Production and characterization of ZrCN-Ag coatings deposited by mganetron sputtering

Tese de Doutoramento (Programa doutoral em Engenharia de Materiais)

Synthetic biology approaches to engineer new pathways for the production of plant secondary metabolites

Secondary metabolites from plants are important sources of high-value chemicals, many of them being pharmacologically active. These metabolites are commonly isolated through inefficient extractions from natural biological sources and are often difficult to synthesize chemically. Therefore, their production using engineered organisms has lately attracted an increased attention. Curcuminoids, an example of such metabolites, are produced in Curcuma longa and exhibit anti-cancer and anti-inflammatory activities. Herein we report the construction of an artificial biosynthetic pathway for the curcuminoids production in Escherichia coli. Different 4-coumaroyl-CoA ligases (4CL) and polyketide synthases (diketide-CoA synthase (DCS), curcumin synthase (CURS) and curcuminoid synthase) were tested. The highest curcumin production (70 mg/L) was obtained by feeding ferulic acid and with the Arabidopsis thaliana 4CL1 and C. longa DCS and CURS enzymes. Other curcuminoids (bisdemethoxy- and demethoxycurcumin) were also produced by feeding coumaric acid or a mixture of coumaric and ferulic acids, respectively. Curcuminoids, including curcumin, were also produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase and 4-coumarate 3-hydroxylase were used. Caffeoyl-CoA O-methyltransferase was used to convert caffeoyl-CoA to feruloyl-CoA. This pathway represents an improvement of the curcuminoids heterologous production. The construction of this pathway in another model organism is being considered, as well as the introduction of alternative enzymes.

Optimization of low-cost nutritional supplementation for lignocellulosic ethanol fermentation

[Excerpt] Bioethanol from lignocellulosic materials (LCM), also called second generation bioethanol, is considered a promising alternative to first generation bioethanol. An efficient production process of lignocellulosic bioethanol involves an effective pretreatment of LCM to improve the accessibility of cellulose and thus enhance the enzymatic saccharification. One interesting approach is to use the whole slurry from treatment, since allows economical and industrial benefits: washing steps are avoided, water consumption is lower and the sugars from liquid phase can be used, increasing ethanol concentration [1]. However, during the pretreatment step some compounds (such as furans, phenolic compounds and weak acids) are produced. These compounds have an inhibitory effect on the microorganisms used for hydrolysate fermentation [2]. To overcome this, the use of a robust industrial strain together with agro-industrial by-products as nutritional supplementation was proposed to increase the ethanol productivities and yields. (...)

An efficient Azorean thermophilic consortium for lignocellulosic biomass degradation

[Excerpt] Lignocellulosic plant biomass is being envisioned by biorefinery industry as an alternative to current petroleum platform because of the large scale availability, low cost and environmentally benign production. The industrial bioprocessing designed to transform lignocellulosic biomass into biofuels are harsh and the enzymatic reactions may be severely compromised reducing the production of fermentable sugars from lignocellulosic biomass. Thermophilic bacteria consortium are a potential source of cellulases and hemicellulases adapted to extreme environmental conditions, which can be exploited as a new source for the development of more robust enzymatic cocktails. (...)

Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...)

Biotechnological valorization of waste cooking oils: lipase and microbial lipids production by Yarrowia lipolytica

[Excerpt] Waste cooking oils (WCO) generated from vegetable oils used at high temperatures in food frying, cause environmental problems and must be reutilized. New strategies to valorize these wastes are attracting a great scientific interest due to the important advantages offered from an economic and environmental point of view. A microbial platform can be established to convert low-value hydrophobic substrates, such as waste cooking oils, to microbial lipids (single cell oil, SCO) and other value-added bioproducts, such as lipase. (...)

Developing novel strains for butanol production

[Excerpt] The growing global demand for new energy sources combined with environmental concerns had motivated the search for alternative fuels, produced from renewable raw materials. During the last decade, ethanol was considered the next generation of biofuels. But more recently, n-butanol gained attention due to its superior fuel properties when compared with ethanol. Although n-butanol is naturally produced by solventogenic bacteria through ABE fermentation, the low productivities obtained with this bioprocess discouraged its use. Thus, most of n-butanol produced nowadays is chemical synthesized via petrochemical routes and its price is extremely sensitive to crude oil’s price. One possible approach to overcome this issue is to express non-native pathways in microbial factories. (...)

Citric acid production by Y. lipolytica W29 from crude glycerol: strain improvement by mutagenesis

[Excerpt] Citric acid, an important and versatile organic acid extensively used in several industries, is originally produced by Aspergillus niger in submerged fermentation from molasses [1]. However, Yarrowia lipolytica have been studied and demonstrate a great potential as citric acid producer from several carbon sources [1–5] including crude glycerol, a low cost byproduct from the biodiesel industry [6]. The simultaneous production of the isomer isocitric acid is the major problem in using this yeast in the citric acid production. (...)

Energy production from lipids by novel anaerobes

[Excerpt] Anaerobic microbial diversity encloses a very high potential that can be used for biotechnological applications. This potential is still largely unexplored, since the majority of the microorganisms in Nature are unknown or poorly characterized. This work is focused on the study of novel anaerobic microorganisms that participate in the metabolism of lipids, long chain fatty acids (LCFA) and glycerol, with the main goal of producing valuable energy-rich organic compounds. For that, conventional anaerobic culturing procedures were combined with continuous bioreactors operation and allied to microbial ecology approaches. Two main examples of the work performed will be presented. (...)

Biosynthesis, detection and toxicology of ochratoxins - optimisation of production

Ochratoxin A (OTA) is a very well known mycotoxin found in several food commodities for which maximum limits are being discussed in EC in other to produce appropriate regulations. OTA is one of several ochratoxins produced by Aspergillus and Penicillium species. All the compounds in this group have a molecular structure very similar to OTA and some were already isolated from natural substrates. Several of these compounds such as ochratoxin , methyl and ethyl ester of ochratoxin A, 4-R and S-hydroxyochratoxin A, 10-hydroxyochratoxin A and ochratoxin A open lactone are commercially unavailable. However, they can be easily synthesized through OTA modification. With the main objective of its application on further research works, OTA production, isolation and purification has been optimised from an A. alliaceus strain grown on wheat medium. Synthesis and purification of some OTA derivatives has been achieved and an HPLC method for their detection was optimised. Data about their production by several species of Aspergillus will be presented. The toxicological properties of ochratoxins are still not very clear and a future EC safety limit for OTA will depend on e.g., a better clarification of its carcinogenity. Could OTA derivatives play a role here?

Bioethanol production by Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis from delignified coconut fibre mature and lignin extraction according to biorefinery concept

In search to increase the offer of liquid, clean, renewable and sustainable energy in the world energy matrix, the use of lignocellulosic materials (LCMs) for bioethanol production arises as a valuable alternative. The objective of this work was to analyze and compare the performance of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the production of bioethanol from coconut fibre mature (CFM) using different strategies: simultaneous saccharification and fermentation (SSF) and semi-simultaneous saccharification and fermentation (SSSF). The CFM was pretreated by hydrothermal pretreatment catalyzed with sodium hydroxide (HPCSH). The pretreated CFM was characterized by X-ray diffractometry and SEM, and the lignin recovered in the liquid phase by FTIR and TGA. After the HPCSH pretreatment (2.5% (v/v) sodium hydroxide at 180 °C for 30 min), the cellulose content was 56.44%, while the hemicellulose and lignin were reduced 69.04% and 89.13%, respectively. Following pretreatment, the obtained cellulosic fraction was submitted to SSF and SSSF. Pichia stipitis allowed for the highest ethanol yield 90.18% in SSSF, 91.17% and 91.03% were obtained with Saccharomyces cerevisiae and Zymomonas mobilis, respectively. It may be concluded that the selection of the most efficient microorganism for the obtention of high bioethanol production yields from cellulose pretreated by HPCSH depends on the operational strategy used and this pretreatment is an interesting alternative for add value of coconut fibre mature compounds (lignin, phenolics) being in accordance with the biorefinery concept.

Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas

Synthesis gas, a mixture of CO, H2, and CO2, is a promising renewable feedstock for bio-based production of organic chemicals. Production of medium-chain fatty acids can be performed via chain elongation, utilizing acetate and ethanol as main substrates. Acetate and ethanol are main products of syngas fermentation by acetogens. Therefore, syngas can be indirectly used as a substrate for the chain elongation process.

The role of marine anaerobic bacteria and archaea in bioenergy production

The development of products from marine bioresources is gaining importance in the biotechnology sector. The global market for Marine Biotechnology products and processes was, in 2010, estimated at 2.8 billion with a cumulative annual growth rate of 510% (Børresen et al., Marine biotechnology: a new vision and strategy for Europe. Marine Board Position Paper 15. Beernem: Marine Board-ESF, 2010). Marine Biotechnology has the potential to make significant contributions towards the sustainable supply of food and energy, the solution of climate change and environmental degradation issues, and the human health. Besides the creation of jobs and wealth, it will contribute to the development of a greener economy. Thus, huge expectations anticipate the global development of marine biotechnology. The marine environment represents more than 70% of the Earths surface and includes the largest ranges of temperature, light and pressure encountered by life. These diverse marine environments still remain largely unexplored, in comparison with terrestrial habitats. Notwithstanding, efforts are being done by the scientific community to widespread the knowledge on oceans microbial life. For example, the J. Craig Venter Institute, in collaboration with the University of California, San Diego (UCSD), and Scripps Institution of Oceanography have built a state-of-the-art computational resource along with software tools to catalogue and interpret microbial life in the worlds oceans. The potential application of the marine biotechnology in the bioenergy sector is wide and, certainly, will evolve far beyond the current interest in marine algae. This chapter revises the current knowledge on marine anaerobic bacteria and archaea with a role in bio-hydrogen production, syngas fermentation and bio-electrochemical processes, three examples of bioenergy production routes.

Production and extraction of polysaccharides and oligosaccharides and their use as new food additives

Polysaccharides and oligosaccharides can improve quality and enhance nutritional value of final food products due to their technological and nutritional features ranging from their capacity to improve texture to their effect as dietary fibers. For this reason, they are among the most studied ingredients in the food industry. The use of natural polysaccharides and oligosaccharides as food additives has been a reality since the food industry understood their potential technological and nutritional applications. Currently, the replacement of traditional ingredients and/or the synergy between traditional ingredients and polysaccharides and oligosaccharides are perceived as promising approaches by the food industry. Traditionally, polysaccharides have been used as thickening, emulsifying, and stabilizing agents, however, at this moment polysaccharides and oligosaccharides claim health and nutritional advantages, thus opening a new market of nutritional and functional foods. Indeed, their use as nutritional food ingredients enabled the food industry to develop a countless number of applications, e.g., fat replacers, prebiotics, dietary fiber, and antiulcer agents. Based on this, among the scientific community and food industry, in the last years many research studies and commercial products showed the possibility of using either new or already used sources (though with changed properties) of polysaccharides for the production of food additives with new and enhanced properties. The increasing interest in such products is clearly illustrated by the market figures and consumption trends. As an example, the sole market of hydrocolloids is estimated to reach $7 billion in 2018. Moreover, oligosaccharides can be found in more than 500 food products resulting in a significant daily consumption. A recent study from the Transparency Market Research on Prebiotic Ingredients Market reported that prebiotics' demand was worth $2.3 billion in 2012 and it is estimated to reach $4.5 billion in 2018, growing at a compound annual growth rate of 11.4% between 2012 and 2018. The entrance of this new generation of food additives in the market, often claiming health and nutritional benefits, imposes an impartial analysis by the legal authorities regarding the accomplishment of requirements that have been established for introducing novel ingredients/food, including new poly- and oligosaccharides. This chapter deals with the potential use of polysaccharides and oligosaccharides as food additives, as well as alternative sources of these compounds and their possible applications in food products. Moreover, the regulation process to introduce novel polysaccharides and oligosaccharides in the market as food additives and to assign them health claims is discussed.