O aumento na produção de carne bovina aumentou a concentração e volume dos resíduos líquidos produzidos durante seu beneficiamento, conhecidos como água residuária de matadouros (ARMV). Isso vem estimulando o desenvolvimento de processos que operem em alta carga com alta eficiência para seu tratamento. Visto o exposto, objetivou-se avaliar um sistema de tratamento para a ARMV operando em condições anaeróbia/aeróbia/anóxica de forma conjugada visando a remoção de matéria orgânica, nutrientes e toxidade. As unidades que compunham o sistema estudado eram três reatores com biofilme denominados: Reator Anaeróbio Híbrido (RAH), Reator Aeróbio de Leito Móvel (MBBR) e Reator Anóxico com Biofilme (RAB). A ARMV in natura era coletada em um frigorífico e matadouro bovino do vale do Paraíba e caracterizada em termos físicos, químicos e toxicológicos a nível agudo e crônico. O experimento durou aproximadamente 370 dias, operando sob variações de carga orgânica. A coleta ao longo do sistema foi feita semanalmente e as amostras caracterizadas em termos físico-químicos e toxicológicos tanto a nível agudo quanto crônico. Nos reatores RAH, MBBR e RAB foram realizadas testes hidrodinâmicos em condições abiótico e biótico, respectivamente. Com os dados obtidos nas três fases estudadas foram levantados os parâmetros cinéticos de crescimento da biomassa dispersa, além da caracterização microbiológica da mesma e do biofilme. Os resultados obtidos com base na caracterização físico-química e toxicológica da ARMV, in natura, revelaram altas concentrações em toda série de sólidos, de ácidos voláteis totais, alcalinidades, macro e micronutrientes, matéria orgânica em termos de DBO520°C e DQO nas formas total, solúvel e particulada e também de COD. Apresentou-se como um efluente extremamente tóxico a nível agudo para os organismos, bactérias P. putida e E. coli e microcrustaceo D. similis, e extremamente tóxico a nível crônico para os organismos, microcrustaceios C. silvestri e C. dúbia, bactérias E. coli e P putida e alga P. subcaptata. Com base nos testes toxicológicos, concluiu-se que os microcrustaceos e algas foram mais sensíveis as toxinas da ARM testada que as bactérias. O reator RAH operando sob choques orgânicos demonstrou ótimo desempenho operacional. As cargas orgânicas (COV) aplicadas ao RAH foram 608,9; 3.030,4 e 9.581,5 mg L-1 d-1 em termos de DQO para as fases I, II e III, estatisticamente diferente entre si. Apresentando para as três fases eficiências estatisticamente iguais. O reator MBBR operando sob choques orgânicos demonstrou ótimo desempenho operacional. As cargas orgânicas (COV) aplicadas ao MBBR foram 286,5; 2085,2 e 3889,6 mg L-1 d-1 em termos de DQO para as fases I, II e III, estatisticamente diferente entre si. Apresentando para as fases I, II e III eficiências de 76,9%; 60% e 81%, respectivamente, e estatisticamente iguais. Conclui-se com a pesquisa realizada que os reatores RAH e MBBR foram capazes de absorver choques orgânicos e hidráulicos submetidos a biomassa mantendo-se em altos valores de eficiência. O reator MBBR também apresentou bom desempenho no processo de nitrificação com eficiências de 61,2%; 68,1%; 50,7% para as fases I, II e III, respectivamente. A concentração média de OD de 3 mg L-1 mantida no MBBR apresentou-se acima do suficiente. O reator RAB operando sob choques orgânicos demonstrou ótimo desempenho operacional. As cargas orgânicas (COV) aplicadas ao RAB foram 194,9; 1769,1 e 2230,6 mg L-1 d-1 em termos de DQO para as fases I, II e III, estatisticamente diferente entre si. As fases I, II e III apresentaram eficiências de 70,7%; 46,4% e 69,8%, respectivamente, e estatisticamente, iguais entre as fases I e III. Em termos de toxidade, os parâmetros estudados nas fases I e II, mostraram-se ideais para remoção de toxidade, sendo que no final de ambas a ARM tratada apresentou-se livre de toxidade a nível agudo e crônico. Os reatores RAH, MBBR e RAB demonstraram ótimo desempenho hidrodinâmico e cinético. Concluiu-se que o sistema anaeróbio/aeróbio/anóxico estudado foi eficiente no tratamento da ARMV, aliando condições de fácil monitoramento, rapidez no processo e ótimo desempenho. / Development in bovine meat production has increased volume and concentration of liquid residues produced during their improvement, known as slaughterhouses wastewaters (SW). This fact has been stimulating the develpment of processes operating in highly charge and efficience for their treatment. As seen, we focused to develop a treatment system for the SW operating in a anaerobic/aerobic/anoxic condition in a conjugated form, aiming the remotion of organic matter, nutrients and toxicity. The units evolving the studied system were three biofilms reators named: Anaerobic Hibrid Reactor (AHR), Moving Bed Biofilm Reactors (MBBR), and Anoxic Biofilm Reactor (AnBR). The SW in natura was collected in the slaughterhouse of the Paraíba Valley, and were characterized in physical, chemical and toxically in a highly sharp and cronically levels. The experiment has lasted for 370 days, operating under organic load rate (OLR). The withdraws along the system were done weekly and the samples were characterized in physical-chemical and toxicological terms and in both sharp and cronical ways. Hidrodinamic tests were realized in reactors AHR, MBBR and AnBR in abiotic and biotic condictions, repectivelly. With the data obtained in the three studied phases, knetics parameters were collected for the dispersal biomass, besides its biofilm and biomass characterization. The results, based on SW physico - chemical and toxicological characterization, showed high concentrations in all solids series, in total volatic acids, alcalinity, macro and micro nutrients, organic matter in terms of BOD520°C and COD in their solute and particulate forms and also in DOC. It was presented as highly toxix effluent in a sharp level for organisms, bacteria P. putida ans E. coli, and D. similis microcrustacean. And extremely toxic to organisms chronic C. silvestri and C. dúbia microceustacean and P. subcaptata algae. According with toxicological tests it was concluded that the microcrustaceos and a algae were more sensitive to the SSW toxines than bacteria. The AHR reator operating under organic shocks showed excelent operational development. The OLR applied to the AHR were 608.9; 3,030.4 and 9,581.5 mg L-1 d-1 in terms of COD for the phases I, II, III, statiscally different among it, showing for the three phases scores of 76,9%; 60% and 81%, respectivelly, and the same, statistically speaking. We may conclude with this research that the AHR and AMBR reators were capable to absorb hidraulic and organic shocks submitted to biomass keeping high levels of efficiency. The MBBR reator has shown also a good performance in nitrification process, scoring 61.2%; 68.1%; 50.7% in effectiveness for phases I, II and III, respectively. The average DO concentration of 3 mg L-1 maintained in MBBR showed over sufficience. The AnBR reator under organic shocks showed highly operational performance. The OLR applied to AnBR were 194.9; 1,769.1 e 2,230.6 mg L-1 d-1 in terms of COD for the phases I, II, III statistically different among them. The phases I, II, III presented efficiences of 70.7%; 46.4% and 69.8%, respectively, and statiscally the same between phases I and III. In terms of toxicity, the studied parameters in phases I and II showed to be ideal to remove the toxicity from both sharp and chronical levels. The reators AHR, MBBR and AnBR showed excelent hidraulic and kinetic performances. We may conclude that the studied anaerobic/aerobic/ anoxic system was efficient in the treatment of SW, joining, easy feasible conditions, velocity in processing and high performance.

D-amino acids (D-AAs) are the α-carbon enantiomers of L-amino acids (L- AAs), the building blocks of proteins in known organisms. It was largely believed that D-AAs are unnatural and must be toxic to most organisms, as they would compete with the L-counterparts for protein synthesis. Recently, new methods have been developed that allow scientists to chromatographically separate the two AA stereoisomers. Since that time, it has been discovered that D-AAs are vital molecules and they have been detected in many organisms. The work of this dissertation focuses on their place in bacterial metabolism. This specific area was selected due to the abundance of D-AAs in bacteria-rich environments and the knowledge of their part in several processes, such as peptidoglycan synthesis, biofilm disassembly, and sporulation. We focused on the bacterium Pseudomonas putida KT2440 which inhabits the densely populated plant rhizosphere. Due to its versatility and cosmopolitan character, this bacterium has provided an excellent system to study D-AA metabolism. In the first chapter, we have developed a new approach to identify specific genes encoding enzymes acting on D-AAs, collectively known as amino acid racemases. Using this novel method, we identified three amino acid racemases encoded by the genome of P. putida KT2440. All of the enzymes were subsequently cloned and purified to homogeneity, followed by a complete biochemical characterization. The aim of the second chapter was to understand the specific role of the peculiar broad-spectrum amino acid racemase Alr identified in chapter one. After constructing a markerless deletion of the cognate gene, we conducted a variety of phenotypic assays that led to a model for a novel catabolic pathway that involves D-ornithine as an intermediate. The work in chapter three identifies for the first time numerous rhizosphere-dwelling bacteria capable of catabolizing D-AAs. Overall, the work in this dissertation contributes a novel understanding of D-AA catabolism in bacteria and aims to stimulate future efforts in this research area.

O acúmulo de poluentes orgânicos persistentes é um dos principais problemas de contaminação do meio ambiente em todo o mundo. As atividades industriais e os avanços tecnológicos na maioria dos setores de produção contribuem com a crescente liberação de compostos poluentes, altamente tóxicos, biocumulativos e resistentes à degradação física, química, fotolítica e biológica. Esses poluentes são o resultado do uso indevido de pesticidas de um modo geral, da subprodução não planejada de sintéticos tóxicos como dioxinas policloradas e de uma lista enorme de atividades que promovem a combustão incompleta da matéria orgânica e despejam no ambiente uma vasta quantidade de hidrocarbonetos aromáticos persistentes. A biorremediação é uma estratégia biotecnológica que vem lançar luz sobre as técnicas de revitalização de sítios contaminados. É baseada na utilização de microorganismos, ou suas enzimas, para reduzir ou eliminar perigos ambientais contaminantes em substâncias inertes, CO2 e água. As oxigenases são uma classe de enzimas amplamente estudadas por suas propriedades catalíticas únicas, sendo a clorocatecol 1,2- dioxigenase de Pseudomonas putida um interessante alvo biotecnológico para a biorremediação devido a sua ampla especificidade a substratos aromáticos, aromáticos halogenados e dialogenados altamente poluentes, biocumulativos e carcinogênicos. Nesse trabalho, o protocolo de expressão heteróloga em E. coli foi implementado em nosso laboratório e a purificação realizada em coluna de afinidade através do uso de resina de quitina. Os cristais de cor marrom avermelhado da enzima clorocatecol 1,2-dioxigenase Pseudomonas putida foram obtidos na presença de polietilenogligol e acetato de magnésio durante o período de 10 dias, utilizando-se a técnica de difusão de vapor em gota sentada. A estrutura cristalográfica da enzima Pp 1,2-CCD foi determinada através da utilização da técnica MR-SAD, utilizando átomos de ferro, cofator da proteína, como agentes espalhadores e as coordenadas da enzima 3-clorocatecol 1,2-dioxigenase de Rhodococcus opacus como modelo de busca. O modelo final, contendo 3 moléculas na unidade assimétrica foi refinado a 3.4 Å de resolução. A enzima se enovela na forma dimérica (Fe3+)2, e apresenta de um modo geral, dois domínios catalíticos compostos de fitas-beta e uma região de loops e um domínio de ligação composto por hélices formando um túnel hidrofóbico. Como a primeira clorocatecol de bactérias gram-negativa a ser descrita para essa classe de enzimas, será possível investigar as diferenças conformacionais que levam aos mecanismos de catálise, amplo a diversos substratos, e avaliar características de seu funcionamento e ativação, como uma importante ferramenta para validação do papel desta proteína dentro do processo de biorremediação. / Accumulation of persistent organic pollutants is one of the most important environmental problems worldwide. Industrial activities and technological advances contribute to the spread of pollutants, highly toxic, bioaccumulative, and resistant to physical, chemical, photolytic and biological degradation. The persistent organic pollutants are consequence of the inappropriate use of pesticides, the production of toxic synthetic compounds such as polychlorinated biphenyls and a large list of activities promote incomplete combustion of which organic matter and release a large amount of persistent aromatic hydrocarbons to the environment. Bioremediation is a biotechnogical strategy that has shed light on revitalization techniques of contaminated sites. It is based on the application of microorganisms, or their enzymes, to eliminate or reduce environmental contaminants into inert substances, CO2 and water. The oxygenases are a class of largely studied enzymes due to their catalytic properties, being chlorocatechol 1,2-dioxygenase from Pseudomonas putida an interesting biotechnological target for bioremediation due to its specificity to a broad spectrum of highly polluted aromatic substrates. In the present work, heterologous expression protocol has been implemented in our laboratory and purification was performed by using affinity column in chitin resin. Brown-reddish crystals of chlorocatechol 1,2-dioxygenase from Pseudomonas putida were obtained in presence of polyethylene glycol (PEG) and magnesium acetate after 10 days, by utilizing vapor diffusion techniques in sitting drops. The crystallographic structure of Pp 1,2-CCD has been solved by MR-SAD technique, using iron atoms, the enzyme cofactor, as scattering centers and the coordinates of 3- chlorocatechol 1,2-dioxygenase of Rhodococcus opacus as the search model. The final model, containing three molecules in the asymmetric unit, has been refined up to 3.4 Å resolution. The enzyme folds in the dimeric form (Fe3+)2, and shows two catalytic domains composed of -strands and a loop region, and a helical domain that pack together forming a hydrophobic channel. As being the first member of the chlorocatechol dioxygenase family of enzymes from a gram-negative bacteria to be solved, it will be possible to explore the crystallographic structure of chlorocatechol 1,2- dioxygenase from Pseudomonas putida in order to investigate the conformational differences that explain its mechanism of action, on a broad spectrum of substrates, and evaluate the functional features, as an important tool to validate the role of this enzyme in the bioremediation process.

Plants often live in association with growth-promoting bacteria, which provide them with several benefits. One such benefit is the lowering of plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves the immediate biosynthetic precursor of ethylene, ACC. The plant hormone ethylene is responsible for many aspects of plant growth and development but under stressful conditions ethylene exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4, isolated from the rhizosphere of reeds, is a potent plant growth-promoting strain and as such was used, along with an ACC deaminase minus mutant of this strain, to study the role of ACC deaminase in plant growth-promotion. Also, transgenic plants expressing a bacterial ACC deaminase gene were used to study the role of this enzyme in plant growth and stress tolerance in the presence and absence of nickel. Transcriptional changes occurring within plant tissues were investigated with the use of an Arabidopsis oligonucleotide microarray. The results showed that transcription of genes involved in hormone regulation, secondary metabolism and the stress response changed in all treatments. In particular, the presence of ACC deaminase caused genes for auxin response factors to be up-regulated in plant tissues suggesting a de-repression of auxin signaling in the absence of high levels of ethylene. Also, transgenic plants had longer roots and grew faster than the non-transformed plants and genes involved in the stress response and secondary metabolism were up-regulated. Plants inoculated with bacteria had lower levels of secondary metabolism gene expression and slightly higher stress response gene expression than uninoculated plants. Yet, inoculation with the ACC deaminase-expressing bacterium caused less up-regulation of plant genes involved in stress and defense responses and the down-regulation of genes involved in nitrogen metabolism in comparison to plants inoculated with the ACC deaminase minus mutant. Nickel stress caused the down-regulation of genes involved in photosynthesis and carbon fixation and the up-regulation of genes involved in stress responses, and amino acid and lipid breakdown suggesting energy starvation. When transgenic plants expressing ACC deaminase in the roots were exposed to nickel stress, plant stress symptoms were significantly lower and biomass was significantly higher suggesting that lowering the level of ethylene relieved many of the stress symptoms. In fact, genes involved in photosynthesis, secondary metabolism and nitrate assimilation were up-regulated in transgenic plants compared with non-transformed plants in the presence of nickel, suggesting that ACC deaminase is effective at reducing the severe effects of this metal stress.

Plants often live in association with growth-promoting bacteria, which provide them with several benefits. One such benefit is the lowering of plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves the immediate biosynthetic precursor of ethylene, ACC. The plant hormone ethylene is responsible for many aspects of plant growth and development but under stressful conditions ethylene exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4, isolated from the rhizosphere of reeds, is a potent plant growth-promoting strain and as such was used, along with an ACC deaminase minus mutant of this strain, to study the role of ACC deaminase in plant growth-promotion. Also, transgenic plants expressing a bacterial ACC deaminase gene were used to study the role of this enzyme in plant growth and stress tolerance in the presence and absence of nickel. Transcriptional changes occurring within plant tissues were investigated with the use of an Arabidopsis oligonucleotide microarray. The results showed that transcription of genes involved in hormone regulation, secondary metabolism and the stress response changed in all treatments. In particular, the presence of ACC deaminase caused genes for auxin response factors to be up-regulated in plant tissues suggesting a de-repression of auxin signaling in the absence of high levels of ethylene. Also, transgenic plants had longer roots and grew faster than the non-transformed plants and genes involved in the stress response and secondary metabolism were up-regulated. Plants inoculated with bacteria had lower levels of secondary metabolism gene expression and slightly higher stress response gene expression than uninoculated plants. Yet, inoculation with the ACC deaminase-expressing bacterium caused less up-regulation of plant genes involved in stress and defense responses and the down-regulation of genes involved in nitrogen metabolism in comparison to plants inoculated with the ACC deaminase minus mutant. Nickel stress caused the down-regulation of genes involved in photosynthesis and carbon fixation and the up-regulation of genes involved in stress responses, and amino acid and lipid breakdown suggesting energy starvation. When transgenic plants expressing ACC deaminase in the roots were exposed to nickel stress, plant stress symptoms were significantly lower and biomass was significantly higher suggesting that lowering the level of ethylene relieved many of the stress symptoms. In fact, genes involved in photosynthesis, secondary metabolism and nitrate assimilation were up-regulated in transgenic plants compared with non-transformed plants in the presence of nickel, suggesting that ACC deaminase is effective at reducing the severe effects of this metal stress.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-27T14:23:05Z No. of bitstreams: 1 texto completo.pdf: 166311 bytes, checksum: 83322a34768e7a94f65989a1722e7533 (MD5) / Made available in DSpace on 2017-04-27T14:23:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 166311 bytes, checksum: 83322a34768e7a94f65989a1722e7533 (MD5) Previous issue date: 2004-08-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Três procariotas obtidos de filoplano de tomateiro (Bacillus cereus, Pseudomonas putida, Novosphingobium capsulatum), previamente selecionados como bons antagonistas de patógenos da cultura foram formulados em cinco formas distintas e avaliados quanto a sua efetividade como agentes de biocontrole. Quatro das cinco formulações, consistiam de produtos comerciais (Agromil-Raiz, Agromil-S, Ecolife 40 and ISR 2000) os quais propágulos dos antagonistas foram incorporados. A quinta formulação foi proposta no presente trabalho e consiste de uma solução de nutrientes com estratos de parede de células de Saccharomyces cerevisiae e goma xantana. Os efeitos deletérios das formulações sobre o crescimento dos antagonistas bem como a sobrevivência foram investigados. Os resultados indicam que Agromil-S e ISR 2000 tem efeito inibitório sobre o crescimento dos antagonistas os outros produtos não interferem significantemente na multiplicação dos antagonistas. A vida de prateleira dos antagonistas na formulação proposta foi estudada e nenhuma foi eficiente em aumentar a sobrevivência quando comparadas com o controle. O biocontrole experimental de doenças do tomateiro induzidas por três patógenos Corynespora cassiicola, Pseudomonas syringae pv. tomato, e oidio respectivamente, em casa de vegetação foi tentado para todos aos antagonistas e para todas as formulações, o controle foi alcançado embora em alguns casos não se encontrou diferença significativa entre o tratamento e o controle. Mais esforços e pesquisas devem ser realizados afim de melhorar a proteção de células a serem dispensadas em plantas. / To improve each antagonist efficiency as control agent of tomato leaf diseases, a study was carried out on five distinct formulae consisting of additives and one out of three autochthonous, isolated prokaryotic residents (Bacillus cereus, Pseudomonas putida, Novosphingobium capsulatum) of the tomato phylloplane and already selected as good antagonists to tomato pathogens. Four of these five formulations consisted of commercial products (Agromil-Raiz, Agromil-S, Ecolife 40 and ISR 2000) to which each antagonist living propagules were incorporated. The fifth of these combinations consisted of a nutrient solution amended with a Saccharomyces cerevisiae cell wall extract along with xanthan gum. Therefore, under consideration were the effects of each formula on each antagonist protective action and survival. Results of in vitro studies indicated that Agromil-S and ISR 2000 inhibited the antagonists' growth while the others didn't interfere in a significant way with the antagonists’ multiplication. In shelf live experiments, none of the studied formulation was efficient enough to increase significantly the antagonist survival when compared to controls. Under greenhouse conditions, every antagonist, in all formulas, controlled the tomato leaf diseases caused by Corynespora cassiicola, Pseudomonas syringae pv. tomato, or Oidium sp., although differences between some treatments and controls were not significant. We feel that with this we have only begun our efforts to find ways to improve protection of the antagonist’s living cells being delivered to tomato plants, and which will render them eventually well protected against leaf pathogens. . / Dissertação importada do Alexandria

O acúmulo de poluentes orgânicos persistentes é um dos principais problemas de contaminação do meio ambiente em todo o mundo. As atividades industriais e os avanços tecnológicos na maioria dos setores de produção contribuem com a crescente liberação de compostos poluentes, altamente tóxicos, biocumulativos e resistentes à degradação física, química, fotolítica e biológica. Esses poluentes são o resultado do uso indevido de pesticidas de um modo geral, da subprodução não planejada de sintéticos tóxicos como dioxinas policloradas e de uma lista enorme de atividades que promovem a combustão incompleta da matéria orgânica e despejam no ambiente uma vasta quantidade de hidrocarbonetos aromáticos persistentes. A biorremediação é uma estratégia biotecnológica que vem lançar luz sobre as técnicas de revitalização de sítios contaminados. É baseada na utilização de microorganismos, ou suas enzimas, para reduzir ou eliminar perigos ambientais contaminantes em substâncias inertes, CO2 e água. As oxigenases são uma classe de enzimas amplamente estudadas por suas propriedades catalíticas únicas, sendo a clorocatecol 1,2- dioxigenase de Pseudomonas putida um interessante alvo biotecnológico para a biorremediação devido a sua ampla especificidade a substratos aromáticos, aromáticos halogenados e dialogenados altamente poluentes, biocumulativos e carcinogênicos. Nesse trabalho, o protocolo de expressão heteróloga em E. coli foi implementado em nosso laboratório e a purificação realizada em coluna de afinidade através do uso de resina de quitina. Os cristais de cor marrom avermelhado da enzima clorocatecol 1,2-dioxigenase Pseudomonas putida foram obtidos na presença de polietilenogligol e acetato de magnésio durante o período de 10 dias, utilizando-se a técnica de difusão de vapor em gota sentada. A estrutura cristalográfica da enzima Pp 1,2-CCD foi determinada através da utilização da técnica MR-SAD, utilizando átomos de ferro, cofator da proteína, como agentes espalhadores e as coordenadas da enzima 3-clorocatecol 1,2-dioxigenase de Rhodococcus opacus como modelo de busca. O modelo final, contendo 3 moléculas na unidade assimétrica foi refinado a 3.4 Å de resolução. A enzima se enovela na forma dimérica (Fe3+)2, e apresenta de um modo geral, dois domínios catalíticos compostos de fitas-beta e uma região de loops e um domínio de ligação composto por hélices formando um túnel hidrofóbico. Como a primeira clorocatecol de bactérias gram-negativa a ser descrita para essa classe de enzimas, será possível investigar as diferenças conformacionais que levam aos mecanismos de catálise, amplo a diversos substratos, e avaliar características de seu funcionamento e ativação, como uma importante ferramenta para validação do papel desta proteína dentro do processo de biorremediação. / Accumulation of persistent organic pollutants is one of the most important environmental problems worldwide. Industrial activities and technological advances contribute to the spread of pollutants, highly toxic, bioaccumulative, and resistant to physical, chemical, photolytic and biological degradation. The persistent organic pollutants are consequence of the inappropriate use of pesticides, the production of toxic synthetic compounds such as polychlorinated biphenyls and a large list of activities promote incomplete combustion of which organic matter and release a large amount of persistent aromatic hydrocarbons to the environment. Bioremediation is a biotechnogical strategy that has shed light on revitalization techniques of contaminated sites. It is based on the application of microorganisms, or their enzymes, to eliminate or reduce environmental contaminants into inert substances, CO2 and water. The oxygenases are a class of largely studied enzymes due to their catalytic properties, being chlorocatechol 1,2-dioxygenase from Pseudomonas putida an interesting biotechnological target for bioremediation due to its specificity to a broad spectrum of highly polluted aromatic substrates. In the present work, heterologous expression protocol has been implemented in our laboratory and purification was performed by using affinity column in chitin resin. Brown-reddish crystals of chlorocatechol 1,2-dioxygenase from Pseudomonas putida were obtained in presence of polyethylene glycol (PEG) and magnesium acetate after 10 days, by utilizing vapor diffusion techniques in sitting drops. The crystallographic structure of Pp 1,2-CCD has been solved by MR-SAD technique, using iron atoms, the enzyme cofactor, as scattering centers and the coordinates of 3- chlorocatechol 1,2-dioxygenase of Rhodococcus opacus as the search model. The final model, containing three molecules in the asymmetric unit, has been refined up to 3.4 Å resolution. The enzyme folds in the dimeric form (Fe3+)2, and shows two catalytic domains composed of -strands and a loop region, and a helical domain that pack together forming a hydrophobic channel. As being the first member of the chlorocatechol dioxygenase family of enzymes from a gram-negative bacteria to be solved, it will be possible to explore the crystallographic structure of chlorocatechol 1,2- dioxygenase from Pseudomonas putida in order to investigate the conformational differences that explain its mechanism of action, on a broad spectrum of substrates, and evaluate the functional features, as an important tool to validate the role of this enzyme in the bioremediation process.

The aim of this work is study of utilization of PHA producing bacteria in bioremediation technologies. For this study were used bacteria Pseudomonas putida KT2440 and two isolates from soil contaminated by petroleum - Pseudomonas gessardii (D2) a Pseudomonas fulva (D3). The experimental part describes especially study of feather biodegradation using selected microbial strains. All the tested bacterial strains were capable of feather degradation and utilization as the sole carbon source. During biodegradation experiment, we monitored weight loss of feather, protease and keratinase activity, concentration of bacterial biomass and PHA content as well as pH. The highest biodegradation ability and keratinase activity was observed in Pseudomonas putida. None of tested bacteria accumulated detectable amount of PHA during growth on waste feather, nevertheless, bacterial biomass grown during feather degradation can be used as an inoculum for PHA production on waste frying oil and octanoic acid. Using this experimental setup, high PHA content (54% of cell dry weight) was achiaved in Pseudomonas putida. Another part of the thesis deals with biodegradation of petroleum oil. The highest capability of growth on this carbon source were determined in Pseudomonas fulva.

With a growing global population, food security and waste management strategies are needed (FAO, 2002b); insects have been promoted for these goals. Black soldier fly larvae (BSFL) are endorsed for their ability in decomposition and use as feed (Bava et al., 2019; Swett Walker, 2018). Studies show the life cycle of BSFL is impacted by bacterial supplementation (Franks et al., 2021; Kooienga et al., 2020). We seek to determine the effects of supplementing BSFL diet with Pseudomonas putida 3P. We conducted two forms of a 10-day bench-top study observing larval mass, frass conversion, C:N content, and ammonia production with supplementation of Pseudomonas putida 3P in single and double-doses. Supplementation with P. putida results in roughly 1.5X larger larval wet and dry mass, variable concentrations of ammonia, and approximately 1.15X smaller C:N ratios in frass. This suggests interruption of larval rearing may affect volatile ammonia concentrations and inhibit P. putida behavior.

Im Rahmen der vorliegenden Arbeit wurden die Wechselwirkungen im System Bakterium –Tensid – Schadstoff mittels kalorimetrischer Untersuchungen (ITC, DSC) sowie mit XPS-Analysen und durch Zeta-Potential-Messungen an Bakterienoberflächen charakterisiert. Für die Untersuchungen wurden zwei Gram-positive Rhodococcus-Stämme und ein Gram-negativer Pseudomonas putida-Stamm verwendet. Als Biotenside wurden das Rhamnolipid JBR 425 und der von Rhodococcus erythropolis B7g produzierte Trehalosetetraester (THL-4) ausgewählt. Das synthetische Tensid SDS diente als Referenzsubstanz. Aus den kalorimetrischen Experimenten konnte eine starke Wechselwirkung zwischen den Tensiden und den aktiven Bakterienkulturen abgeleitet werden. THL-4 führte beim Wachstum der Rhodococcen auf n-Hexadecan zur Verkürzung der lag-Phase. SDS wies hingegen eine toxische Wirkung für die Bakterienstämme auf. Thermodynamische Betrachtungen ergaben, dass Wechselwirkungen des SDS mit den Bakterienzellen gegenüber der Mizellbildung bevorzugt werden.