate change. CO2-to-CH4 conversion represents a cutting-edge solution for CO2 capture and use, contributing to the reduction of GHG emission. Catalytic conversion of CO2-to-CH4 have been investigated, however, the high cost associated to the catalysts employed limits their use on a large scale. Biological CO2 methanation can overcome the significant technical and economic challenges of catalytic CO2 methanation. The biological production of CH4 using CO2-rich gases together with H2 is a promising strategy for the production of bioproducts. Hydrogenotrophic methanogens have a crucial role on the direct conversion of CO2+H2 into CH4, hence the importance to study the specific hydrogenotrophic methanogenic activity (SHMA). Methodology: In this work, the effect of initial substrate (H2/CO2) pressure, from 100 to 500 kPa, on the SHMA, on CH4 production rate and on developed microbial communities were evaluated. Two different pressurized bioreactors were studied using anaerobic granular sludge as the biocatalyst and H2/CO2 (80:20, v/v) as sole carbon and energy source. Gaseous compounds were analyzed by GC and archaeal diversity within granular sludge was monitored by 16S r-RNA based techniques. Results: The results showed an increase in the SHMA as well as in the CH4 production rate with the increase of the initial H2/CO2 pressure. This results are very interesting since no inhibitory effects were observed on the microbial activity, demonstrating the resistance of the anaerobic granular sludge. The Illumina results showed that Methanosarcinales, Methanobacteriales and Methanomicrobiales were the three orders that prevailed in the pressurized system, for all the pressures tested. However, hydrogenotrophic methanogens from Methanobacterium and Methanospirillum genera slightly increased their relative abundance, varying from 38% (100 kPa) to 41% (500 kPa) and from 8% (100 kPa) to 12% (500 kPa), respectively. Conclusions: In conclusion, the archaeal community seems to be very stable when submitted to increasing H2/CO2 pressures, highlighting the potential of the anaerobic granular sludge as an efficient microbial platform for the production of added-value compounds from gaseous carbon waste streams.Portuguese Foundation for Science and Technology (FCT): POCI-01-0145-FEDER-031377; strategic funding of UIDB/04469/2020 unit; BioTecNorte operation (NORTE-01-0145-FEDER-000004); FCT doctoral grant PD/BD/128030/2016.info:eu-repo/semantics/publishedVersio

CH4 production at moderate H2/CO2 pressures - insights on the specific hydrogenotrophic methanogenic activity

CO2 is one of the main contributors to greenhouse gases (GHGs), being its emission to the atmosphere one of the major driver of global climate change. Biological methanation of CO2 using renewable H2 provides a promising approach to use of superplus renewable electrical power to produce a gaseous fuel. CH4 is considered an important renewable energy carrier, that has a wide range of applications such as natural gas for distribution. Hydrogenotrophic methanogens are key elements in the CO2/H2 methanation process. Thus the importance to study the specific hydrogenotrophic methanogenic activity (SHMA). The effect of the initial substrate (H2/CO2) pressure on the SHMA was investigated in two different pressurized bioreactors. The results suggest that in addition to the increase of the initial substrate pressure, also the bioreactor configuration influence the SHMA, which is crucial for the success of biological CO2 methanation technologies but also in anaerobic bioreactors treating wastewaters.info:eu-repo/semantics/publishedVersio

Physiological characterization and promoter engineering of Acetobacterium wieringae for acetone production via gas fermentation

The pressing need to mitigate environmental concerns has driven research into sustainable energy and chemical production methods that reduce carbon emissions. Gas fermentation offers a promising avenue for low-carbon fuel and chemical synthesis. Acetobacterium wieringae, particularly strain A. wieringae JM, has emerged as an attractive host for gas-based biorefineries due to its unique abilities, including growth in diverse gas compositions and pH ranges, and efficient growth on carbon monoxide without co-substrates. This study focuses on enhancing the potential of A. wieringae for acetone production through genetic modification. A transformation protocol was developed, and the acetone production operon from Clostridium acetobutylicum was introduced. Novel promoters were explored to widen gene expression possibilities in A. wieringae. The stability of the plasmid backbone pMTL83151 carrying replicon pCB102 was assessed. Additionally, the tolerance of A. wieringae to gas synthesis derived from biogenic residue gasification was evaluated for potential industrial application. Gas composition significantly influenced acetone production by A. wieringae, with distinct physiological effects observed between strain A. wieringae DSM 1911 and A. wieringae JM. Four constitutive promoters from A. wieringae JM and four from C. autoethanogenum were successfully expressed, exhibiting stronger activity than the reference Pthl promoter from C. acetobutylicum. Notably, A. wieringae JM demonstrated robust growth in synthesis gas from biomass gasification, though with physiological variations. This study unveils the intricate relationship between gas composition, physiological attributes, and acetone production in A. wieringae. The expanded promoter repertoire enhances genetic manipulation potential, propelling the strain's capacity for versatile gene expression. Moreover, the resilience of A. wieringae JM to gasification-derived gas synthesis highlights its viability for industrial implementation. These findings contribute to advancing the development of gas-based biorefineries, paving the way for sustainable chemical production with reduced environmental impact.info:eu-repo/semantics/publishedVersio

Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways

Developing new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock. Taken together, the production of bio commodities from low-cost gas streams containing CO, CO2, and H2, obtained from the gasification of any carbon-containing waste streams or off-gases from heavy industry (steel mills, processing plants, or refineries), embodies an opportunity for affordable and renewable chemical production. To achieve this, by studying non-model autotrophic acetogens, current limitations concerning low growth rates, toxicity by gas streams, and low productivity may be overcome. The Acetobacterium wieringae strain JM is a novel autotrophic acetogen that is capable of producing acetate and ethanol. It exhibits faster growth rates on various gaseous compounds, including carbon monoxide, compared to other Acetobacterium species, making it potentially useful for industrial applications. The species A. wieringae has not been genetically modified, therefore developing a genetic engineering method is important for expanding its product portfolio from gas fermentation and overall improving the characteristics of this acetogen for industrial demands.The involved research was fnancially supported by the Portuguese Foundation for Science and Technology (FCT): POCI-01-0145-FEDER-031377; strategic funding of UIDB/04469/2020 unit; BioTecNorte operation (NORTE-01-0145- FEDER-000004); FCT doctoral grant PD/BD/150583/2020info:eu-repo/semantics/publishedVersio

Editorial: The microbiology of the biogas process

[Excerpt] The world is facing unprecedent challenges, related with energy crisis and climate change. Intensification of renewable energy production, with special focus on sustainable biogas and biomethane, is one of the front-line topics today. Biogas/biomethane will play a role in the transition toward climate-neutral and secure energy system. Ensuring that biomethane is produced from organic waste/wastewater is essential to support circularity and sustainability. Scaling up biomethane production and assuring its economic competitiveness are current key challenges. Biogas generation occurs in natural and engineered environments, such as anaerobic bioreactors, and involves a cascade of reactions catalyzed by complex microbial communities. To unlock and boost the full potential of waste-based biomethane production, bioprocess optimization is needed, which requires deep knowledge on microbial diversity and physiology, as well as on the complex microbial interactions and metabolic networks occurring in biogas processes. This was the motivation for launching the Research Topic “The Microbiology of the Biogas Process,” which comprises six original research articles by 48 authors, addressing different facets of the theme and resorting to diverse approaches, reflecting the complexity of the topic. [...]This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit.info:eu-repo/semantics/publishedVersio

Oxidação electroquímica de compostos lipídicos como precursora do tratamento anaeróbio

A digestão anaeróbia é um processo de tratamento de efluentes e simultaneamente uma fonte de energia, através da produção de biogás. A presença de compostos lipídicos nos efluentes não só causa problemas de inibição dos microorganismos como pode levar à lavagem do sistema, sendo frequente a aplicação de pré-tratamentos. Os tratamentos físico-químicos mostram-se, por vezes, insuficientes e originam lamas indesejáveis. Em alternativa, a oxidação electroquímica deste tipo de compostos orgânicos apresenta-se como um processo prometedor na medida em que proporciona a alteração dos lípidos e promove a respectiva conversão em biogás. No presente trabalho, as transformações electroquímicas do ácido oleico, em solução aquosa com eléctrodos de platina (Pt), chumbo (Pb) e grafite, são analisadas numa vasta gama de potenciais. Os dados obtidos permitem estabelecer as condições operativas para proceder à electrooxidação do lípido sob controlo potenciostático. Os rendimentos da oxidação electroquímica do composto lipídico sobre os diferentes substratos e de acordo com os parâmetros electroquímicos empregues são discutidos.Fundação para a Ciência e a Tecnologia (FCT

Microbial propionate production from carbon monoxide a novel bioprocess

Introduction: The fermentation of CO-rich gases by carboxidotrophic microbes is a promising way to produce valuable organic compounds. Propionate is a value-added compound with numerous industrial applications, e.g. as an antifungal agent in food and feed, and as a building block to produce plastics and herbicides. Propionate is currently produced by petrochemical processes, but it can be produced from ethanol and acetate by some propionogenic bacteria. Ethanol and acetate are usually formed by acetogenic bacteria from CO-rich gases. Accordingly, propionate can be indirectly produced from CO-rich gases, representing a new approach on the realm of microbial CO conversion. Methodology: Four distinct synthetic co-cultures were constructed, consisting of: Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T); A. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T); A. wieringae strain JM and P. propionicus (DSM 2379T); A. wieringae strain JM and A. neopropionicum (DSM 3847T). The physiology of CO conversion to propionate was accessed and a proteogenomic analysis was performed in the best performing co-culture to get insight into the involved biochemical pathways and microbial interactions within the synthetic consortium. Results: Propionate was produced by all the co-cultures, with the highest titer (~24 mM) measured in the co-culture composed of A. wieringae strain JM + A. neopropionicum, which also produced isovalerate (~4 mM), butyrate (~1 mM), and isobutyrate (~0.3 mM). In this synthetic consortium, A. wieringae strain JM converts CO to a acetate and ethanol via the Wood-Ljungdahl pathway; acetate can also be converted to ethanol through the action of aldehyde oxidoreductase (AOR); A. neopropionicum converts ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during co-cultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, and this results in isovalerate and isobutyrate production. Conclusions: This synthetic co-culture represents a new bioprocess for the microbial production of propionate from carbon monoxide, that couples the Wood-Ljungdahl and acrylate pathways. Furthermore, this symbiosis engages an interesting perspective on how C1-fixing and C3-producing microorganisms can be used to expand the product scope of gas fermentation.Portuguese Foundation for Science and Technology (FCT): POCI-01-0145-FEDER-031377; strategic funding of UIDB/04469/2020 unit; BioTecNorte operation (NORTE-01-0145-FEDER-000004); FCT doctoral grants PD/BD/128030/2016 and PD/BD/150583/2020. Netherlands Science Foundation (NWO): Project NWO-GK-07; Perspectief Programma P16-10; Gravitation Grant, Project 024.002.002.info:eu-repo/semantics/publishedVersio

Bioprocess optimization for generation of hepatocytes derived from hiPSC and its application in primary hyperoxaluria type 1 disease modelling

Primary hyperoxaluria type 1 (PH1) is a rare metabolic disorder caused by mutations in the hepatic alanine-glyoxylate aminotransferase (AGT). Defective AGT in PH1 patients is characterized by excessive oxalate synthesis, which leads to a broad range of kidney complications including the end-stage renal disease [1]. Combined liver-kidney transplantation remains the only effective treatment; however significant morbidity, mortality and costs encouraged the development of advanced cell- and gene-based therapies for PH1. Thus, our aim was to implement a novel strategy to generate high numbers of functional hepatocyte-like cells (HLC) from PH1 patient derived human induced pluripotent stem cells (PH1.hiPSC), for PH1 disease modelling and further application in drug and therapeutics development. PH1.HLC were differentiated as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion, according to the integrated bioprocess previously developed by our group [2,3]. Briefly, PH1.hiPSC were aggregated and expanded in STB for 4 days preceding the hepatic differentiation. hiPSC to HLC commitment begin by culturing the 3D aggregates in different medium formulations (from Takara BioEurope AB). Two different dissolved oxygen (pO2) conditions were explored: a normoxia (pO2: uncontrolled, 95% air, 5% CO2) throughout the differentiation process (21 days) and a hypoxia with a low oxygen (pO2: 4% O2) environment between day 4 and day 14 of the differentiation. Our results showed that PH1-hiPSC successfully proliferated as 3D aggregates with an expansion factor of 6-fold after 4 days in culture while maintaining their pluripotent phenotype. Low dissolved oxygen concentration during hepatic specification, generate higher yields of HLC and improve gene expression levels of ALB, A1AT and CYP3A4 hepatic markers when compared with HLC differentiated under uncontrolled pO2 conditions. Moreover, Flow cytometry analysis, revealed a higher hepatocyte content of 80% (low pO2) vs 43% (uncontrolled pO2) for albumin, showing a higher process efficiency. Transcriptomic analysis using RNAseq confirmed that hepatocyte differentiation was enhanced in the low dissolved oxygen condition. In addition, these PH1.HLC showed functional characteristics typical of hepatocytes including production of important hepatic proteins (albumin, alpha 1 antitrypsin), urea and bile acids. PH1.HLC also display drug metabolization capacity, CYP450 activity and, by histological assessment, glycogen storage and positive staining for albumin and AFP markers. To further characterize the PH1 disease features, we performed a detailed metabolomic analysis and demonstrated that PH1.HLC show defective AGT activity with significantly higher production and secretion of oxalate for PH1.HLC when compared with HLC generated from healthy counterparts. Overall, controlling the dissolved oxygen concentration at key stages of the hepatic differentiation process improved cell yield and the maturation status of HLC. The bioprocess developed and optimized in this work offers high relevance not only for generation of more accurate in vitro models to study PH1 rare disease, but also towards the development of novel therapies. Acknowledgements & Funding: this study was funded by a grant from ERA-NET E-Rare 3 research program, JTC ERAdicatPH (E-Rare3/0002/2015) and Fundação para a Ciência e Tecnologia project MetaCardio (PTDC/BTM-SAL/32566/2017); iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior, through national funds is acknowledged. P. V., J. I. A. were supported by FCT fellowships SFRH/BD/145767/2019, SFRH/BD/116780/2016 respectively. [1] P. Cochat, N. Engl. J. Med., vol. 369, no. 7, pp. 649–658, 2013. [2] B. Abecasis, J. Biotechnol., vol. 246, pp. 81–93, 2017. [3] I. Isidro, Biotechnol Bioeng, vol. 118, 3610–3617, 2021

Propionate production from carbon monoxide by synthetic cocultures of acetobacterium wieringae and propionigenic bacteria

Gas fermentation is a promising way to convert CO-rich gases to chemicals. We studied the use of synthetic cocultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far, isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore, this cocultivation approach was investigated. Four distinct synthetic cocultures were constructed, consisting of Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T), Ac. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T), Ac. wieringae strain JM and P. propionicus (DSM 2379T), and Ac. wieringae strain JM and An. neopropionicum (DSM 3847T). Propionate was produced by all the cocultures, with the highest titer (;24mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (;4mM), butyrate (;1mM), and isobutyrate (0.3mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior.This research was financially supported by Project NWO-GK-07 from the Netherlands Science Foundation (NWO), the Perspectief Programma P16-10 from NWO Applied and Engineering Sciences (AGS), by a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and by the Portuguese Foundation for Science and Technology (FCT) under the scope 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. The financial support from FCT and European Social Fund (POPH-QREN) through the grant PD/BD/128030/2016 (given to Ana Luísa Arantes) and PD/BD/150583/2020 (given to João P. C. Moreira) and through the project INNOVsyn—Innovative strategies for syngas fermentation (POCI-01-0145-FEDER-031377) are gratefully acknowledged.info:eu-repo/semantics/publishedVersio