Os polissacarídeos possuem diversas aplicações industriais devido a sua ampla variedade de propriedades físico-químicas. Além desses empregos tradicionais, pesquisas mais recentes estão impulsionando o uso de polissacarídeos para novas aplicações, principalmente na área de terapia farmacêutica. Com o objetivo de ampliar a produção industrial de polissacarídeos microbianos, pesquisas têm se concentrado nos exopolissacarídeos (EPS), que apresentam produtividade elevada e processos de extração e purificação mais simples quando comparados aos polímeros tradicionais. A lasiodiplodana, uma ?-(1->6)-D-glucana, é um EPS produzido por Lasiodiplodia theobromae, um fungo filamentoso característico de regiões tropicais e patógeno de mais de 500 espécies vegetais. Na produção de EPS observa-se que, como resultado do crescimento e da produção do biopolímero, o meio de cultivo torna-se mais viscoso, tornando difícil a manutenção da homogeneidade no biorreator e, consequentemente, prejudicando a transferência de massa e oxigênio no meio. Nos cultivos em que ocorrem mudanças na reologia do meio e nos quais são utilizados fungos filamentosos, sensíveis ao cisalhamento, o uso de biorreatores convencionais, como o Stirred Tank Reactor (STR) e o Airlift, não é adequado. Nesse contexto, no presente estudo tem-se como objetivo estudar a cinética de crescimento, consumo de substrato e produção de EPS pelo fungo filamentoso Lasiodiplodia theobromae, a partir de glicose e de glicerol, no Biorreator Agitado e Aerado de Baixo Cisalhamento (BAABC) e compará-lo com o STR. No estudo inicial, realizado em frascos agitados, a maior produção de EPS (6,49 ± 0,03 g/L) foi alcançada pelo ensaio G, que continha a maior concentração glicose testada (50 g/L) e a menor concentração de extrato de levedura (3 g/L). Quando se utilizou glicerol como fonte de carbono, a maior produção de EPS (3,39 ± 0,06 g/L) foi observada no Ensaio O, que continha 30 g/L de glicerol e a maior concentração de fonte de nitrogênio testada (12 g/L de extrato de levedura). Nos ensaios em biorreatores, quando utilizou-se glicose como fonte de carbono observou-se que o BAABC com controle de temperatura proporcionou uma produção de 3,17 ± 0,16 g/L de EPS, concentração inferior à obtida em frascos, porém bastante superior à obtida no biorreator STR (0,70 ± 0,12 g/L). Para os meios contendo glicerol, o biorreator STR proporcionou uma produção de EPS de 3,02 ± 0,19 g/L, enquanto no BAABC a concentração de EPS obtida foi muito menor (1,45 ± 0,25 g/L). Analisando-se, porém, a concentração máxima de biomassa obtida (28,86 ± 1,46 g/L) bem como os parâmetros cinéticos relacionados (YX/S e QX), nota-se que a produção de biomassa foi muito superior e mais eficiente no BAABC. Apesar das diferenças morfológicas visualmente observadas e confirmadas por meio de microscopia óptica nos EPS obtidos a partir de glicose e de glicerol, as análises de Difração de raios-X e Espectroscopia de absorção na região do infravermelho com Transformada de Fourier permitiram evidenciar a similaridade estrutural entre ambos os EPS. / Polysaccharides have several industrial applications because of their wide variety of physicochemical properties. In addition to these traditional applications, recent research is driving the use of polysaccharides towards new applications, especially in the field of pharmaceutical therapy. With the aim of increasing the industrial production of microbial polysaccharides, research has focused on exopolysaccharides (EPS), which have high productivity and simpler extraction and purification processes when compared to traditional polymers. Lasiodiplodana, a ?-(1->6)-D-glucan, is an EPS produced by Lasiodiplodia theobromae, a filamentous fungus characteristic of tropical areas and pathogenic of more than 500 plant species. In EPS production, as a result of the growth and biopolymer production, the culture medium becomes more viscous, making it difficult to maintain homogeneity inside the bioreactor and, consequently, harming the transfer of mass and oxygen in the medium. In cultures where changes in media rheology occur and in which shear-sensitive filamentous fungi are used, the use of conventional bioreactors, such as Stirred Tank Reactor (STR) and Airlift, is not appropriate. In this context, the objective of the present study was to study the growth kinetics, substrate consumption and EPS production by the Lasiodiplodia theobromae filamentous fungus, from glucose and glycerol, in the Low-Shear Aerated-Agitated Bioreactor (LSAAB) and compare it with the STR. In the initial study, conducted in shaken flasks, the highest EPS production (6.49 ± 0.03 g/L) was achieved by the G test, which contained the highest glucose concentration tested (50 g/L) and the lowest concentration of yeast extract (3 g/L). When glycerol was used as the carbon source, the highest EPS yield (3.39 ± 0.06 g/L) was observed in Test O, which contained 30 g/L glycerol and the highest concentration of nitrogen source tested (12 g/L of yeast extract). In the bioreactor trials, when glucose was used as the carbon source, it was observed that the LSAAB with temperature control provided a production of 3.17 ± 0.16 g/L of EPS, a concentration lower than that obtained in flasks, but rather higher than that obtained in the STR bioreactor (0.70 ± 0.12 g/L). For the glycerol-containing media, the STR bioreactor produced 3.02 ± 0.19 g/L of EPS, while in LSAAB the EPS concentration obtained was much lower (1.45 ± 0.25 g/L). However, analyzing the maximum concentration of biomass obtained (28.86 ± 1.46 g/L) as well as the related kinetic parameters (YX/S and QX), biomass production was much higher and more efficient in LSAAB. In spite of the visually observed morphological differences confirmed by optical microscopy in EPS obtained from glucose and glycerol, X-ray diffraction and absorption spectroscopy analyzes in the infrared region with Fourier transform showed the structural similarity between both EPS.

Os polissacarídeos possuem diversas aplicações industriais devido a sua ampla variedade de propriedades físico-químicas. Além desses empregos tradicionais, pesquisas mais recentes estão impulsionando o uso de polissacarídeos para novas aplicações, principalmente na área de terapia farmacêutica. Com o objetivo de ampliar a produção industrial de polissacarídeos microbianos, pesquisas têm se concentrado nos exopolissacarídeos (EPS), que apresentam produtividade elevada e processos de extração e purificação mais simples quando comparados aos polímeros tradicionais. A lasiodiplodana, uma ?-(1->6)-D-glucana, é um EPS produzido por Lasiodiplodia theobromae, um fungo filamentoso característico de regiões tropicais e patógeno de mais de 500 espécies vegetais. Na produção de EPS observa-se que, como resultado do crescimento e da produção do biopolímero, o meio de cultivo torna-se mais viscoso, tornando difícil a manutenção da homogeneidade no biorreator e, consequentemente, prejudicando a transferência de massa e oxigênio no meio. Nos cultivos em que ocorrem mudanças na reologia do meio e nos quais são utilizados fungos filamentosos, sensíveis ao cisalhamento, o uso de biorreatores convencionais, como o Stirred Tank Reactor (STR) e o Airlift, não é adequado. Nesse contexto, no presente estudo tem-se como objetivo estudar a cinética de crescimento, consumo de substrato e produção de EPS pelo fungo filamentoso Lasiodiplodia theobromae, a partir de glicose e de glicerol, no Biorreator Agitado e Aerado de Baixo Cisalhamento (BAABC) e compará-lo com o STR. No estudo inicial, realizado em frascos agitados, a maior produção de EPS (6,49 ± 0,03 g/L) foi alcançada pelo ensaio G, que continha a maior concentração glicose testada (50 g/L) e a menor concentração de extrato de levedura (3 g/L). Quando se utilizou glicerol como fonte de carbono, a maior produção de EPS (3,39 ± 0,06 g/L) foi observada no Ensaio O, que continha 30 g/L de glicerol e a maior concentração de fonte de nitrogênio testada (12 g/L de extrato de levedura). Nos ensaios em biorreatores, quando utilizou-se glicose como fonte de carbono observou-se que o BAABC com controle de temperatura proporcionou uma produção de 3,17 ± 0,16 g/L de EPS, concentração inferior à obtida em frascos, porém bastante superior à obtida no biorreator STR (0,70 ± 0,12 g/L). Para os meios contendo glicerol, o biorreator STR proporcionou uma produção de EPS de 3,02 ± 0,19 g/L, enquanto no BAABC a concentração de EPS obtida foi muito menor (1,45 ± 0,25 g/L). Analisando-se, porém, a concentração máxima de biomassa obtida (28,86 ± 1,46 g/L) bem como os parâmetros cinéticos relacionados (YX/S e QX), nota-se que a produção de biomassa foi muito superior e mais eficiente no BAABC. Apesar das diferenças morfológicas visualmente observadas e confirmadas por meio de microscopia óptica nos EPS obtidos a partir de glicose e de glicerol, as análises de Difração de raios-X e Espectroscopia de absorção na região do infravermelho com Transformada de Fourier permitiram evidenciar a similaridade estrutural entre ambos os EPS. / Polysaccharides have several industrial applications because of their wide variety of physicochemical properties. In addition to these traditional applications, recent research is driving the use of polysaccharides towards new applications, especially in the field of pharmaceutical therapy. With the aim of increasing the industrial production of microbial polysaccharides, research has focused on exopolysaccharides (EPS), which have high productivity and simpler extraction and purification processes when compared to traditional polymers. Lasiodiplodana, a ?-(1->6)-D-glucan, is an EPS produced by Lasiodiplodia theobromae, a filamentous fungus characteristic of tropical areas and pathogenic of more than 500 plant species. In EPS production, as a result of the growth and biopolymer production, the culture medium becomes more viscous, making it difficult to maintain homogeneity inside the bioreactor and, consequently, harming the transfer of mass and oxygen in the medium. In cultures where changes in media rheology occur and in which shear-sensitive filamentous fungi are used, the use of conventional bioreactors, such as Stirred Tank Reactor (STR) and Airlift, is not appropriate. In this context, the objective of the present study was to study the growth kinetics, substrate consumption and EPS production by the Lasiodiplodia theobromae filamentous fungus, from glucose and glycerol, in the Low-Shear Aerated-Agitated Bioreactor (LSAAB) and compare it with the STR. In the initial study, conducted in shaken flasks, the highest EPS production (6.49 ± 0.03 g/L) was achieved by the G test, which contained the highest glucose concentration tested (50 g/L) and the lowest concentration of yeast extract (3 g/L). When glycerol was used as the carbon source, the highest EPS yield (3.39 ± 0.06 g/L) was observed in Test O, which contained 30 g/L glycerol and the highest concentration of nitrogen source tested (12 g/L of yeast extract). In the bioreactor trials, when glucose was used as the carbon source, it was observed that the LSAAB with temperature control provided a production of 3.17 ± 0.16 g/L of EPS, a concentration lower than that obtained in flasks, but rather higher than that obtained in the STR bioreactor (0.70 ± 0.12 g/L). For the glycerol-containing media, the STR bioreactor produced 3.02 ± 0.19 g/L of EPS, while in LSAAB the EPS concentration obtained was much lower (1.45 ± 0.25 g/L). However, analyzing the maximum concentration of biomass obtained (28.86 ± 1.46 g/L) as well as the related kinetic parameters (YX/S and QX), biomass production was much higher and more efficient in LSAAB. In spite of the visually observed morphological differences confirmed by optical microscopy in EPS obtained from glucose and glycerol, X-ray diffraction and absorption spectroscopy analyzes in the infrared region with Fourier transform showed the structural similarity between both EPS.

The formation of the Salmonella extracellular matrix is a multicellular behavior important for environmental persistence. It is comprised of uniquely but ill-defined assembled thin aggregative fimbriae (Tafi), cellulose and uncharacterized polysaccharides. Consequently, investigations were launched into further clarifying Tafi assembly and the polysaccharide constituents of the extracellular matrix. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC has been elusive. In this study using the clinical isolate, Salmonella Enteritidis 27655-3b, agfBAC transcripts were detected using a reverse transcriptase and transcription was not enhanced by replacement of a stem-loop structure immediately preceding agfC. AgfChis was purified, localized to the periplasm, and found to specifically bind noncrystalline cellulose suggesting an association with the extracellular matrix. An inframe ΔagfC mutant displayed an abundance of 20 nm fibers, which could be complemented with agfC in trans, in addition to Tafi and an increase in cell hydrophobicity. Depolymerization of purified 20 nm fibers required exceptionally stringent conditions to release what proved to be AgfA subunits revealing the 20 nm fibers as AgfA assemblages of unique morphology. The role of AgfC in Tafi assembly was investigated further via a novel, quantitative antibody-capture assay of in-frame agf mutants. A soluble antibody-accessible form of AgfA was captured in wt, ΔagfB and ΔagfF strains in support of the extracellular nucleation-precipitation pathway of Tafi assembly, but not in ΔagfC or ΔagfE mutants. These results suggest that AgfC and AgfE are required for AgfA’s extracellular assembly and thus may act as atypical AgfAspecific chaperones which facilitate Tafi assembly. The implications of these results are presented in an assembly model for Tafi. Additional investigations revealed that Salmonella produces an O-Antigen capsule co-regulated with the extracellular matrix. Structural analysis of purified extracellular polysaccharides (EPS) yielded a repeating oligosaccharide unit similar to iv that of lipopolysaccharide O-Antigen with modifications. Putative carbohydrate transport and regulatory operons important for capsule expression, designated emcA-H and emcIJ, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various in-frame Δemc mutants where emcG and emcE were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that, AgfD differentially regulated the emc operons in coordination with extracellular matrix genes. Survival assays demonstrated the capsule is important for desiccation tolerance. The emc genes were found to be conserved in Salmonellae and thus, the O-Antigen extracellular matrix capsule may be a conserved survival strategy important for environmental persistence. Finally, a compositionally unique acidic EPS was found associated with the extracellular matrix. In-frame ΔbcsA, ΔemcG and ΔagfA mutants but neither ΔagfAΔbcsA nor ΔagfD mutants bound calcofluor, a β-glucan binding fluorescent agent, suggesting that multicellular behavior itself and not necessarily AgfD alone was influencing EPS expression. A transposon library was screened by ELISA using serum generated against purified EPS. This identified mutations inactivating genes involved in quorum sensing AI-2 degradation, flagella repression and Tafi and TolA expression. All mutations resulted in the loss of multicellular behavior and immunologically decreased levels of Tafi. This is the first report that implicates quorum sensing AI-2 degradation and flagella repression as part of the regulatory circuit for Tafi expression. Together, the results reveal Tafi uses assembly factors to facilitate extracellular polymerization which likely assists the formation of a network of branched, amorphous fimbriae. Tafi together with EPS form the extracellular matrix: Tafi stabilizes the EPS on the microbial communities; EPS imparts it with physical properties such as hydration, charge and diffusion barriers that protect it from adverse environmental conditions such as desiccation and antimicrobials. This probably contributes to Salmonella survival in the environment and facilitates its cyclic lifestyle.

Universiẗat, Diss., 2004--Bielefeld.

Orientador: Maria de Lourdes Corradi Custodio da Silva / Banca: Gil Valdo José da Silva / Banca: Maurício Boscolo / Resumo: Bactérias do gênero Rhizobium são conhecidas por manterem uma relação simbiótica com determinadas plantas leguminosas e atuam como fixadores biológicos de nitrogênio. Elas vêm sendo estudadas em relação à produção de exopolissacarídeos (EPS), o qual acredita-se estar relacionado com o processo de reconhecimento e invasão do microrganismo na planta hospedeira. Estas moléculas apresentam propriedades físico-químicas e reológicas interessantes para a indústria alimentícia, podendo ser aplicadas como estabilizantes, emulsificantes ou gelificantes. Estudos da estrutura química realizados em quatro EPS produzidos por Rhizobuim tropici (em dois meios de cultivos diferentes), Mesorhizobium e Rhizobium sp os caracterizaram como sendo heteropolímeros. Esses polímeros são constituídos principalmente por glucose e galactose, com traços de manose, apresentando ácido urônico, acetila e piruvato com substituintes. Por cromatografia de gel permeação e eletroforese em gel de poliacrilamida apenas os polímeros produzidos por Rhizobium tropici (R2) e Mesorhizobium (R3) comportaram-se como moléculas homogêneas com baixo grau de polidispersividade. Estes EPS foram paracialmente caracterizados utilizando-se técnicas de FT-IR, RMN de 1H e 13C / Abstract: Bacteria of the Rhizobium genus are known to maintain a symbiotic relationship with certain leguminous plants and they act as biological nitrogen fixation. They have been studied as exopolysaccharides (EPS) producers, which may be related to the recognition process and to the microorganism invasion into the host plant. These molecules exhibit interesting physical-chemical and rheological properties for the food industry since they can be used as stabilizers, emulsifiers or gelling agents. Studies of the chemical structure of four EPS produced by Rhizobium tropici (in two different culture media), Mesorhizobium and Rhizobium sp have characterized them as heteropolymers. The EPS are constituted mainly by glucose and galactose with traces of mannose besides uronic acid, pyruvate and acetyl as substituents. Gel permeation chromatography and polyacrylamide gel electrophoresis showed that the polymers produced by Rhizobium tropici (R2) and Mesorhizobium (R3) behaved as homogeneous molecules with low polydispersity. Thus these two EPS were partially characterized by the techniques of FT-IR, 1H and 13C NMR / Mestre

Orientador: Manoel Victor Franco Lemos / Banca: Maria Inês Tiraboshi Ferro / Banca: Maria José Tavares Ranzani de Paiva / Resumo: Dentre as enfermidades de importância na piscicultura, destaca-se a columnariose, cujo agente etiológico é a Flavobacterium columnare, bactéria de ampla distribuição geográfica, responsável por um elevado número de mortalidade em peixes de várias espécies, principalmente em condições intensivas de criação. Visando o melhor conhecimento desta bactéria para desenvolvimento de métodos de diagnóstico e controle da doença, os objetivos deste estudo foram isolar, caracterizar bioquímica e molecularmente por PCR-RFLP do gene 16S rDNA de F. columnare, detectar fenotipicamente a formação de cápsulas destes isolados pelo teste Agar vermelho congo, e avaliar a composição do EPS quando produzidos por meio de cromatografia líquida de alta eficiência. Ao todo foram obtidos 37 isolados e a caracterização bioquímica indica que os isolamentos são classificados como F. columnare. O filograma gerado pela técnica de PCR-RFLP mostrou três principais ramificações entre os isolados de F. columnare. Os testes comprovaram que a presença de cápsula na célula bacteriana não está diretamente relacionada à formação de biofilme, e o monossacarídeo preponderante em F. columnare é a glicose.Portanto, a utilização da PCR-RFLP para a identificação da bactéria apresentou-se como ferramenta mais rápida que as técnicas bioquímicas atuais e os dados referentes a produção de biofilme são relevantes para futuros estudos que busquem métodos enzimáticos para impedimento da aderência e formação de biofilmes destes patógenos aquáticos em sistemas de aqüicultura e consequentemente a prevenção da columnariose / Abstract: Columnaris disease stands out among the illnesses of importance in fish breeding, its etiological agent is Flavobacterium columnare, which has been recognized as a worldwide pathogen, responsible for high degree of mortality in many fish species, especially in conditions of intensive breed. Looking for a better knowledge of this bacteria and aiming to develop diagnosis methods and disease control, the objectives of this study were to isolate, to biochemistry and molecularly characterize by 16S rDNA gene PCR-RFLP of F. columnare, to detect phenotipically the formation of capsules by the agar Congo red method, and to evaluate the EPS composition by high-performance liquid chromatography. There were obtained 37 isolates and the biochemistry characterization indicated that the isolates were classified as F. columnare. The phylogenetic tree generated by PCR-RFLP technique showed three main branches among the F. columnare isolates. The presence of capsule on the bacterial cells has not a direct relationship to biofilm formation, and considering its composition it was observed that the preponderant monosaccharide is glucose. Therefore, the PCR-RFLP alternative to identify this bacteria presented itself as a faster tool than actual biochemical techniques and the results regarding to biofilm production are relevant to future studies that search for enzymatic methods to abolish the adherence and biofilm formation by this aquatic pathogen in aquaculture systems, and, consequently, columnaris disease prevention / Mestre

Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-19Bitstream added on 2014-06-13T20:10:49Z : No. of bitstreams: 1 monteiro_nk_me_sjrp.pdf: 687247 bytes, checksum: 5856bbc04fe8d3e52218193110c45156 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Bactérias do gênero Rhizobium são conhecidas por manterem uma relação simbiótica com determinadas plantas leguminosas e atuam como fixadores biológicos de nitrogênio. Elas vêm sendo estudadas em relação à produção de exopolissacarídeos (EPS), o qual acredita-se estar relacionado com o processo de reconhecimento e invasão do microrganismo na planta hospedeira. Estas moléculas apresentam propriedades físico-químicas e reológicas interessantes para a indústria alimentícia, podendo ser aplicadas como estabilizantes, emulsificantes ou gelificantes. Estudos da estrutura química realizados em quatro EPS produzidos por Rhizobuim tropici (em dois meios de cultivos diferentes), Mesorhizobium e Rhizobium sp os caracterizaram como sendo heteropolímeros. Esses polímeros são constituídos principalmente por glucose e galactose, com traços de manose, apresentando ácido urônico, acetila e piruvato com substituintes. Por cromatografia de gel permeação e eletroforese em gel de poliacrilamida apenas os polímeros produzidos por Rhizobium tropici (R2) e Mesorhizobium (R3) comportaram-se como moléculas homogêneas com baixo grau de polidispersividade. Estes EPS foram paracialmente caracterizados utilizando-se técnicas de FT-IR, RMN de 1H e 13C / Bacteria of the Rhizobium genus are known to maintain a symbiotic relationship with certain leguminous plants and they act as biological nitrogen fixation. They have been studied as exopolysaccharides (EPS) producers, which may be related to the recognition process and to the microorganism invasion into the host plant. These molecules exhibit interesting physical-chemical and rheological properties for the food industry since they can be used as stabilizers, emulsifiers or gelling agents. Studies of the chemical structure of four EPS produced by Rhizobium tropici (in two different culture media), Mesorhizobium and Rhizobium sp have characterized them as heteropolymers. The EPS are constituted mainly by glucose and galactose with traces of mannose besides uronic acid, pyruvate and acetyl as substituents. Gel permeation chromatography and polyacrylamide gel electrophoresis showed that the polymers produced by Rhizobium tropici (R2) and Mesorhizobium (R3) behaved as homogeneous molecules with low polydispersity. Thus these two EPS were partially characterized by the techniques of FT-IR, 1H and 13C NMR

Submitted by AYLAN KENER MENEGHINE (aylankm@gmail.com) on 2017-01-10T01:14:02Z No. of bitstreams: 1 Tese_Aylan_Kener_Meneghine.pdf: 2274173 bytes, checksum: 058fc5159e6fa34a7c970ce061ce70c5 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-12T19:08:55Z (GMT) No. of bitstreams: 1 meneghine_ak_dr_jabo.pdf: 2274173 bytes, checksum: 058fc5159e6fa34a7c970ce061ce70c5 (MD5) / Made available in DSpace on 2017-01-12T19:08:55Z (GMT). No. of bitstreams: 1 meneghine_ak_dr_jabo.pdf: 2274173 bytes, checksum: 058fc5159e6fa34a7c970ce061ce70c5 (MD5) Previous issue date: 2016-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O composto orgânico produzido a partir de carcaças, resíduos animais e vegetais é uma alternativa viável para a substituição total ou parcial dos fertilizantes minerais utilizados na atualidade. No processo de compostagem participam diferentes populações microbianas, e com isso o composto torna-se um sistema rico para utilização como fertilizante no solo, complementando assim as necessidades nutricionais e microbianas do meio ambiente. Entretanto, há poucos trabalhos envolvendo análise da diversidade bacteriana em solos sob uso de composto orgânico feito a partir de carcaças, e também pouco se conhece sobre o impacto ambiental do uso agrícola de composto orgânico na qualidade da água. Existe também a questão se há influência da água utilizada para irrigação na qualidade do solo. O objetivo central desse trabalho foi analisar a diversidade bacteriana e perfil funcional de um solo de horta e da água de um córrego utilizada para irrigação. E como objetivo secundário, através do isolamento de bactérias da água verificar o potencial biotecnológico de produção e uso de exopolissacarídeo como bioemulsificante de óleo e hidrocarbonetos. As amostras de solo e água utilizadas nesse trabalho foram coletadas na área do departamento rural da Fundação Parque Zoológico de São Paulo, em setembro de 2014. Todo material coletado foi transportado até o Laboratório de Bioquímica de Micro-organismos e de Plantas, onde realizou-se a extração de DNA total e sequenciamento através de tecnologia Ion Proton (Life Technologies), isolamento bacteriano, produção e aplicação de exopolissacarídeo como bioemulsificante. Ao analisar o DNA metagenômico observou-se que tanto na água quanto no solo há uma abundância de bactérias encontradas normalmente em áreas agrícolas sob influência de composto orgânico feito a partir de outras fontes. Verificou-se também, através da análise dos genes relacionados com os ciclos biogeoquímicos, abundância de genes do processo de desnitrificação na amostra de solo. Além de se observar que na análise da estrutura taxonômica dos ciclos biogeoquímicos, não há bactérias em comum entre as amostras de solo e água com do composto orgânico. Porém ao avaliar o perfil funcional total da água, solo e outros dez metagenomas, através de uma análise de agrupamento hierárquico, notou-se que a água causa maior influência no solo do que o próprio composto orgânico. Com relação ao isolamento bacteriano, foi possível isolar a bactéria Sphingomonas sp., sendo que o isolado identificado demonstrou produzir grande quantidade de exopolissacarídeo (EPS). O EPS formou emulsões estáveis após 24 horas e 168 horas. Além disso o EPS foi um melhor agente emulsificante para hexano e querosene dentre todos os óleos analisados. Os resultados mostraram contribuição significativa com relação ao potencial do EPS como agente bioemulsificante que aumentaria a degradação de hidrocarbonetos e efeitos de emulsificação em biotecnologia ambiental. / The organic compost produced from carcasses, animal and vegetable waste is a viable alternative to full or partial replacement of mineral fertilizers used nowadays. In the composting process there are involved different microbial populations, and the compost becomes a rich system for use as a fertilizer in the soil, thereby supplementing the nutritional and microbial requirements of the medium. However, there are few studies involving the analysis of bacterial diversity in soil under use of organic compost made from carcasses, and also little is known about the environmental impact of agricultural use of organic compost in water quality. Furthermore, there is also the question of whether there is influence of the water used for irrigation on soil quality. Because of these questions, the central objective of this study was to analyze the bacterial diversity and functional profile of a soil from vegetable garden and freshwater used for irrigation from a local stream. As a secondary objective, we aimed to verify the production and biotechnology potential of a bacterial exopolysaccharide as oil and hydrocarbons bioemulsifier. Soil and freshwater samples used in this study were collected at rural department of the Zoo Foundation Park of São Paulo, in September 2014. All material collected was transported to the Laboratório de Bioquímica de Micro-organismos e de Plantas where we proceeded with the total DNA extraction experiments, sequencing through Ion Proton technology (Life Technologies), bacterial isolation, production and application of exopolysaccharide as bioemulsifier. By analyzing the metagenomic DNA it was observed that both freshwater and soil were plenty of bacterial communities normally found in agricultural areas under influence of organic amendments. Through the analysis of genes related to biogeochemical cycles, it was found abundance of genes about denitrification process in the soil sample. Also it can be noted by the analysis of the taxonomic structure of biogeochemical cycles, there was no bacteria shared between soil and freshwater samples with the organic compost. But when evaluating the functional profile of freshwater, soil and other ten metagenomes, we observed that freshwater causes greater influence on the soil than the organic compost. Regarding the bacterial isolation, it was possible to identify the bacteria Sphingomonas sp., and it has been shown to produce large amount of exopolysaccharide (EPS). The EPS has shown stability in its emulsions after 24 hours and 168 hours. Additionally, it’s a better emulsifying agent for hexane and kerosene among other oils analysed. Furthermore, our findings promoted significant contribution by showing EPS potential as a bioemulsifier agent that would enhance hydrocarbon degradation and emulsification effects in environmental biotechnology.

Our lab discovered that approximately 1 in 10,000 Escherichia coli cells in stationary phase remain in suspension after a high g-force centrifuge event. To establish the mechanism behind this curious phenotype, multiple mutant strains of E. coli were independently evolved such that the majority of their populations resisted migration when exposed to high centrifugal forces. Genomic DNA sequencing of the mutants' revealed unique, isolated mutations in genes involved in capsule synthesis and exopolysaccharide (EPS) production. Each mutant exhibits a novel mechanism that allows them to remain in suspension. The mutants were further characterized by determining their growth rates, strengths of resistance to various centrifugal forces, the phenotype's dependence on a carbon source, and timing of the phenotype's presentation. The results revealed: comparable mutant generation times to the wild-type strain, variable resistance to centrifugal force, phenotype dependence on carbon source, and phenotype presentation during early stationary phase. To interrogate the mechanism by which these cells stay in suspension the production of EPS was quantified, and gene knock-outs were performed. Quantification of the EPS revealed approximately a seventeen-fold increase in EPS in the mutants' compared to the wild-type strain. Gene knock-outs revealed the EPS produced can be attached to the outer-membrane or freely secreted into the media by different mechanisms. In addition, this mechanism was further confirmed to be responsible for the centrifuge resistant trait by attaching extracted EPS to polystyrene microspheres. Experimental results show that mutant extracted EPS treated beads caused increased bead retention in suspension compared to wild-type EPS treated beads. These results reveal that E. coli is using a novel mechanism to adapt to a new environmental factor introduced to remove the bacteria. With the discovery of this mechanism and the transferability to inorganic objects industrial applications are now envisioned where particle sedimentation is controllable and mixtures remain homogenized by attaching optically transparent biomolecules.

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive. / Master of Science