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 of polyhydroxyalkanoates from oil-containing substrates

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.

Bioproduction of succinic acid from renewable feedstocks by Actinobacillus succinogenes 130Z

Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)

Improvement of a photoautotrophic chassis robustness for synthetic biology applications

Cyanobacteria are photoautotrophic microorganisms with great potential for the biotechnological industry due to their low nutrient requirements, photosynthetic capacities and metabolic plasticity. In biotechnology, the energy sector is one of the main targets for their utilization, especially to produce the so called third generation biofuels, which are regarded as one of the best replacements for petroleum-based fuels. Although, several issues could be solved, others arise from the use of cyanobacteria, namely the need for high amounts of freshwater and contamination/predation by other microorganisms that affect cultivation efficiencies. The cultivation of cyanobacteria in seawater could solve this issue, since it has a very stable and rich chemical composition. Among cyanobacteria, the model microorganism Synechocystis sp. PCC 6803 is one of the most studied with its genome fully sequenced and genomic, transcriptomic and proteomic data available to better predict its phenotypic behaviors/characteristics. Despite suitable for genetic engineering and implementation as a microbial cell factory, Synechocystis’ growth rate is negatively affected by increasing salinity levels. Therefore, it is important to improve. To achieve this, several strategies involving the constitutive overexpression of the native genes encoding the proteins involved in the production of the compatible solute glucosylglycerol were implemented, following synthetic biology principles. A preliminary transcription analysis of selected mutants revealed that the assembled synthetic devices are functional at the transcriptional level. However, under different salinities, the mutants did not show improved robustness to salinity in terms of growth, compared with the wild-type. Nevertheless, some mutants carrying synthetic devices appear to have a better physiological response under seawater’s NaCl concentration than in 0% (w/v) NaCl.

Forest biomass and wood consumption In the lower course of the Amazon: a case study of rhe Urubueua Island

Várzea and terra-firme forests in the lower course of the Amazon were compared in terms of forest structure, wood volume increments and forest biomass. The wood volume of várzea forests was smaller than that of terra-firme forests, particularly when severe human intervention such as the cultivation of açaí palm occurred. The difference was even greater in the forest weight comparison because of the lower wood density of várzea trees. These trees are not directly influenced by water stress during the dry season, while late wood with a high density is formed in the terra-firme trees. The annual forest disappearance area due to firewood for tile factories was estimated to be about 276 ha on the island investigated, which had an area of 36,200 ha. Assuming that the forests are rotatively cultivated every 25 to 30 years, the total deforestation area is 6,870-6,948 ha in 25 years and 8,244~8,337 ha in 30 years. This result means that the balance between forest biomass and utilization is not in crisis, however, this balance might be lost as long as substitutive energy such as electricity is not supplied.

Sex determination in the Giant fish of Amazon Basin, Arapaima gigas (Osteoglossiformes, Arapaimatidae), using laparoscopy

The Giant of Amazon basin, pirarucu, Arapaima gigas, is the largest scaled freshwater fish in the world. pirarucu cultivation has recently started, driven by the decline in natural populations and high market value. Currently, there are no reliable methods for sexual differentiation in this species other than direct examination of gonads, which requires dissection of specimens. A non-lethal and less invasive method for sexual identification is highly desirable in order to properly group broodstock for mating and offspring production. We utilized laparoscopic examination in anesthetized pirarucu to differentiate between male and female individuals. This method allowed for the observation and differentiation of the reproductive organs within an individual. Our results suggest that laparoscopy is an efficient method for sex differentiation in pirarucu causing minimal stress to the fish.

Fine-root production in two secondary forest sites with distinct ages in Eastern Amazon

The objective of this work was to assess the fine-root (≤ 2 mm diameter) production dynamics of two forest regrowths at different ages. Fine-root production was monitored by the ingrowth core method in one 18-year-old site (2 ha) and one 10-year-old site (0.5 ha), both localized in the Apeú region, Northern Pará State, Brazil. The sites were abandoned after successive shifting cultivation, beginning in 1940. Monthly production of live fine-root was similar between sites and was influenced by rainfall seasonality, with higher production during the dry season than the wet season for mass and length. However, mortality in terms of mass was higher in the 10-year-old site than in the 18-year-old site. The seasonality influenced mortality only in the 18-year old site following the pattern observed for live fine-root. The influence seasonal on mortality in terms of length was different between sites, with higher mortality during the wet season in the 10-year-old site and higher mortality during the dry season in the 18-year-old site. Specific root length was higher during the wet season and at the 10-year-old site. Fine-root production was not influenced by the chronosequence of the sites studied, probably fine-root production may have already stabilized in the sites or it depended more on climate and soil conditions. The production of fine-roots mass and length were indicators that generally showed the same pattern.

[Re]ativar os terrenos abandonados e a vegetação nas margens do rio Tâmega e das Lagoas em Chaves

Dissertação de mestrado integrado em Arquitectura

Hydrocarbonoclastic bacteria: from bioremediation to bioenergy feedstock

Tese de Doutoramento em Engenharia Química e Biológica.

The family haloplasmataceae

Haloplasmataceae is a family within the order Haloplasmatales, which currently includes one single genus and species: Haloplasma contractile. This family has unusual phenotypic features the most noticeable being a unique morphology and cellular contractility cycle and a distinct phylogenetic position between the Firmicutes and the Tenericutes (Mollicutes). Members of the Haloplasmataceae have been isolated from the upper sediments of a deep-sea anoxic brine in the Red Sea, but cultivation-independent studies have found related sequences in a wide range of biotopes including other extreme environments, contaminated soils and marine sediments, as well as intestinal samples. The isolation and description of new representatives of this family might therefore result in significant changes to the current description.

Seleção de microalgas com potencial de produção de biocombustíveis

Dissertação de mestrado em Bioengenharia

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

Dissertação de mestrado em Bioengenharia

Producción de factores de virulencia por fitopatógenos bacterianos de soja. Utilización de productos naturales como biocontrol. Production of virulence factors by soybean bacterial phytopathogens. The use of natural products as biocontrolers.

En Argentina el cultivo de soja ocupa el primer lugar en superficie sembrada. El 90% de la producción se obtiene en la zona central del pais. La siembra directa favorece la multiplicación y supervivencia de fitopatógenos causantes de tizón y pústula bacterianos. El tizón es producido por Pseudomonas syringae pv. glycinea observándose manchas marrones en las hojas. Produce gran variedad de toxinas: coronatina, faseolotoxina, siringomicina, tabtoxina, proteínas “nucleation ice”, entre otras, las cuales contribuyen a la clorosis y necrosis. En la infección, además, están involucrados exopolisacáridos (levano y alginato). La celulosa ha sido relacionada en la adhesión bacteriana y en la formación de biofilm. La pústula es causada por Xanthomonas axonopodis pv. glycines. Produce manchas pequeñas con una pequeña pústula de color claro. Libera enzimas como α-amilasa, proteasa, endo β-mannanasa, actividad peptolítica, que degradan componentes vegetales. Xantan, producido por X. axonopodis es uno de los componentes necesarios para la formación de biofilm. Este último es considerado un importante factor de virulencia porque proporciona una estrategia de colonización que otorga mayor resistencia a ambientes desfavorables, tolerancia a antimicrobianos, producción de metabolitos y exoenzimas, etc. Actualmente el control de bacterias fitopatógenas se realiza mediante pesticidas con alta toxicidad para los consumidores y el ambiente. Para evitar las bacteriosis en la práctica se sugiere la rotación de cultivos y utilizar semillas certificadas. Se están probando compuestos naturales derivados de plantas medicinales como pesticidas; estos se pueden dividir en varias categorías fitoquímicas. Varios estudios confirman la actividad antibacteriana, antifúngica y antiviral de estos productos. Extractos vegetales con alto contenido de flavonoides y aceite esenciales poseen una importante actividad antibacteriana. Además, algunos aceites esenciales podrían estar incidiendo en la liberación y/o producción de biofilm, exopolisacáridos y exoproteínas. La gran incidencia de las infecciones por fitopatógenos y las pérdidas económicas que estas acarrean hacen que su control presente grandes dificultades para la agricultura sustentable en soja de nuestro país. En este trabajo se propone estudiar los diferentes factores de virulencia de cepas bacterianas fitopatógenas y evaluar el rol que cumplen en el proceso de la enfermedad en cultivos de soja y desarrollar estrategias para el control de bacteriosis vegetales aplicando productos naturales aislados de plantas aromáticas. La correcta utilización de productos antimicrobianos de origen natural aplicados sobre el cultivo y/o sobre las semillas evitaría la dispersión de la enfermedad y la eliminación al medio ambiente de productos contaminantes no deseados. In Argentina, soybean cultivation occupies the first place; 90% of this cereal is produced in the central region of the country. Intensive tillage practices favour multiplication and survival of bacterial phytopathogens causing blight and pustule diseases. Pseudomonas syringae pv. glycinea produce several toxins like coronatine, faseolotoxine, siringomicine, tabtoxine and proteins of nucleation ice that contribute to the develop of chlorosis and necrosis, characteristic of bacterial blight. It also produces levan and alginate, cellulose and biofilm. Pustule disease is caused by Xanthomonas axonopodis pv glycines, which produce enzymes like α-amilase, protease, endo β-mannanase, peptolitic activity, xanthan and biofilm. Nowadays the control of phytopathogenic bacteria consists in the application of pesticides that are toxic for the environment and man. Natural products from medicinal plants are a new alternative for the treatment of phytopathogens. Researches made with phytochemical compounds (flavonoids, phenols, quinones, cummarines, essential oils, terpenes) support the antimicrobial activity of these natural products. What is more, these substances could suppress the biofilm, exoproteins and exopolisaccharides formation and release of them. The infections caused by phytopathogens provoke economical loses and its control presents big difficulties in our country. The proposal of this work is the characterization of phytopatoghenic strains, its virulence factors and the role they play in the disease process. The development of a new alternative for the control of vegetable bacteriosis using natural products obtained from aromatic plants and the correct application of them on sown fields or on seeds is also an objective in this work.

Caracterización, procesamiento e industrialización del grano de quinoa, cultivado en la provincia de Córdoba - Argentina. Characterization, processing and industrialization of quinoa seed harvested in the province of Córdoba - Argentina

La quinoa (Chenopodium quinoa Willd), es un pseudocereal originario de la región Andina. Fue utilizada como alimento básico por los pueblos nativos. La quinoa, la papa y el maíz constituyeron el trinomio base de la alimentación indígena de este continente. La colonización española fue desplazando su cultivo a favor del trigo europeo y otros cereales, quedando reducida a las zonas altas de la región andina. La Quínoa ha adquirido una considerable atención en los últimos tiempos, principalmente por la calidad de sus proteínas y la ausencia de gluten en ella. Su empleo está ampliamente difundido en los países andinos, especialmente Bolivia y Perú, con un notable crecimiento de la superficie sembrada. En nuestro país la explotación de este cultivo se ubica principalmente en las provincias norteñas de Salta y Jujuy. En estos últimos años se ha reivindicado su cultivo y los granos privados de saponinas son considerados como un excelente alimento, reconocido por la OMS, la FAO y la NASA. Además de la calidad de sus lípidos y vitaminas, y al elevado contenido en almidón, la quinoa posee una proteína de excelente calidad nutricional y libre de gluten, lo que hace a este grano especialmente indicado para la alimentación de personas que sufren de la enfermedad celíaca o del síndrome de intestino irritado. El presente proyecto está orientado al aprovechamiento integral del grano de quinoa. Es nuestra intensión aquí, demostrar que dicho grano, cultivado en la provincia de Córdoba, permitirá elaborar productos alimenticios asi como también derivados de su industrialización. Para este objetivo se cuenta con las instalaciones de la Planta Piloto del Instituto de Ciencia y Tecnología de los Alimentos (ICTA), de la UNC, así como de intrumental moderno y acorde, como HPLC, GC, Espectrofotómetro UV-Vis, rotavapores de laboratorio e industrial, cámara fría, balanzas analíticas y de precisión, muflas, estufas, molinos y tamices, así como también, contamos con profesionales, algunos de ellos realizando su tesis doctoral en este tema. En cuanto a los objetivos que se persiguen, se espera obtener productos tales como sopas, papillas, productos para panadería y galletería y salsas. En el plano industrial, se pretende elaborar concentrados proteicos, almidón y saponinas. Como se dijo más arriba, a nivel internacional la quinoa ha comenzado a extender sus fronteras, y es así que hoy el principal productor mundial de este grano, Bolivia, destina un porcentaje importante de su producción a la exportación. La creciente demanda mundial de quinoa a hecho que se constituya en un cultivo estratégico y de alto valor, con precios internacionales que rondan los U$S 1200 la tonelada. Si a esto unimos que la planta presenta una gran resistencia a la sequía, que se adapta bien a terrenos salitrosos, arenosos y pobres, podemos comprender la importancia que adquiere para nuestra provincia, toda vez que en la misma existen zonas geográficas potencialmente aptas para su cultivo. Quinoa (Chenopodium quinoa Willd) is a pseudocereal originating in the Andean region. It was used as a staple food by native peoples. Quinoa, potatoes and corn were the tree most important indigenous staple food to this part of South America. Spanish colonization was marginalized cultivation in favor of European wheat and other grains, displacing it to the highlands of the Andean region. Quinoa has recently gained considerable attention, mainly by its protein quality and lack of gluten. Its use is widespread in the Andean countries, especially Bolivia and Peru, with a notable increase in plantings. In our country, the exploitation of this crop is located mainly in the northern provinces of Salta and Jujuy. In recent years its cultivation has been promoted, and the grains once free of saponins are considered an excellent food, recognized by WHO, FAO and NASA. In addition to its lipid and vitamins, and high starch contain, quinoa protein has an excellent nutritional value and it is free of gluten, making it particularly suitable for this grain to feed people with celiac disease or irritable bowel syndrome. This project aims at an integral development of quinoa grain. It is our intention here to demonstrate that this grain grown in the province of Córdoba, can produce food products resulting from local industrialization. This team has access to the facilities of the Pilot Plant of the Institute of Science and Food Technology (ICTA) of the UNC, and the modern equipments in it, as HPLC, GC, UV-Vis spectrophotometer, laboratory and industrial rotary evaporators, cold storage, analytical and precision balances, flasks, ovens, grinders and screens. Also, we have an important professional staff, some of them doing their thesis on this subject. With regard to the objectives pursued, we expect to obtain products such as soups, baby food, bakery products and biscuits and sauces. At the industrial level, it aims at producing protein concentrates, starch and saponins.

A comparative study of the physical, chemical and microbiological status of soils, as influenced by conventional and reduced tillage

Historically, shifts to reduced and no-tillage management for production of crops were fostered by needs to decrease soil erosion and loss of organic matter, reduce fuel and labour costs and conserve soil water, as compared with conventional fallow tillage management. Recent interest in maintaining soil quality has been stimulated by a renewed awareness of the importance of soil condition to both the sustainability of agricultural production systems and environmental quality (Doran and Parkin, 1996). The aim of this project was to determine the impact on the physical, chemical and microbiological status of the soil of conventional and reduced tillage. It has been suggested that the reduced soil disturbance associated with the tine cultivator improves soil structure, increases nutrient content in the top 10cm of soil, increases microbial activity and improves physical characteristics. From this study it was determined that the environmental benefits linked to reduced tillage in literature, did not develop in the first two years of this programmes implementation. The results of this study determined that soil nutrients did not increase in concentration in the top 10 cm of soil under reduced cultivation. The only exception was exchangeable potassium. As potassium is not a mobile nutrient its movement is dependent on soil disturbance, therefore under reduced cultivation its concentration was allowed to accumulate in the upper horizon of the soil profile. Microbial activity was greater in the conventionally tilled treatments, as determined by total aerobic bacterial numbers. This could be due to the increased rates of soil aeration in this treatment. Numbers of aerobic bacteria were greater in the conventional tillage treatments at both incubation temperatures of 22 and 32° C. The physical characteristics of the soil determined, indicate that below the depth of soil cultivation, cone penetration resistance increases. Therefore the reduced cultivation treatments would be more prone to soil compaction, higher in the soil profile.