Impact of carbon/nitrogen feeding strategy on polyhydroxyalkanoates production using mixed microbial cultures

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.

Exploring the relationship between toxin and spore production in the human enteric pathogen Clostridium difficile

RESUMO: Clostridium difficile é presentemente a principal causa de doença gastrointestinal associada à utilização de antibióticos em adultos. C. difficile é uma bactéria Gram-positiva, obrigatoriamente anaeróbica, capaz de formar endósporos. Tem-se verificado um aumento dos casos de doença associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrência, em parte, devido à emergência de estirpes mais virulentas, mas também devido à má gestão do uso de antibióticos. C. difficile produz duas toxinas, TcdA e TcdB, que são os principais fatores de virulência e responsáveis pelos sintomas da doença. Estas são codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxigénio são essenciais para a transmissão do organismo e recorrência da doença. A expressão dos genes do PaLoc ocorre em células vegetativas, no final da fase de crescimento exponencial, e em células em esporulação. Neste trabalho construímos dois mutantes de eliminação em fase dos genes tcdR e tcdE. Mostrámos que a auto-regulação do gene tcdR não é significativa. No entanto, tcdR é sempre necessário para a expressão dos genes presentes no PaLoc. Trabalho anterior mostrou que, com a exceção de tcdC, os demais genes do PaLoc são expressos no pré-esporo. Mostrámos aqui que TcdA é detectada à superfície do esporo maduro e que a eliminação do tcdE não influencia a acumulação de TcdA no meio de cultura ou em associação às células ou ao esporo. Estas observações têm consequências para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser também um veículo para a entrega da toxina nos estágios iniciais da infecção, que TcdA possa ser libertada durante a germinação do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligação à mucosa do cólon.---------------------------ABSTRACT: Clostridium difficile is currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract. Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of “hypervirulent” strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence. The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation of tcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes. A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Production and characterization of electrospun composite fibers: confinement of thermosensitive microgels

Materials engineering focuses on the assembly of materials´ properties to design new products with the best performance. By using sub-micrometer size materials in the production of composites, it is possible to obtain objects with properties that none of their compounds show individually. Once three-dimensional materials can be easily customized to obtain desired properties, much interest has been paid to nanostructured poly-mers in order to build biocompatible devices. Over the past years, the thermosensitive microgels have become more common in the framework of bio-materials with potential applicability in therapy and/or diagnostics. In addition, high aspect ratio biopolymers fibers have been produced using the cost-effective method called electrospinning. Taking advantage of both microgels and electrospun fibers, surfaces with enhanced functionalities can be obtained and, therefore employed in a wide range of applications. This dissertation reports on the confinement of stimuli-responsive microgels through the colloidal electro-spinning process. The process mainly depends on the composition, properties and patterning of the precur-sor materials within the polymer jet. Microgels as well as the electrospun non-woven mats were investigated to correlate the starting materials with the final morphology of the composite fibers. PNIPAAm and PNIPAAm/Chitosan thermosensitive microgels with different compositions were obtained via surfactant free emulsion polymerization (SFEP) and characterized in terms of chemical structure, morphology, thermal sta-bility, swelling properties and thermosensitivity. Finally, the colloidal electrospinning method was carried out from spinning solutions composed of the stable microgel dispersions (up to a concentration of about 35 wt. % microgels) and a polymer solution of PEO/water/ethanol mixture acting as fiber template solution. The confinement of microgels was confirmed by Scanning Electron Microscopy (SEM). The electrospinning process was statistically analysed providing the optimum set of parameters aimed to minimize the fiber diameter, which give rise to electrospun nanofibers of PNIPAAm microgels/PEO with a mean fiber diameter of 63 ± 25 nm.

Optimization of production of anti-cancer vaccines based on dendritic cells

Cancer remains as one of the top killing diseases in first world countries. It’s not a single, but a set of various diseases for which different treatment approaches have been taken over the years. Cancer immunotherapy comes as a “new” breath on cancer treatment, taking use of the patients’ immune system to induce anti-cancer responses. Dendritic Cell (DC) vaccines use the extraordinary capacity of DCs’ antigen presentation so that specific T cell responses may be generated against cancer. In this work, we report the ex vivo generation of DCs from precursors isolated from clinical-grade cryopreserved umbilical cord blood (UCB) samples. After the thawing protocol for cryopreserved samples was optimized, the generation of DCs from CD14+ monocytes, i.e., moDCs, or CD34+ hematopoietic stem cells (HSCs), i.e, CD34-derived DCs, was followed and their phenotype and function evaluated. Functional testing included the ability to respond to maturation stimuli (including enzymatic removal of surface sialic acids), Ovalbumin-FITC endocytic capacity, cytokine secretion and T cell priming ability. In order to evaluate the feasibility of using DCs derived from UCB precursors to induce immune responses, they were compared to peripheral blood (PB) moDCs. We observed an increased endocytosis capacity after moDCs were differentiated from monocyte precursors, but almost 10-fold lower than that of PB moDCs. Maturation markers were absent, low levels of inflammatory cytokines were seen and T cell stimulatory capacity was reduced. Sialidase enzymatic treatment was able to mature these cells, diminishing endocytosis and promoting higher T cell stimulation. CD34-derived DCs showed higher capacity for both maturation and endocytic capacity than moDCs. Although much more information was acquired from moDCs than from CD34-derived DCs, we conclude the last as probably the best suited for generating an immune response against cancer, but of course much more research has to be performed.

Risk of employing an evolvable production system

Nowadays manufacturing companies are facing a more challenging environment due to the unpredictability of the markets in order to survive. Enterprises need to keep innovating and deliver products with new internal or external characteristics. There are strategies and solutions, to different organisational level from strategic to operational, when technology is growing faster in operational level, more specifically in manufacturing system. This means that companies have to deal with the changes of the emergent manufacturing systems while it can be expensive and not easy to be implement. An agile manufacturing system can help to cope with the markets changeability. Evolvable Production Systems (EPS) is an emergent paradigm which aims to bring new solutions to deal with changeability. The proposed paradigm is characterised by modularity and intends to introduce high flexibility and dynamism at shop floor level through the use of the evolution of new computational devices and technology. This new approach brings to enterprises the ability to plug and unplug new devices and allowing fast reformulation of the production line without reprogramming. There is no doubt about the advantages and benefits of this emerging technology but the feasibility and applicability is still under questioned. Most researches in this area are focused on technical side, explaining the advantages of those systems while there are no sufficient works discussing the implementation risks from different perspective, including business owner. The main objective of this work is to propose a methodology and model to identify, classify and measure potential risk associated with an implementation of this emergent paradigm. To quantify the proposed comprehensive risk model, an Intelligent Decision system is developed employing Fuzzy Inference System to deal with the knowledge of experts, as there are no historical data and sufficient research on this area. The result can be the vulnerability assessment of implementing EPS technology in manufacturing companies when the focus is more on SMEs. The present dissertation used the experts’ knowledge and experiences, who were involved in FP7 project IDEAS, which is one of the leading projects in this area.

Bio-valorization of crude glycerol through polyhydroxyalkanoates production

Due to the prospective partial replacement of fossil fuels by biodiesel, its production has continuously grown in the last decade. The increase in global biodiesel production demands the development of sustainable applications of its main by-product, crude glycerol. In this thesis the feasibility of producing polyhydroxyalkanoates (PHA) by a mixed microbial community using crude glycerol as feedstock was investigated. Several incubation conditions were studied in order to maximize PHA production. The microbial population selected under aerobic dynamic feeding conditions had the ability to consume both major carbon fractions present in the crude, glycerol and methanol. Two biopolymers were stored, poly-3-hydroxybutyrate (PHB) and glucose biopolymer (GB), apparently using glycerol as the only carbon source for their production. The microbial enrichment obtained was able to accumulate up to 47% PHB of cell dry weight with a productivity of 0.24 g HA/L d. The overall PHA yield on total substrate consumed (0.32 g COD HB/g COD crude glycerol) was in the middle range of those reported in literature (0.08–0.58 g COD PHA/g COD real waste). The increase of temperature from 23ºC to 30ºC favored the culture fraction that accumulates glucose biopolymer with a maximum accumulation value of 25% of cell dry weight, which is an interesting value but not the main goal of this thesis. The fact that crude glycerol can be used to produce PHA without any pre-treatment step, makes the overall production process economically more competitive, reducing polymer final cost. This was the first study that demonstrates the valorization of the glycerol fraction present in the crude glycerol into PHA using an aerobic mixed microbial consortium.

Modeling and optimization of extracellular polysaccharides production by Enterobacter A47

Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters.

Practice variations in the production of health

This study investigates three questions related to medical practice variation. First, it tests whether average length of stay across Portuguese National Health Service hospitals varies when controlling for differences in patients’ characteristics. Second, it looks at hospital-level characteristics in order to find out whether these are able to explain differences in average length of stay across hospitals. Finally, it proposes a best practice average length of stay for each of the six episodes of care analyzed. To perform the analysis, administrative data from the Diagnosis-Related groups’ data set for the year of 2012 was used. A replication of a hierarchical two-stage model with hospital fixed effects was carried out. The results show that after taking patients’ characteristics into account, variation in average length of stay across hospitals exists. This variation cannot be explained by hospital-level characteristics.

Polyhydroxyalkanoates production by photosynthetic mixed cultures

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.

Coupling organic substrate removal to methane production in a fully biological microbial electrolysis cell

Microbial electrolysis cells (MECs) are an innovative and emerging technique based on the use of solid-state electrodes to stimulate microbial metabolism for wastewater treatment and simultaneous production of value-added compounds (such as methane). This research studied the performance of a two-chamber MEC in terms of organic matter oxidation (at the anode) and methane production (at the cathode). MEC‟s anode had been previously inoculated with an activated sludge, whereas the cathode chamber inoculum was an anaerobic sludge (containing methanogenic microorganisms). During the experimentation, the bioanode was continuously fed with synthetic solutions in anaerobic basal medium, at an organic load rate (OLR) of around 1 g L-1 d-1, referred to the chemical oxygen demand (COD). At the beginning (Run I), the feeding solution contained acetate and subsequently (Run II) it was replaced with a more complex solution containing soluble organic compounds other than acetate. For both conditions, the anode potential was controlled at -0.1 V vs. standard hydrogen electrode, by means of a potentiostat. During Run I, over 80% of the influent acetate was anaerobically oxidized at the anode, and the resulting electric current was recovered as methane at the cathode (with a cathode capture efficiency, CCE, accounting around 115 %). The average energy efficiency of the system (i.e., the energy captured into methane relative to the electrical energy input) under these conditions was over 170%. However, reactor‟s performance decreased over time during this run. Throughout Run II, a substrate oxidation over 60% (on COD basis) was observed. The electric current produced (57% of coulombic efficiency) was also recovered as methane, with a CCE of 90%. For this run the MEC‟s average energy efficiency accounted for almost 170 %. During all the experimentation, a very low biomass growth was observed at the anode whereas ammonium was transferred through the cationic membrane and concentrated at the cathode. Tracer experiments and scanning electron microscopy analyses were also carried out to gain a deeper insight into the reactor performance and also to investigate the possible reasons for partial loss of performance. In conclusion, this research suggests the great potential of MEC to successfully treat low-strength wastewaters, with high energy efficiency and very low sludge production.

Utilização de colas naturais para placas de derivados de madeira – uma síntese

Resumo: Nos últimos anos tem-se verificado um interesse crescente pela área das colas naturais, em particular na produção de placas de derivados de madeira para utilização no interior das construções. Estas colas parecem ser uma boa alternativa às colas sintéticas, derivadas do petróleo, pois, ao contribuírem para uma melhor qualidade do ar interior associada a uma menor incorporação de energia no produto final, apresentam vantagens interessantes em termos ambientais e de saúde. Nesse sentido fez-se uma revisão da bibliografia sobre a utilização laboratorial e mesmo semi-industrial de colas naturais em placas de derivados de madeira. São apresentados e discutidos alguns exemplos de materiais que estas colas aglutinam, bem como as suas características finais com vista a futuros desenvolvimentos experimentais de placas fabricadas a partir de resíduos agrícolas comuns. Abstract: An increased interest for bio-based adhesives for wood-based panels for building purposes has been arising. This type of adhesives could be a good alternative for petroleum based synthetic adhesives as bio-based adhesives can contribute for healthier indoor environments and, simultaneously, present lower embodied energy. For the previous reasons a bibliographic review was conducted, on the use of bio-based adhesives for the production of wood-based building panels. Some examples of the adhesives, the type of applications and the characterization of the resulting products are presented and compared, bearing in mind contributing for a future increase of research in this area.

MANGROVE BARK: A RENEWABLE RESIN SOURCE FOR WOOD ADHESIVES

From four solutions tested to extract tannins from mangrove bark for wood adhesives, hot water is recommended. Hot water extracted 21.4% of formaldehyde-hydrochloric acid reactive polyphenols on oven-dry bark basis.

The reasons behind the progression in PISA scores: An education production function approach using semi-parametric techniques

Research Masters