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.

Conversion of Cn-unsaturated into Cn-2-saturated LCFA can occur uncoupled from methanogenesis in anaerobic bioreactors

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50\% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.

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.

Microbial production of potent phenolic-antioxidants through solid state fermentation

The agroindustrial residues including plant tissues rich in polyphenols were explored for microbial production of potent phenolics under solid state fermentation processes. The fungal strains capable of hydrolyzing tannin-rich materials were isolated from Mexican semidesert zones. These microorganisms have been employed to release potent phenolic antioxidants during the solid state fermentation of different materials (pomegranate peels, pecan nut shells, creosote bush and tar bush). This chapter includes the critical parameters for antioxidants production from selective microbes. Technical aspects of the microbial fermentation of antioxidants have also been discussed.

Valorization of agro-industrial wastes towards the production of rhamnolipids

In this work, oil mill wastewater (OMW), a residue generated during olive oil extraction, was evaluated as an inducer of rhamnolipid production. Using a medium containing as sole ingredients corn steep liquor (10%, v/v), sugarcane molasses (10%, w/v) and OMW (25%, v/v), Pseudomonas aeruginosa #112 produced 4.5 and 5.1 g of rhamnolipid per liter in flasks and reactor, respectively, with critical micelle concentrations as low as 13 mg/l. Furthermore, in the medium supplemented with OMW, a higher proportion of more hydrophobic rhamnolipid congeners was observed comparing with the same medium without OMW. OMW is a hazardous waste which disposal represents a serious environmental problem; therefore, its valorization as a substrate for the production of added-value compounds such as rhamnolipids is of great interest. This is the first report of rhamnolipid production using a mixture of these three agro-industrial by-products, which can be useful for the sustainable production of rhamnolipids.

Production of whey protein-based aggregates under ohmic heating

Formation of whey protein isolate protein aggregates under the influence of moderate electric fields upon ohmic heating (OH) has been monitored through evaluation of molecular protein unfolding, loss of its solubility, and aggregation. To shed more light on the microstructure of the protein aggregates produced by OH, samples were assayed by transmission electron microscopy (TEM). Results show that during early steps of an OH thermal treatment, aggregation of whey proteins can be reduced with a concomitant reduction of the heating chargeby reducing the come-up time (CUT) needed to reach a target temperatureand increase of the electric field applied (from 6 to 12 V cm1). Exposure of reactive free thiol groups involved in molecular unfolding of -lactoglobulin (-lg) can be reduced from 10 to 20 %, when a CUT of 10 s is combined with an electric field of 12 V cm1. Kinetic and multivariate analysis evidenced that the presence of an electric field during heating contributes to a change in the amplitude of aggregation, as well as in the shape of the produced aggregates. TEM discloses the appearance of small fibrillar aggregates upon the influence of OH, which have recognized potential in the functionalization of food protein networks. This study demonstrated that OH technology can be used to tailor denaturation and aggregation behavior of whey proteins due to the presence of a constant electric field together with the ability to provide a very fast heating, thus overcoming heat transfer limitations that naturally occur during conventional thermal treatments.

Candida tropicalis biofilms: biomass, metabolic activity and secreted aspartyl proteinase production

According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation.

Characterization of late antique and early medieval pottery production of the city of Braga and its territory

The fundamental goal we set ourselves when developing this study is to try to characterize, both technically and formally, ceramics made in the city of Braga and its territory from the initial moments of the Late Antiquity to the Middle Ages. Thus, we will focus on analyzing some own productions that appear attached to the phases of late antique occupation —ceramics of red engobes and late gray—, as well as in the early medieval containers identified in different archaeological interventions practiced in the Braga environment. Concretely, we will analyze the material from various excavations conducted recently at the Theatre in the solar number 20/28 and 36/56 from the Afonso Henriques Street and the former District Hostel as well as the church of São Martinho de Dume.

Production of biohydrogen from dark fermentation of organic wastes

Dissertação de mestrado em Bioengenharia

Development of a biorefinery–based bioprocess for pigment and lipid production by oleaginous yeasts

Dissertação de mestrado em Bioengenharia