Dissertations / Theses on the topic 'Bombicola'

Sophorolipid production was studied using Candida bombicola ATCC 22214. Solid and liquid alkanes or fatty acids were used as lipophilic substrates in shake flask experiments. With a few of these substrates, there was a significant amount of direct incorporation into the sophorolipid products. Of all substrates tested, only hexadecane and heptadecane resulted in the exclusive production of crystalline, diacetylated lactone sophorolipids that contained a single type of hydroxy fatty acid. It was determined that the presence of a single type of sophorolipid structure was necessary to obtain crystalline products.
It was also possible to obtain the crystalline product using larger scale experiments with hexadecane. The direct incorporation of hexadecane was shown to result in increased sophorolipid production efficiency with hexadecane being used as the sole source of the hydroxy fatty acids needed for sophorolipid production and the hydrophilic substrate being used as a source of energy. With the other lipophilic substrates, this efficiency was lost because there was appreciable de novo synthesis of the fatty acids.

The production of sophorolipids from the yeast Candida bombicola (ATCC 22214) was studied. Heptadecane resulted in the highest percentage of direct incorporation into the sophorolipid products of all the hydrocarbon substrates used. The diacetylated lactone form of the sophorolipid product was favoured when the hydroxycarboxylic acid was sixteen or seventeen carbon atoms long. Crystalline product was only obtained when a high degree of uniformity of both the type of sophorolipid and the number of carbon atoms in the fatty acid part of this lipid was achieved. These conditions were only met when the hydrocarbon substrate was hexadecane or heptadecane and the specific products in these cases were diacetylated lactones.
The surface properties of the sophorolipids were analyzed using a modified evaporating drop technique. The lactone sophorolipids were more effective at reducing the surface tension of water than the acid sophorolipids. This could be part of the explanation for the fact that the lactones have been shown to be more effective than the acid form of the sophorolipids for several different applications.

The yeast Candida bombicola (ATCC 22214) was grown in the self-cycling fermentor (SCF). A medium with both glucose and hexadecane was used to examine sophorolipid production. The sophorolipid was collected in a crystalline form. A modified control scheme that monitored the rate of dissolved oxygen (DO) consumption was successfully implemented to account for the weak or nonexistent minima in the DO.
The effect of harvesting with the circulation pump off was also examined. An improvement in the usage of hexadecane due to increased carry over to subsequent cycles was observed. Production rates of sophorolipid were increased due to the higher initial hexadecane concentrations.
Harvesting with the pump off also allowed the sophorolipid to precipitate, which improved the recovery of product. The yield of sophorolipid (g sophorolipid/g hexadecane consumed) was found to be inversely proportional to the initial sophorolipid concentration.

Tall oil, a mixture of long-chain oleic and linoleic acids, was found to be a suitable lipophilic substrate for the production of sophorolipid, an extracellular glycolipid surfactant, by Candida bombicola ATCC 22214. Oxygen limitation was found to decrease sophorolipid production in this system. Partitioning experiments with sophorolipid revealed that the surfactant increases the affinity of oleic acid for the aqueous phase of a two phase system. C. bombicola therefore has potential applications in the remediation of pulp and paper effluent containing recalcitrant long-chain fatty acids; both for its ability to degrade the acids and for the tendency of sophorolipid to make fatty acids more bioavailable to organisms involved in downstream activated sludge treatment. A novel capillary gas chromatography method was developed to rapidly and quantitatively analyze long-chain fatty acids.

Nombreux sont les microorganismes connus capables de synthétiser des caroténoi͏̈des d'intérêt industriel. Ce travail s'intéresse essentiellement à la production d'un pigment, peu étudié et non commercialisé, par une espèce de levure également peu connue, à partir d'un milieu de culture peu coûteux. Dans ce cadre, l'étude réalisée comprend trois parties: la première décrit la stratégie suivie pour l'isolement et l'identification d'une nouvelle souche de Sporobolomyces : SP. Ruberrimus H110. La seconde partie concerne l'extraction et l'identification des pigments synthétisés par cette souche. L'efficacité de méthodes, soit chimiques, soit physiques, pour la déstructuration des parois de la levure a été déterminée. Parmi ces méthodes, le broyage mécanique suivi d'une récupération-dissolution des pigments par l'éthanol a montré les meilleurs résultats. Toutefois, la récupération des pigments s'accompagne d'une extraction de lipides polyinsaturés qui peuvent être éliminés. L'identification de ces pigments a été validée après la mise au point d'une méthode analytique précise mettant en jeu un couplage C. L. H. P. -U. V. / A. P. C. I. -S. M. Et par l'utilisation de l'I. R. T. F. ; il s'agit de la torularhodine et du β-carotène. Dans la troisième partie, les études cinétiques au cours desquelles l'influence de la température et de la concentration de sulfate d'ammonium (source d'azote) notamment, ont montré que la torularhodine accumulée dans les cellules est très largement majoritaire avec plus de 95 %. Sans être optimales, les concentrations de torularhodine obtenues sont importantes; elles peuvent atteindre 3,7 mg. G-1 de levure soit environ 130 mg. L-1 à partir de glycérol technique (substrat carboné)
Many microorganisms are able to synthesize carotenoids for industrial using. The aim of the present work is to study the production of an uncommon red pigment by a quasiunknown yeast growing in an inexpensive liquid medium. This study is divided mainly into three parts: the first one describes how a new strain of Sporobolomyces, SP. Ruberrimus H11O, was isolated and identified. The second part is dealing with the breaking of the cell wall when using different chemical and physical methods. Among the methods tested, the crushing of the cells was the most efficient to recover the pigment by further ethanol extraction. But a low part of cellular poly-unsatured fatty acids are extracted too and needs to be separated. The carotenoids were identified as torularhodin and β-carotene with the aid of an analytical method specially developed using a H. P. L. C. -U. V. / A. P. C. I. -M. S. Tandem and co-use F. T. I. R. In the last part, after the carbon source choice done, the kinetic parameters of the yeast growth and pigment production were determined when varying temperature or ammonium sulphate (nitrogen source) notably. Thus, technical glycerol is uptaken by the new strain to produce about 35 g dry cells L-1 and 3. 7 mg torularhodin g-1 of cells that is to say 130 mg. L-1 in non-optimal culture conditions. One of the interests of this strain use is that torularhodin represents about 95 % of total carotenoids

The Self-Cycling Fermentation (SCF) technique and an agar diffusion assay were used to follow the production of lipase by Candida bombicola. Repeatable lipase production profiles were determined for two different media. Each medium gave a considerably different profile.
When the broth contained oil, 60 to 80% of the lipase activity remained after freezing. All activity was lost when the broth did not contain oil.
The lipase was found to be associated with the cell. Up to a certain concentration of lipase, the presence of agar caused the lipase to disassociate from the cell and diffuse into the agar. Beyond this concentration of lipase the agar was found to saturate.
A new technique of level control in the cyclone reactor was used. A pressure transducer was found to be an accurate and inexpensive device for level control.

Mestrado em Biotecnologia
Sophorolipids are a type of biosurfactants produced by several microorganisms, including the yeast Starmerella bombicola. They find application mainly due to their emulsifying activity and antimicrobial properties. Sophorolipids produced by Starmerella bombicola are composed by one sophorose molecule and a hydroxylated fatty acid of 16 or 18 carbon atoms. One or two acetylations can occur, one on each glucose, and a lactonization as well. Despite the interesting characteristics of classic sophorolipids produced by the referred yeast, there are indications that sophorolipids with branched a hydrophobic tail have higher activity at lower temperatures. These sophorolipids are produced by Rhodotorula bogoriensis, however the yields obtained with this yeast are low. By producing these sophorolipids in Starmerella bombicola, the yields obtained could be higher. Furthermore, the production of these novel sophorolipids will allow broadening of the biosurfactants applications. This thesis describes two approaches to produce sophorolipids with a branched hydrophobic tail: use of Guerbet alcohols as substrates for the Starmerella bombicola culture, and the introduction of genes that will take care of the in-chain hydroxylation of the sophorolipids. Guerbet alcohols are branched molecules, and are expected to be incorporated in the sophorolipids molecules. The genes to be used are from Elizabethkingia meningoseptica and Rhodococcus rhodochrous and encode for an oleate hydratase, an enzyme responsible for the conversion of oleic acid into 10-hydroxystearic acid.
Soforolípidos são um tipo de biosurfactantes produzidos por vários microorganismos, incluindo a levedura Starmerella bombicola. As suas principais aplicações devem-se à sua sua atividade emulsificante e propriedades antimicrobianas. Soforolípidos produzidos por Starmerella bombicola são compostos por uma molécula de soforose e um ácido gordo hidroxilado de 16 ou 18 átomos de carbono. Apesar das interessantes caraterísticas dos soforolípidos clássicos produzidos pela levedura referida, há indicações de que os soforolípidos com cauda hidrofóbica ramificada têm maior atividade a temperaturas mais elevadas. Estes soforolípidos são produzidos por Rhodotorula bogoriensis, no entanto o rendimento obtido por esta levedura é baixo. Ao produzir estes soforolípidos em Starmerella bombicola, o rendimento obtido poderá ser superior. A produção destes soforolípidos irá também permitir o alargamento das aplicações dos biosurfactantes. Esta tese descreve duas medidas para a produção de soforolípidos com cauda hidrofóbica ramificada: uso de álcoois de Guerbet como substrato para a cultura de Starmerella bombicola, e a introdução de genes que serão responsáveis pela hidroxilação no meio da cadeia hidrofóbica dos soforolípidos. Álcoois de Guerbet são moléculas ramificadas, sendo esperado a sua incorporação nas moléculas de soforolípidos. Os genes usados são provenientes de Elizabethkingia meningoseptica e Rhodococcus rhodochrous, e codificam para uma oleate hidratase, enzima responsável pela conversão do ácido oleico no ácido 10-hidroxistearico.

L'etude d'une fermentation produisant un biosurfactant glycolipidique, les sophorolipides de candida bombicola a ete effectuee. Dans des conditions optimisees mettant en uvre une alimentation controlee en glucose et en un substrat lipidique, les esters ethyliques de l'huile de colza, des concentrations de sophorolipides superieures a 300 g. L##1 ont ete obtenues. On decrit dans une premiere partie, la cinetique et le bilan detailles de cette fermentation ou le produit, apparaissant en limitation azotee apres la croissance de la levure, se separe du milieu aqueux. Une deuxieme partie concerne la mise au point d'une technique permettant la determination qualitative et quantitative des sophorolipides individuels (20 a 30 composes) produits. La chromatographie liquide haute performance avec elution par un gradient eau/acetonitrile associee a un detecteur evaporatif a diffusion de lumiere a permis d'obtenir ce resultat. L'identification des composes a mis en uvre un ensemble de techniques variees comme diverses conversions chimiques des composes, chromatographie en phase gazeuse, spectrometrie de masse et resonance magnetique nucleaire. Dans une troisieme partie, l'outil analytique developpe a ete utilise pour le suivi de la composition en sophorolipides de fermentations effectuees dans differentes conditions. Deux points ont ete etudies: l'influence du mode de conduite de la fermentation (conditions physico-chimiques, mode d'apport des sources de carbone); effet de la nature du substrat lipidique (n-alcanes, huiles vegetales, esters d'acides gras). Des conclusions relatives au mode de biosynthese des sophorolipides en sont tirees

El present treball representa la continuació a la línia de recerca per a la producció de soforolípids (SLs) per fermentació en estat sòlid (FES) que va ser inciada en nuna tesi aneterior. Els SLs són un tipus de surfactant biològic produït per microorganismes i representa una gran alternativa per a la substitució dels surfactants químics, els quals presenten una gran demanda. Els estudis es van realitzar per FES utilitzant com a substrats residus agroindustrials (residu de winterització de l’oli de gira-sol i melassa de remolatxa sucrera) i el llevat Starmerella bombicola. La primera etapa de la investigació va consistir en l’avaluació de 9 materials de diferent naturalesa (biològics i inerts) per a ser utilitzats com a suports per a la producció d’SLs per FES a una escala de 0.5 L. Els materials biològics (lignocel·lulòsics) van presentar millors resultats en comparació als materials inerts. La palla de blat (PT) va resultar ser el suport amb la major producció de SLs (0.20 gSL g-1MS), el qual va ser seleccionat per a la segona etapa relacionada a l’avaluació de la producció de SLs a escales més grans. Així mateix, es va comprovar que és possible produir SLs utilitzant altres residus anàlegs el residu de winterització, però, cal tenir en compte el contingut d’impureses dels mateixos i el procés de recuperació del compost per evitar les interferències d’aquestes mateixes en la qualitat final del producte. L’avaluació de l’escalat de la producció de SLs es va dur a terme en dos reactors de diferent grandària (22 i 100 L), on es va estudiar l’efecte de la temperatura durant el procés i la producció de SLs. Es va observar que mitjançant una temperatura ambient controlada i la implementació d’estratègies de ventilació la temperatura pot ser controlada. Així mateix, es va implementar un procés de diferents cicles d’esterilització que va permetre la reducció del creixement d’altres microorganismes dins el reactor, els quals podrien interferir en el procés metabòlic del llevat per produir SLs. Els resultats van demostrar que el llevat té la capacitat de produir SLs tot i que es modifiquin les seves condicions òptimes de temperatura. Finalment, com a part d’una estada de recerca que es va realitzar a l’Institut Tecnològic d’Estudis Superiors de Monterrey (ITESM) a Monterrey, Nuevo León, Mèxic, es van avaluar dos diferents tecnologies per a l’extracció i recuperació de SLs com a alternativa per a la substitució al mètode convencional amb acetat d’etil. Es van estudiar els sistemes de dues fases aquoses (ATPs) i l’extracció amb fluids supercrítics (FSC). L’extracció amb ATPs va permetre incrementar el rendiment de SLs de 0.20 a 0.34 gSL g-1MS mentre que amb FSC el rendiment va ser de 0.13 gSL g-1MS. Els resultats demostren que hi ha alternatives amigables amb el medi ambient i amb potencial per extreure SLs produïts a partir del procés de FES. Aquesta tesi representa un camí per explorar a més detall altres estratègies per a la producció, recuperació i purificació de SLs mitjançant FES i continuar amb la línia d’investigació que es va iniciar en el Grup de Recerca en Compostatge (GICOM) del Departament d’Enginyeria Química, Biològica i Ambiental de la Universitat Autònoma de Barcelona.
El presente trabajo representa la continuación a la línea de investigación iniciada en una tesis anterior, para la producción de soforolípidos (SLs) por fermentación en estado sólido (FES). Los SLs son un tipo de surfactante biológico producido por microorganismos y representa una gran alternativa para la sustitución de los surfactantes químicos, los cuales presentan una gran demanda. Los estudios se realizaron por FES utilizando como sustratos residuos agroindustriales (residuo de winterización del aceite de girasol y melaza de remolacha azucarera), y su fermentación por una cepa de la levadura Starmerella bombicola. La primera etapa de esta investigación consistió en la evaluación de 9 materiales de diferente naturaleza (biológicos e inertes) para ser utilizados como soportes para la producción de SLs por FES a una escala de 0.5 L. Los materiales biológicos (lignocelulósicos) presentaron mejores resultados en comparación a los materiales inertes. La paja de trigo (PT) resultó ser el soporte con la mayor producción de SLs (0.20 gSL g-1MS), el cual fue seleccionado para la segunda etapa relacionada a la evaluación de la producción de SLs a escalas mayores. Así mismo, se comprobó que es posible producir SLs utilizando otros residuos análogos al residuo de winterización, sin embargo, es necesario tomar en cuenta el contenido de impurezas de los mismos y el proceso de recuperación del compuesto para evitar las interferencias de estas mismas en la calidad final del producto. El escalamiento de la producción de SLs se llevó a cabo en dos reactores de diferente tamaño (22 y 100 L), en donde se estudió el efecto de la temperatura durante el proceso y la producción de SLs. Se observó que mediante una temperatura ambiente controlada y la implementación de estrategias de aireación la temperatura puede ser controlada. Así mismo, se implementó un proceso de diferentes ciclos de esterilización que permitió disminuir el crecimiento de otros microorganismos dentro del reactor, los cuales podrían interferir en el proceso metabólico de la levadura para producir SLs. Los resultados demostraron la levadura tiene la capacidad de producir SLs a pesar de que se modifiquen sus condiciones óptimas de temperatura. Finalmente, como parte de una estancia de investigación que se realizó en el Instituto Tecnológico de Estudios Superiores de Monterrey (ITESM) en Monterrey, Nuevo León México se evaluaron dos diferentes tecnologías para la extracción y recuperación de SLs como alternativa para la sustitución al método convencional con acetato de etilo, se estudiaron los sistemas de dos fases acuosas (ATPS) y la extracción con fluidos supercríticos (FSC). La extracción con ATPS permitió incrementar el rendimiento de SLs de 0.20 a 0.34 gSL g-1MS, mientras que con FSC el rendimiento fue de 0.13 gSL g-1MS. Los resultados demuestran que existen alternativas amigables con el medio ambiente y con potencial para extraer SLs producidos a partir del proceso de FES. Esta tesis representa un camino para explorar a mayor detalle otras estrategias para la producción, recuperación y purificación de SLs mediante FES y continuar con la línea de investigación que se inició en el Grupo de Investigación en Compostaje (GICOM) del Departamento de Ingeniería Química, Biológica y Ambiental de la Universitat Autònoma de Barcelona.
The present work represents the continuation of the research line for the production of sophorolipids (SLs) by solid state fermentation (FES), that was started in a previous PhD thesis. SLs are a type of biological surfactant produced by microorganisms and represent a great alternative for the substitution of chemical surfactants, which are in great demand. The studies were carried out by FES using agro-industrial residues (winterization oil cake and sugar beet molasses) as substrates, and its fermentation with the strain of yeast Starmerella bombicola. The first stage of this investigation consisted in the evaluation of 9 materials of different nature (biological and inert) to be used as supports for the production of SLs by FES at a 0.5 L scale. The biological materials (lignocellulosic) presented better results in comparison to inert materials. Wheat straw (PT) resulted the support with the highest production of SLs (0.20 gSL g-1MS), which was selected for the second stage related to the evaluation of the production of SLs at larger scales. Likewise, it was found that it is possible to produce SLs using other residues analogous to the winterization residue, however, it is necessary to take into account their impurity content and the compound recovery process to avoid their interferences in the final quality of the product. The scaling of the production of SLs was carried out in two reactors of different sizes (22 and 100 L), where the effect of temperature during the process and the production of SLs was studied. It was observed that through a controlled room temperature and the implementation of aeration strategies, the temperature can be controlled. Likewise, a process of different sterilization cycles was implemented that allowed the growth of other microorganisms within the reactor to be reduced, which could interfere in the metabolic process of the yeast to produce SLs. The results demonstrated that the yeast has the ability to produce SLs despite its optimal temperature conditions being modified. Finally, as part of a research stay that was carried out at the Instituto Tecnológico de Estudios Superiores de Monterrey (ITESM) in Monterrey, Nuevo León México, two different technologies for the extraction and recovery of SLs were evaluated as an alternative to replace the conventional method. with ethyl acetate, two phase aqueous systems (ATPS) and supercritical fluid extraction (FSC) were studied. Extraction with ATPS allowed to increase the performance of SLs from 0.20 to 0.34 gSL g-1MS, while with FSC the yield was 0.13 gSL g-1MS. The results show that there are environmentally friendly alternatives with the potential to extract SLs produced from the FES process. This thesis represents a way to explore in greater detail other strategies for the production, recovery and purification of SLs by means of FES and to continue with the line of research that began in the Composting Research Group (GICOM) of the Department of Chemical, Biological Engineering and Environmental of the Autonomous University of Barcelona.
Universitat Autònoma de Barcelona. Programa de Doctorat en Ciència i Tecnologia Ambientals

This diploma thesis deals with the microbial production of sophorolipids by the Starmerella genus yeasts. The theoretical part of the thesis includes general characteristics of biosurfactants with the focus on sophorolipids. There are described the options of biotechnological production of sophorolipids and fields of their possible applications. Furthermore, the theoretical part deals with the describtion of Starmerella bombicola yeast, which is often used for biotechnological production of sophorolipids. Six strains of the Starmerella genus were cultured in the basic medium suitable for sophorolipid produsction. The amount of produced sophorolipids was tested by the emulsification capacity assay, solubilization of crystalline anthracene assay, measuring the surface tension by the Du-Noüy-Ring method and determination of the sophorolipid concentration by extraction with ethyl acetate. The purity of the extracted sophorolipids was inspected by the Fourier Transform infrared spectrosopy (FT-IR) Based on the results, the Starmerella bombicola CBS 6009 and the Starmerella anomalae CBS 14178 strains were studied in greater detail. They were cultured in several production media of different composition and the effect of the individual components on the ability of the sophorolipid production was monitored. Based on the results, it was evaluated that the composition of the medium has a great influence on the production ability of the strains. The highest production rate of sophorolipids was achieved in the basic production medium and so was in the medium containing molasses and Indian waste oil. The cultivation mode has great effect on the sophotolipid production rate. It has been found that when cultured in a bioreactor, the strains produced sophorolipids in a larger amount and of a different quality than in the shaker flasks.

Os soforolipídios são biossurfactantes compostos por uma sefarose ligada a um ácido graxo. As suas propriedades tensoativas possibilitam um amplo potencial de aplicação. Esses compostos são sintetizados por diversas cepas do gênero Candida na presença de uma fonte hidrofílica e hidrofóbica de carbono. Neste trabalho foi avaliada a produção de soforolipídios por Candida bombicola ATCC2214 utilizando o melaço de cana de açúcar, caldo de cana, sacarose ou glicose como fonte hidrofílica e a gordura de frango ou óleo de girassol como fonte hidrofóbica. A composição da melhor combinação foi otimizada utilizando um Box-Behnken. Os soforolipídios produzidos foram caracterizados estruturalmente por espectroscopia de infravermelho com transformada de Fourier (FTIR) e por ressonância magnética nuclear (RMN). Foram determinadas propriedades tensoativas: tensão superficial, concentração micelar crítica (CMC) e índice de emulsificação. Os soforolipídios produzidos foram aplicados na biorremediação de solos contaminados com óleo lubrificante, juntamente com consórcio bacteriano comercial (Bacillus subtilis e Bacillus licheniformis). A melhor produção de soforolipídios foi de 16,83 g L-1 em gordura de frango com glicose. A condição otimizada para a produção dos SLP foi 75 g L-1 de gordura de frango, 77,5 g L-1 de glicose, 2,5 g L-1 de extrato de levedura e sem ureia, atingindo 41,63 g L-1. A composição dos soforolipídios produzidos são da forma lactônica diacetilada, com capacidade de diminuir a tensão superficial da água de 70 mN/m para de 35 mN/m, CMC de 65 mg L-1 e mostraram capacidade emulsificante em óleo lubrificante, n-heptano e tolueno. Os resultados obtidos sugerem que esses compostos possuem potencial na aplicação em biorremediação de solos contaminados. Os soforolipídios ao serem adicionados ao consórcio bacteriano (Bacillus subtilis e Bacillus licheniformis) apresentaram degradação mais alta do que apenas com os microrganismos comerciais. A melhor eficiência de biodegradabilidade do óleo lubrificante foi de 4%, onde foi adicionado 1 g de soforolipídios e 4g do consórcio bacteriano por Kg de solo.
The sophorolipidis are biosurfactants composed of a sepharose linked to a chain hydroxy fatty acid. Their surface-active properties allow a wide application potential. The sophorolipids are synthesized by several strains of Candida in the presence of hydrophilic and hydrophobic sources. In this work, was evaluated the production of sophorolipids produced by Candida bombicola ATCC2214, using sugar cane molasses, cane juice, sucrose or glucose as hydrophilic source and the chicken fat or sunflower oil as hydrophobic source. The composition of the best combination was optimized using a Box-Behnken design. The sophorolipids produced were characterized by Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). The surface-active properties such as surface tension, critical micelle concentration (CMC) and emulsification index were also determined. Finally, the sophorolipidis were applied in the bioremediation lubricating oil contaminated soils by increasing the availability of this contaminant to be degraded by commercial bacterial consortium (Bacillus subtilis and Bacillus licheniformis). The highest yield was 16,83 g L-1 in chicken fat with glucose. The optimal condition get 41,63 g L-1 of sophorolipids in 75 g L-1 of chicken fat, 7,5 g L-1 of glucose, 2,5 g L-1 of yeast extract and without urea. The sophorolipids produced were diacetilada lactonic form, capable of reducing the surface tension of water from 70 mN / m to 35 mN / m, CMC 65 mg L-1 and showed to be good emulsifiers for lubricating oil, n-heptane and toluene. The results suggest that these glycolipids have the great potential to be used in bioremediation of contaminated soils. The sophorolipids together with the bacterial consortium showed the highest degradation of the lubricating oil when compared to the commercial microorganisms. The best efficiency of biodegradability of the lubricating oil was 4% by adding 1 g Kg-1 of soforolipídios and 4 g Kg-1 of bacterial consortium.

The present study investigated the feasibility of production of sophorolipids (SLs) using yeast Candida bombicola grown on hydrolysates derived lignocellulosic feedstock either with or without supplementing oil as extra carbon source. Several researchers have reported using pure sugars and various oil sources for producing SLs which makes them expensive for scale-up and commercial production. In order to make the production process truly sustainable and renewable, we used feedstocks such as sweet sorghum bagasse, corn fiber and corn stover. Without oil supplementation, the cell densities at the end of day-8 was recorded as 9.2, 9.8 and 10.8 g/L for hydrolysate derived from sorghum bagasse, corn fiber, and corn fiber with the addition of yeast extract (YE) during fermentation, respectively. At the end of fermentation, the SL concentration was 3.6 g/L for bagasse and 1.0 g/L for corn fiber hydrolysate. Among the three major sugars utilized by C. bombicola in the bagasse cultures, glucose was consumed at a rate of 9.1 g/L-day; xylose at 1.8 g/L-day; and arabinose at 0.98 g/L-day. With the addition of soybean oil at 100 g/L, cultures with bagasse hydrolysates, corn fiber hydrolysates and standard medium had a cell content of 7.7 g/L; 7.9 g/L; and 8.9 g/L, respectively after 10 days. The yield of SLs from bagasse hydrolysate was 84.6 g/L and corn fiber hydrolysate was15.6 g/L. In the same order, the residual oil in cultures with these two hydrolysates was 52.3 g/L and 41.0 g/L. For this set of experiment; in the cultures with bagasse hydrolysate; utilization rates for glucose, xylose and arabinose was recorded as 9.5, 1.04 and 0.08 g/L-day respectively. Surprisingly, C. bombicola consumed all monomeric sugars and non-sugar compounds in the hydrolysates and cultures with bagasse hydrolysates had higher yield of SLs than those from a standard medium which contained pure glucose at the same concentration. Based on the SL concentrations and considering all sugars consumed, the yield of SLs was 0.55 g/g carbon (sugars plus oil) for cultures with bagasse hydrolysates. Further, SL production was investigated using sweet sorghum bagasse and corn stover hydrolysates derived from different pretreatment conditions. For the former and latter sugar sources, yellow grease or soybean oil was supplemented at different doses to enhance sophorolipid yield. 14-day batch fermentation on bagasse hydrolysates with 10, 40 and 60 g/L of yellow grease had cell densities of 5.7 g/L, 6.4 g/L and 7.8 g/L, respectively. The study also revealed that the yield of SLs on bagasse hydrolysate decreased from 0.67 to 0.61 and to 0.44 g/g carbon when yellow grease was dosed at 10, 40 and 60 g/L. With aforementioned increasing yellow grease concentration, the residual oil left after 14 days was recorded as 3.2 g/L, 8.5 g/L and 19.9 g/L. For similar experimental conditions, the cell densities observed for corn stover hydrolysate combined with soybean oil at 10, 20 and 40 g/L concentration were 6.1 g/L, 5.9 g/L, and 5.4 g/L respectively. Also, in the same order of oil dose supplemented, the residual oil recovered after 14-day was 8.5 g/L, 8.9 g/L, and 26.9 g/L. Corn stover hydrolysate mixed with the 10, 20 and 40 g/L soybean oil, the SL yield was 0.19, 0.11 and 0.09 g/g carbon. Overall, both hydrolysates supported cell growth and sophorolipid production. The results from this research show that hydrolysates derived from the different lignocellulosic biomass feedstocks can be utilized by C. bombicola to achieve substantial yields of SLs. Based upon the results revealed by several batch-stage experiments, it can be stated that there is great potential for scaling up and industrial scale production of these high value products in future.

The use/valorization agro-industrial waste is a priority worldwide. The objective of this work was a novel valorization of white grape pomace (WGP), a lignocellulosic waste feed-stock rich in carbohydrates (around 42 wt. % of free soluble sugars) and oil (around 13 wt. %). The objective was to use the WGP free sugars and oil as carbon sources for the production of sophorolipids (SL) by Starmerella bombicola, a yeast species known for thriving in sugar-rich environments and being fructophilic, while equally comfortable with the presence of oils in growth medium. The goal was to produce sophorolipids (SL), bio-surfactants known to be biodegradable, biocompatible and with high added value surfactant characteristics, which also exhibit anti-tumor and antimicrobial activity. Free sugars of WGP were obtained by water extraction and oils of WGP was obtained by supercritical CO2 extraction and also by Soxhlet extraction. Yeast growth experiments led to a maximum sophorolipid yield of 34.9 g/L with a medium containing both WGP free carbohydrates and oil. Sophorolipids were purified by n-hexane extraction and characterized using LC-MS, 1H-NMR and surface tension. Both types of sophorolipids, lactonic and acidic, with both linoleic acid and oleic acid moieties, were detected in LC-MS analysis. These moieties were the most abundant in WGP oil: linoleic acid (62 %) and oleic acid (17 %). 1H-NMR also detected the presence of C18 hydrocarbon chain lengths and sophorose. The SL obtained had a minimum surface tension (MST) of 35.62 mN/m for a critical micellar concentration (CMC) of 53.79 mg/L.

Tese de mestrado em Microbiologia Aplicada, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018
A frutofília é uma característica pouco comum entre os microrganismos, que consiste na preferência de frutose como fonte de carbono e energia. Em leveduras, até agora, esta característica foi reportada no género Zygossacharomyces e no clado Wickerhamiella/Starmerella (W/S). O comportamento frutofílico está relacionado com a presença do transportador de frutose, Ffz1, porém, o papel da frutose no metabolismo destas leveduras ainda não está completamente elucidado. Uma das leveduras que se destaca no clado W/S é St. bombicola não só pela facilidade em manipular esta espécie geneticamente, mas também por ser produtora de elevadas quantidades de soforolípidos. Estes são biosurfactantes que têm características favoráveis do ponto de vista ambiental e, são aplicados em áreas como a cosmética e produtos de limpeza. Além disso, St. bombicola assim como outras espécies pertencentes ao clado W/S foram reportadas como produtoras de elevadas quantidades de manitol a partir de frutose através da enzima manitol desidrogenase (Mtdh) utilizando NADPH como cofator, regenerando assim NADP+. Tendo em conta que a produção de manitol é uma via metabólica dependente de frutose, o objetivo desta tese foi tentar perceber qual o papel do manitol na levedura St. bombicola. Como St. bombicola possui dois genes que codificam para duas desidrogenases do manitol foram utilizados três mutantes de deleção previamente construídos: um mutante onde o gene que codificava para a enzima Mtdh1 (mtdh1Δ) foi interrompido, outro para a enzima Mtdh2 (mtdh2Δ) e, finalmente um mutante duplo (mtdh1Δmtdh2Δ). A estirpe selvagem e mutantes de deleção foram cultivados, num meio onde o comportamento frutofílico é visível designado por 20FG (contendo 10 % (w/v) de frutose e 10 % (w/v) de glucose) onde a produção de manitol foi estudada. Não foi detetada produção de manitol pelos mutantes mtdh1Δ e mtdh1Δmtdh2Δ, porém foi detetada produção de manitol semelhante à estirpe selvagem pelo mutante mtdh2Δ, o que permitiu inferir que a principal enzima responsável pela conversão de frutose em manitol é a Mtdh1. Curiosamente, quando crescidos nesse mesmo meio a várias temperaturas (25 ºC, 30 ºC e 32.5 ºC), os mutantes de deleção (mtdh1Δ e mtdh1Δmtdh2Δ) não atingiam as mesmas densidades celulares que a estirpe selvagem às temperaturas mais elevadas. Além disso, excluímos também a possibilidade de os mutantes de deleção terem algum tipo de dificuldade em consumir especificamente um dos açúcares usados, glucose e frutose, cultivando-os em 20 % (w/v) glucose e em 20 % (w/v) de frutose uma vez que nestes meios os mutantes continuavam sem atingir as mesmas densidades celulares que a estirpe selvagem. Tendo em conta a elevada osmolaridade do meio utilizado assim como as diversas descrições do papel do manitol como osmoprotetor, foi avaliado o crescimento dos mutantes de deleção num meio onde a pressão osmótica é baixa, 2FG contendo 1 % (w/v) de frutose e 1 % (w/v) de glucose, a 30 ºC e 32.5 ºC. Curiosamente, a 32.5 ºC os mutantes de deleção não conseguiam sequer crescer. O facto de o defeito no crescimento ser ainda mais pronunciado neste meio de cultura permitiu-nos excluir a possibilidade de o manitol funcionar como osmoprotetor em St. bombicola. Além disso, a taxa de crescimento da estirpe selvagem e dos mutantes de deleção foi também analisada a 25 ºC, 30 ºC e 32.5 ºC. Porém, não foi detetada qualquer diferença na taxa de crescimento entre a estirpe selvagem e os mutantes de deleção a 25 ºC e a 30 ºC o que sugere que as diferenças que observamos são de facto no rendimento em biomassa e não na velocidade de crescimento. A adição de sorbitol (18% (w/v) ao meio 2FG a 32.5 ºC repõe a capacidade de crescimento dos mutantes. Contudo, o sorbitol pode ser metabolizado quando presente em elevadas concentrações. No entanto, quando cultivámos um mutante duplo – onde o gene que codifica para uma enzima necessária para a metabolização do sorbitol (SOR1) foi interrompido assim como o gene MtDH1, mtdh1Δsor1Δ - verificamos que esse mutante ainda consegue crescer o que indica que o sorbitol poderá substituir o papel do manitol na célula e que o poliol em si tem de facto um papel no crescimento a elevadas temperaturas. Esta conclusão é corroborada quando a estirpe selvagem e os mutantes de deleção foram cultivados no meio contendo 1 % (w/v) de glucose e 1 % (w/v) de frutose ao qual foi adicionado 18% (w/v) manitol e os mutantes de deleção atingiam densidades celulares semelhantes à estirpe selvagem a 32.5 ºC. Tendo em conta que os mutantes de deleção não atingem as mesmas densidades celulares que a estirpe selvagem e, que é algo que parece estar intimamente relacionado com a temperatura a produção intracelular e extracelular de manitol foi analisada no meio 20FG a 20 ºC, 25 ºC e 30 ºC. Curiosamente, um aumento do rendimento intracelular de manitol é observado assim como extracelular o que sugere que o manitol desempenha um efeito protetor na célula contra o stress térmico, possivelmente tendo um papel estabilizador da membrana como a trealose em S. cerevisiae, em que os grupos hidroxilo formam pontes de hidrogénio com a bicamada lipídica da membrana. Esta explicação coaduna-se com o facto de, em S. cerevisiae a trealose ser necessária dos dois lados da membrana para garantir proteção uma vez que, em St. bombicola, não só a produção de manitol intracelular, mas também a quantidade extracelular de manitol aumenta com a temperatura. Em suma, durante esta dissertação foi possível fazer uma contribuição significativa relativamente ao papel da produção de manitol em St. bombicola. Esse papel poderá residir no efeito termoprotector do próprio manitol, mas também na manutenção do equilíbrio redox das vias do metabolismo central.
Fructophily is a rare trait that consists in the preference of fructose over glucose as carbon and energy source. In spite of the fact that it is already known that fructophily in yeasts is intimately related with the presence of a high capacity fructose transporter called Ffz1, the importance of fructose in the metabolism of fructophilic yeasts is not well established yet. St. bombicola converts fructose directly into mannitol through a mannitol dehydrogenase (Mtdh) which uses NADPH as a cofactor. In order to understand the role of mannitol in this yeast, three deletion mutants previously constructed, where the genes encoding for Mtdh were disrupted (mtdh1Δ, mtdh2Δ, mtdh1Δmtdh2Δ) were used. Growth assays were performed at three temperatures 25 ºC, 30 ºC and 32.5 ºC and it was possible to observe that the MtDH deletion mutants did not reach the same cell densities as the wild-type. Also, at the highest temperature tested (32.5 ºC) and at low sugar concentrations, the MtDH deletion mutants were not even able to grow. The ability to grow was restored through the addition of exogenous polyols like mannitol and sorbitol, suggesting that these polyols could be replacing the function of mannitol produced by the cell. Also, the intracellular mannitol content produced by the wild-type increased at higher temperatures, suggesting a role as thermoprotector. It seems possible that mannitol, like trehalose in S. cerevisiae, can stabilize the lipid bilayer of the plasma membrane through hydrogen bonding. In this thesis we were able to make a significant contribution to elucidate the role of mannitol production in St. bombicola. The first role may be as thermoprotector in the cell and the second role may be related with redox homeostasis.