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An estimated 1000 trillion cubic meters of gas in the unconventional hydrocarbon resource of gas hydrates in the world?s ocean floors far exceeds the known hydrocarbons in conventional reserves like coal, petroleum, and natural gas. These hydrate deposits also contain massive amounts of the greenhouse gases like methane and carbon dioxide. As relatively little is known about the oceanloor natural gas hydrates, mechanisms leading to the formation of these hydrates in ocean sediments need to be investigated before the significant technical challenges of recovery and environmental hazards are addressed. The subject research focuses on possible catalytic effects of biosurfactants on the formation of natural gas hydrates in oceanloor sediments. Sand/clay packs were saturated with seawater containing 1000 ppm of biosurfactant and pressurized with natural gas of 90 mole% methane, 6 mole% ethane and 4 mole% propane. The experimental results showed that gas hydrates formation in porous media is catalyzed by biosurfactants at very low concentrations. Commercially available representatives from the five biosurfactant classifications that microbes produce were purchased and evaluated in sand/clay packs at hydrateorming conditions. The rate of formation and induction time differed in the presence of bentonite and kaolin. The surface activities of biosurfactants were either specific to sand or clay surfaces. While in the presence of bentonite, Surfactin decreased hydrate induction time by 71% over a reference test with no biosurfactant in the seawater; Surfactin lowered induction time by 25% in the presence of kaolin. Rhamnolipid reduced the induction time by 58% in the presence of bentonite and by 66% in the presence of kaolin. Snomax and Emulsan, belonging to the classification of polysaccharide lipid complexes, reduced induction time by 30 to 40% in the presence of both kaolin and bentonite. Fatty acids reduced the induction time by 55% in the presence of bentonite and by 20% in the presence of kaolin. Surfactin enhanced the rate of formation by 400% in the presence of bentonite, but it had minimal effect in the presence of kaolin. Emulsan and Snomax increased the rate of formation by 250%, while rhamnolipid and phospholipids doubled formation rate in the presence of bentonite. Emulsan increased the rate of formation by 800%. In seawater, at hydrateorming conditions, rhamnolipid was found to have a critical micellar concentration of 12 ppm. This very low value of CMC suggests that minimal bacterial activity in ocean sediments could greatly catalyze hydrate formation. The recent analysis by Lanoil et al. (2001) of sediments from around gas hydrate mounds in the Gulf of Mexico gives a direct association between microbes and gas hydrates and supports the conclusions of the subject work.

Les circuits et réseaux d’eau subissent épisodiquement des problèmes de contamination par des microorganismes tels que Legionella pneumophila, conduisant à la dégradation de la qualité microbiologique de l’eau circulante. De nouveaux moyens de traitements doivent être mis en place de façon notamment à limiter l’usage de biocides chimiques. Ce travail de thèse s’inscrit dans cette problématique et a pour objectif d’identifier de nouveaux composés actifs contre L. pneumophila en tenant compte de son comportement et de ses interactions au sein de son microenvironnement. De manière à obtenir des composés produits par des souches bactériennes appartenant à la même niche écologique que L. pneumophila, une campagne de prélèvement d’eau a été réalisée. Un total de 273 isolats environnementaux ont été isolés et testés contre L. pneumophila. Les résultats obtenus indiquent que 178 de ces isolats (65%) présentent une activité anti-Legionella. Quatre souches ont ensuite été sélectionnées (Aeromonas bestiarum SW257, Rahnella aquatilis SW265, Flavobacterium spp. PW52 et Pseudomonas spp PW329) et les composés actifs produits ont été caractérisés. A. bestiarum SW257 produit un peptide anti-Legionella. Flavobacterium sp. PW52 produit un mélange de composés anti-Legionella ayant des propriétés tensioactives, les flavolipides. Pseudomonas sp. PW329 produit des composés organiques volatiles, identifiés par SPME-GC-MS, actifs contre L. pneumophila. Enfin, R. aquatilis SW265 produit un sidérophore à activité anti-Legionella. / Water is essential to sustain life and water sources used for human consumption must be biologically safe, to avoid any risk for health. Indeed, the most common and widespread health risk associated with drinking water are infectious diseases caused by pathogenic microorganisms such as Legionella pneumophila. However, more efforts are needed to control disinfection by-products and minimize people exposure to potentially hazardous chemicals while maintaining adequate disinfection to ensure good water quality. Thus, this work aimed to find natural antibacterial compounds to control L. pneumophila growth using bacteria from freshwater environments. Environmental aquatic bacteria were sampled from five freshwater sources to get a large culturable bacterial collection. A total of 273 bacterial isolates were recovered and screened for their ability to produce anti-Legionella compounds. Among those, 178 (65%) were shown to be active against L. pneumophila. Four strains (Aeromonas bestiarum SW257, Rahnella aquatilis SW265, Flavobacterium spp. PW52, and Pseudomonas spp PW329) were next selected for the characterization of their active compounds. A. bestiarum SW257 produces an anti-Legionella peptide, and Flavobacterium spp PW52 produces a mixture of anti-Legionella compounds with surface active properties, named flavolipids. Finally Pseudomonas sp. PW329 delivers many volatile organic compounds, and R. aquatilis SW26 produces a anti-Legionella siderophore.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009. / Surfactants are compounds that reduce interfacial surface tension, resulting in detergency, emulsifying, foaming and dispersing properties. Surfactants produced via biochemical processes (biosurfactants) form a niche market with their low toxicity, biodegradability and high specificity attributes. Biosurfactants have recently received considerable attention owing to their potential as biomedical molecules. In this study a knowledge base was established for the development of a process which produces biosurfactants for use as antimicrobial agents. Specifically, rhamnolipid biosurfactants were produced from Pseudomonas aeruginosa and tested for antimicrobial activity against target organisms. Accurate and reproducible analyses for the quantification of rhamnolipids and antimicrobial activity were developed. The amount of rhamnolipid was determined indirectly by measuring the rhamnose concentration. A novel HPLC method as well as an orcinol colorimetric method were developed for rhamnose measurement. In order to obtain accuracy with the orcinol method it was found that samples must be extracted at least three times prior to the analysis. An examination of literature on rhamnolipid production showed that many studies used colorimetric methods without extraction. Antibacterial activity was quantified by zone clearing around wells of supernatant in soft agar containing the target organism Mycobacterium aurum. This target organism is especially important in a South African context, since it is used to indicate possible susceptibility of tuberculosis to antibiotics. This method was developed for antibacterial testing, after a standard disk diffusion method proved to be ineffective. Antifungal activity of rhamnolipids was evaluated against the fungus Botrytis cinerea, by growing a lawn of fungus on a plate and adding rhamnolipid. The factors influencing rhamnolipid production were studied by growing different Pseudomonas aeruginosa strains from the ATCC culture collection, namely ATCC 9027 and ATCC 27853 as well as a locally isolated strain under different media conditions. The initial focus was on production of biosurfactants in media containing glucose as substrate. Alkanes were subsequently investigated as an alternative substrate, since they are readily available in South Africa as byproducts from the petrochemical industry. The rhamnolipids produced from the culture collection strains were evaluated for their antibacterial activity against Mycobacterium aurum. A number of key factors were identified which were important for the development of a rhamnolipid production process. Of critical importance were the media conditions. Good production was achieved on glucose media containing a phosphate limitation, pH buffering around neutral pH and a high carbon concentration (2 % carbon). When Pseudomonas aeruginosa ATCC 9027 was cultured on this medium (a minimal salts phosphate limited medium with a Tris buffer), it produced 1.31 g/l rhamnose, equivalent to 4.0 g/l rhamnolipid. This rhamnolipid concentration is 2.7-fold higher that of 1.47 g/l reported in the literature with the same strain (cultured on a different phosphate limited medium The particular strain also proved to be a factor which influenced the yield of rhamnolipids. A rhamnose concentration of 0.43 g/l was obtained with Pseudomonas aeruginosa ATCC 27853 grown on MSM+Tris medium, compared to 1.31 g/l produced by Pseudomonas aeruginosa ATCC 9027 on the same medium. The most promising strain and medium, Pseudomonas aeruginosa ATCC 9027 and MSM+Tris medium, were evaluated under controlled conditions in an instrumented bioreactor. Nearly double the rate of growth and production were obtained in the bioreactor, indicating that production time can be shortened considerably under controlled conditions. However, when compared to shake flask studies, only a 4 % increase in growth and a 5 % increase in rhamnolipid production were achieved in the bioreactor, indicating that the yield was limited by the media components or process conditions. With media containing hexadecane as sole carbon source, negligible rhamnolipid production was achieved. Slow growth was observed and the stationary phase had not been reached even after 2 weeks of growth. It was shown that in glucose media rhamnolipid production only commenced in the stationary phase. Since the stationary phase was not reached during growth on hexadecane, rhamnolipids, which are known to increase the availability of alkanes through emulsification and solubilisation, could not be produced. A strategy was devised to accelerate growth on alkane media. A dual substrate medium containing both glucose and hexadecane was investigated. It was hypothesised that growth would be promoted by glucose leading to rhamnolipid production, which would then increase the uptake of hexadecane. Rhamnolipid was produced in the dual substrate experiments, but the hexadecane uptake was still poor. This was suggested to be due to the exposure of the cells to glucose in the inoculum or test flask, which hampered the ability of the cells to utilise hexadecane. It was reasoned that the ability to utilise hexadecane was determined by the cell hydrophobicity, which was influenced by the exposure to hydrophilic or hydrophobic substrates. Rhamnolipids from Pseudomonas aeruginosa ATCC 9027 and ATCC 27853 were shown to have antibacterial activity against Mycobacterium aurum. The largest zone of clearing of 45 mm was obtained with 4 g/l rhamnolipid from Pseudomonas aeruginosa ATCC 9027. The activity was shown to be directly related to the rhamnolipid concentration, highlighting the importance of maximising the biosurfactant yield when developing a process for the production of rhamnolipids as antimicrobial agents. Antifungal activity tests against Botrytis cinerea were inconclusive. Future studies should expand the antimicrobial application of rhamnolipids by testing their activity against a larger range of target organisms. In order to maximise the rhamnolipid yield in future studies, a fed batch process is proposed which would increase the cell density thereby increasing rhamnolipid production and prolonging the stationary phase, which was found to be the phase associated with rhamnolipid production. Different feeding strategies should be investigated, depending on the kinetics of substrate consumption. It is desirable to feed the smallest volume of substrate that is necessary with a high concentration in order to keep the dilution rate low and maximise the product concentration. A factorial design is recommended for this purpose. Further studies with alkanes as carbon source should be conducted using strains that have been maintained and cultured on media containing alkanes as sole carbon source. Alternative biosurfactant producing strains should also be investigated, which have higher natural cell hydrophobicities.

Aproximadamente 85% do leite utilizado para manufatura de queijos é descartado em forma soro, um subproduto com alto valor nutricional, que, embora seja usado na alimentação animal, como fertilizante e em suplementos alimentares, é considerado um poluente ambiental quando descartado em efluentes sem o devido tratamento. No Brasil, 40% do soro de leite produzido pelas indústrias de laticínios são descartados como efluentes sem tratamento adequado e, uma alternativa de melhor aproveitamento é a sua utilização como meio de cultivo alternativo para obtenção de micro-organismos e biomoléculas de interesse farmacêutico e industrial. Esporos de Bacillus atrophaeus são amplamente utilizados como bioindicadores para esterilização, e também são conhecidos por expressar biossurfactantes, que possuem potencial atividade antimicrobiana e anti-adesiva. Devido a sua importância e ampla aplicabilidade, estudos que viabilizem a obtenção deste micro-organismo e seus subprodutos devem ser melhor explorados. O objetivo deste trabalho foi avaliar a utilização de soro de leite como meio de cultivo para obtenção de esporos de B. atrophaeus e de biossurfactantes, como forma de reaproveitamento do que seria descartado. B. atrophaeus foi cultivado em meios contendo de 2,5g/L a 40 g/L de carboidratos expressos em soro de leite, a 37°C por 24 horas, 3 e 6 dias, em meios líquido (em água) e sólido (1% ágar). Foram determinados a biomasssa celular, a concentração de biossurfactantes e a resistência térmica dos esporos obtidos. Para o cultivo de 20g/L em meio líquido, a biomassa de 0,59 (± 0,24) g/L, expressou 30,0 (± 1,40) mg/L de biossurfactantes, o que corresponde a população de 4,8*108 esporos/mL e valor D = 2,73 min. Em meio sólido, em 20g/L, a biomassa de 0,65 (± 0,01) g/L, expressou 48,1 (± 2,84) mg/L de biossurfactantes, o que corresponde a população de 2,0*108 esporos/mL e valor D = 1,50 min, valor similar ao cultivo em PCA de no qual a biomassa de 1,22 (± 0,01) g/L, expressou 59,3 (± 0,4) mg/L de biossurfactantes, o que corresponde a população de 4,6*108 esporos/mL e valor D = 1,20 min. Os resultados demonstram a viabilidade do uso de soro de leite como meio para crescimento e esporulação de B. atrophaeus e biossurfactantes, podendo ser reaproveitado para obtenção de produtos de interesse farmacêutico e industrial. / Approximately 85% of the milk used to manufacture cheese is discarded as whey, a byproduct with high nutritional value, which, it is used in animal in feeding, fertilizer and in food supplements, whey is considered an environmental pollutant when discarded without proper treatment. In Brazil, 40% of the whey produced by dairy industries is discarded as effluent without the necessary treatment and, an alternative for its use is the utilization as an alternative culture media to obtain microorganisms and biomolecules of pharmaceutical and industrial interest. Spores of Bacillus atrophaeus are widely used as bioindicators for sterilization, and are also known for biosurfactant expression, which possess antimicrobial and anti-adhesive activity. Due to its importance and broad applicability, studies that enable the obtainment of this microorganism and its subproducts must be explored. The objective of this study was to evaluate the utilization of whey as culture media to obtain B. atrophaeus spores and biosurfactants, in a way to reuse what it would be discarded. B. atrophaeus was cultivated in medias containing 2.5 g/L to 40.0 g/l of carbohydrates expressed in whey, at 37ºC for 24 hours, 3 and 6 days, in medias reconstituted in liquid (water) and in solid state (1% agar). It was determined the biomass, biosurfactants concentration and thermal resistance of the obtained spores. For cultivation of 20.0 g/L in liquid state, the biomass was 0.59 (± 0.24) g/L, and it has expressed 30.0 (± 1.40) mg/L of biosurfactants, which corresponds a spore population of 4.8*108 spores/mL and D-value= 2.73 min. In 20.0 g/L at solid state, the biomass of 0.65 (± 0.01) g/L, expressed 48.1 (± 2.84) mg/L of biosurfactants, which corresponds to a spore population of 2.0*108 spores/mL and D-value= 1.50 min, similar value when cultivated in PCA which the biomass of 1.22 (± 0.01) g/L, expressed 59.3 (± 0.4) mg/L of biosurfactants, which corresponds to a spore population of 4.6*108 spores/mL and D-value = 1.20 min. The results have shown the viability of whey as culture media for growth and sporulation of B. atrophaeus and biosurfactants, which can be reused to obtain products of pharmaceutical and industrial interest.

Na natureza, os microorganismos podem apresentar forma de vida planctônica ou podem estar aderidos a superfícies formando comunidades conhecidas como biofilmes. A formação de biofilmes na indústria alimentícia é uma constante preocupação visto que os microorganismos aderidos podem causar contaminações persistentes, levando a deterioração do alimento e a transmissão de doenças. Uma alternativa para evitar a adesão bacteriana e a formação de biofilmes é o pré-condicionamento de superfícies com biosurfatantes, que são compostos tensoativos de origem microbiana capazes de alterar as propriedades físico-químicas e conseqüentemente modificar as interações entre a bactéria e a superfície. Os biosurfatantes, surfactina obtida de Bacillus subtilis e ramnolipídeo de Pseudomonas aeruginosa, foram testados quanto a capacidade de evitar a adesão e remover biofilmes de bactérias patogênicas de interesse alimentar. Foram avaliadas culturas individuais e mistas de Staphylococcus aureus, Listeria monocytogenes, e Salmonella Enteritidis utilizando-se como modelo superfícies de poliestireno. O pré-condicionamento da superfície com surfactina na concentração de 0,25% reduziu a adesão de Salmonella Enteritidis e Listeria monocytogenes em 42%, enquanto que o tratamento com ramnolipídeo a 1% reduziu a adesão de Listeria monocytogenes e Staphylococcus aureus ao poliestireno em 57,8% e 67,8% respectivamente. O condicionamento com os biosurfatantes não se mostrou eficiente na redução da adesão das culturas mistas das bactérias se comparado aos resultados obtidos para as culturas individuais. O poliestireno condicionado com os biosurfatantes apresentou redução na hidrofobicidade devido ao caráter aniônico destas moléculas. A repulsão eletrostática e a redução das interações hidrofóbicas promovidas pelo condicionamento do poliestireno com ramnolipídeo foram fatores determinantes na atividade antiadesiva observada para L. monocytogenes e S. aureus, entretanto os resultados obtidos para a superfície tratada com surfactina sugerem que outros parâmetros influenciaram nos resultados observados. Após 2 h de contato a surfactina na concentração de 0,1% promoveu a remoção de 63,7% do biofilme de S. aureus, 95,9% do biofilme de L. monocytogenes, 35,5% do biofilme de S. Enteritidis e 58,5% do biofilme da cultura mista das três bactérias. Já o ramnolipídeo na concentração de 0,25% removeu 58,5% do biofilme de S. aureus, 26,5% do biofilme de L. monocytogenes, 23,0 % do biofilme de S. Enteritidis e 24% do biofilme da cultura mista após 2 h contato. De modo geral, o aumento do tempo de contato e da concentração dos biosurfatantes reduziu a remoção dos biofilmes. A surfactina e o ramnolipídeo demonstraram potencial para uso como agentes anti-adesivos assim como para a remoção de biofilmes de bactérias patogênicas de importância alimentar. / In nature, microrganisms can live as planktonic cells or can be found living in communities attached in surfaces forming biofilms. Biofilm represents a great concern for food industry, since it can be a source of persistent contamination that can lead to food spoilage and the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants that are microbial compounds that can modify the physico-chemical properties of the surfaces changing bacterial interactions and consequently adhesion. The biosurfactants, surfactin obtained from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated as agents to avoid the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes e Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0,25% reduces in 42,0% the adhesion of L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1,0% reduced in 57,8% the adhesion of L. monocytogenes and in 67,8% the adhesion of S. aureus to polystyrene. The conditioning of surface with biosurfactants was less effective to avoid adhesion of mixed cultures of the bacteria when compared with the results obtained for individual cultures. The polystyrene surface conditioned with the biosurfactants showed a reduction in the hydrophobicity due to the anionic character of the molecules. The electrostatic repulsion and the reduction on hydrophobic interactions promoted by the conditioning of surface with rhamnolipids were determinant factors to explain the anti-adhesive activity observed for L. monocytogenes and S. aureus, however, the data obtained with surfactin suggest that other parameters have influenced the results observed. After 2 h contact with surfactin at 0,1% concentration, the pre-formed biofilms of S. aureus were reduced by 63,7%, L. monocytogenes biofilms were reduce by 95,9% , S. Enteritidis biofilms by 35,5% and the mixed culture biofilm by 58,5%. The rhamnolipids at 0,25% concentration removed 58,5% of biofilm of S. aureus, 26,5% of the biofilm of L. monocytogenes, 23,0% the biofilm of S. Enteritidis and 24,0% the biofilm of the mixed culture after 2 h of contact. In general, the increase in concentration of biosurfactants and in the time of contact decreases the biofilm remove percentage. These results demonstrate that surfactin and rhamnolipids present potential to be used as agents to control the attachment and to disrupt biofilms of food-borne pathogens.

Intoxicações alimentares são uma das causas mais significativas de mortalidade em países desenvolvidos e em desenvolvimento, sendo as contaminações bacterianas as responsáveis pela maioria dos casos. Dentre elas destacam-se as causadas por espécies de Staphylococcus, que são nocivas tanto pela infecção do organismo hospedeiro, quanto pela intoxicação por enterotoxinas termoestáveis presentes em alimentos contaminados e mal acondicionados. Além disso, a capacidade de S. aureus formarem biofilmes confere maior proteção contra agentes de controle. Neste contexto, torna-se importante desenvolver novos métodos visando inibir estes agentes patogênicos. Uma alternativa ao emprego de conservantes sintéticos é a utilização de biossurfatantes, como os ramnolipídeos (RL) que, além de apresentarem alta estabilidade a temperatura, pH e concentração salina, apresentam baixa toxicidade e são biodegradáveis. O objetivo deste trabalho foi avaliar o efeito do pH na atividade antimicrobiana dos RL sobre células planctônicas e sésseis de Staphylococcus aureus (ATCC 8095) e comparar seu efeito ao do dodecil sulfato de sódio (SDS). Os resultados mostraram que o pH exerce grande influência na ação dos surfatantes, sendo mais efetiva em valores de pH menores. Em pH 5, os RL apresentaram ação bactericida (CBM=19,5 mg L-1) superior ao SDS (CBM=39,1 mg L-1), sendo capazes de inibir e remover 80% dos biofilmes em concentrações de 156,2 mg L-1 e reduzir em cerca de 40 % a hidrofobicidade da superfície celular de S. aureus. Em valores de pH maiores a ação dos RL não superou a do SDS, porém estes ainda mostraram resultados promissores, principalmente na remoção de biofilmes evidenciado pela microscopia confocal. A espectroscopia FTIR revelou que quando em pH 6, 7 e 8 S. aureus, possivelmente, induz alterações em sua membrana celular a fim de diminuir a sensibilidade aos surfatantes. As imagens de MEV mostraram que os RL promovem deformações nas células, podendo levá-las a ruptura. O aumento da força iônica, promovido pela adição de NaCl no meio, favoreceu a ação antimicrobiana dos RL, o que torna a aplicação dos RL em alimentos bastante promissora. / Food poisoning can be considered one of the most significant causes of mortality in developed and developing countries with bacterial contamination being responsible for the majority of cases. Among them are those caused by Staphylococcus species that can beharmful, both from the infection of the host organism as the intoxication by thermostable enterotoxins present in contaminated and poorly conditioned food. In addition, the ability to form biofilms gives S. aureus greater protection against control agents. Under this context, it becomes important to develop new methods to inhibit those pathogens. An alternative to the use of synthetic preservatives are biosurfactants such as rhamnolipids (RL), which shows high stability to temperature, pH and salt concentration along with the fact that they have low toxicity and are biodegradable. The aim of this study was to evaluate the effect of pH on the antimicrobial activity of RL on planktonic and sessile Staphylococcus aureus cells (ATCC 8095) and compare its effect to that of the sodium dodecyl sulfate (SDS). The results showed that pH exerts a great influence on the action of surfactants, with greater effectiveness at lower pH. At pH 5, RL presented higher bactericidal action (CBM = 19.5 mg L-1) than SDS (CBM = 39.1 mg L-1), also being able to inhibit and remove 80% of biofilms at concentrations of 156. 2 mg L- 1 and reduce the hydrophobicity of S. aureus cell surface by about 40%. At higher pH values, the action of RL did not exceed that of SDS, neverthelessthey showed promising results, mainly on the removal of biofilms evidenced by confocal microscopy. FTIR spectroscopy revealed that at pH 6, 7 and 8,S. aureus possibly induces changes in its cell membrane in order to decrease the sensitivity to surfactants. The MEV images showed that the RL cause deformations in the cells, which can lead to rupture. The increase in ionic strength, promoted by the addition of NaCl in the medium, favored the antimicrobial action of RL, which makes the application of RL in foods very promising.

Os biossurfactantes são compostos de origem vegetal e microbiana, com propriedades tensoativas e/ou emulsificantes, que devido ao seu apelo ambiental em relação aos surfactantes sintéticos vêm se destacando nos últimos anos. São considerados produtos ecologicamente corretos, por serem biodegradáveis e exibirem reduzida/nula toxicidade. Além disso, a possibilidade de biossíntese utilizando processos fermentativos, empregando subprodutos agroindustriais como fonte de nutrientes é outra vantagem destes bioprodutos. O presente trabalho avaliou a produção de biossurfactantes por leveduras utilizando o hidrolisado hemicelulósico de bagaço de cana-de-açúcar como fonte de carbono. Durante o estudo utilizou-se processos fermentativos em frascos na biossíntese dos biossurfactantes e métodos cromatográficos, espectrofotométricos, espectrométricos na caracterização dos hidrolisados e dos biossurfactantes. Observou-se que as 30 leveduras estudadas produziram biossurfactantes glicolipídicos em meio de cultivo suplementado com hidrolisado hemicelulósico de bagaço de cana-de-açúcar, utilizando entre 14 e 99 % da xilose e exibiram produtividades variando de 0.0035 a 0,1667 g/L.h. Os glicolipídeos produzidos pelas leveduras SSS1, SSS5, SSS6, SSS13, SSS18, SSS27 e SSS28 apresentaram potencial larvicida quando testados frente a Aedes aegypti, eliminando entre 20 - 100 % das larvas. Dentre as leveduras estudadas, a Trichosporon mucoides (SSS11) mostrou-se como melhor produtora de biossurfactante glicolipídico (produção de 11,334 ± 2,169 g/L - produtividade de 0,1667 g/L.h), produzindo um soforolipídeo com propriedades emulsificantes destacadas em diversas condições ambientais, onde os parâmetros temperatura, salinidade e pH foram variados. Após a otimização da produção tendo como base o meio suplementado com hidrolisado hemicelulósico, o biossurfactante produzido pela levedura apresentou um índice de emulsificação variando entre 86 - 90 % e uma tensão superficial de 34 mN/m, sendo considerado um promissor bioproduto para aplicação industrial. / Biosurfactants are plant and microbial metabolites with surface-active properties and / or emulsifiers, which due to environmental concerns about synthetic surfactants, have been highlighted in recent years. This intrinsic biodegradability and non-toxity of biosurfactants are responsible for their increased importance when compared to synthetic analogues. More than that, additionally the possibility of using alternative substracts for its synthesis, like biodegradable agroindustrial subproducts and the use of a bioprocess, reduce their the final cost of the production. Considering this, the present research evaluated biosurfactants production by yeasts using sugar cane bagasse hemicellulosic hydrolysate as carbon source, suggesting these compounds are potential alternative products for lignocellulosic biorefineries. The 30 yeasts studied produced glycolipid biosurfactants in supplemented sugar cane bagasse hydrolysate growth media, with 14 - 99 % of xylose and 0,0035 -0,1667 g/L.h productivity. The glycolipids produced by yeasts SSS1, SSS5, SSS6, SSS13, SSS18, SSS27 and SSS28 presented high larvicidas properties when tested against Aedes aegypti, killing 20 - 100 % of larvaes. Among the analyzed yeasts, Trichosporon mucoides (SSS11) was the best glycolipid biosurfactant productor (11,334 ± 2,169 g/L - productivity of 0,1667 g/L.h), allowing the isolation of a sophorolipid with emulsifier properties, tested in many physico-chemical conditions like variation of temperature, salt concentrations and pH. After nutritional otimization using the suplemented hemicellulosic hydrolysate media growth, the biosurfactant produced by Trichosporon mucoides presented emulsification levels from 86 - 90 % and a superficial tension of 34 mN/m, being considered an important bioproduct for industrial application.

Aproximadamente 85% do leite utilizado para manufatura de queijos é descartado em forma soro, um subproduto com alto valor nutricional, que, embora seja usado na alimentação animal, como fertilizante e em suplementos alimentares, é considerado um poluente ambiental quando descartado em efluentes sem o devido tratamento. No Brasil, 40% do soro de leite produzido pelas indústrias de laticínios são descartados como efluentes sem tratamento adequado e, uma alternativa de melhor aproveitamento é a sua utilização como meio de cultivo alternativo para obtenção de micro-organismos e biomoléculas de interesse farmacêutico e industrial. Esporos de Bacillus atrophaeus são amplamente utilizados como bioindicadores para esterilização, e também são conhecidos por expressar biossurfactantes, que possuem potencial atividade antimicrobiana e anti-adesiva. Devido a sua importância e ampla aplicabilidade, estudos que viabilizem a obtenção deste micro-organismo e seus subprodutos devem ser melhor explorados. O objetivo deste trabalho foi avaliar a utilização de soro de leite como meio de cultivo para obtenção de esporos de B. atrophaeus e de biossurfactantes, como forma de reaproveitamento do que seria descartado. B. atrophaeus foi cultivado em meios contendo de 2,5g/L a 40 g/L de carboidratos expressos em soro de leite, a 37°C por 24 horas, 3 e 6 dias, em meios líquido (em água) e sólido (1% ágar). Foram determinados a biomasssa celular, a concentração de biossurfactantes e a resistência térmica dos esporos obtidos. Para o cultivo de 20g/L em meio líquido, a biomassa de 0,59 (± 0,24) g/L, expressou 30,0 (± 1,40) mg/L de biossurfactantes, o que corresponde a população de 4,8*108 esporos/mL e valor D = 2,73 min. Em meio sólido, em 20g/L, a biomassa de 0,65 (± 0,01) g/L, expressou 48,1 (± 2,84) mg/L de biossurfactantes, o que corresponde a população de 2,0*108 esporos/mL e valor D = 1,50 min, valor similar ao cultivo em PCA de no qual a biomassa de 1,22 (± 0,01) g/L, expressou 59,3 (± 0,4) mg/L de biossurfactantes, o que corresponde a população de 4,6*108 esporos/mL e valor D = 1,20 min. Os resultados demonstram a viabilidade do uso de soro de leite como meio para crescimento e esporulação de B. atrophaeus e biossurfactantes, podendo ser reaproveitado para obtenção de produtos de interesse farmacêutico e industrial. / Approximately 85% of the milk used to manufacture cheese is discarded as whey, a byproduct with high nutritional value, which, it is used in animal in feeding, fertilizer and in food supplements, whey is considered an environmental pollutant when discarded without proper treatment. In Brazil, 40% of the whey produced by dairy industries is discarded as effluent without the necessary treatment and, an alternative for its use is the utilization as an alternative culture media to obtain microorganisms and biomolecules of pharmaceutical and industrial interest. Spores of Bacillus atrophaeus are widely used as bioindicators for sterilization, and are also known for biosurfactant expression, which possess antimicrobial and anti-adhesive activity. Due to its importance and broad applicability, studies that enable the obtainment of this microorganism and its subproducts must be explored. The objective of this study was to evaluate the utilization of whey as culture media to obtain B. atrophaeus spores and biosurfactants, in a way to reuse what it would be discarded. B. atrophaeus was cultivated in medias containing 2.5 g/L to 40.0 g/l of carbohydrates expressed in whey, at 37ºC for 24 hours, 3 and 6 days, in medias reconstituted in liquid (water) and in solid state (1% agar). It was determined the biomass, biosurfactants concentration and thermal resistance of the obtained spores. For cultivation of 20.0 g/L in liquid state, the biomass was 0.59 (± 0.24) g/L, and it has expressed 30.0 (± 1.40) mg/L of biosurfactants, which corresponds a spore population of 4.8*108 spores/mL and D-value= 2.73 min. In 20.0 g/L at solid state, the biomass of 0.65 (± 0.01) g/L, expressed 48.1 (± 2.84) mg/L of biosurfactants, which corresponds to a spore population of 2.0*108 spores/mL and D-value= 1.50 min, similar value when cultivated in PCA which the biomass of 1.22 (± 0.01) g/L, expressed 59.3 (± 0.4) mg/L of biosurfactants, which corresponds to a spore population of 4.6*108 spores/mL and D-value = 1.20 min. The results have shown the viability of whey as culture media for growth and sporulation of B. atrophaeus and biosurfactants, which can be reused to obtain products of pharmaceutical and industrial interest.

Os biossurfactantes são compostos de origem vegetal e microbiana, com propriedades tensoativas e/ou emulsificantes, que devido ao seu apelo ambiental em relação aos surfactantes sintéticos vêm se destacando nos últimos anos. São considerados produtos ecologicamente corretos, por serem biodegradáveis e exibirem reduzida/nula toxicidade. Além disso, a possibilidade de biossíntese utilizando processos fermentativos, empregando subprodutos agroindustriais como fonte de nutrientes é outra vantagem destes bioprodutos. O presente trabalho avaliou a produção de biossurfactantes por leveduras utilizando o hidrolisado hemicelulósico de bagaço de cana-de-açúcar como fonte de carbono. Durante o estudo utilizou-se processos fermentativos em frascos na biossíntese dos biossurfactantes e métodos cromatográficos, espectrofotométricos, espectrométricos na caracterização dos hidrolisados e dos biossurfactantes. Observou-se que as 30 leveduras estudadas produziram biossurfactantes glicolipídicos em meio de cultivo suplementado com hidrolisado hemicelulósico de bagaço de cana-de-açúcar, utilizando entre 14 e 99 % da xilose e exibiram produtividades variando de 0.0035 a 0,1667 g/L.h. Os glicolipídeos produzidos pelas leveduras SSS1, SSS5, SSS6, SSS13, SSS18, SSS27 e SSS28 apresentaram potencial larvicida quando testados frente a Aedes aegypti, eliminando entre 20 - 100 % das larvas. Dentre as leveduras estudadas, a Trichosporon mucoides (SSS11) mostrou-se como melhor produtora de biossurfactante glicolipídico (produção de 11,334 ± 2,169 g/L - produtividade de 0,1667 g/L.h), produzindo um soforolipídeo com propriedades emulsificantes destacadas em diversas condições ambientais, onde os parâmetros temperatura, salinidade e pH foram variados. Após a otimização da produção tendo como base o meio suplementado com hidrolisado hemicelulósico, o biossurfactante produzido pela levedura apresentou um índice de emulsificação variando entre 86 - 90 % e uma tensão superficial de 34 mN/m, sendo considerado um promissor bioproduto para aplicação industrial. / Biosurfactants are plant and microbial metabolites with surface-active properties and / or emulsifiers, which due to environmental concerns about synthetic surfactants, have been highlighted in recent years. This intrinsic biodegradability and non-toxity of biosurfactants are responsible for their increased importance when compared to synthetic analogues. More than that, additionally the possibility of using alternative substracts for its synthesis, like biodegradable agroindustrial subproducts and the use of a bioprocess, reduce their the final cost of the production. Considering this, the present research evaluated biosurfactants production by yeasts using sugar cane bagasse hemicellulosic hydrolysate as carbon source, suggesting these compounds are potential alternative products for lignocellulosic biorefineries. The 30 yeasts studied produced glycolipid biosurfactants in supplemented sugar cane bagasse hydrolysate growth media, with 14 - 99 % of xylose and 0,0035 -0,1667 g/L.h productivity. The glycolipids produced by yeasts SSS1, SSS5, SSS6, SSS13, SSS18, SSS27 and SSS28 presented high larvicidas properties when tested against Aedes aegypti, killing 20 - 100 % of larvaes. Among the analyzed yeasts, Trichosporon mucoides (SSS11) was the best glycolipid biosurfactant productor (11,334 ± 2,169 g/L - productivity of 0,1667 g/L.h), allowing the isolation of a sophorolipid with emulsifier properties, tested in many physico-chemical conditions like variation of temperature, salt concentrations and pH. After nutritional otimization using the suplemented hemicellulosic hydrolysate media growth, the biosurfactant produced by Trichosporon mucoides presented emulsification levels from 86 - 90 % and a superficial tension of 34 mN/m, being considered an important bioproduct for industrial application.