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"Available online 29 April 2022", Zymomonas mobilis ZM4 presents interesting features for the biotechnological production of prebiotics. In this work, a Box-Behnken design approach was used to optimize the medium composition and maximize the fructooligosaccharides (FOS) production, in a one-step process. Afterwards, the scale-up to bioreactor allowed a high yield, titer and productivity of FOS (0.52 ± 0.05 gFOS gSuc-1, 156.50 ± 0.58gL-1, 4.89 ± 0.88gL-1 h-1). Furthermore, after 72h, 86.69 ± 3.70gL-1 of ethanol, 8.0 ± 0.9gL-1 of levan and 45.62 ± 2.70gL-1 of sorbitol were also produced. After purification (purity degree of 80.9%), an in vitro model using human fecal inoculum was used to assess the prebiotic potential of the FOS-enriched mixture. Herein we describe for the first time a one-step approach to produce a prebiotic mixture with market potential as a functional food ingredient using Z. mobilis ZM4., The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project FoSynBio (POCI-01-0145-FEDER-029549)., info:eu-repo/semantics/publishedVersion

Biogas microbiology involves microorganisms with diverse physiological traits, establishing interactions and networks in microbial communities. It is therefore a complex and broad topic. Biogas generation occurs in natural and engineered man-made systems. Knowledge on physiology and diversity of these microorganisms is pivotal to better control the biogas generation in those systems impacting both renewable energy production and methane emissions in nature. The 4th International Conference on Biogas Microbiology will bring together scientists from microbiology and engineering domains, linking life and technology to shape the future of biogas. This conference is the 4th of a series of conferences on biogas microbiology (Leipzig, Germany 2011, Uppasala, Sweden 2014, and Wageningen, The Netherlands 2017). In 2022, it takes place in Braga, Portugal, at Altice Forum Braga, in a joint organization of University of Minho and University of Santiago de Compostela. Welcome to Braga!, info:eu-repo/semantics/publishedVersion

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/publishedVersion

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/publishedVersion

Until now the prebiotic potential of pure trisaccharide raffinose on human health remains poorly investigated. In particular, studies following the guidelines recently established by the International Scientific Association for Probiotics and Prebiotics are scarce [1]. Raffinose is non-digestible to humans but can be metabolized by colon bacteria [2], therefore being considered a prebiotic candidate. Different approaches for testing the prebiotic potential of new compounds have been described. However, an in vitro model is considered a more suitable first-stage strategy to screen prebiotics candidates [3], contrarily to in vivo studies which are expensive and time-consuming. In this work, an in vitro model using human fecal inocula of two healthy volunteers (D1 and D2) was used to study the prebiotic potential of raffinose and compare it with the well-stablished and commercial prebiotic lactulose. The intestinal microbiota showed preference for raffinose as substrate presenting the highest consumption value at 48 h (96.0 ± 0.9 % D1 and 95.3 ± 0.7 % D2). The fermentation of raffinose decreased the medium pH, the ammonia concentration and the relative amount of Proteobacteria, including Escherichia coli, while increasing the total production of lactate and short chain fatty acids, viz. acetate and propionate, (129.9±2.6 mM D1 and 179.6±0.6 mM D2), CO2 (10.8±0.8 mmol/Lmedium D1 and 5.2±0.3 mmol/Lmedium D2) and the relative amount of Bifidobacterium and Lactobacillus. This study strongly suggests that raffinose holds potential prebiotic properties for human health and, subsequently, potential to be commercialized as functional food ingredient., CA an BBC acknowledge their grants (2020.00293.CEECIND and SFRH/BD/132324/2017) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2020 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Projects FoSynBio (POCI-01-0145-FEDER 029549) and NewFood – Food Technologies Valorization (NORTE-324 01-0246-FEDER-000043). The authors also acknowledge 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/publishedVersion

Gas fermentation is a promising way to convert CO-rich gases to chemicals. In this work we aimed to develop an electrotransformation protocol for the promising gas-converting Acetobacterium wieringae strain JM (sJM) [1], to further implement heterologous pathways for the production of new compounds from gas streams. In this matter, Clostridial species are the most genetic manipulated homoacetogens, and within the Acetobacterium genus, only A. woodii has been used. Therefore, there is a strong need and opportunity to expand the molecular toolkit for sJM and other Acetobacterium species. To achieve this, we tested reported electrotransformation procedures for A. woodii, using available molecular tools from Clostridial species, namely the plasmid vectors pMTL82151, pMTL83151, pMTL84151, and pMTL84151 containing the Gram-positive replicons pBP1, pCB102, pCD6, and pIM13 with the thiamphenicol resistance gene (catP). The utilization reported electrotransformation procedures did not result in sJM transformants. Therefore, we developed a new protocol which we report to be applicable to sJM, A. wieringae DSM 1911 and A. woodii DSM 1030. The most important changes for higher expeditiously and reproducibility were the use of fixed cell density of competent cells resulting in electroporation cell-plasmid ratios of 2 to 3 (OD600.mL/g), the use of an electric field strength of 10 kV/cm, and the selection of transformants in serum anaerobic bottles with molten agar of our optimized basal media. All plasmid vectors were transformed, having pMTL83151 yielded the highest transformation efficiency in all tested Acetobacterium strains, reaching efficiencies up to 5.0 × 102, 2.0 × 103, and 5.1 × 103 transformants g-1 DNA for sJM, A. wieringae DSM 1911 and A. woodii DSM 1030, respectively. Key factors affecting the electrotransformation efficiency include cell-wallweakening using D-threonine, pH of wash buffer, field strength of the electric pulse, plasmid amount, and sucrose osmoprotection. The electrotransformation procedure and tools reported here unlock the genetic manipulation of this biotechnologically relevant sJM strain, which will be directed towards the expansion of the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol., 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/150583/2020., info:eu-repo/semantics/publishedVersion

info:eu-repo/semantics/publishedVersion

Xylooligosaccharides (XOS) are considered emergent prebiotics with potential for several industrial applications. Here, we investigated the prebiotic activity of XOS-containing hydrolysates produced by different processes: direct fermentation of beechwood xylan (BXL) and enzymatic treatment of beechwood (XLH) or rice husk (RHH) xylans. BXL was obtained by fermentation of beechwood xylan (2.5 g/L increased up to 5.0 g/L after 3 h) using a cloned Bacillus subtilis 3610 containing the xylanase gene xyn2. XLH and RHH were obtained by enzymatic hydrolysis (40 °C, 180 rpm, 24 h) of xylans using a crude xylanase extract from Aureobasidium pullulans CCT 1261 as follows: RHH 3% (w/v) rice husk xylan in pH 5.3 and enzyme load of 200 U/gxylan; XLH 6% (w/v) beechwood xylan in pH 6.0 and enzyme load of 260 U/gxylan. The hydrolysates obtained from each strategy were lyophilized and further used as substrates (10 g/L) in anaerobic fermentation (37 °C, 48h) with fecal inoculum (11%, v/v) to evaluate the prebiotic activity. During the in vitro fermentation, liquid and gas samples were taken periodically for further analysis, as biomass samples for DNA extraction and sequencing analysis. The reduction of both the pH and the ammonia concentration together with the production of short-chain fatty acids, lactate (BLX: 80.4±2.2 mM; RHH: 104.7±4.9 mM; XLH 107.9±5.9 mM) and CO2 (BLX: 12.3±0.5 mmol/L; RHH: 18.1±0.3 mmol/L; XLH: 18.8±1.2 mmol/L) suggested the prebiotic potential of the XOS-containing hydrolysates. Additionally, the fermentation of these hydrolysates decreased the relative abundance of Clostridium, Desulfovibrionales, and Methanobacteriaceae and stimulated the growth of Bacteroides, Megamonas funiformis, and Lactobacillus reuteri. Based on these results, we concluded that the non-purified XOS-containing hydrolysates have promising prebiotic activities for further applications in food and cosmetic areas., CAPES (finance code 001); CNPq (grant numbers 423285/2018-1 and 304857/2018-1); FoSynBio (POCI-01-0145-FEDER-029549); LIGNOZYMES (POCI-01-0145-FEDER-029773). Gabrielle Gautério acknowledge the financial support Postgraduate Student Mobility Program (PROPESP/FURG). Beatriz Cardoso and Claudia Amorim acknowledge their grants (SFRH/BD/132324/2017 and 2020.00293.CEECIND) from Foundation for Science and Technology (FCT)., info:eu-repo/semantics/publishedVersion

Unprecedented environmental concerns related to the intensive use of fossil fuels has led to the use of alternative energy carriers for the production of biofuels/biochemicals. In this context, the development of alternative technologies for carbon capture and utilization has attracting more and more attention. Gas fermentation is a promising strategy for the production of bioproducts through the conversion of carbon-containing gases, contributing at the same time, to the reduction of greenhouses gases emission. Microbial CO2 conversion with renewable H2, for production of alternative fuels, such as CH4, open perspectives to solve different environmental problems. 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). Objectives: In this work, a pressurized bioreactor was operated in batch mode, under increased H2/CO2 pressure from 100kPa to 500 kPa, to investigate its potential effect on SHMA, on CH4 production rate and on microbial communities. Anaerobic granular sludge was used as biocatalyst. A mixture of H2/CO2 (80% H2 and 20% CO2, v/v) was used as sole carbon and energy source. Gaseous compounds were analyzed by gas chromatography, liquid products were analyzed by HPLC and archaeal diversity within granular sludge was monitored by 16S r-RNA based techniques. Results: An increase in the SHMA as well as in the CH4 production rate was observed 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 for CH4 production at moderate H2/CO2 pressures (up to 500 kPa). The Illumina sequence results showed that Methanosarcinales, Methanobacteriales and Methanomicrobiales were the three orders that prevailed in the pressurized system, for all the different 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/ Impact of the work: 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., info:eu-repo/semantics/publishedVersion

As the end of fossil fuels-era is approaching, new biotechnological alternatives for biofuels and biochemicals production are emerging. Here we focus on the use of gaseous substrates, namely H2/CO2 and CO/H2/CO2 (syngas) as carbon and energy sources for the production of compounds with economical value, such as biomethane and fatty-acids by anaerobic granular sludge at moderate pressures up to 5×105 Pa. It was proven that anaerobic granular sludge can stand increased H2/CO2 pressure maintaining a high rate of methane production. On the other hand, the rise of syngas pressure up to 5×105 Pa led to a reduction on CO and H2 consumption rates and to a shift towards the formation of propionate and butyrate. The increase of initial H2/CO2 or syngas total pressure resulted in a specialization of the microbial communities. Anaerobic granular sludge was a stable and efficient microbial platform for the conversion of gaseous substrates., This work was financially supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the following programmes: project INNOVsyn (POCI-01-0145-FEDER-031377; PTDC/BTA-BTA/31377/2017), strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). The authors thank the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte, BioTecNorte operation (NORTE 01- 0145-FEDER-000004). The FCT postdoctoral grants (SFRH/BPD/101034/2014 and SFRH/BPD/104837/2014) attributed to Marlene Lopes and Joana Alves are also gratefully acknowledged., info:eu-repo/semantics/publishedVersion

Dissertação de mestrado em Biotecnologia, Global energy demands and environmental concerns are accelerating the development of renewable energy sources and promoting the replacement of conventional manufacturing processes. In this context, low-biodegradable materials assume a huge importance since it serves as the feedstock for these new bio-based processes. Gasification is an alternative technology for biomass and wastes treatment, and originates a carbon-rich gas (called syngas, mainly composed of carbon monoxide (CO), carbon dioxide (CO2) and hydrogen (H2)), which can be further converted into a large spectrum of chemicals and fuels. Syngas fermentation uses microorganisms as biocatalysts to assimilate 1-carbon molecules and produce value-added compounds, such as acetic and n-butyric acids, ethanol, butanol, among others. Butanol has emerged as a high valuable compound for the chemical and energy industries due to its characteristics (higher energy content, lower toxicity, among others). Although it is nowadays produced from petrochemical processes, some acetogens can produce it from syngas fermentation. From the previous, Clostridium carboxidivorans is considered a promising species for industrial application due to its unique capacity of producing higher alcohols (butanol and hexanol) from syngas at specific experimental conditions. The first part of this thesis focused on the physiological characterization of C. carboxidivorans when converting different substrates (glucose, syngas and CO) and the dependence of yeast extract as a co-factor during syngas fermentation. The second part aimed to evaluate different operational strategies to promote alcohol production (specially butanol): cultivation at sub-optimal temperature and testing different CO partial pressures within the syngas mixture. The main results and conclusions from this thesis are: 1) the versatility of C. carboxidivorans, as it grew well in all the conditions tested, showing no inhibition when cultivated at different temperatures (30 and 37 ºC), pressures (100, 170 and 200 kPa), different gaseous substrates and even in the absence of yeast extract; it was also shown that a higher CO concentration was crucial for n-butyric acid synthesis and that yeast extract is important to accelerate growth but do not influence acetic acid titers; 2) regarding the strategies to promote solventogenesis, the use of above-atmospheric pressures showed to be more promising than cultivation at 30 ºC, since higher pressures (170 and 200 kPa) lead to an increase in CO consumption rate and accelerated n-butyric acid synthesis, which suggests the microorganism was following the metabolic pathway that further leads to butanol production., O crescimento económico e as alterações climáticas têm promovido o desenvolvimento das energias renováveis e de novos processos biotecnológicos. Os resíduos (biomassa e resíduos urbanos) constituem a matéria–prima necessária para esta transição. A gaseificação é uma tecnologia utilizada no tratamento de resíduos, cujo produto final é um gás rico em carbono (monóxido de carbono (CO) e dióxido de carbono (CO2)) e hidrogénio (H2), denominado gás de síntese (ou syngas) que pode ser convertido em vários compostos de elevado interesse económico. A fermentação de syngas recorre à capacidade de certos microrganismos para utilizar estes gases e convertê-los em compostos de valor acrescentado, como os ácidos acético e n-butírico, etanol, butanol, entre outros. O butanol, por exemplo, tem elevado valor para a indústria química e para o sector da energia, devido às suas características (elevado conteúdo energético, baixa toxicidade, entre outros). Apesar de ser atualmente produzido por processos petroquímicos, alguns microrganismos têm a capacidade de o produzir a partir da fermentação de syngas. A espécie Clostridium carboxidivorans destaca-se devido à sua capacidade única de produzir álcoois de maior peso molecular (butanol e hexanol) a partir de syngas em condições operacionais específicas. A primeira parte desta tese focou-se na caracterização fisiológica de C. carboxidivorans na conversão de diferentes substratos (glucose, syngas e CO), bem como na dependência do extrato de levedura como co-factor de crescimento, durante a fermentação de syngas. A segunda parte teve como objetivo estudar estratégias operacionais para promover a produção de álcoois (ex. butanol): temperatura e pressão. Os principais resultados e conclusões são: 1) a versatilidade de C. carboxidivorans, dado ter crescido em todas as condições testadas, não mostrando inibição do crescimento quando cultivado a diferentes temperaturas (30 e 37 ºC), pressões (100, 170 e 200 kPa), misturas gasosas e mesmo na ausência de extrato de levedura no meio de cultura; verificou-se também que uma concentração de CO superior na mistura gasosa foi crucial para a síntese de ácido n-butírico e que o extrato de levedura é importante para acelerar o crescimento, mas não influencia a produção de ácido acético; 2) quanto às diferentes estratégias estudadas, a utilização de pressões superiores (> 100 kPa) mostrou ser mais promissora do que alterar a temperatura de fermentação, pois pressões mais elevadas (170 e 200 kPa) levaram a um aumento da taxa de consumo de CO e produção de ácido n-butírico, o que sugere que o microrganismo estaria a seguir a via metabólica que originará butanol.