Rhamnolipid biosurfactants are investigated to determine their role in biological processes, and for discovery of novel, more biocompatible applications in areas related to medicine, agriculture, and environment. Fundamental understanding of the physical and chemical properties of rhamnolipids is needed. Thus, systematic studies on solution and interfacial properties on rhamnolipids from P.aeruginosa ATCC 9027 were undertaken. A purification protocol was developed and a thorough qualitative and quantitative speciation analysis was performed with high performance liquid chromatography-mass spectrometry (HPLC-MS), nuclear magnetic resonance (NMR) spectroscopy and HPLC with evaporative light scattering detection (ELSD). Acid-base properties of the mixture of 30 monorhamnolipid congeners, including both saturated and unsaturated species, were characterized at concentrations below and above the CMC at fixed ionic strength using potentiometry and attenuated total reflectance-Fourier transform infrared (ATRFTIR) spectroscopy. A pKₐ of 4.3 was found for concentrations below 50 μM and a pKₐ of 5.6 above 100 μM. The pKₐ is dictated by aggregation in solution. Molecular areas of monorhamnolid monolayers at the air-water interface are strongly influenced by protonation state, and increase from 31 to 109 Ų/molecule as pH increases from 4 to 8.5 and as ionic strength decreases. Adsorption isotherms of monorhamnolipids on γ-Al₂O₃ were investigated as a function of pH using ATR-FTIR spectroscopy and Frumkin model fits, from which K(ads) values of 1.20 (± 0.10) x 10⁵ M⁻¹ at pH 4.0, 2.14 (± 0.51) x 10⁴ M⁻¹ at pH 6.3 and 1.31 (± 0.09) x 10³ M⁻¹ at pH 8.6 were obtained. Interaction parameters were positive at all pH values. Cooperative adsorption is driven by hydrophobic interactions (physisorption) at any pH including hydrogen bonding and electrostatic interactions. Chemisorption was also observed at high pH values. Formation constants for monorhamnolipid-Pb²⁺ and monorhamnolipid-Cd²⁺ complexes were determined using differential pulse polarography in the low μM concentration range. A modified Lingane equation was developed to account for monorhamnolipid adsorption on the Hg surface. β values for adsorbed metal complexes are ~10^3.2 and ~10^0.8 for Pb²⁺ and Cd²⁺, respectively, compared to previously published β values of 10^8.58 and 10^6.89, respectively. Evidence for 1:1 and 2:1 monorhamnolipid-metal complexes was provided by electrospray ionization-mass spectrometry.

Des microorganismes produisant des molécules tensioactives ont été isolés à partir d’échantillons de sols contaminés par des huiles, en provenance des provinces de Songkhla et Chiangmai (Thaïlande) et de Shianghai (Chine). Les différentes souches ont été sélectionnées de façon à obtenir les biosurfactants ayant les meilleures propriétés tensioactives et d’émulsification. Parmi 102 souches isolées, 6 microorganismes produisaient des biosurfactants. La souche SK80 a conduit aux meilleures propriétés tensioactives. Des observations morphologiques macroscopiques et microscopiques ont permis de caractériser la souche SK80. L’analyse de la séquence ARNr 28S indique que cette souche appartient à la famille Exophiala Dermatitidis. La composition du milieu de culture (source de carbone et d’azote) et les conditions de culture de ce microorganisme ont été adaptées de façon à obtenir des quantités importantes de biosurfactant. Des analyses spectroscopiques (RMN 1H, RMN 13C, COSY et de masse, APCI MS) ont révélé que ce biosurfactant était un monooléate de glycérol. La monomyristine a été choisie comme constituent synthétique modèle dans des études d’encapsulation. Deux méthodes de préparation, émulsion/évaporation de solvant, nanoprécipitation, ont été employées pour encapsuler la monomyristine dans des nanoparticules recouvertes de dextrane et dont le cœur était constitué de poly(acide lactique) ou de dextrane hydrophobisé. Les conditions d’encapsulation ont été variées afin de maximiser le rendement d’encapsulation et la stabilité colloïdale des particules / Biosurfactant producing microorganisms were isolated from oil contaminated soils collected from Songkhla and Chiangmai province, Thailand and Shianghai, China. Their culture broths were screened for obtaining biosurfactants with the highest surface activity and emulsification ability. Among 102 isolates, 6 microorganisms produced biosurfactants. The culture supernatant of SK80 strain exhibited the highest surface activity. SK80 was identified by macroscopic morphology, microscopic morphology and showed that it is a black mold. The 28S rRNA sequence homology analysis suggested that SK80 belongs to Exophiala dermatitidis. The composition of culture medium such as carbon source, nitrogen source, and culture condition of this microorganism was optimized to obtain high amounts of biosurfactant. 1H NMR, 13C NMR, COSY and Mass Spectrometer (APCI MS) results indicated that this biosurfactant was monoolein (oleoyl glycerol), a kind of monoacylglycerol. Monomyristin was chosen as a monoacylglycerol model to be synthesized and used as nanoparticle encapsulated drug. Two preparation methods, emulsion/solvent evaporation and nanoprecipitation, were used to encapsulate monomyristin in dextran-covered nanoparticles with poly(lactic acid) of hydrophobized dextran as the core material. Encapsulation conditions were optimized with regard to the yield encapsulation and the colloidal stability

No description available.

Petroleum hydrocarbons are organic pollutants of major concern due to their wide distribution, persistence, complex composition, and toxicity. They can bioaccumulate in food chains where they disrupt biochemical or physiological activities and can affect genetic integrity of many organisms, resulting in carcinogenesis, mutagenesis and impairment of reproductive capacity. Polycyclic aromatic hydrocarbons (PAHs) have been recognized as priority pollutants due to their carcinogenic, mutagenic and teratogenic properties. Bioremediation, which utilizes the metabolic versatility of microorganisms such as bacteria and fungi to degrade or detoxify hazardous wastes into harmless substances has been recognized as a sustainable, economic, environmentally friendly and versatile alternative for the remediation of many contaminated environments; however its effectiveness is limited by low bioavailability of nonaquous phase and soil-bound PAHs and petroleum hydrocarbons due to their low aqueous solubility, high hydrophobicity and strong sorption to soil. The purpose of this study was to investigate the PAHs and petroleum hydrocarbons bioavailability and subsequent biodegradation enhancement potential of biosurfactants. Biosurfactants have steadily gained increased significance in environmental applications such as bioremediation dueto several advantages over surfactants of chemical origin, such as biodegradability, environmental compatibility, low toxicity, high selectivity and specific activity at extreme temperature, pH and salinity. A series of experiments was designed to investigate the bioavailability and subsequent biodegradation enhancement potential of the biosurfactants produced by the bacterial strains Bacillus subtilis CN2, Ochrobactrum intermedium CN3, Paenibacillus dendritiformis CN5 and Bacillus cereus SPL_4 in liquid culture and soil microcosms with PAH-enriched microbial consortium from chronically contaminated sites. The biosurfactants exhibited a high level of thermal stability, tolerance to extreme levels of salinity and a positive effect for increasing pH. They were identified after Fourier Transform Infrared (FT-IR) spectrometry, Thin Layer Chromatography (TLC) and Liquid Chromatography/Tandem Mass Spectrometry (LC MS/MS) analyses. The biosurfactants physicochemical characterization displayed vast structural diversity and potent surface active properties of surface tension reduction and emulsion formation with a range of hydrocarbons. The lipopeptide biosurfactants produced by CN3 and CN2 enhanced degradations of used motor oil and petroleum sludge in liquid culture. In a shake flask pyrene degradation study, lipopeptide supplementations at 600 and 300 mg L-1 enhanced pyrene degradation to 83.5% and 67% respectively in 24 days compared to 16% degradation in its absence. However, degradation of pyrene was reduced to 57% as the lipopeptide supplementation was raised to 900 mg L 1. This demonstrates that the biodegradation of pyrene was found to increase with an increase in the lipopeptide concentration up to a threshold level. In a soil bioremedial study, microcosms supplemented with 0.2 and 0.6% (w/w) lipopeptide, 51.2% of 4-ring and 55% of 5- and 6-ring PAHs, 64.1% of 4-ring and 79% of 5- and 6-ring PAHs were removed respectively, compared to, 29% of 4-ring and 25.5% of 5- and 6-ring PAHs removal in the surfactant free control after 64 days of incubation. However, there was no statistically significant change in the degradation rates of low molecular weight PAHs in surfactant amended and surfactant free controls. The degradation of 5 and 6 ring PAHs was significantly enhanced (p < 0.05) in the higher surfactant dosage compared to the lower dosage. The results of this work demonstrated that the use of biosurfactants is a viable option to reduce clean-up time and for effective remediation of soil and aqueous media contaminated with polycyclic aromatic and petroleum hydrocarbons. The study demonstrated potential applications of microbial surfactants and provided an insight for further investigation of their large scale production for commercial applications. / Thesis (PhD)--University of Pretoria, 2016. / Chemical Engineering / PhD / Unrestricted

La pollution des écosystèmes marins côtiers par les hydrocarbures, en particulier les HAPs, est un problème environnemental majeur. Le même constat est fait pour le littoral Sud de Sfax (Tunisie) dont la pollution presque généralisée menace sérieusement les ressources naturelles existantes dans la région. La caractérisation physico-chimique des échantillons marins prélevés à partir des trois ports (plaisance, commerce et pêche) de la ville de Sfax, prouve une contamination par des micropolluants organiques (hydrocarbures) et inorganiques (métaux) qui sont considérés comme des excellents traceurs de la pollution urbaine et industrielle et ils font partie des composés les plus toxiques étant donné leur faible biodégradation. La méthode d’empreinte moléculaire (PCR-SSCP) montre une dominance du domaine Bacteria suivie des Eucarya et des Archaea au sein des échantillons marins étudiés. Les analyses statistiques par le logiciel R ont montré l’absence de corrélation entre la communauté bactérienne identifiée par PCR-SSCP et les paramètres physico-chimiques étudiés.Dans une autre partie de travail, quatre souches bactériennes marines hydrocarbonoclastes ont été isolées et caractérisées sur les plans phénotypique et phylogénétique, après des enrichissements sur des HAPs et sur le pétrole brut: FLU5 de Bacillus stratosphericus, NAPH6 de Pseudomonas aeruginosa, PYR2 de Bacillus licheniformis isolées sur le fluoranthène, le naphtalène et le pyrène, respectivement, en présence de 30 g/l NaCl; et CO100 de Staphylococcus sp., isolée sur le pétrole brut, en présence de 100 g/l NaCl. Les analyses chromatographiques, GC-MS ou GC-FID, montrent les capacités biodégradatives intéressantes de ces composés récalcitrants par les bactéries isolées. En outre, ces quatre souches bactériennes, sont capables de produire des biosurfactants nommés BS-FLU5, BS-NAPH6, BS-PYR2 et BS-CO100, sur plusieurs sources de carbones, y compris l’huile de friture résiduelle, un substrat bon marché, minimisant ainsi le coût élevé de production de ces tensioactifs. Les analyses MALDI-TOF/MS, des biosurfactants BS-FLU5, BS-PYR2 et BS-CO100 purifiés, montrent qu’il s’agit des lipopeptides, les biosurfactants BS-NAPH6 sont de nature rhamnolipidique, sur la base des analyses FTIR. Ces quatre biosurfactants sont caractérisés par des propriétés tensiactives intéressantes : une faible CMC, une importante réduction de la tension de surface... Ils sont stables vis-à-vis d’une large gamme de pH, de température et de salinité. De plus, ces agents tensioactifs sont doués d’activité de remobilisation des hydrocarbures contenus dans des sols pollués par. Les biosurfactants BS-FLU5, BS-PYR2 et BS-CO100, présentent des activités anti-adhésives et anti-biofilms intéressantes contre des biofilms de certains microorganismes pathogènes. Par ailleurs, une propriété cicatrisante remarquable sur des plaies d’excision chez un modèle expérimental de rats de race Wistar, a été montrée par les quatre biosurfactants pour des concentrations de l’ordre de 5 et 10 mg/ml, en comparaison avec un cicatrisant de référence (CICAFLORA®). A noter que, l’évaluation de la cytotoxicité des biosurfactants étudiés, a montré qu’ils n’ont pas des effets toxiques sur des cellules rénales humaines HEK-239 à des concentrations jusqu’à 1000 µg/ml pour BS-FLU5 et BS-CO100 et jusqu’à 200 µg/ml pour BS-NAPH6 et BS-PYR2. La production des biosurfactants de la souche FLU5 à l’échelle pilote (deux fermenteurs de 20 et 100 litres, volume total), en présence d’un milieu économique, montre une augmentation des quantités des biosurfactants produits par rapport à l’échelle laboratoire (erlenmeyer, 1 litre). L’ensemble de ces résultats prometteurs, montrent que les souches marines isolées FLU5, NAPH6, PYR2 et CO100, ainsi que leurs biosurfactants demeurent d’intérêts biotechnologiques pour divers types d’applications, tels que la bioremédiation, l’agroalimentaire, la cosmétique... / Pollution of coastal marine ecosystems by hydrocarbons, in particular polycyclic aromatic hydrocarbons (PAHs), is a major environmental problem. The South coast of Sfax (Tunisia) is an example of a polluted ecosystem subject to both urbanization and industrialization including the outfall of untreated domestic sewage and wastewaters, fishery activities, as well as ship traffic and boat pollution. The physico-chemical characterization of the seawater taken from three harbours (pleasure, commercial and fishing) of the city of Sfax, showed a heavy contamination by organic and inorganic micropollutants. These are excellent tracers of urban and industrial pollution, and they are among the most toxic compounds due to their low biodegradation.The molecular fingerprinting technique (PCR-SSCP) showed the dominance of the Bacteria domain followed by Eucarya and Archaea within the studied marine samples. Statistical analysis using the R software, showed that no correlation was identified between the bacterial community identified by PCR-SSCP and the studied physico-chemical parameters.In another part, four marine, aerobic and hydrocarbonoclastic strains: Bacillus stratosphericus FLU5, Pseudomonas aeruginosa NAPH6, Bacillus licheniformis PYR2, isolated after enrichments on fluoranthene, naphthalene and pyrene, respectively, and in the presence of 30 g/l NaCl. Strain Staphylococcus sp. CO100 was isolated after enrichment on crude oil, in the presence of 100 g/l. Chromatographic analysis (GC-MS or GC-FID), showed the interesting biodegradative capacities of these recalcitrant compounds by the isolated bacteria.Besides, these strains showed their capacity to produce efficient surface active agents BS-FLU5, BS-NAPH6, BS-PYR2 and BS-CO100, on several substrates and in particular the residual frying oil, which is a cheap and renewable carbon source alternative, thus minimizing the high cost of producing surfactants. The MALDI-TOF/MS analysis of the purified BS-FLU5, BS-PYR2 and BS-CO100 biosurfactants revealed that they are belonging to lipopeptide family. FTIR analysis showed the glycolipid nature, more precisely the rhamnolipid type, of biosurfactant BS-NAPH6.These four biosurfactants are characterized by interesting tensioactive properties (low CMC, important surface tension reduction...). Furthermore, these surface active agents showed interest stability against a broad range of pH, temperature and salinity. The application of these biosurfactants, in oil recovery, from hydrocarbons-contaminated soil, showed that they were more effective on the hydrocarbon-remobilization than some tested synthetic surfactants. The biosurfactants BS-FLU5, BS-PYR2 and BS-CO100, were found to have notable anti-adhesif and anti-biofilm activities, being able to prevent and eliminate the biofilm formation by pathogenic microorganisms. Moreover, the four tested biosurfactants showed an interesting healing activity, on the wound site in a rat model. They increased significantly the percentage of wound closure when compared to the untreated and CICAFLORA® (a reference pharmaceutical product) treated groups, using two different concentrations (5 and 10 mg/l). Interestingly, the evaluation of the cytotoxicity of the studied biosurfactants, showed that they have no toxic effects on human HEK-239 cells at concentrations up to 1000 μg/ml for BS-FLU5 and BS-CO100 and up to 200 μg/ml for BS-NAPH6 and BS-PYR2. An attempt to produce biosurfactant produce by strain FLU5 on a pilot-scale (fermentors of 20 and 100 liter, as total volume), using a cost-effective medium, was also performed. Preliminary results showed an increase in the quantities of biosurfactantsBS-FLU5 produced on a pilot-scale compared to the lab-scale (Erlenmeyer of 1 liter).These results highlight the interest for potential use of strains FLU5, NAPH6, PYR2 and CO100, as well as their biosurfactants, in a wide variety of industrial, environmental and biotechnological applications.

Biosurfactants are compounds that exhibit surface activity (e.g., reduce surface and interfacial tension) and are derived from natural, biological sources. They are considered green substances due to their natural derivation, biodegradability, and relatively low toxicity. Biosurfactants from multiple classes have been shown to interact with metals, and a review of these interactions is provided. Rhamnolipids produced by Pseudomonas aeruginosa are attracting attention for metal remediation applications. The purpose of this dissertation is to evaluate rhamnolipids' ability to complex rare earth elements, determine the environmental compatibility of novel rhamnolipid diastereomers, and assess the efficacy of rhamnolipid as a collector in ion flotation. Previous research shows rhamnolipids selectively bind elements of environmental concern over common soil and water cations, but there had been no examination of transition metals from the f-block of the periodic table. The f-block elements include the rare earth elements, which are a vital component of nearly every modern technology and subject to supply risk. The interaction between monorhamnolipids and the rare earth elements was investigated by determining conditional stability constants using a resin-based ion exchange method. For the 27 metals examined, the conditional stability constants could be divided into three groups, albeit somewhat subjectively: weakly, moderately, and strongly bound. UO22+, Eu3+, Nd3+, Tb3+, Dy3+, La3+, Cu2+, Al3+, Pb2+, Y3+, Pr3+, and Lu3+are strongly bound with conditional stability constants ranging from 9.82 to 8.20; Cd2+, In3+, Zn2+, Fe3+, Hg2+, and Ca2+ are moderately bound with stability constants ranging from 7.17 to 4.10; and Sr2+, Co2+, Ni2+, UO22+, Cs+, Ba2+, Mn2+, Mg2+, Rb+, and K+ are weakly bound with stability constants ranging from 3.95 to 0.96. The uranyl ion is reported twice due to the ion demonstrating two distinct binding regions. The conditional stability constants were demonstrated to be an effective predictor of metal removal order. The metal parameters of enthalpy of hydration and ionic charge to radius ratio were shown to be determinants of complexation strength. Naturally produced rhamnolipids are a mixture of congeners. Synthetic rhamnolipid synthesis has recently enabled production of four monorhamnolipid diastereomers of a single congener. The biodegradability, acute toxicity (Microtox assay), embryo toxicity (Zebrafish assay), and metal binding capacity of the diastereomers was investigated and compared to natural monorhamnolipid. Biodegradability testing showed all the diastereomers were inherently biodegradable. By the Microtox assay, all of the monorhamnolipids were categorized as slightly toxic by Environmental Protection Agency ecotoxicity categories. Out of 22 parameters tested, the zebrafish toxicity assay showed only diastereomer toxicity for the mortality parameter, except for diastereomer R,R which showed no toxic effects. All the monorhamnolipids interacted with both Cd2+ and Pb2+. Ion flotation is one possible technology for metal recovery and remediation of metal contaminated waters. Ion flotation utilizes charged surfactants to collect and concentrate non-surface active ions at the surface of an aerated solution. Rhamnolipid's suitability as a collector in ion flotation was investigated. A flotation column was designed to test monorhamnolipid efficacy as a collector. Monorhamnolipids form foams and effectively remove Cs+, Cd2+, and La3+ from solution. The efficacy of the flotation process relies on the collector:colligend ratio and valency of the colligend. Flotation of metal solutions showed a removal order of Cd2+>La3+>>Cs+ when the metals were present individually and mixed at equimolar concentrations. When mixed at order of magnitude different concentrations, the flotation order was Cd2+>>Cs+>>La3+. These studies show rhamnolipid has potential to be used for environmentally-compatible metal recovery and metalliferous water remediation, especially for the rare earth elements.

Οι επιφανειοδραστικοί παράγοντες είναι μόρια αμφίφιλα (αποτελούνται δηλ. από μια υδρόφιλη κεφαλή και μία υδρόφοβη ουρά). Λόγω της δομής τους αυτής παρέχεται η δυνατότητα, μέσω προσθήκης τους σε διάλυμα, να προκαλείται μείωση της επιφανειακής (υγρού-αέρα) και της διεπιφανειακής τάσης (υγρού-υγρού) επιτρέποντας στις δύο φάσεις να αναμιχθούν και να αλληλεπιδράσουν. Η ιδιότητά τους αυτή σε συνδυασμό με την ικανότητά τους να σχηματίζουν γαλακτώματα καθιστά τις ουσίες αυτές ιδιαίτερα σημαντικές, με πάρα πολλές εφαρμογές στη σύγχρονη βιομηχανία τροφίμων αλλά και σε περιβαλλοντικές εφαρμογές, όπως στην αποκατάσταση αποδεκτών ρυπασμένων με υδρογονάνθρακες ή μέταλλα, στη μικροβιακή αύξηση ανάκτησης πετρελαίου, στην ιατρική, στη γεωργία, στη βιομηχανία καλλυντικών κ.ά. Εκτός από τους χημικά συντιθέμενους επιφανειοδραστικούς παράγοντες έχουν αναφερθεί σήμερα αντίστοιχες ουσίες μικροβιακής προέλευσης, που είναι γνωστές ως βιοεπιφανειοδραστικές. Παρόλη την ευρεία χρήση των χημικά συντιθέμενων επιφανειοδραστικών ουσιών, λόγω του χαμηλού κόστους παραγωγής τους, τα βιολογικά επιφανειοδραστικά έχουν λάβει εκτεταμένη προσοχή τα τελευταία χρόνια λόγω των μοναδικών ιδιοτήτων τους. Έτσι, εκτός του ότι είναι μη-τοξικά, ορισμένα από αυτά έχουν επιδείξει πιο ειδικές και αποτελεσματικότερες επιφανειοδραστικές ιδιότητες σε σύγκριση με πολλές συμβατικές συνθετικές επιφανειοδραστικές ουσίες, παρέχοντας νέες δυνατότητες για βιομηχανικές εφαρμογές. Οι βιοεπιφανειοδραστικοί παράγοντες ταξινομούνται σε διάφορες κατηγορίες ανάλογα με τη χημική τους σύνθεση, το φορτίο τους και τη μικροβιακή τους προέλευση. Η σύνθεσή τους γίνεται από μεγάλη ποικιλία μικροοργανισμών και θρεπτικών μέσων. Μια από τις σημαντικότερες κατηγορίες βιολογικά συντιθέμενων επιφανειοδραστικών παραγόντων είναι τα ραμνολιπίδια. Η πλειοψηφία των στελεχών που έχει αναφερθεί ότι παρουσιάζουν δυνατότητα παραγωγής βιοεπιφανειοδραστικών παραγόντων ανήκει στο γένος Pseudomonas και τα περισσότερα από αυτά έχουν ταυτοποιηθεί ως Ρ. aeruginosa. Ένα πρόσφατα ανακαλυφθέν βακτήριο, το οποίο ανήκει στο γένος Tetragenococcus, το οποίο βρέθηκε να παράγει βιοεπιφανειοδραστικούς παράγοντες, αποτελεί ο Tetragenococcus koreensis που αποτελεί και το αντικείμενο μελέτης της παρούσας εργασίας. Στην παρούσα εργασία χρησιμοποιήθηκαν διαφορετικής σύστασης συνθετικά θρεπτικά μέσα και μελετήθηκε η δυνατότητα ανάπτυξης του μικροοργανισμού καθώς και η ικανότητά του να συνθέσει βιοεπιφανειοδραστικούς παράγοντες. Επίσης μελετήθηκε η ικανότητα των προϊόντων στο σχηματισμό γαλακτωμάτων, στη μείωση της επιφανειακής τάσης, ενώ επίσης αναπτύχθηκε μεθοδολογία για τον ποσοτικό προσδιορισμό τους. Η συμπεριφορά του μικροοργανισμού παρουσίασε αρκετές διαφοροποιήσεις στα θρεπτικά μέσα που χρησιμοποιήθηκαν τόσο ως προς τον ρυθμό ανάπτυξής του αλλά και ως προς την ικανότητα σύνθεσης προϊόντων. / Surfactants are amphiphilic molecules (i.e., consisting of a hydrophilic head and a hydrophobic tail). Due to this structure it is possible, by adding them in solution, to cause reduction of surface (air-liquid) and the interfacial tension (liquid-liquid), allowing the two phases to be mixed and to interact. The capacity is combined with the ability to form emulsions which renders these substances particularly important to many applications in modern food industry and in environmental applications, such as to restore recipients contaminated with hydrocarbons or metals, microbial growth in oil recovery in medicine, agriculture, industry cosmetics etc. Besides chemically synthesized surfactants reported today relevant substances of microbial origin, which are known as biosurfactants. Despite the widespread use of chemically synthesized surfactants, because of its low production costs, organic surfactants have received extensive attention in recent years because of their unique properties. Thus, in addition to being non-toxic, some of them have shown more specific and effective surfactant properties compared to many conventional synthetic surfactants, providing new possibilities for industrial applications. The biosurfactant agents are classified into various categories according to their chemical composition, their charge and microbial origin. The composition is made of a wide variety of microorganisms and nutrients. One of the major classes of biologically synthesized surfactants are rhamnolipids. The majority of the strains have been reported to exhibit potential biosurfactant production factors belonging to the genus Pseudomonas and most of them were identified as P. aeruginosa. A newly discovered bacterium, which belongs to the genus Tetragenococcus, which was found to produce biosurfactants is the Tetragenococcus koreensis which is the subject of the present work. In this work we used different composition synthetic media and studied the possibility of growth of the microorganism and the ability to synthesize biosurfactants. Furthermore, we studied the ability of the products in the formation of emulsions, the decrease of surface tension, and also developed a methodology to quantify them. The behavior of the organism showed several differences in nutrient media used both in its growth rate but also in the ability to synthesize products.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-19Bitstream added on 2014-06-13T20:24:23Z : No. of bitstreams: 1 lovaglio_rb_dr_rcla.pdf: 1731223 bytes, checksum: 43dfc85c18edb74e4fc520e7b8f963ac (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os biossurfactantes são metabólitos secundários que apresentam atividade de superfície, podendo ser aplicados em diferentes processos industriais, como produção de fármacos, compostos químicos, cosméticos, biorremediação de ambientes poluídos por petróleo e derivados e recuperação terciária do petróleo. A maior vantagem destes compostos quando comparado aos surfactantes sintéticos reside na sua diversidade estrutural, baixa toxicidade e alta biodegradabilidade. Apesar das inúmeras vantagens apresentadas pelos biotensoativos, eles não são comercialmente utilizados devido ao alto custo de produção. O objetivo deste trabalho foi estudar a produção de ramnolipídios por mutantes de P. aeruginosa a partir de resíduos industriais e óleos vegetais, bem como caracterizar as propriedades de solução desses biossurfactante, além de verificar a influência da fonte de carbono na produção de homólogos de ramnolipídios por P. aeruginosa. Na primeira etapa as fermentações foram conduzidas em tubos de ensaio, após a seleção dos micro-organismos com potencial para alta produção, os mesmos foram testados em frascos Erlenmeyer. O mutante com potencial para elevada produção foi avaliado, junto com a linhagem selvagem, em fermentadores. A linhagem selecionada e a utilização do meio de cultura livre de cálcio contendo óleo de girassol como fonte de carbono acarretou em um aumento de 70% na produção de ramnolipídios. A adição de etanol às soluções de ramnolipídios comprovou o potencial desse tensoativo para serem aplicados na indústria de cosméticos, já que as propriedades de tensão superficial, agregação e emulsificação foram mantidas nas três concentrações de álcool adicionadas. Além disso, verificou-se que a mudança da fonte de carbono acarreta alteração nos tipos ácidos graxos e proporções de homólogos de ramnolipídios produzidos por P. aeruginosa LBI 2A1 / The biosurfactants are secondary metabolites that exhibit surface activity and can be applied in different industrial processes such as production of pharmaceuticals, chemicals, cosmetics, bioremediation of environments polluted by oil and derivatives and tertiary recovery of oil. The greatest advantage of these compounds compared to synthetic surfactants lies in their structural diversity, low toxicity and high biodegradability. Despite the numerous advantages provided by biosurfactants, they are not commercially used due to the high cost of production. The present study aimed to evaluate the production of rhamnolipids by mutants of P. aeruginosa using industrial waste and vegetable oils as carbon source, as well as to characterize the properties of these biosurfactant solutions and investigate the influence of carbon source in the production of homologous rhamnolipds by P. aeruginosa. In the first stage fermentations were conducted in test tubes, after selection of microorganisms with potential for high production they were tested in Erlenmeyer flasks. The mutant with the potential for increased production was assessed, along with the wild strain in bioreactors. The selected strain and the calcium free medium used with sunflower oil as carbon source resulted in an 70% increase in the production of rhamnolipids. The addition of ethanol to the rhamnolipids solutions proved the potential of this biosurfactant to be applied in the cosmetic industry, since the properties of surface tension, emulsification and aggregation were held in all three concentrations of ethanol added. Moreover, it was found that changing the carbon source caused alteration in the types of fatty acids and rhamnolipids counterparts proportions produced by P. aeruginosa LBI 2A1

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-24T18:44:08Z No. of bitstreams: 1 dissertao - celia f.c. da rosa.pdf: 4567448 bytes, checksum: 6d91b7c9dd8c2b9a808ad10d387dd9f0 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-12-01T16:12:48Z (GMT) No. of bitstreams: 1 dissertao - celia f.c. da rosa.pdf: 4567448 bytes, checksum: 6d91b7c9dd8c2b9a808ad10d387dd9f0 (MD5) / Made available in DSpace on 2012-12-01T16:12:48Z (GMT). No. of bitstreams: 1 dissertao - celia f.c. da rosa.pdf: 4567448 bytes, checksum: 6d91b7c9dd8c2b9a808ad10d387dd9f0 (MD5) Previous issue date: 2008 / A reação de transesterificação de óleos vegetais tem como produto principal o biodiesel, porém o glicerol que é subproduto desta reação pode ser transformado em resíduo devido ao aumento da produção deste biocombustível. Estudos revelam que o glicerol pode ser utilizado como substrato em processos fermentativos juntamente com microrganismos capazes de utilizá-lo como fonte de carbono. A bactéria Pseudomonas aeruginosa demonstra capacidade de assimilação de glicerol para produção de biossurfactantes, que são moléculas produzidas por microrganismos capazes de diminuir a tensão superficial. A maior vantagem de utilização dos biossurfactantes é a aceitabilidade ambiental já que são biodegradáveis e podem ser sintetizados a partir de fontes renováveis. Atualmente a maior aplicabilidade dos biossurfactantes está focada na biorremediação de poluentes, no entanto possui várias propriedades de interesse para inúmeras aplicações na agricultura, cosméticos, produtos farmacêuticos, detergentes, processamento de alimentos e outros. A maioria dos biossurfactantes são moléculas complexas e compreendem uma grande variedade de estruturas químicas, tais como:glicolipídios, lipopeptídios e lipoproteínas, biossurfactantes poliméricos, fosfolipídios, lipídios neutros e ácidos graxos. Dentre os glicolipídios, os mais estudados são os ramnolipídios, produzidos pela bactéria Pseudomonas aeruginosa. O presente trabalho teve como principal objetivo avaliar a composição do meio de produção para obtenção de ramnolipídio a partir da bactéria Pseudomonas aeruginosa LBM10 por fementação submersa, utilizando a técnica de planejamento experimental e análise de superfície de resposta. A cepa bacteriana, isolada de resíduos de pescado capturados na região costeira do sul do Brasil, foi utilizada para produção de ramnolipídio em meio mineral tendo o glicerol como única fonte de carbono. Foi realizado um planejamento completo central rotacional a fim de determinar a melhor condição de estudo, sendo as variáveis do planejamento a concentração de glicerol (13,2 a 46,8 g/L), a relação C/N (12,8 a 147,2) e a relação C/P (12,8 a 147,2), tendo-se como respostas a concentração de ramnolipídio, expressa como ramnose (g/L), índice de emulsificação (IE24, %), redução da tensão superficial (%) e os fatores de conversão de substrato em produto (YP/S), substrato em célula (YX/S) e célula em produto (YP/X). Os ensaios de fermentação foram realizados em incubadora rotatória a 30°C e 180 rpm, por um período de 144 h. De acordo com a validação dos modelos empíricos e análise das superfícies de respostas, a condição que permite obter maior produção de ramnolipídios (2,25 g/L) associada a um alto índice de emulsificação (65 %) é a que utiliza concentração de glicerol de 13,2 g/L, xv relação C/N de 12,8 e relação C/P na faixa de 40, apresentando YP/S de 0,26 g/g e redução da tensão superficial de 38,33 %. / The reaction of transesterification of vegetal oils has the biodiesel as main product. However, glycerol, the by-product of this reaction, can become an important feedstock with the increase in the production of this biofuel. Studies show that glycerol can be used as substrate in fermentatiion processes with microorganisms able to use it as carbon source. The bacteria Pseudomonas aeruginosa demonstrates capability of assimilation of glycerol for production of biosurfactants, which are molecules produced by microorganisms which reduce the surface tension. The most important advantage of biosurfactants is their ecological acceptance: they are biodegradable and can be produced from renewable substrates. Most work on biosurfactants applications has been focusing on bioremediation of pollutants however these microbial compounds exhibit a variety of useful properties for several applications in agriculture, cosmetics, pharmaceuticals, detergents, food processing and others. Most microbial surfactants are complex molecules, comprising a wide variety of chemical structures, such as glycolipids, lipopeptides and lipoprotein, polymeric biosurfactants, phospholipids, neutral lipids and fatty acids. Among glycolipid-type biosurfactants, rhamnolipids produced by Pseudomonas aeruginosa have been widely studied. The main objective of this work was to evaluate the culture medium for rhamnolipid production from Pseudomanas aeruginosa LBM10 by submerged fermentation using experimental design and surface response methodology. The strain, isolated from fish samples captured in a southern coastal zone in Brazil, was used to produce rhamnolipids using mineral media with glycerol as the only carbon source. A rotatable central composite design was used to establish the best medium composition. The parameters were: glycerol concentration (13.2 to 46.8 g/L), C/N ratio (12.8 to 147.2) and C/P ratio (12.8 to 147.2), and the responses were: rhamnolipid concentration, expressed as rhamnose (g/L), emulsification index (IE24, %), surface tension reduction (%) and yield factors YP/S, YX/S and YP/X. The fermentation assays were carried out in a rotary shaker at 30ºC and 180 rpm by 144 h. According to validation of the empirical models and response surfaces analysis, the condition that allows to get greater production of rhamnolipids (2.25 g/L) associate to high emulsification index (65 %) is that uses glycerol concentration of 13.2 g/L, C/N ratio of 12.8 and C/P ratio of 40, showing YP/S of 0.26 g/g and surface tension reduction of 38.33 %.

Orientador: Glaucia Maria Pastore / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-09T21:05:05Z (GMT). No. of bitstreams: 1 Mano_MarioCezarRodrigues_M.pdf: 478580 bytes, checksum: 01364784fc41427aff6972b83ce42886 (MD5) Previous issue date: 2008 / Resumo: Biossurfactantes são compostos anfifílicos produzidos por microrganismos que possuem atividade superficial, ou seja, a capacidade de reduzir a tensão superficial e interfacial entre dois líquidos imiscíveis. Devido a suas características, esses compostos possuem uma ampla gama de potenciais utilizações que vão desde a indústria de alimentos, cosméticos, farmacêutica, petroquímica entre outras. O processo de recuperação e purificação de biossurfactantes é alvo de constantes aperfeiçoamentos, pois compreende, em alguns casos, até 60% do custo de produção dos mesmos. Alguns processos utilizados com mais freqüência para recuperação de biossurfactantes são as técnicas de fracionamento de espuma, precipitação e extração, além da ultrafiltração. O objetivo deste trabalho foi avaliar a viabilidade dos processos de ultrafiltração e diafiltração para recuperação e purificação de biossurfactantes produzidos por Bacillus subtilis. O processo foi conduzido com a produção de espuma via fermentação, que continha alta concentração de biossurfactantes. Após pré-tratamento, a espuma passou pelos processos de ultrafiltração e diafiltração, com parâmetros fixos e pré-estabelecidos. Os resultados da ultrafiltração mostram um aumento na concentração de biossurfactantes de 0,7 para aproximadamente 1,1 g/L, porém com um grau de pureza ainda abaixo de 50%. Já no processo de diafiltração, os resultados apontam para uma purificação de valores de pureza superiores a 50%. Uma análise final indica que ambos os processos, de concentração de biossurfactantes por ultrafiltração, e de purificação por diafiltração são viáveis e conseguem um bom rendimento, a custo baixo, sendo uma boa alternativa no ¿downstream¿ de tais produtos / Abstract: Biosurfactant are amphifilic compounds produced by microorganisms that have superficial activity, or capacities of reducing superficial and interfacial tension between two immiscible liquids. Besides these characteristics, this compound has a large utility, going since the food industry, cosmetic, pharmacy, petrochemical among others. The biosurfactant recovery and purification process is the target of constant improvements, because it means until 60% of total production cost. Some process used with more frequency for biosurfactant recovery are foam fractionation, precipitation and extraction, besides ultrafiltration. The aim of this work was to evaluate the viability of ultrafiltration and diafiltration process for Bacillus subtilis biosurfactant recovery and purification. The process was conduced with a foam production by aerobic fermentation, which contained high biosurfactant concentration. After pre-treatment, the foam passes to ultrafiltration and diafiltration process, with fixed and pre-established parameters. The ultrafiltration results show an increase in the biosurfactant concentration from 0,7 to approximately 1,1 g/L, but with a lower 50% purity level. In the diafiltration process already, the results show purity levels higher than 50%. Final analysis indicates in both, the biosurfactant concentration by ultrafiltration and the biosurfactant purification by diafiltration, viability and good yield, with low cost, it might be a good alternative in ¿downstream process¿ of such products / Mestrado / Mestre em Ciência de Alimentos