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Polyhydroxyalkanoates (PHA) have been pointed as an alternative to reduce the use of conventional plastics. PHA are natural polyesters that are produced by microorganisms, are biodegradable, biocompatible and have thermoplastics/elastomeric properties similar to those of petrochemical plastics. PHA are currently produced based on pure cultures, but the costs of investment (equipment) and production (energy and refined substrates) have discouraged the widespread utilization of PHA as commodity plastics. The production of PHA by mixed microbial cultures (MMC) can reduce the costs of investment and production, and consequently the PHA price, through use of open systems (less sophisticated equipment) and low-cost substrates, such as agro-industrial wastes/by- products. The MMC PHA production process consists generally of three stages: acidogenic fermentation to obtain VFA as PHA precursors, culture selection to enrich a culture in PHA- storing microorganisms with high capacity to accumulate PHA and a high growth rate, and PHA accumulation to maximize the PHA content in the biomass. The culture selection stage is considered the key step in this process since it determines the biomass productivity and its capacity of storing PHA. This PhD project has focused on the study of the impact of biomass withdrawal strategy on the performance of the selection reactor and consequently on biomass productivity and global PHA productivity. An experimental setup composed by two sequencing batch reactors (SBR) was operated under similar conditions, at an organic loading rate (OLR) of 100 Cmmol.L-1.d-1 and solids retention time (SRT) of 4 days, except for the timing of biomass withdrawal. Reactor 1 (R1) operated under a conventional strategy of biomass withdrawal (BW) at the end of the famine phase, while Reactor 2 (R2) performed the BW at the end of the feast phase. A synthetic mixture of acetic and propionic acid was selected as a representative carbon source composition from a fermented feedstock. The first study in this PhD project aimed to elucidate the effect of two different BW strategies on the performance of the culture selected. Both selection reactors had similar PHA storing capacity as proven by similar specific PHA storage rates of 0.44 ± 0.09 Cmol-PHA.Cmol-XA-1.h-1 in R1 versus 0.36 ± 0.09 Cmol-PHA.Cmol- XA-1.h-1 in R2 and similar PHA content of the biomass during the accumulation assays (51.7 ± 1.5 wt.% in R1-Ac versus 56.1 ± 1.0 wt.% in R2-Ac). However, R1 presented a much higher biomass productivity (about 4-fold higher than R2), due to higher biomass concentration (2.8 ± 0.4 g-XA.L-1 in R1 and 0.83 ± 0.2 g-XA.L-1 in R2), thus resulting in a much higher global PHA productivity. This study demonstrated that removing the excess biomass at the end of the famine phase resulted in a much higher global PHA productivity than removing the excess biomass at the end of the feast phase, providing important insight into how MMC systems can be best operated to maximize PHA productivity. In the second study of this PhD project, the same system was used to assess the influence of the OLR and SRT in tandem with the BW strategy to maximize the PHA productivity. It was demonstrated that increasing the OLR led to an improvement of biomass productivity and consequently to an improvement of the PHA productivity in both selection reactors, while the reduction of SRT to less than 1 d resulted in the reduction of the storage response in both reactors, compromising the overall PHA productivity. Additionally, at a SRT lower than 1.7 d, the accumulation stage did not improve the productivity of the PHA producing process in R2, and so, the biomass removed at the end of the feast phase could be forwarded directly to downstream processing, reducing investment and operational costs. A preliminary cost assessment of the two different operational modes was then performed, comparing: (1) the utilization of both selection and accumulation reactors and the conventional BW; and (2) a single selection reactor was used with the feast phase BW. The results demonstrated that the utilization of a culture selection reactor with the feast phase BW at the SRT of 0.8 d was the configuration resulting in the lowest total annual costs and lowest break-even price (1.88 €/kg PHA-crude). Also, this configuration met the minimum limit of 40% wt.% PHA content in the biomass, which has been previously indicated as a viable threshold for cost-effective downstream processing. Os polihidroxialcanoatos (PHAs) têm sido apontados como uma alternativa para reduzir a utilização de plásticos convencionais. Os PHAs são poliésteres naturais produzidos por microrganismos, são biodegradáveis, biocompatíveis e têm propriedades termoplásticas/elastoméricas semelhantes às dos plásticos de origem petroquímica. Os PHA são atualmente produzidos com base em culturas microbianas puras, mas os custos de investimento (equipamento) e de produção (energia e uso de substratos refinados) têm desencorajado a utilização generalizada dos PHAs como “plásticos de comodidade”. A produção de PHA por culturas microbianas mistas (MMC) pode reduzir os custos de investimento e de produção, e consequentemente o preço final dos PHAs, através da utilização de reatores menos sofisticados e substratos de baixo custo, tais como resíduos/subprodutos agroindustriais. O processo de produção de PHA com base em MMC consiste geralmente em três fases: a fermentação acidogénica para obter VFA que são precursores de PHA, a seleção da cultura para enriquecer uma cultura em microrganismos com elevada capacidade de acumular PHA e uma elevada taxa de crescimento, e a produção de PHA para maximizar o conteúdo de PHA na biomassa. A fase de seleção da cultura é considerada a etapa chave neste processo, uma vez que determina a produtividade em biomassa e a sua capacidade de acumulação de PHA. Este projeto de doutoramento centrou-se no estudo do impacto da estratégia de remoção da biomassa no desempenho do reator de seleção e, consequentemente, na produtividade em biomassa e na produtividade global de PHAs. Uma instalação experimental composta por dois reatores descontínuos sequenciais (SBR) foi operada em condições semelhantes, com uma carga orgânica (OLR) de 100 Cmmol.L-1.d-1 e um tempo de retenção de sólidos (SRT) de 4 dias, exceto no que diz respeito à altura do ciclo em que foi efetuada a purga de biomassa. O Reator 1 (R1) foi operado com uma estratégia convencional de remoção de biomassa (RB) no final da fase de fome, enquanto o Reator 2 (R2) xviii realizou a RB no final da fase de fartura. Para efeitos de simplificação do substrato, foi utilizada como fonte de carbono uma mistura sintética de ácido acético e propiónico. O primeiro estudo neste projeto de doutoramento teve como objetivo elucidar o efeito de duas estratégias diferentes de RB no desempenho da cultura selecionada. Ambos os reatores de seleção apresentaram uma capacidade de acumulação de PHAs semelhante, comprovada por taxas específicas de acumulação de PHAs semelhantes de 0,44 ± 0,09 Cmol-PHA.Cmol-XA-1.h-1 no R1 comparado com 0,36 ± 0,09 Cmol-PHA.Cmol-XA-1.h-1 no R2, e conteúdo de PHA na biomassa semelhante durante os ensaios de acumulação (51,7 ± 1,5 %(g/g) no R1-Ac comparado a 56,1 ± 1,0 %(g/g) no R2-Ac). Contudo, o R1 apresentou uma produtividade em biomassa muito superior (cerca de 4 vezes superior à do R2), devido a uma maior concentração de biomassa (2,8 ± 0,4 g-XA.L-1 no R1 e 0,83 ± 0,2 g-XA.L-1 no R2), resultando assim numa produtividade global de PHA muito superior. Este estudo demonstrou que a remoção do excesso de biomassa no final da fase de fome resultou numa produtividade global de PHAs muito mais elevada do que a remoção do excesso de biomassa no final da fase de fartura, o que permite tirar conclusões importantes sobre a forma de operação dos sistemas operados com MMC de modo a maximizar a produtividade de PHAs. No segundo estudo deste projeto de doutoramento, o mesmo sistema foi utilizado para avaliar a influência da OLR e SRT em conjunto com a estratégia de RB para maximizar a produtividade de PHA. Foi demonstrado que o aumento da OLR levou a uma melhoria da produtividade de biomassa e consequentemente a uma melhoria da produtividade de PHAs em ambos os reatores de seleção, enquanto a redução do SRT para menos de 1 dia resultou na redução da resposta de acumulação de PHA em ambos os reatores, comprometendo a produtividade global de PHAs. Além disso, a um SRT inferior a 1,7 dias, a fase de acumulação não melhorou a produtividade do processo de produção de PHAs no R2, pelo que a biomassa removida no final da fase de fartura pode ser encaminhada diretamente para a etapa de extração/purificação, reduzindo os custos de investimento e operação. Foi realizada uma avaliação económica com o objetivo de quantificar o impacto dos dois processos: (1) o sistema constituído pelo reator de seleção e de acumulação com RB convencional; e (2) um único reator de seleção com a estratégia de RB na fase de fartura. Os resultados demonstraram que a utilização de um reator de seleção com remoção de biomassa na fase de fartura e SRT de 0,8 dias foi a configuração que resultou no menor custo total anual e no menor preço de equilíbrio (1,88 €/kg PHA). Além disso, esta configuração atingiu o limite mínimo de 40% de PHAs na biomassa, valor que é considerado como viável na etapa de extração.

Bio-based and biodegradable plastics are an ecological alternative to conventional petroleum-derived polyolefins. Polyhydroxyalkanoates (PHA) have drawn significant attention as one of the most promising biopolymers due to its biocompatibility and biodegradable character. In particular, the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) – P(3HB-co-3HV) – has exceptional thermal and mechanical properties, which can be manipulated by varying its monomeric composition. PHA production using non-aseptic mixed microbial cultures (MMCs) enables the use of waste/surplus feedstocks, contributing simultaneously to the implementation of a circular economy approach and to reduce the process operational costs associated to the traditional pure cultures’ PHA production. The MMC process usually comprises three steps: the acidogenic fermentation, the enrichment of a MMC in PHA-storers and finally the PHA production. The monomeric composition of the P(3HB-co-3HV) produced by MMCs largely depends on the fraction of each soluble fermentation product (SFP) produced in the first step of the process, which can be precursors of either 3-hydroxybutyrate (3-HB) or 3-hydroxyvalerate (3-HV). This Thesis project was focused on solving, at pilot scale, the main difficulties (at the production level) that currently prevent industrial implementation of the PHA production process by MMC: the high manufacturing costs associated to the low process performance and the ability to consistently manipulate and tailor the polymer composition (and thus its properties). A three-stage MMC process was implemented at pilot scale. Fruit waste (FW) was selected as feedstock, since it contains high concentration of carbon and is globally generated in large volumes, causing several environmental and economic problems. FW is a nutrient-deficient feedstock, thus enabling the implementation of an uncoupled carbon and nitrogen feeding strategy in the selection reactor. The first study conducted in this PhD assesses, for the first time, the impact of the sludge retention time (SRT) (2 and 4 days) and the organic loading rate (OLR) (from 2.6 to 14.5 gCOD.L-1.d-1) on the growth versus polymer storage dynamics of PHA-storers selected under the uncoupled feeding strategy. The results showed that, similar biomass volumetric productivities were achieved for each OLR tested, regardless the SRT. However, the culture selected at 4 d SRT showed superior specific storage rates and accumulation capacity resulting in a global PHA productivity (4.6 ± 0.3 g.L-1.d-1) that was 80% above that of 2 d SRT (2.6 ± 0.2 g.L-1.d-1 ). This study underlined the importance of achieving a good balance between culture growth and accumulation capacity to increase the viability of the PHA-producing process from wastes. The same pilot plant set-up and feedstock (FW) were used to incorporate different effective operating conditions in the three stages of the process with the objective of boosting the overall PHA production performance (namely, PHA content on biomass, global productivity and overall yield). The OLR and pH of the acidogenic reactor were successfully adjusted targeting a high fermentation yield (0.74 gCOD.gCOD-1) and the production of a fermentate highly enriched in butyrate (56.8%, gCOD-basis), resulting in enhanced PHA production steps. A well selected MMC was obtained as a result of uncoupling the carbon to the nitrogen feeding, and the biomass volumetric productivity attained the unprecedented value of 1.96 g.L-1.d-1 as a response to the high OLR (8.7 gCOD.L-1.d-1) imposed. The culture selected at the optimal OLR achieved a high storage yield (0.98 gCOD.gCOD-1), and the continuous feeding strategy led to a maximum PHA content of 80.5% (w/w) at the end of accumulation assays. The obtained global PHA productivity (8.1 g-PHA.L-1.d-1) and overall process yield (0.45 gCOD.gCOD-1) are the highest values reported for MMC using a real waste feedstock at pilot scale. Moreover, a P(3HB-co-3HV) copolymer with a 3-HV content of 0.24 (g-basis) and a molecular weight of 311 KDa was produced, making this material an ideal candidate for packaging applications, the largest market of plastic usage. Lastly, the possibility of tailoring the precursors that influence the P(3HB-co-3HV) composition was investigated, by controlling the operating pH (between 4.69 and 6.34) of a continuous upflow anaerobic sludge blanket (UASB) reactor fed with FW. The impact of the operating conditions imposed was assessed by evaluating the microbial community profile, the corresponding performance and the impact on polymer composition. The fermentation yield of the UASB was maintained quite stable (between 0.72 and 0.79 gCOD.gCOD-1) during the entire operational period. On the other hand, the 3-HV bioprecursors fraction in effluent was highly affected by pH, resulting in the production of P(3HB-co-3HV) copolymers with quite different monomeric compositions. Overall, the 3-HV content of the produced polymers varied from 0.16 to a maximum of 0.44 (gCOD-basis) when the pH of the acidogenic reactor increased from 4.69 to 5.92. Moreover, the end-stream SFP composition were similar for identical operational pH values tested in different periods, showing that despite the changes occurring in the system, the selected cultures were resilient and able to produce a consistent profile of fermentation products (and thus a constant PHA monomeric composition). Additionally, the IWA Anaerobic Digestion Model No. 1 (ADM1) was expanded to include the pathway of valerate production from lactate, aiming to dynamically predict the profile of the PHA bioprecursors produced. Calibration and validation procedures were done against data from two distinct pilot-scale UASB reactors fed with FW and describing different pH and OLR dynamics. The model was capable to predict the different PHA bioprecursors production in the UASB reactors with overall strong correlations with the experimental data for different OLRs over the pH range between 4.20 and 5.16, providing a useful tool for process optimisation and tailoring of the PHA monomer composition. This thesis highlights the importance of understanding in-depth the impact of key operating conditions on the PHA production process with MMC to increase its overall viability. Moreover, it shows that predicting and regulating the acidogenic process is essential to promote an adequate PHA bioprecursors composition production, which are both promising results towards the full-scale implementation of the PHA production from MMC. Plásticos biodegradáveis de origem biológica são uma alternativa ecológica às poliolefinas convencionais derivadas do petróleo. Os polihidroxialcanoatos (PHAs) têm vido a ganhar prestígio como um dos bioplásticos mais promissores devido ao seu carater biocompatível e biodegradável. Em particular, o copolímero poli(3-hidroxibutirato-co-3-hidroxivalerato) – P(3HB-co-3HV) – tem excelentes propriedades mecânicas e térmicas, que podem ser manipuladas variando a sua composição monomérica. A produção de PHA usando culturas microbianas mistas (MMCs) não assépticas permite o uso de resíduos/sub-produtos como substratos, contribuindo simultaneamente para a implementação de uma economia circular e para reduzir os custos operacionais associados ao tradicional processo de produção de PHA com culturas puras. O processo de produção com MMC envolve normalmente 3 passos: uma fermentação acidogénica, o enriquecimento de uma MMC em bactérias acumuladoras de PHA e, por último, a produção de PHA. A composição monomérica do P(3HB-co-3HV) produzido por MMCs depende fortemente da fração de cada produto de fermentação solúvel (SFP) produzido no primeiro passo do processo, que podem ser precursores de 3-hidroxibutirato (3-HB) ou de 3-hidroxivalerato (3-HV) Este projeto de Tese teve como foco principal resolver, à escala piloto, as principais dificuldades (ao nível da produção) que impendem a implementação do processo de produção de PHAs a partir de MMCs à escala industrial: os custos de produção altos associados à baixa performance do processo e a habilidade de manipular consistentemente a composição do polímero (e portanto as suas propriedades). O processo de três passos de produção de PHAs foi implementado à escala piloto. Resíduos de fruta estragada (FW) foram selecionados como substrato, visto que contêm altas concentrações de carbono e são globalmente gerados em grandes volumes, causando diversos problemas ambientais e económicos. FW são um substrato deficiente em nutrientes, o que permite implementar uma estratégia de alimentação em que o fornecimento de carbono é desacoplado do azoto no reator de seleção. O primeiro estudo realizado durante este PhD teve como objetivo estudar, pela primeira vez, o impacto do tempo de retenção de lamas (SRT) (2 e 4 dias) e da taxa de carga orgânica (OLR) (de 2.6 a 14.5 gCOD.L-1.d-1) nas dinâmicas de crescimento versus armazenamento de polímero de culturas produtoras de PHA sujeitas ao regime de alimentação desacoplado. Os resultados mostraram que, semelhantes produtividades volumétricas de biomassa são observadas para cada OLR imposta, independentemente do SRT. No entanto, a cultura selecionada a um SRT de 4 d mostrou uma taxa especifica de produção de polímero e uma capacidade de acumulação superiores, apresentando uma produtividade global de PHA (4.6 ± 0.3 g.L-1.d-1) 80% superior ao atingido pela cultura selecionada a um SRT de 2 d (2.6 ± 0.2 g.L-1.d-1 ). Este estudo realçou a importância de conseguir um bom equilíbrio entre o crescimento da cultura e a sua capacidade de acumulação, de forma a aumentar a viabilidade do processo de produção de PHA a partir de resíduos. A mesma instalação piloto e substrato (FW) foram usados no seguinte estudo, onde várias condições de operação eficazes foram incorporadas de uma só vez nos três passos do processo com o objetivo de impulsionar a performance global da produção de PHA (nomeadamente, o conteúdo de PHA na biomassa, a produtividade global e o rendimento global). A OLR e o pH do reator acidogénico foram ajustados com sucesso de forma a atingir um elevado rendimento de fermentação (0.74 gCOD.gCOD-1) e produzir um fermentado enriquecido em butirato (56.8%, base gCOD), que irá beneficiar a performance da cultura nos próximos passos do processo. Uma MMC bem selecionada foi obtida como resultado da aplicação da estratégia de desacoplamento do carbono do azoto, e a produtividade volumétrica de biomassa atingiu o valor sem precedentes de 1.96 g.L-1.d-1, como resposta à alta OLR (8.7 gCOD.L-1.d-1) imposta. A cultura selecionada à OLR ótima mostrou um elevado rendimento de produção de PHA (0.98 gCOD.gCOD-1), e a estratégia de alimentação continua levou à obtenção de um conteúdo máximo de PHA na biomassa de 80.5% (w/w) no final dos ensaios de acumulação. A produtividade global de PHA (8.1 g-PHA.L-1.d-1) e o rendimento global do processo (0.45 gCOD.gCOD-1) obtidos foram os valores mais altos reportados para MMC usando um resíduo real como substrato à escala piloto. Alem disso, foi produzido um copolímero P(3HB-co-3HV) com 0.24 (base g) de monómeros 3-HV e um peso molecular de 311 KDa, o que faz deste material um excelente candidato para ser usado em embalagens, o sector de mercado que apresenta o consumo de plástico mais elevado. Por último, a possibilidade de controlar a produção dos percursores que influenciam a composição do P(3HB-co-3HV) foi investigada através da manipulação do pH (entre 4.69 e 6.34) de um reator anaeróbio de fluxo ascendente (UASB) alimentado com FW. O impacto das diferentes condições de operação impostas foi avaliado através da observação do perfil da comunidade microbiana selecionada, da sua performance e do impacto na composição dos diferentes polímeros produzidos. O rendimento de fermentação do UASB manteve-se relativamente estável (entre 0.72 e 0.79 gCOD.gCOD-1) durante todo o período de operação. Por outro lado, a fração de precursores de 3-HV no efluente foi bastante afetada pelo pH, o que resultou na produção de copolímeros P(3HB-co-3HV) com diferentes composições monoméricas. De forma global, o conteúdo de 3-HV nos polímeros produzidos variou de 0.16 até um máximo de 0.44 (base gCOD) quando o pH do reator acidogénico aumentou de 4.69 para 5.92. Além disso, a composição das correntes de SFP produzidas a valores de pH semelhantes, mas testados em diferentes períodos da operação, foram idênticas, mostrando que, apesar das mudanças que ocorrem no sistema, as culturas selecionadas foram resilientes a capazes de produzir um perfil de produtos de fermentação consistente (resultando numa composição monomérica de polímero constante). Além do trabalho experimental relatado, o modelo de digestão anaeróbia No. 1 (ADM1) foi expandido de forma a incluir a via de produção de valerato a partir de lactato, com o objetivo de se prever dinamicamente o perfil de precursores de PHA produzido no reator UASB descrito. Os procedimentos de calibração e validação foram realizados usando dados de dois reatores UASB piloto distintos, ambos alimentados com FW e sujeitos a diferentes dinâmicas de pH e OLR. O modelo foi capaz de prever a produção dos diferentes precursores de PHA nos reatores UASB e atingir fortes correlações com os dados experimentais obtidos para várias OLRs dentro do intervalo de pH de 4.20 a 5.16, providenciando uma ferramenta útil para otimização do processo e para manipular a composição monomérica de PHA. Esta tese realça a importância de efetivamente perceber o impacto de condições de operação chave no processo de produção de PHA com MMC de forma a aumentar a sua viabilidade global. Alem disso, mostrou que prever e regular o processo acidogénico é essencial para promover a produção de precursores de PHA com uma composição adequada, o que são ambos resultados promissores no sentido da implementação à escala industrial do processo de produção de PHA a partir de MMC.

Nutrient and micropollutant removal, as well as resource recovery, are some of the major current concerns in the wastewater treatment field. Wastewater treatment plants (WWTPs) need solutions to quickly tackle these emerging problems and operate as sustainably as possible. Therefore, this thesis focused on providing insight into some of the major challenges in these fields, such as: i) factors influencing efficient enhanced biological phosphorus removal (EBPR) and its microbial community structure; ii) the biotransformation of an important micropollutant (diclofenac) in the EBPR process and iii) phosphorus and carbon solubilisation from WWTP sludge for resource recovery. EBPR is a complex process where specific bacteria are used for phosphorus removal from the bulk liquid. The most important polyphosphate accumulating organism (PAO) is “Candidatus Accumulibacter phosphatis”, that comprises of phylogenetically different Types, clades and subclades, whose metabolism can be influenced by specific conditions. A reactor enriched with Accumulibacter (> 85 %) was operated for over a one-year period, and identification at the sub-clade level was necessary to correlate the specific identity of the Accumulibacter group with the observed reactor performance. The carbon feeding rate proved to be crucial for the expression of different metabolisms, where a fast-feeding rate resulted in a mixed phosphate/glycogen accumulating metabolism leading to poor P removal (< 30 %) that correlated with clusters ii and iii of Accumulibacter IIc, while a slow carbon feeding rate resulted in complete phosphorus removal and an abundance of cluster i. This work showed that some organisms commonly recognised as PAO do not promote efficient phosphorus removal and there is a need to recharacterize the organisms within the Accumulibacter group. Removal of micropollutants is another emerging concern, especially since some micropollutants such as diclofenac have been shown to be near-recalcitrant in conventional WWTPs. Additionally, some physical/chemical processes have been shown to produce transformation products that are more toxic than the parent compound. Diclofenac biotransformation was investigated in the EBPR process, where the metabolic pathway and toxicity of diclofenac by-products were also assessed. Although Accumulibacter did not appear to transform diclofenac as efficiently as other bacteria (e.g., nitrifiers), it was found to still contribute towards its detoxification. Furthermore, a correlation was observed between the quantity of diclofenac biotransformation and Accumulibacter Type, which could suggest that enrichment of Type II Accumulibacter can stimulate diclofenac biodegradation in wastewater treatment. Finally, resource recovery is one of the major initiatives in the wastewater treatment field. WWTPs are no longer seen as just treatment facilities, but also present opportunities to recover other added-value products and provide more sustainable solutions. Phosphorus is an irreplaceable nutrient and a scarce resource that must be recovered from phosphorus rich waste streams. Biological acidification was tested in this study as an additional step within conventional WWTPs and was shown to have great potential to solubilise up to 80 % of phosphorus and produce on average 0.35 g CODVFA/gVS that could be used in subsequent processes for the production of phosphorus rich fertilizers or bioplastics, respectively. A remoção de nutrientes e micropoluentes, bem como a recuperação de recursos, são algumas das principais preocupações atuais na área do tratamento de águas residuais. As estações de tratamento de águas residuais precisam de soluções para combater rapidamente esses problemas emergentes e operar da forma mais sustentável possível. Por esse motivo, o objectivo desta tese foi identificar alguns dos principais desafios nestas áreas, tais como: i) fatores que influenciam a remoção biológica eficiente de fósforo e respetiva composição da comunidade microbiana; ii) a biotransformação de um importante micropoluente (diclofenac) no processo de remoção biológica de fósforo e iii) solubilização de fósforo e carbono de lamas de tratamento de águas residuais com objectivo de recuperação de recursos. A remoção biológica do fósforo é um processo complexo em que bactérias específicas são usadas para a remoção do fósforo da parte liquida. O organismo acumulador de polifosfato (PAO) mais importante é a “Candidatus Accumulibacter phosphatis”, composta por diferentes tipos filogenéticos, clades e subclades, cujo metabolismo das diferentes espécies é influenciado por condições especificas. Neste trabalho, um reator enriquecido em Accumulibacter (> 85%) foi operado por um período superior a um ano, durante o qual a identificação de uma espécie , ao nível do subclade, foi feita de forma a correlacionar a identidade específica do grupo Accumulibacter com o desempenho do reator. A taxa de alimentação de carbono provou ser crucial para a expressão de diferentes metabolismos. Uma taxa de alimentação rápida resultou em um metabolismo misto de acumulação de fosfato/glicogênio, levando a uma baixa remoção de P (< 30%), o que se correlacionou com os clusters ii e iii, enquanto uma taxa de alimentação de carbono lenta levou a um metabolismo de acumulação de polifosfato típico, resultando na remoção completa do fósforo e de uma abundância do cluster i dentro da mesma Accumulibacter IIc. Este trabalho mostrou que alguns organismos habitualmente reconhecidos como PAO não promovem uma remoção eficiente de fósforo, verificando-se assim a necessidade de reclassificar os organismos que são PAOs dentro do grupo Accumulibacter. A remoção de micropoluentes é outra preocupação emergente, especialmente porque alguns micropoluentes, como o diclofenaco, mostraram ser recalcitrantes em estações de tratamento de águas residuais convencionais. Foi demonstrado que alguns processos físicos/químicos produzem produtos de transformação mais tóxicos do que o composto original. A biotransformação do diclofenaco foi investigada simultaneamente com a remoção biológica do fósforo, onde a via metabólica e a toxicidade dos subprodutos do diclofenaco também foram avaliadas. Embora a Accumulibacter não pareça biotransformar o diclofenaco tão eficientemente quanto outras bactérias (por exemplo, nitrificantes), ainda assim contribui para a sua desintoxicação ao produzir subprodutos de menor toxicidade do que o próprio diclofenaco. Além disso, foi observada uma correlação entre a quantidade de biotransformação de diclofenaco e o tipo de Accumulibacter, o que pode sugerir que o enriquecimento de Accumulibacter do tipo II pode estimular a biodegradação do diclofenaco no tratamento de águas residuais. Por fim, a recuperação de recursos é uma das principais iniciativas na área de tratamento de águas residuais. As estações de tratamento de águas residuais não são apenas vistas como estações de tratamento, apresentando também oportunidades de recuperação de outros produtos de valor acrescentado e de soluções mais sustentáveis. O fósforo é um nutriente insubstituível e um recurso escasso que deve ser recuperado de correntes de resíduos ricos em fósforo. A acidificação biológica foi testada neste estudo como uma etapa adicional a incluir em estações de tratamento de águas residuais convencionais e demonstrou ter o potencial de solubilizar até 80 % do fósforo e produzir 0,35 g CODVFA/g VS, que poderá ser usado em processos subsequentes para a produção de fertilizantes ricos em fósforo ou bioplásticos, respetivamente.

Polyhydroxyalkanoates (PHA) are considered a promising alternative to oil-based plas- tics. They are naturally occurring polyesters, biocompatible and biodegradable into CO2 and H2O. Nowadays, PHA are produced through a costly process using single cultures at full-scale, which has prevented the widespread use of this plastics for cheaper applications. An alternative bioprocess with mixed microbial cultures (MMC) has been considered as an alternative approach using wastes as feedstock. This process generally consists of acido- genic fermentation of a waste, selection of a PHA-accumulating culture and PHA accumulation. This thesis has mostly focused in the last two steps, particularly on overcoming the complexity of fermented wastes by valorizing them into PHA with a focus pilot-scale experiments. In the first experimental study, the typical 3-stage process was setup at pilot scale. A caproate-rich fermentate was produced from fruit waste and used in the subsequent stages to produce a 3-hydroxyhexanoate-rich PHA, containing up to 66% (wt.) of this medium chain length (mcl) monomer. The metabolism of caproate was further elucidated and a metabolic pathway to produce this mcl monomer was proposed. A second study was carried out using a nitrogen-rich fermented food waste as substrate for bench-scale and pilot-scale PHA processes, which resulted in the production of a 3-hy- droxyoctanoate-containing tetra-polymer. A mass balance was performed to understand the fermentation products' metabolism and a projection to latent structures (PLS) model was cre- ated to predict PHA production. In the third study, ammonia was stripped out from fermented sewage sludge and fed to a bench-scale selection reactor during the famine phase as nitrogen source in a partially un- coupled carbon and nitrogen feeding strategy. This additional step resulted in an increase in 77% of productivity compared to similar operating conditions without nitrogen removal be- forehand. The impact of using a nitrogen-rich fermented food waste as nitrogen source in the fam- ine phase, while a fermented fruit waste was used as carbon source in the feast phase was evaluated in the fourth study. Although a slight decrease in PHA content and productivity was observed, the change in operating conditions allowed the use of a low-cost waste instead of a supplementation of nutrients. This Thesis demonstrates how to overcome process challenges through process adapta- tions to produce an added-value product from highly abundant wastes. Polihidroxialcanoatos (PHA) são considerados uma alternativa promissora aos plásticos derivados do petróleo. Os PHA são poliésteres naturais, biocompatíveis e biodegradáveis em CO2 e H2O. Hoje em dia, os PHA são produzidos através de um processo caro usando culturas puras à escala industrial, o que tem impedido um uso mais abrangente destes plásticos em aplicações de baixo custo. Um bioprocesso alternativo usando culturas microbianas mistas (MMC) tem sido consi- derado com alternativa usando resíduos como substratos. Este processo consiste geralmente na fermentação acidogénica do resíduo, seleção da cultura bacteriana acumuladora de PHA e a produção de PHA. Esta tese foca-se nos últimos dois passos, particularmente em ultrapassar as barreiras que os resíduos fermentados possuem, valorizando-os a PHA em experiências à escala piloto. No primeiro estudo experimental, o processo convencional de três passos foi implemen- tado à escala piloto. Um efluente rico em caproato foi produzido a partir de resíduo de fruta e usado nos passos subsequentes para produzir um PHA rico em 3-hidroxihexanoato, contendo até 66% (wt.) deste monómero de cadeia média (mcl). O metabolismo do caproato foi eluci- dado e a via metabólica da produção deste monómero mcl foi proposta. Um segundo estudo foi conduzido usando resíduo de comida rico em azoto como subs- trato em processos de escala laboratorial e piloto e que resultou na produção de um tetra polímero de PHA que continha 3-hidroxioctanoato. Um balanço de massa foi feito de forma a compreender o metabolismo dos produtos de fermentação e um criou-se um modelo de pro- jeção das estruturas latentes (PLS) para prever a produção de PHA. No terceiro estudo, a amónia foi removida de lamas de esgoto fermentadas e alimentada durante a fase de fome como fonte de azoto numa estratégia de alimentação de carbono e azoto desacoplada. Este passo adicional resultou num aumento de produtividade em 77% comparativamente a condições de operação similares sem o passo de remoção de azoto. O impacto do uso de um resíduo de comida rico em azoto como fonte de azoto na fase de fome, enquanto um resíduo de fruta fermentada foi usado como fonte de carbono na fase de fartura foi estudado no quarto estudo. Apesar do pequeno decréscimo no conteúdo final de PHA e produtividade, a mudança de condições de operação permitiu o uso de um resíduo fermentado de baixo custo como alternativa à suplementação de nutrientes. Esta tese demonstra como ultrapassar desafios de processo através de adaptações ao processo tradicional de forma a produzir um produto de valor acrescimento a partir de resí- duos abundantes.

The arising of novel bio-based products and processes is vital for the sustainable development of society. The exploitation of marine resources towards the development of new ecological products with commercial interest is a promising solution. In this thesis, marine biodiversity was explored for the development of new and improved biomaterials and/or bioactive compounds based on marine exopolysaccharides (EPS). Marine microorganisms, including both microalgae and bacteria, were prospected for their EPS production capacity. The results obtained in this study showed that marine microalgae have an enormous potential as producers of EPS with unique chemical compositions, including high contents in sulphate and unusual sugars. These characteristics are often associated with biological activities, which supports the potential of microalgal EPS to be employed as bioactive compounds. Indeed, in this work, the EPS produced by microalga Heterocapsa AC210 demonstrated antioxidant and anti-inflammatory properties. Additionally, the EPS produced by bacteria isolated from unusual marine environments showed interesting compositions, including high uronic acids contents, and relevant functional properties. These biopolymers presented thickening, gel and film forming capacities, suggesting that they might be successfully used as structuring biomaterials. This work showed that it was possible to use low-cost feedstocks for the cultivation of the bacterium Alteromonas macleodii Mo169, resulting in higher productivities and distinctive EPS compositions. The EPS produced by this strain also revealed potential in the nanotechnology field, as it might be used for the ecological synthesis of gold, silver, and selenium nanoparticles with wound healing and antioxidant properties. Overall, this work supports that natural EPS bioprospected from marine microorganisms can be used for the development of bio-based products with application in high-value markets, thus, contributing to a sustainable future powered by marine biotechnology. O aparecimento de novos produtos e processos de origem biológica é vital para o desenvolvimento sustentável da sociedade. Uma solução promissora é a exploração dos recursos marinhos para o desenvolvimento de novos produtos ecológicos com interesse comercial. Esta tese explora a biodiversidade marinha com o objetivo de desenvolver novos biomateriais e/ou compostos bioactivos a partir de exopolissacáridos (EPS) marinhos. A capacidade de produzir EPS foi avaliada para vários microrganismos marinhos, incluindo microalgas e bactérias. Os resultados obtidos neste trabalho demonstraram que as microalgas marinhas têm um enorme potencial na produção de EPS com composições químicas únicas, que incluem um elevado conteúdo em sulfato e monossacáridos invulgares. Estas características estão frequentemente associadas a atividades biológicas, reforçando a ideia de que os EPS das microalgas têm potencial para serem utilizados como compostos bioactivos. De facto, neste trabalho, o EPS produzido pela microalga Heterocapsa AC210 demonstrou ter propriedades antioxidantes e anti-inflamatórias. Além disso, os EPS produzidos por bactérias isoladas de ambientes marinhos incomuns apresentaram composições interessantes, incluindo altos teores de ácidos urónicos, e propriedades funcionais relevantes. Estes biopolímeros mostraram ter capacidade espessante, bem como a capacidade de formar géis e filmes, o que sugere que possam ser utilizados como biomateriais estruturais. Este trabalho demonstrou ser possível utilizar substratos de baixo custo no cultivo da bactéria Alteromonas macleodii Mo169, o que resultou em produtividades superiores e EPS com composições distintas. O EPS produzido por esta cultura também revelou ter potencial na área da nanotecnologia, dado que pode ser utilizado na síntese ecológica de nanopartículas de ouro, prata e selénio com propriedades regenerantes e antioxidantes. No geral, este trabalho apoia a convicção de que os EPS naturais obtidos de microrganismos marinhos podem ser utilizados para o desenvolvimento de bioprodutos com aplicações em mercados de elevado valor comercial, contribuindo assim para um futuro sustentável baseado na biotecnologia marinha.

Enhanced biological phosphorus removal (EBPR) facilities achieve low effluent phosphorus (P) levels (below 1 g P.m-3) for long periods of time. However, these facilities are often affected by unpredictable upsets that increase their operational costs and reduce the potential to recover P from downstream processes. Thereby, these facilities need to have access to reliable tools, capable of dynamically predicting EBPR performance and diagnosing plant upsets. To address this need, a novel integrated metabolic activated sludge model, the META-ASM, was developed with a robust single set of default parameters to describe the activity of the key organisms and processes relevant to EBPR systems. The advances regarding EBPR mechanisms investigated over the last twenty years were integrated in the META-ASM model to overcome various shortcomings of existing EBPR models. Special attention is given to the effect of operational conditions on the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), along with the capability of PAOs and GAOs to denitrify, the metabolic shifts as a function of storage polymer concentration for each group, the role of these polymers in endogenous processes, and a better description of the fermentation process. The model was calibrated and validated against 34 data sets describing different EBPR dynamics obtained from bench-scale batch tests inoculated with lab-scale enriched PAO-GAO cultures and full- scale sludge from different EBPR facilities. The overall strong correlations obtained between the predicted and the measured EBPR profiles demonstrated that this new model reduces calibration efforts and is capable of predicting the microbial and chemical transformations over a wide range of operational and environmental conditions, supporting the robustness of the unique default parameter set that was generated. A performance comparison between META-ASM and literature models also demonstrated that existing models require extensive parameter changes and have limited predictive power to describe different EBPR dynamics. The capacity of the META-ASM model to describe the long-term performance of a full-scale 3- stage Phoredox (A2/O) EBPR system and to be used as an operational diagnostic tool was evaluated in a 1336-day long-term dynamic simulation, while its performance was compared with the ASM-inCTRL model, a version based on the Barker & Dold model. Overall, the META-ASM provided a better description of PAOs active biomass and storage polymers and was a more powerful operational diagnostic tool for plant upsets. Viable troubleshooting scenarios were simulated to mitigate the upsets caused by the high aerobic hydraulic retention times (HRTs) and low organic loading rates (OLRs) of the plant. This thesis demonstrates that the META-ASM model is a powerful operational diagnostic tool for EBPR systems, capable of predicting plant upsets, optimising performance and evaluating new process designs.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics with potential for fossil-based plastics’ replacement, that can be produced by mixed microbial cultures (MMC) from wastes. In this thesis, the possibility of PHAs synthesis from pulp and paper wastes, for the production of films and flowerpots for plants transport, was assessed. The PHAs production by MMC was carried out in a 3-stage process. After selection of the residues able to produce volatile fatty acids, the acidogenic fermentation stage consisted in their fermentation in reactor under different operating conditions. In the PHAs accumulating MMC enrichment stage, a microbial community with good PHAs storing capacity was selected through the use of a feast and famine regime, being also analysed by fluorescence in situ hybridization (FISH). Finally, in the accumulation stage the enriched MMC was fed with fermented wastes from stage 1, aiming to reach its maximum PHAs accumulation capacity. The produced polymer was extracted and evaluated for use in films and flowerpots, together with two others produced by Bioeng group, with different monomers content and purity.

Food wastes are essentially the organic material discarded, lost or degraded during the process of food processing or consumption and represent almost a third of all food produced. The present work aims to optimise the valorisation of food wastes in order to produce bioplastics which is an interesting way to valorise this waste. The polyhydroxyalkanoates (PHA) are biopolymers naturally produced by microorganisms that is considered an important alternative to conventional fossil-based plastics since they have similar properties. However, PHA production costs still high, mainly due to the use of pure cultures and costly substrates. The use of mixed microbial cultures and low-cost renewable feedstocks contribute to lower the PHA production costs. This process is divided in three steps: substrate acidogenic fermentation, culture selection and polymer accumulation. Acidogenic fermentation convert the food waste into volatile fatty acids that are used as substrate for the selection phase. Here, PHA accumulating bacteria are cultivated under selective conditions and accumulation is used for those selected bacteria producing the maximum PHA possible. The following work focus on the first step, i.e. studying the impact of optimal operation conditions in terms of pH and dissolved hydrogen concentrations at acidogenic stage on fermentation products profiles. The variation of those profiles induces changes in the final bioplastic which ultimately could lead to new strategies to control the process in order of targeting a bioplastic with specific physical/thermal characteristic. Os resíduos alimentares são essencialmente o material orgânico descartado, perdido ou degradado durante o processo de produção ou consumo de alimentos e representa quase um terço de todos os alimentos produzidos. O presente trabalho tem como objetivo otimizar a valorização de resíduos alimentares, a fim de produzir bioplásticos, sendo esta uma interessante forma de valorizar esses resíduos. Os poli-hidroxialcanoatos (PHA) são biopolímeros produzidos naturalmente por microrganismos e são considerados uma alternativa importante aos plásticos convencionais de origem fóssil, pois possuem propriedades semelhantes. No entanto, os custos de produção de PHA ainda são altos, principalmente devido ao uso de culturas puras e substratos caros. O uso de culturas microbianas mistas e matérias-primas renováveis de baixo custo contribui para reduzir os custos de produção de PHA. Esse processo é dividido em três etapas: fermentação acidogénea do substrato, seleção da cultura e acumulação de polímero. A fermentação acidogénea converte os resíduos alimentares em ácidos gordos voláteis que são usados como substrato na fase de seleção da cultura. Aqui, as bactérias acumuladoras de PHA são produzidas em condições seletivas e a acumulação é usada para as bactérias selecionadas para produzir o máximo de PHA possível. O presente trabalho é focado na primeira etapa estudando o impacto das condições ideais de operação em termos de pH e concentrações de hidrogénio dissolvido nos perfis de produtos de fermentação. A variação desses perfis induz mudanças no bioplástico final, o que pode levar a novas estratégias para controlar o processo, a fim de atingir um bioplástico com características físicas / térmicas específicas.

One of the major challenges of transdermal drug delivery is the creation of methods that allow greater bioavailability of drugs. Many techniques have been proposed in order to address this issue such as hypodermic injections and transdermal patches. However, all presented several disadvantages, such as the requirement of administration by trained people and poor bioavailability. Thus, microneedles have emerged as a promising alternative, since they are less invasive and a painless method for delivering drugs through the skin. In this dissertation, a low-cost procedure was proposed, without resorting to microfabrication techniques for the production of hollow microneedles on laser technology and 3D printing using biodegradable and biocompatible polymers such as commercially available poly(lactic acid) (PLA) and the poly(hydroxybutyrate-cohydroxyvalerate) (P(HB-co-HV)) extracted from biomass. Laser technology was used to carve specific patterns on the PDMS that served as a mould for the production of needles, while 3D printing was used to produce a counter-mould in order to create a hollow profile in the microneedle when it is pressed onto the assembled mould-molten polymer. The best condition for the production of the moulds was obtained with the spiral pattern, a power of 50 W and a speed of 0.051 m/s. The first step of the production was performed successfully, and the microneedles produced were characterized by SEM, XRD and mechanical compression tests while the biomass was submitted to SEM-EDS, FTIR and XRD analysis. To demonstrate the feasibility of the second production step, the geometry of the counter-mould was optimized, and a macroscale proof of concept was performed using an insulin syringe to produce the hollow needle profiles. At the end of this work, the performance of P(HB-co-HV) in the production of microneedles was satisfactory, leaving good indicators on the feasibility of its use in biomedical applications such as the one studied.

Petroleum-based plastics have massively contributed to a substantial increase in environmental pollution. Biobased plastics have emerged as a much-needed alternative solution to fossil dependent synthetic polymers. Polyhydroxyalkanoates (PHA) are a promising group of biobased and biodegradable displaying similar features to conventional plastics. Their high-cost production has become a hindrance to its economical applicability. Alternative low-cost processes of production can be achieved by using renewable raw materials (as feedstock) combined with the use of mixed microbial cultures (MMCs). Extensive research aiming the decrease of PHA production costs focused on the use of phototropic mixed cultures (PMC) enriched in purple bacteria is being developed (Fradinho et al. (2014)). The culture is selected under a feast and famine regime (FF) being able to reach up to 30% PHA / VSS using acetate as a carbon source in light/dark cycles. The permanent feast regime occurs as an alternative to the well-studied FF strategy and consists of a constant presence of carbon and the absence of electron acceptors. Ultimately, microorganisms such as purple bacteria will dissipate the reducing power (generated by the consumption of carbon source) and PHA will be formed. Using this strategy, Fradinho et al. (2016) obtained a 60% PHA/VSS content in a test with high light intensity. This work comes in the continuity of a previous study integrating a European project, NoAW, that aims for the valorisation of agro-industrial residues and for the production of valuable biobased products while exploring the phototrophic capacity of PMC under a permanent feast regime. PMC selection was done in an SBR under 24h cycles (12h of light and 12h dark) operating in a continuous feeding mode during the light phase. The culture was fed synthetic medium composed of a mixture of volatile fatty acids (VFAs) simulating fermented waste of cow manure and maize silage. During the reactor operation, several operating conditions were tested and the most promising results were obtained under the double limitation of phosphorus and sulfur. A decrease in the amount of algae population (phototrophic organisms that do not produce PHA) was observed which contributed to a higher percentage of PHA production. Indeed, the culture attained a PHA content of 31% PHA / VSS (25 Cmmol/L) in accumulation tests with optimized light intensity and acetate as carbon source. This highly attractive photosynthetic cultures’ ability opens new doors in its applicability in wastewater treatment plants (WWTP) that are under the direct illumination of sunlight. Wastewater could become a viable source of carbon for the production of valuable bioproducts, giving a highly desirable contribute to the implementation of a European circular economy.

Concrete is a strong, durable and cheap material. Its main problem is cracking, inducing to major problems like sulphate and chloride attacks, carbonation and decalcification. Repairing concrete can be very costly. The solution is the Self Healing Concrete. A sustainable, reliable, economical, in long term, and stable concept. Based on an aerobic spore-forming bacteria introduced in concrete and whenever there was a crack and water came in, the bacteria would start precipitating CaCO3. Concrete can be found also in oil wells. Due to high temperatures and pressures, suffers a lot of stresses and tensions. In Aarhus University, researchers decided to apply the Self Healing Cement in this anaerobic environment. A proper bacteria needed to be found to handle such conditions. Natranaerobaculum magadiense (N. magadiense) was the bacteria to be studied. The project is centred on the precipitation of CaCO3 induced by N. magadiense. The main goal of this work was to study the physiology of N. magadiense at different conditions. It was investigated its behaviour in terms of extreme conditions, since it is a polyextremophile. The project tests involved a reduction of carbonate (HCO –3 ) from the optimum media, that conflict with microbial CaCO3 precipitation. N. magadiense’s behaviour when the pH was changed. A carbon source replacement to measure the CO2=CO 2–3 production and extreme conditions were applied and the results were evaluated. We proved that N. magadiense cannot grow without carbonates. It was not found a good replacement carbon source. [HCO –3 ] cannot be reduced a lot, and [NaCl] cannot be a replacement compound, since [Cl– ] inhibited the cells, and it is not recommended to change the pH. The iron reduction process is more noticeable at lower temperatures and the fermentative process is at higher temperatures. In conclusion, N. magadiense is a good candidate to be used in the oil industry but more research is needed to move to its application.

Existem várias técnicas de referência para deteção precisa e segura de doenças e agentes patogénicos. No entanto estas técnicas ou requerem preparações de amostras com várias etapas, ou então de métodos de análise dependentes de equipamento de laboratório de custo elevado e de técnicos qualificados (que levem a cabo o procedimento). Quando as circunstâncias são de baixos recursos, como junto das populações que mais necessitam de meios de deteção de doenças, estas técnicas tornam-se impraticáveis. Desta forma, nas últimas duas décadas, investigação e desenvolvimentos no ramo da microfluídica trouxeram inovações na miniaturização, automação e redução dos custos dos métodos de referência dependentes de laboratórios, com o objetivo de produzir dispositivos descartáveis adequados a circunstâncias ambulatórias de baixos recursos – dispositivos de diagnóstico rápido. Neste trabalho foram desenvolvidas membranas microfluídicas para deteção colorimétrica de glucose a partir dos biopolímeros poli(3-hidroxibutirato) (P(3HB)) e poli(3-hidroxibutirato-co-valerato) (P(3HB-co-HV)), com as características de dispositivos de diagnóstico rápido e a vantagem de serem biodegradáveis. A fabricação destas membranas baseou-se no uso dos biopolímeros para criação de estruturas porosas capazes de transportar líquidos por ação capilar, isto é, sem o recurso a forças exteriores. Testaram-se diferentes metodologias no processo de fabricação, obtendo-se os resultados mais promissores com a técnica de lixiviação com sal, aliada a uma exposição a plasma de oxigénio, como forma de tornar os biopolímeros, - inerentemente hidrofóbicos - em matrizes porosas microfluídicas. Utilizando as membranas produzidas como substrato, alcançou-se a produção de um biossensor colorimétrico de glucose em soluções aquosas, baseado em reações enzimáticas.

One of the most important issues in creating new drug delivery methods is improving drug permeation into the skin. Therefore, many techniques have been proposed, such as oral administration, intradermal vaccines, transdermal patches, among others, but all of them present several limitations. In the past few years, a new effective, innovative and safe drug delivery system was proposed. This technology is named as microneedles (MNs) and it is a hybrid combination of hypodermic injections and transdermal drug delivery systems, which consists in micro-scale needles that can pierce the skin by a simple, minimally invasive and painless route, enabling to transport drugs and macromolecules into the human body. This dissertation reports the development of a biopolymer-based microneedle patch, using biodegradable and biocompatible polymers, polylactic acid (PLA) and poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)), through a low-cost and maskless laser technology. The laser technology was used to engrave specific patterns on a substrate that served as mold for the MNs production. The best results were obtained with a laser power of 30 W at 0.15 m/s, with the spiral model as pattern. The respective MNs had a length of 0.69 mm and a diameter of 0.33 mm, ideal for painless penetration of skin. Moreover, P(HB-co-HV) demonstrated a higher mechanical stability than commercial PLA, confirming its promising use as MNs biomaterial.

Antibiotics are intensively used in medical therapy, veterinary medicine, and the farming indus-try, resulting in continuous releases of the compounds into WWTPs and fresh water resources. Since conventional WWTPs are not effective to completely remove antibiotics, there are growing concerns related to the influence of its mode of action to microbial communities, as well as the risk to human health, by promoting and spreading antibiotic resistance. The aim of this research is to develop an ef-fective and practical bio-augmentation strategy for the removal of the antibiotic sulfamethoxazole SMX - one of the most frequently detected synthetic sulfonamide antibiotics in wastewater. A strain identified as Achromobacter denitrificans PR1, previously isolated from activated sludge (AS), was found as a potential organism for bio-augmentation for SMX removal in polluted waters. The SMX degradation kinetics of this organism are stimulated in the presence of biogenic substrates, e.g. ace-tate/succinate, and are two to three orders of magnitude higher than the degradation kinetics of activat-ed sludge at environmentally relevant concentrations. Bioaugmentation of AS with the strain PR1 (batch experiments) led to superior biotransformation rates of SMX (by sixty times) compared to AS, within a complex carbon environment in WWTPs. Biological degradation models were calibrated to describe accurately the fates of sulfamethoxazole and the two human metabolites, e.g. N4-acetyl-SMX and SMX-N1-Glucuronide, in the systems, under various redox conditions. The strain was subsequently bioaugmented into membrane bioreactors operated under aerobic conditions, which led to the enhancement and stabilization of the SMX removal, especially when SMX shock loads occurred. Changing hydraulic retention times, and thus the availability of primary substrates, was found to affect the cometabolism of SMX by the bioaugmented strain in activated sludge. After the bioaugmentation, the loss of viability of the introduced strains was observed and re-inoculation of the degrading strain seems to be a logical solution to maintain removal efficiency of the target compound.

Polyhydroxyalkanoates (PHA), are a biodegradable polymer, naturally synthesized by several bacteria and with similar characteristics to conventional plastics. Recently, phototrophic PHA production has also been achieved in photosynthetic mixed cultures (PMCs) under an open, non-aerated and using direct sunlight system, lowering the operational costs commonly associated to the PHA production by pure and aerated mixed cultures. The photo-bioreactors (PBR) are also commonly used as wastewater (WW) treatment systems along with a consortium microalgae and bacteria communities. The main goals of this thesis were precisely to select a PHA accumulating PMC to achieve a new added-value bioproduct. The enrichment process of the PMC was performed by two different selection strategies - permanent feast regime and feast and famine regime - that were carried out under the same operational conditions of High Rate Algae Ponds (HRAPs) from Chiclana Wastewater Treatment Plant, using artificial illumination and fed with real wastewaters, establishing the effect of using wastewater on the culture growth and PHA production stability, and furthermore to evaluate the impact of operational conditions (OLR, pH, temperature and light intensity) on the PHA production performance of the system. This will be the first time that the PHA production through PMC will be tested at demonstration scale and using real wastewater.

Polyhydroxyalkanoates (PHA) production using mixed microbial cultures (MMC) requires a multi-stage process involving the microbial selection of PHA-storing microorganisms, typically operated in sequencing batch reactors (SBR), and an accumulation reactor. Since low-cost renewable feedstocks used as process feedstock are often nitrogen-deficient, nutrient supply in the selection stage is required to allow for microbial growth. In this context, the possibility to uncouple nitrogen supply from carbon feeding within the SBR cycle has been investigated in this study. Moreover, three different COD:N ratios (100:3.79, 100:3.03 and 100:2.43) were tested in three different runs which also allowed the study of COD:N ratio on the SBR performance. For each run, a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5 gCOD L-1 d-1 was used as carbon feedstock, whereas ammonium sulfate was the nitrogen source in a lab-scale sequence batch reactor (SBR) with 1 L of working volume. Besides, a sludge retention time (SRT) of 1 d was used as well as a 6 h cycle length. The uncoupled feeding strategy significantly enhanced the selective pressure towards PHA-storing microorganisms, resulting in a two-fold increase in the PHA production (up to about 1.3 gCOD L-1). A high storage response was observed for the two runs with the COD:N ratios (gCOD:gN) of 100:3.79 and 100:3.03, whereas the lowest investigated nitrogen load resulted in very poor performance in terms of polymer production. In fact, strong nitrogen limitation caused fungi to grow and a very poor storage ability by microorganisms that thrived in those conditions. The COD:N ratio also affected the polymer composition, indeed the produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) showed a variable HV content (1-20 %, w/w) among the three runs, lessening as the COD:N increased. This clearly suggests the possibility to use the COD:N ratio as a tool for tuning polymer properties regardless the composition of the feedstock.

Single cell protein (SCP) defines the dried cells of microorganisms that can be used as a protein supplement both in animal feed or human food. SCP could represent an effective alterna-tive to meet the high and growing demand of feed products. This work aimed to develop a simple and economically feasible process to obtain SCP by adding value to an industrial waste stream. Spent sulfite liquor (SSL), pure glycerol and crude glycerol and two different types of wood oils were selected. However, the wood oils were early discharged (after characterization) as possible substrates due to their low chemical oxygen de-mand (COD) content and an unpleasant and undesirable smell. Thus, only SSL and pure and crude glycerol were tested. Crude glycerol proved to be the most viable choice given the high COD content – - g O2/L – and the low price per ton of COD – 200 €/ton COD. In continuous mode it was possible to achieve biomass concentrations of - g CDW/L (- g CDW/g COD) while efficiently removing - % of the COD of the influent stream. When tested in batch mode, biomass concentration reached a maximum of - g CDW/L (- g CDW/g COD) after - days of operation. After - days of operation, CDW per liter started to decrease which could indicate some sort of inhibition by fermentation by-products or other toxic compounds. Considering a batch operation mode, it was possible to produce SCP (- %w/w of protein content) with a commercial price of - €/ton protein. Thus, SCP production was considered feasible since it was possible to obtain a product that can highly compete with other feed products, for example, fishmeal (market price: 1.940 €/ton protein).

Assegurar a qualidade de um produto farmacêutico implica garantir a conformidade de todas as etapas, ao longo de todo o seu ciclo de vida, desde a aquisição das matérias-primas até à libertação do produto acabado, assegurando a validação dos equipamentos, instalações e processos. Quando uma indústria farmacêutica assegura a conformidade de todos os passos envolventes (e que possam influenciar o processo de fabrico) é capaz de demonstrar, perante entidades responsáveis e clientes, que os seus produtos apresentam a qualidade pré-estabelecida na autorização de introdução no mercado (AIM) e que, consequentemente, irão ter o desempenho pretendido. Esta dissertação insere-se na garantia da qualidade dos Laboratórios Atral, do grupo AtralCipan, mais propriamente na qualificação de equipamentos no setor Formas Sólidas Orais Cefalosporínicas (FSO3) por forma a assegurar a qualidade dos produtos acabados produzidos. O objetivo deste trabalho é a qualificação dos principais equipamentos existentes no setor FSO3 por forma a assegurar a qualidade dos medicamentos lá fabricados. Para a qualificação do desempenho dos equipamentos (à exceção do tamisador, compactador e detetor de metais) foram utilizados dados históricos presentes nos registos de lotes dos principais produtos do setor por forma a efetuar uma avaliação retrospetiva. Para isso efetuou-se uma análise de risco FMEA (análise do modo de falha e consequência), aos equipamentos existentes no setor, com o objetivo de estabelecer os parâmetros dos equipamentos que pudessem influenciar negativamente a qualidade do produto final. À exceção da qualificação do desempenho da máquina de blisterar 308 PBL3, uma vez que faltavam alguns dados de lotes que ainda não tinham sido analisados pelo setor de Controlo da Qualidade, os principais equipamentos do FSO3 encontram-se atualmente qualificados. A conclusão da qualificação dos equipamentos presentes no setor FSO3, bem como da análise de risco efetuada irá contribuir para um melhoramento da qualidade e da credibilidade do setor perante clientes e entidades responsáveis.

Enhanced biological phosphorus removal (EBPR) is the most economic and sustainable option used in wastewater treatment plants (WWTPs) for phosphorus removal. In this process it is important to control the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), since EBPR deterioration or failure can be related with the proliferation of GAOs over PAOs. This thesis is focused on the effect of operational conditions (volatile fatty acid (VFA) composition, dissolved oxygen (DO) concentration and organic carbon loading) on PAO and GAO metabolism. The knowledge about the effect of these operational conditions on EBPR metabolism is very important, since they represent key factors that impact WWTPs performance and sustainability. Substrate competition between the anaerobic uptake of acetate and propionate (the main VFAs present in WWTPs) was shown in this work to be a relevant factor affecting PAO metabolism, and a metabolic model was developed that successfully describes this effect. Interestingly, the aerobic metabolism of PAOs was not affected by different VFA compositions, since the aerobic kinetic parameters for phosphorus uptake, polyhydroxyalkanoates (PHAs) degradation and glycogen production were relatively independent of acetate or propionate concentration. This is very relevant for WWTPs, since it will simplify the calibration procedure for metabolic models, facilitating their use for full-scale systems. The DO concentration and aerobic hydraulic retention time (HRT) affected the PAO-GAO competition, where low DO levels or lower aerobic HRT was more favourable for PAOs than GAOs. Indeed, the oxygen affinity coefficient was significantly higher for GAOs than PAOs, showing that PAOs were far superior at scavenging for the often limited oxygen levels in WWTPs. The operation of WWTPs with low aeration is of high importance for full-scale systems, since it decreases the energetic costs and can potentially improve WWTP sustainability. Extended periods of low organic carbon load, which are the most common conditions that exist in full-scale WWTPs, also had an impact on PAO and GAO activity. GAOs exhibited a substantially higher biomass decay rate as compared to PAOs under these conditions, which revealed a higher survival capacity for PAOs, representing an advantage for PAOs in EBPR processes. This superior survival capacity of PAOs under conditions more closely resembling a full-scale environment was linked with their ability to maintain a residual level of PHA reserves for longer than GAOs, providing them with an effective energy source for aerobic maintenance processes. Overall, this work shows that each of these key operational conditions play an important role in the PAO-GAO competition and should be considered in WWTP models in order to improve EBPR processes.

Water is a limited resource for which demand is growing. Contaminated water from inadequate wastewater treatment provides one of the greatest health challenges as it restricts development and increases poverty in emerging and developing countries. Therefore, the connection between wastewater and human health is linked to access to sanitation and to human waste disposal. Adequate sanitation is expected to create a barrier between disposed human excreta and sources of drinking water. Different approaches to wastewater management are required for different geographical regions and different stages of economic governance depending on the capacity to manage wastewater. Effective wastewater management can contribute to overcome the challenges of water scarcity. Separate collection of human urine at its source is one promising approach that strongly reduces the economic and load demands on wastewater treatment plants (WWTP). Treatment of source-separated urine appears as a sanitation system that is affordable, produces a valuable fertiliser, reduces pollution of water resources and promotes health. However, the technical realisation of urine separation still faces challenges. Biological hydrolysis of urea causes a strong increase of ammonia and pH. Under these conditions ammonia volatilises which can cause odour problems and significant nitrogen losses. The above problems can be avoided by urine stabilisation. Biological nitrification is a suitable process for stabilisation of urine. Urine is a highly concentrated nutrient solution which can lead to strong inhibition effects during bacterial nitrification. This can further lead to process instabilities. The major cause of instability is accumulation of the inhibitory intermediate compound nitrite, which could lead to process breakdown. Enhanced on-line nitrite monitoring can be applied in biological source-separated urine nitrification reactors as a sustainable and efficient way to improve the reactor performance, avoiding reactor failures and eventual loss of biological activity. Spectrophotometry appears as a promising candidate for the development and application of on-line nitrite monitoring. Spectroscopic methods together with chemometrics are presented in this work as a powerful tool for estimation of nitrite concentrations. Principal component regression (PCR) is applied for the estimation of nitrite concentrations using an immersible UV sensor and off-line spectra acquisition. The effect of particles and the effect of saturation, respectively, on the UV absorbance spectra are investigated. The analysis allows to conclude that (i) saturation has a substantial effect on nitrite estimation; (ii) particles appear to have less impact on nitrite estimation. In addition, improper mixing together with instabilities in the urine nitrification process appears to significantly reduce the performance of the estimation model.

Dissertation to obtain the degree of Master in Chemical and Biochemical Engineering The work present in this thesis was conducted in Portucel Soporcel mill, in the industrial complex of Setúbal, and had as main objective the modelling of the treatment process of the effluents from this industry, using for this purpose the software GPS-X. This program has a clear-cut graphical interface and uses a specialized translator that converts the graphical process into material balance equations, based on dynamic models. These models allow, besides the kinetic descripton of the treatment process carried out at the WWTP, to simulate new scenarios towards the study of critical parameters for the process as well as optimization and control of the WWTP. The effluent that arrives to Portucel’s WWTP, from the pulp and paper mills of the complex, is particularly rich on fibers (solids), lignin, chlorinated and sulphur compounds, resin acids, phenols and starch. It has a brown colour due to the presence of lignin and has a high oxygen chemical demand (about 1,095 g O2/m3). The WWTP uses the activated sludge process with extended aeration. This method allows an efficient removal of organics at the same time as it minimizes the sludge production. For the modelling of the process it was necessary to collect historical data related to the WWTP’s performance over the last 3 years. This data was used as input values for the influent characterisation and as output values to achieve the treated effluent characterisation. Since the first simulation did not lead to the desired output results, it was necessary to proceed to the model calibration, by means of a more detailed study concerning the nutrient and organic fractions of the influent. Once the model was calibrated, a study of the urea flowrate was conducted. The urea is added to the influent, before the beginning of the biological oxidation, as a way to satisfy the nitrogen requirements along the treatment process. However, this flowrate was never submitted to a study that evaluated, in a higher detail, the effective requirements of this nutrient. Thus, some simulations were done using the software, by decreasing successively the value of the urea flowrate and the results obtained were analyzed. Furthermore, these simulations were validated in the WWTP itself, at Portucel, through the decrease of the urea flowrate to half the normal value. Both the simulations and Portucel’s results showed that, actually, the addition of urea is not necessary because it does not affect the treatment process in a significant way, namely in terms of the removal of chemical oxygen demand. The simulations have also showed that the concentration of nitrogen in the final effluent diminishes significantly with the reduction of the urea flowrate, which could be advantageous in an environmental point of view.

Polyhydroxyalkanoates (PHAs) are natural biologically synthesized polymers that have been the subject of much interest in the last decades due to their biodegradability. Thus far, its microbial production is associated with high operational costs, which increases PHA prices and limits its marketability. To address this situation, this thesis’ work proposes the utilization of photosynthetic mixed cultures (PMC) as a new PHA production system that may lead to a reduction in operational costs. In fact, the operational strategies developed in this work led to the selection of PHA accumulating PMCs that, unlike the traditional mixed microbial cultures, do not require aeration, thus permitting savings in this significant operational cost. In particular, the first PHA accumulating PMC tested in this work was selected under non-aerated illuminated conditions in a feast and famine regime, being obtained a consortium of bacteria and algae, where photosynthetic bacteria accumulated PHA during the feast phase and consumed it for growth during the famine phase, using the oxygen produced by algae. In this symbiotic system, a maximum PHA content of 20% cell dry weight (cdw) was reached, proving for the first time, the capacity of a PMC to accumulate PHA. During adaptation to dark/light alternating conditions, the culture decreased its algae content but maintained its viability, achieving a PHA content of 30% cdw. Also, the PMC was found to be able to utilize different volatile fatty acids for PHA production, accumulating up to 20% cdw of a PHA co-polymer composed of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (HV) monomers. Finally, a new selective approach for the enrichment of PMCs in PHA accumulating bacteria was tested. Instead of imposing a feast and famine regime, a permanent feast regime was used, thus selecting a PMC that was capable of simultaneously growing and accumulating PHA, being attained a maximum PHA content of 60% cdw, the highest value reported for a PMC thus far. The results presented in this thesis prospect the utilization of cheap, VFA-rich fermented wastes as substrates for PHA production, which combined with this new photosynthetic technology opens up the possibility for direct sunlight illumination, leading to a more cost-effective and environmentally sustainable PHA production process.

Dissertation for the Degree of Master in Biotechnology The yeast Pichia pastoris produces chitin-glucan complex (CGC), as a cell wall component. CGC is composed of two types of biopolymers, chitin and β-glucans, which confer it great potential for use in the food, cosmetic and pharmaceutical industries. CGC hydrolysis, allows obtaining chitin/chitosan and glucans individually. The chitin and chitosan obtained from CGC have the considerable advantage of being of non-animal origin, which further extends their applications. In last year’s, the production of CGC by Pichia pastoris was realized with glycerol as sole carbon source, achieving high cell density. In this study, different substrates were tested for cultivation of P. pastoris and CGC production. In the first part, mixtures of glucose and xylose, in varying proportions, were tested. Since glucose and xylose are two of the main sugar components of lignocellulosic wastes, the ability of P. pastoris to use them as carbon sources would allow their valorization into value-added products. In the second part, several wastes and byproducts generated by different industries were tested for their suitability as substrates for P. pastoris cultivation. In study of the glucose/xylose mixtures, the best performance was achieved in batch bioreactor experiments with 25% xylose in the medium (20 gL-1 of xylose and 60 gL-1 of glucose), where 35.25 gL-1 biomass was obtained in 64 hours of cultivation. CGC content in the cell wall reached 15% with a volumetric productivity of 0.085 gL-1.h-1. The molar ratio of chitin:β-glucan in the extracted biopolymer was 47:53, higher than obtained with crude glycerol (16:84). In the second part of study, several wastes and byproducts (used cooking oil, sugarcane molasses, cheese whey, waste paper and spent coffee grounds) were tested. The results show that P. pastoris presented low biomass concentration using any of these substrates. Nevertheless, in batch bioreactor experiments the best results were achieved with sugarcane molasses, where 17.78 gL-1 biomass were obtained with a CGC content of 17%. Among the tested substrates, the mixtures of glucose/xylose appear to be the most promising due the good CGC production obtained and the high glucosamine molar fraction in produced polymer. This study opens the hypothesis of utilization of lignocellulosic materials with xylose percentages up to 50%.

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica The combined use of mixed microbial cultures (MMC) and fermented feedstock as substrate may significantly decrease polyhydroxyalkanoates (PHA) production costs and make them more competitive in relation to conventional petroleum-based polymers. However, there still exists a lack of knowledge at metabolic level that limits the development of strategies to make this process more effective. In this thesis, system biology computational tools were developed and applied to PHA production by MMC from fermented sugar cane molasses, rich in volatile fatty acids (VFA). Firstly, a metabolic network able to describe the uptake of complex mixtures of VFA and PHA production was defined. This metabolic network was applied to metabolic flux analysis (MFA) to describe substrate uptake and PHA production fluxes over the enrichment time of a culture submitted to the feast and famine regimen. Then, the minimization of the tricarboxylic acid cycle (TCA) fluxes was identified as the key metabolic objective of a MMC subjected to this regimen by flux balance analysis (FBA). This model enabled to predict, with an acceptable accuracy, the PHA fluxes and biopolymer composition. Subsequently, data gathered from microautoradiography-fluorescence in situ hybridization (MAR-FISH) was used to develop a segregated FBA model able to predict the flux distribution for the three populations identified in the enriched culture. These results were slightly better than those obtained by the non-segregated FBA and were consistent with MFA results. Finally, a dynamic metabolic model was proposed based on the previous models and on a regulatory factor for VFA uptake and PHA production. This model allowed to identify the dynamics of the process and regulatory factor as well as to validate the previous results. Globally, this thesis enabled to demonstrate the potential of using computational tools to understand and optimize PHA production by MMC.

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Dissertação para obtenção do Grau de Mestre em Biotecnologia FucoPol is a fucose-containing biopolymer produced by the bacterium Gram-negative, Enterobacter A47 DSM 23139. It is an exopolysaccharide (EPS) composed of neutral sugars (fucose, galactose, glucose), an acidic sugar (glucuronic acid), and also non-saccharide substituents (acetate, pyruvate and succinate). In this work, two primary objectives were considered: the optimization of EPS production by varying two different parameters, and the functional properties’ assessment of different EPS produced by the bacterium. In the optimization study, the influence of different dissolved oxygen concentrations (controlled at 10, 30 and 60% of air saturation), and the impact of phosphate concentration reduction in the culture medium (5.25 and 3.81 g L-1) were assessed. The objective was to evaluate not only the effect of both parameters on cellular growth and exopolysaccharide-synthesis, but also to determine their influence in EPS chemical composition. The best results were obtained with DO at 10%, with the highest EPS production (6.11 g L-1), and although a reduction of phosphate concentration didn’t affect the EPS production, it reduced the exopolysaccharide’s fucose content to 24%mol. Secondly, the functional properties of nine distinct exopolysaccharides synthesized by Enterobacter A47 under different cultivation conditions were evaluated. Rheologically, most of the EPS polymer solutions showed shear-thinning behavior, wherein EPS-s, EPS-g and GNEX achieved the highest apparent viscosity (0.2 - 0.3 Pa.s) at lower shear rates (0.3 to 1 s-1). Also, most of the polymers presented emulsifying capacity at different temperatures. Concerning the film-forming capacity, EPS-s, EPS-g and EPS-x films demonstrated to have good mechanical properties and high water vapour permeability. And, all polymers described flocculating activity at 0.01% of EPS, which was significantly diminished at lower concentration, 0.001%.

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica Fundação para a Ciência e Tecnologia

Dissertação para obtenção do Grau de Doutor em Engenharia Química, especialidade de Engenharia Bioquímica The present work aimed at studying the treatment of drinking water supplies contaminated with perchlorate and nitrate, using the Ion Exchange Membrane Bioreactor (IEMB) concept. This system combines the transport of these two anions from contaminated water, through an anion exchange membrane, with their biological reduction in a separate compartment. In the IEMB, the mass transport is dependent not only from membrane properties but also from the biocompartment conditions. Multivariate statistical techniques allowed determining the most important process parameters related mainly to the compositions of the polluted water stream and biomedium and to the fluid dynamics operating conditions. The combination of statistical techniques with mechanistic modelling was a major achievement since the counterion transport across the membrane was successfully simulated and predicted under biological reactions ratelimiting conditions. Since nitrate is present in the contaminated water in much higher concentration than that of perchlorate, the IEMB process rate was mainly limited by the perchlorate bioreduction kinetics. This difference influenced organisation of microbial communities in the biofilm. This organization allows sequentially reduction of nitrate and perchlorate thus minimizing perchlorate inhibition by nitrate. Considering a possible large-scale application, it is essential to determine the effect of the key process variables. In this work, the performance of a plate and-frame module configuration, consisting of a series of anion-exchange membranes was investigated. It was found that contaminated water streams are effectively treated and that secondary contamination of treated water by the carbon source used was avoided by a start-up procedure involving a gradual increase of ethanol feeding to the IEMB biocompartment. Fundação para a Ciência e Tecnologia - SFRH/BD/25275/2005

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestrado em Engenharia Química e Bioquímica Os polihidroxialcanoatos (PHAs) são biopolímeros biodegradáveis produzidos a partir de fontes renováveis, constituindo uma alternativa promissora aos plásticos convencionais. No entanto, são produzidos por fermentação de culturas microbianas puras usando substratos de custo elevado. Nos últimos anos, têm vindo à desenvolver-se estratégias alternativas com vista a redução dos custos de produção, nomeadamente o uso de culturas microbianas mistas e substratos de baixo custo. Apesar do potencial demonstrado por estas culturas mistas, existem ainda algumas limitações. Esta tese tem como objectivo o aperfeiçoamento do processo de produção de PHAs por culturas mistas. A produção de PHAs por culturas mistas baseia-se no enriquecimento das culturas em organismos acumuladores de PHAs em condições de “Fome e Fartura” (FF). A cultura seleccionada é então utilizada para produção de PHAs. Foi estudado um processo em 3 etapas: (1) fermentação do melaço (conversão de açúcares em ácidos orgânicos voláteis – VFAs); (2) selecção da cultura em condições FF usando fermentado de melaço como fonte de carbono; (3) produção de PHAs usando a cultura seleccionada em (2) e o fermentado de melaço produzido em (1). Este estudo focou em particular a terceira etapa do processo com vista a aumentar a sua produtividade volumétrica e demonstrar a possibilidade de manipular a composição dos polímeros produzidos. Foram estudados os efeitos do regime de alimentação (contínuo e por pulsos) e do perfil de VFAs na composição do polímero. A alimentação em contínuo permitiu elevar a produtividade volumétrica até 1.2 gPHA /L.h, e ao mesmo tempo aumentar o conteúdo em hidroxivalerato em 11%. Os diferentes ensaios levaram à produção de co-polímeros de hidroxibutirato e hidroxivalerato (P(HB-co-HV))com conteúdo de HV numa gama de 15 a 39%.

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Química e Bioquímica Como é do conhecimento geral, as necessidades energéticas mundiais têm aumentado exponencialmente, as reservas de combustíveis fosseis têm vindo a diminuir e a sua combustão tem sérios efeitos nefastos ambientais, devido às emissões de CO2. Tendo tudo isto em consideração, o desenvolvimento de um mundo energeticamente sustentável requer a redução da dependência dos combustíveis fósseis e a diminuição da poluição por eles gerada. O biohidrogénio é considerado uma alternativa “limpa”, viável e um vector energético do futuro. Assim, com vista à sua utilização como biocombustível, a Directiva Europeia CE/30/2003 propõe uma incorporação de 8% de biocombustíveis, no sector dos transportes, até 2020. O principal objectivo deste estudo foi o desenvolvimento de um processo microbiológico anaeróbio de conversão do glicerol em biohidrogénio, com valorização simultânea deste subproduto da produção de biodiesel. Além disso, tendo em conta a forma reduzida do carbono no glicerol e o custo dos processos anaeróbios, o metabolismo fermentativo do glicerol tem especial interesse do ponto de vista de viabilidade económica. Neste trabalho, comparou-se a produção de H2 por uma estirpe da Enterobacter aerogenes, utilizando glicerol puro e glicerol contido nos resíduos da produção do biodiesel, como substrato. O estudo do efeito de parâmetros físico-químicos como, temperatura do processo e a concentração inicial de substrato e de biomassa, na produção de biohidrogénio, foi também levado a cabo pela metodologia do Desenho Composto Central para determinar as concentrações óptimas de substrato e de biomassa para o processo. O efeito da remoção da fase gasosa, durante a fermentação, foi também avaliado. Os resultados obtidos mostraram que a diminuição da temperatura de operação de 37 para 30ºC, conduziu a um aumento da taxa de biohidrogénio produzido e a uma redução no tempo de equilíbrio do processo. Além disso, foi também observado que a utilização de 10 g/L de glicerol puro ou de resíduos contendo a mesma concentração de glicerol, conduziu a valores de rendimento produção de H2 similares (cerca de 2,5 L H2/L meio de cultura), provando que o desempenho da estirpe de E. aerogenes utilizada,não foi influenciado pela presença de outros compostos presentes nos resíduos, que não o glicerol, pelo menos para a concentração de resíduos de biodiesel estudada. A remoção simultânea de gases formados, principalmente H2 e CO2, ao longo da sua produção, mostrou ser bastante eficiente conduzindo a um aumento do valor da razão volumétrica mL H2/mL CO2, podendo esse atingir 18 na headspace, 6 no saco de recolha e 7 no sistema global, o que é bastante promissor, tendo em conta os custos envolvidos nas tecnologias existentes para a purificação do hidrogénio contido na fase gasosa.

Dissertation presented in partial fulfillment of the requirements for the degree of Master in Biotechnology Polyhydroxyalkanoates (PHAs) are polyesters produced by a variety of microorganisms. Due to the similarity of chemical and physical properties with the conventional plastics, and full biodegradability, PHAs constitute one of the best alternatives for synthetic polymers replacement. However, the production costs of these biopolymers are very high when compared to synthetic polymers production. One way to reduce the production costs is the utilization of low cost raw materials, such as industrial wastes and by-products as carbon source. An example of raw material is cheese whey, a by-product form cheese industry rich in lactose (4-5%). In this work, cheese whey was supplied to Escherichia coli strains harbouring the PHB synthesis genes from Cupriavidus necator for the production of poly(3hydroxybutyrate) (PHB). During this study, diverse reactor operating strategies were tested: feeding controlled by pH under oxygen limitation, feeding without oxygen limitation and continuous feeding. The best results were achieved in a fed-batch system with feeding controlled by pH and oxygen limitation, where 44.93% of PHB content,33.76 g/L of PHB concentration, 78.65 g/L of active biomass concentration and a volumetric productivity of 0.57 gPHB/L.h, were obtained.

Bio-based and biodegradable polymers are regarded as potential replacements of traditional fossil based plastics. Polyhydroxyalkanoates (PHAs) are widely studied biopolymers due to their broad range of thermal and mechanical properties. The impacts of operational conditions, such as pH, temperature, organic loading rate and sludge retention time, in PHA production process by mixed microbial cultures (MMCs) have been evaluated by previous studies, but the influence of dissolved oxygen (DO), which is directly related to process energetic demand, was rarely discussed. This thesis is focused on the effects of DO on the microbial culture selection and PHA accumulation stages, as well as the impact of DO on a combined PHA production and nutrient removal process for wastewater treatment. Efficient Plasticicumulans dominating microbial cultures were enriched under both high DO (3.47 ± 1.12 mg/L) and low DO (0.86 ± 0.50 mg/L) conditions in the feast phase containing mostly the same populations but with different relative abundance. Butyrate and valerate were found to be the preferred substrates as compared to acetate and propionate regardless of DO concentrations. Compared to acetate and propionate, the butyrate and valerate uptakes were not significantly impacted by low DO levels in the PHA accumulation stage. A metabolic model was developed for the first time to describe the substrate preference among multiple volatile fatty acids (VFAs), providing a successful approach for PHA composition prediction and process efficiency optimization when four competing VFAs are supplied. Further, DO level control through both the feast and famine phases of culture selection was applied, at 3.79±0.65 mg/L and 0.48±0.29 mg/L respectively. The low DO level in both the feast and famine phases proved to be insufficient for successful MMC enrichment. By characterizing the microbial evolution under the unlimited DO conditions, it was found that Paracoccus was the dominating population (>50%) in the selected cultures, and substrate competition was correlated with the abundance of Plasticicumulans during culture selection. In order to optimize the integrated PHA production and nutrient removal process fed with ammonia-rich feedstocks in full scale implementation, the impact of DO on both processes was investigated. Much higher DO affinity for VFA consumption was observed as compared to nitrification. A DO control strategy was proposed based on the observation that the PHA production was not influenced while nitrogen was removed by simultaneous nitrification and denitrification processes when controlling DO at low levels (e.g. 0.4-0.8 mg/L).

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica, Especialidade em Engenharia Bioquímica Os biorreactores de membranas (MBRs, ‘membrane bioreactors’) para o tratamento de águas residuais combinam o processo de lamas activadas com um passo de filtração para obtenção de um efluente limpo, livre de sólidos. Os MBRs representam uma tecnologia em expansão no tratamento de águas residuais sobretudo devido ao reduzido espaço que requerem e à elevada qualidade do efluente obtido. No entanto, a colmatação das membranas pode reduzir o desempenho do MBR. Por este motivo, no presente trabalho, pretendeu-se estudar a monitorização dos MBRs, com o objectivo de minimizar o número de parâmetros de monitorização necessários para descrever o desempenho do processo e obter uma monitorização em tempo real com recurso mínimo a técnicas laboratoriais demoradas. Para este fim, estudou-se a aplicabilidade da fluorescência bidimensional em meios biológicos complexos, tais como as lamas activadas utilizadas para o tratamento de águas residuais. A fluorescência bidimensional mostrou ser uma técnica abrangente, capaz de recolher informação relevante sobre o estado do sistema em tempo real. Devido à complexidade da informação contida nos espectros de fluorescência, usaram-se técnicas de estatística multivariada, tais como análise de componentes principais e projecção de estruturas latentes (PLS, ‘projection to latent structures’), para extrair a informação dos espectros e correlacioná-la com parâmetros de operação e de desempenho do MBR. O uso de modelos estatísticos permitiu a previsão de parâmetros chave para o desempenho do MBR usando somente dados de processo impostos ou facilmente adquiríveis em tempo real. Adicionalmente, a modelação estatística foi combinada com um modelo mecanístico, numa estrutura híbrida, de forma a melhorar a previsão mecanística. Este estudo demonstrou ser possível usar modelos PLS para incorporar dados de fluorescência obtidos em tempo real, de modo a melhorar a previsão mecanística sem requerer análises laboratoriais adicionais.

Different oil-containing substrates, namely, used cooking oil (UCO), fatty acids-byproduct from biodiesel production (FAB) and olive oil deodorizer distillate (OODD) were tested as inexpensive carbon sources for the production of polyhydroxyalkanoates (PHA) using twelve bacterial strains, in batch experiments. The OODD and FAB were exploited for the first time as alternative substrates for PHA production. Among the tested bacterial strains, Cupriavidus necator and Pseudomonas resinovorans exhibited the most promising results, producing poly-3-hydroxybutyrate, P(3HB), form UCO and OODD and mcl-PHA mainly composed of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) monomers from OODD, respectively. Afterwards, these bacterial strains were cultivated in bioreactor. C. necator were cultivated in bioreactor using UCO as carbon source. Different feeding strategies were tested for the bioreactor cultivation of C. necator, namely, batch, exponential feeding and DO-stat mode. The highest overall PHA productivity (12.6±0.78 g L-1 day-1) was obtained using DO-stat mode. Apparently, the different feeding regimes had no impact on polymer thermal properties. However, differences in polymer‟s molecular mass distribution were observed. C. necator was also tested in batch and fed-batch modes using a different type of oil-containing substrate, extracted from spent coffee grounds (SCG) by super critical carbon dioxide (sc-CO2). Under fed-batch mode (DO-stat), the overall PHA productivity were 4.7 g L-1 day-1 with a storage yield of 0.77 g g-1. Results showed that SCG can be a bioresource for production of PHA with interesting properties. Furthermore, P. resinovorans was cultivated using OODD as substrate in bioreactor under fed-batch mode (pulse feeding regime). The polymer was highly amorphous, as shown by its low crystallinity of 6±0.2%, with low melting and glass transition temperatures of 36±1.2 and -16±0.8 ºC, respectively. Due to its sticky behavior at room temperature, adhesiveness and mechanical properties were also studied. Its shear bond strength for wood (67±9.4 kPa) and glass (65±7.3 kPa) suggests it may be used for the development of biobased glues. Bioreactor operation and monitoring with oil-containing substrates is very challenging, since this substrate is water immiscible. Thus, near-infrared spectroscopy (NIR) was implemented for online monitoring of the C. necator cultivation with UCO, using a transflectance probe. Partial least squares (PLS) regression was applied to relate NIR spectra with biomass, UCO and PHA concentrations in the broth. The NIR predictions were compared with values obtained by offline reference methods. Prediction errors to these parameters were 1.18 g L-1, 2.37 g L-1 and 1.58 g L-1 for biomass, UCO and PHA, respectively, which indicates the suitability of the NIR spectroscopy method for online monitoring and as a method to assist bioreactor control. UCO and OODD are low cost substrates with potential to be used in PHA batch and fed-batch production. The use of NIR in this bioprocess also opened an opportunity for optimization and control of PHA production process.

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

This thesis is focused on the production of value-added bacterial exopolysaccharides (EPS) by the bacterium Enterobacter A47 (DSM 23139), using industrial by-products/wastes as sub-strates. Enterobacter A47 has demonstrated the ability to synthesize a high molecular weight (Mw) fucose-rich EPS, namely, the heteropolysacharide named FucoPol. This new EPS is com-posed of fucose, glucose, galactose and glucuronic acid, which present interesting functional properties. Cheese whey was the first industrial by-product studied. Under standard controlled param-eters for FucoPol’s production, Enterobacter A47 successfully grew using lactose as carbon source and an EPS concentration of 6.40 g L-1 was reached within 3.2 days of cultivation, corre-sponding to a volumetric productivity of 2.00 g L-1 d-1. The produced EPS was mainly composed of glucuronic acid and fucose, which confers it a great potential for use in high-value applications, such as cosmetics and pharmaceuticals. The use of out-of-specification tomato paste as substrate resulted in the highest production (8.77 g L-1) and overall volumetric productivity (2.92 g L-1 d-1), which were obtained with con-tinuous substrate feeding at a constant flow rate of 11 g h-1. The polymer produced had the typical FucoPol composition: 30-36 mol% of fucose; 22-29 mol% of galactose; 25-34 mol% of glucose; 9-10 mol% of glucuronic acid and 12-22 wt.% of acyl groups. The ability of Enterobacter A47 to grow and produce EPS using as carbon source a mixture of glucose and xylose (75:25%) was also tested using pure sugars and a brewer’s spent grain (BSG) hydrolysate. The use of BSG as substrate resulted in an EPS concentration of 2.30 g L-1 with a low volumetric productivity (0.57 g L-1 d-1). The produced EPS was mainly composed of glucose. On the other hand, 5.71 g L-1 of EPS was achieved after 4 days using commercial glu-cose/xylose mixture, giving an overall volumetric productivity of 1.43 g L-1 d-1. The EPS pro-duced revealed to be similar to FucoPol composition. Although that the ability of Enterobacter A47 to use BSG hydrolysate as sole substrate to grow and produce EPS was demonstrated, further studies need to be developed to increase the polymer’s productivity. In order to evaluate the potential of the different EPS produced by Enterobacter A47 in this study, their properties in aqueous solutions, emulsion forming and stabilizing capacity, and film-forming capacity were studied. EPSCW presented the lowest apparent viscosity (ηa), but it was able to stabilize emulsions for over 4 weeks with olive oil, cedarwood oil and paraffin oil and its films had higher elongation capacity. EPSTP showed the lowest intrinsic viscosity ([η]) and angular frequency at dependence of storage (G’) and loss moduli (G’’) cross-over, good emulsion capacity and stabilization with peanut oil, almond oil and olive oil, and the produced films are slightly more rigid. In contrast, EPSGX reached the highest [η] and ηa, the emulsions formed were very strong with most of the tested oils at low O/W ratios, although having low stability. Moreover, EPSGX films were slight stiffer.

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Química e Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia FCT/MCTES projects PTDC/AMB/65702/2006 and SFRH/PROTEC/49449/2009 and SFRH/BPD/30800/2006 ; COST Action 636

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology Polyhydroxyalkanoates (PHAs) constitute a group of biobased and biodegradable polymers, which have been recognized as good substitutes for petroleum-based polymers in many applications. The large-scale production of PHAs is limited by the high cost of the most commonly used carbon sources (e.g. glucose, sucrose). However, the food industry generates large amounts of wastes, including fat-containing materials that can be used as low cost carbon sources for microbial cultivation, due their high carbon content. In this study, several bacterial strains (Cupriavidus necator, Comamonas testosteroni, Pseudomonas oleovorans, P. resinovorans, P. stutzeri, and P. citronellolis) were evaluated for their ability to grow and produce PHAs using fat-containing wastes generated by the food industry. The materials used in this study were mainly composed of free fatty acids, namely mystiric, oleic, linoleic and stearic acid. In the preliminary shake flask experiments, C. necator, C. testosteroni, P. oleovorans and P. citronellolis were able to grow and produce PHA polymer on margarine waste with the highest content. Those strains were selected for batch bioreactor experiments, wherein C. necator reached the highest polymer content (56%, wt/wt) and volumetric productivity (0.33 gPHA/L.h), Lower PHA contents were achieved by P. citronellolis and P. oleovorans (7.0 and 8.5%, wt/wt, respectively). However, in contrast with C. necator that synthesized polyhydroxybutyrate [P3(HB)], those strains produced medium chain length polyesters (mcl-PHA) containing monomers of 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD). C. necator was also cultivated in two different fed-batch strategies. The first cultivation achieved 76% (wt/wt) of P(3HB), while high cell densities were obtained in the second cultivation (48 g/L of active biomass concentration). Finally, the P(3HB) and mcl-PHA polymers had a glass transition temperature of 0.5–7.9ºC and -45.6, a melting point of 169.3–173.4ºC and 60.9ºC, and degree of crystallinity of 48.7–56.6% and 0.7%, respectively.

Dissertação para obtenção do Grau de Mestre em Biotecnologia The current high demand for plastics has become unsustainable. Polyhydroxyalkanoates are biopolymers stored by bacteria that can potentially replace modern plastics due to: wide range of applications; biodegradability; use of renewable resources as feedstock. High costs of current Polyhydroxyalkanoates production can be reduced using mixed cultures of organisms. Activated sludge from wastewater treatment plants is selected for Polyhydroxyalkanoates production through the imposition of cycles of intermittent feeding. In this study, the acclimation of activated sludge using synthetic volatile fatty acids (VFAs) as substrate resulted in a culture rich in Paracoccus spp. and unidentified filamentous bacteria. Low cost substrates such as sugarcane molasses (SM) or cheese whey (CW) can be employed as feedstock for further cost reduction. This requires an additional step before the microbial selection to ferment the feedstock into VFAs. In this work, the feedstock was changed from SM to CW. The population fed with SM was rich in Actinomycetaceae, while the population fed with CW was rich in Streptococcaceae, affecting the VFA composition. Consequently, the PHA-storing population and the polymer were affected. In the fermented SM (fSM) phase, the population was rich in Azoarcus (41.5 - 64.6%) and in the fCW phase the population was more diverse. Changing the pH in the fermentation reactor also affected the selection stage with an increase in Thauera and Azoarcus and a decrease in Paracoccus. A significant unidentified population of one layer sheet- forming bacteria was observed. Lastly, the occurrence of cell-to-cell communication (QS) in the selection stage was investigated. Possibly, QS molecules were detected when the carbon source was depleted. All steps of polyhydroxyalkanoate production are interconnected and for optimization, all stages must be studied and improved. Moreover, if QS proves to be involved in polyhydroxyalkanoate storage, the addition of QS molecules to the process may be explored for further optimization.