A biosensor is proposed that contains bacteria that naturally effluxes potassium ions when threatened by electrophilic species. Pseudomonas aeruginosa is an activated sludge isolate and possesses the characteristic potassium efflux response. It has been immobilized in calcium alginate beads, photopolymer disks, and a thermally reversible gel in order to ultimately incorporate the immobilized system into a functional biosensor. The potassium efflux and cell viability were measured in the immobilized matrices. Wastewater treatment is of utmost importance; however, processes are easily upset. Upsets can be caused by various electrophiles found in the environment, and can cause serious health effects to people or the environment downstream from an upset. Electrophiles can cause the activated sludge in wastewater treatment facilities to deflocculate, and untreated water can be lost downstream. Devising a detection system for proactively sensing electrophiles prior to an upset is an important complementary goal. Immobilization systems have been evaluated including photopolymer coated alginate beads and sol gel coated alginate beads. The thermally reversible gel, NIPA-co-AAc (N-isopropylacrylamide-co-acrylic acid), shows promise as an immobilization matrix for the bacteria; however its high lower critical solution temperature (LCST) of ~33oC is problematic for typical, ambient applications. Another thermally reversible copolymer, N-isopropylacrylamide-co-N-acryloyl-6-amino caproic acid (NIPA-co-AcACA) was synthesized; however, it did not form a continuous matrix; making it useless as an immobilization scheme for biosensors. Alginate beads fall apart easily in bacteria media, but are structurally stable in potassium solutions. Cells immobilized in alginate beads seemed to efflux four times less potassium than did planktonic controls, while cells in thermally reversible gels effluxed a comparable amount of potassium as planktonic controls. This result may indicate a tighter matrix around the alginate immobilized cells, not allowing proper diffusion of potassium out of the matrix. / Master of Science

To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux. / Master of Science

O tetracloroeteno (PCE), um dos principais contaminantes de águas subterrâneas, é uma molécula recalcitrante, com toxicidade elevada. Processos de biorremediação de água ou solo contaminados com PCE são normalmente limitados pela baixa eficiência de microrganismos sabidamente envolvidos em sua degradação. No entanto, a prospecção de novos microrganismos, mais eficientes na degradação do PCE é uma alternativa para otimizar esses processos. Os objetivos deste estudo foram desenvolver uma técnica de biorremediação utilizando um reator horizontal de leito fixo (RHLF) contendo consórcios de microrganismos eficientes na degradação do PCE, e caracterizar a via de degradação do PCE utilizada pelo consórcio selecionado. Para tanto, amostras de sedimento de dois poços de monitoramento de água foram coletadas de uma metalúrgica com histórico de contaminação com PCE. Os sedimentos foram imobilizados, acondicionados em RHLFs específicos e submetidos a cultivo de enriquecimento em meio mínimo suplementado com PCE. A estrutura das comunidades e a diversidade bacteriana dos RHLFs foram avaliadas e comparadas com as amostras do PM1 e PM2, por PCR-DGGE e sequenciamento de bibliotecas de clones do gene rRNA 16S. Os resultados evidenciaram a seleção de populações bacterianas no RHLF contendo o inóculo do PM1 (In1) após o cultivo de enriquecimento, enquanto no RHLF contendo o inóculo do PM2 (In2), a estrutura da comunidade bacteriana não diferiu daquela observada no PM2. Ensaios de degradação do PCE nos RHLFs, usando cromatografia gasosa associada à espectrometria de massas (CG/EM), mostraram, após 12 horas, uma eficiência de 87 % na degradação do PCE no reator com In1 e 96 % no reator com In2. Foi feito o isolamento e identificação, por sequenciamento do gene rRNA 16S, das bactérias dos RHLFs, sendo identificados 4 isolados do In1, similares a Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans e Stenotrophomonas maltophilia e 7 isolados do In2, similares a Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii e Comamonas sp. Compostos orgânicos voláteis nos reatores com In1 e In2 foram analisados por CG/EM, identificando a produção de clorofórmio (TCM) e 1,1,1-tricloroetano (TCA) como produtos da degradação do PCE. Consórcios formados por bactérias isoladas dos reatores In1 (IIn1) e In2 (IIn2) foram imobilizados e acondicionados em RHLFs distintos para avaliar o potencial dos mesmos na degradação do PCE. Após 12 horas, 92 % do PCE foi degradado nos reatores com IIn1 e IIn2, com produção de TCM e TCA. Testes de degradação usando células em suspensão foram conduzidos para avaliar a eficiência de cada isolado na degradação do PCE. O isolado I8 do In2 (I8In2), identificado como Comamonas sp., teve 68 % de eficiência na degradação do PCE. Ensaios com inibidor de monoxigenases do citocromo P-450 (1-aminobenzotriazole) mostraram que a degradação do PCE nos RHLFs, contendo IIn1, IIn2 e I8In2, foram dependentes dessa enzima. Como conclusão, nós identificamos uma nova via de degradação do PCE altamente eficiente, aeróbia e mediada por monoxigenases e isolamos cepas bacterianas que podem ser usadas como consórcios imobilizados nos RHLFs como uma alternativa eficiente na remediação de áreas contaminadas com PCE. / Tetrachloroethene (PCE), one of the main contaminants of groundwater, is a recalcitrant molecule with high toxicity. Bioremediation processes of water or soil contaminated with PCE are usually limited by the low efficiency of microorganisms known to be involved in its degradation. However, the exploration of new and more efficient microorganisms in the degradation of PCE is an alternative to optimize these processes. The objectives of these studies were to develop a bioremediation technique using horizontal fixed bed reactor (HFBR) containing microbial consortia effective in the PCE degradation, and to characterize the PCE degradation pathway used by the selected consortium. For that, sediment samples of two groundwater monitoring wells were collected from a metallurgical plant with historical of PCE contamination. The sediments were immobilized, packed in specific HFBRs and subjected to enrichment in minimal medium supplemented with PCE. The bacterial community structure and diversity in the HFBRs were evaluated and compared to samples from the MW1 and MW2, by PCR-DGGE and sequencing of 16S rRNA gene clone libraries. The results revealed the selection of bacterial populations in the HFBR containing inoculum from MW1 (In1) after enrichment, while in the HFBRs containing inoculum from MW2 (In2), the bacterial community structure did not differ from that observed in MW2. Tests of PCE degradation in HFBRs using gas chromatography-mass spectrometry (GC/MS) showed, after 12 hours, an efficiency of 87 % in the PCE degradation in the In1 reactor and 96 % in the In2 reactor. Bacteria from HFBR were isolated and identified by sequencing of 16S rRNA gene, and 4 isolates from In1, similar to Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans and Stenotrophomonas maltophilia, and 7 isolates from In2, similar to Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii and Comamonas sp. were identified. Volatile organic compounds in the reactors with In1 and In2 were analyzed by GC/MS, showing the production of chloroform (TCM) and 1,1,1-trichloroethane (TCA) as PCE degradation products. Consortia composed of bacteria isolated from the In1 (IIn1) and In2 (IIn2) reactors were immobilized and packed in distinct HFBRs to evaluate the potential of specific consortia in PCE degradation. After 12 hours, 92 % of PCE was degraded in reactors with IIn1 and IIn2, with the production of TCM and TCA. Degradation tests using cells suspension were conducted to evaluate the efficiency of each isolate in PCE degradation. Isolate I8 from In2 (I8In2), identified as Comamonas sp., showed 68 % efficiency in the PCE degradation. Assays using inhibitor of monooxygenases cytochrome P-450 (1-aminobenzotriazole) showed that the PCE degradation in HFBRs containing IIn1, IIn2 and I8In2 were dependent of this enzyme. In conclusion, we have identified a new highly efficient PCE degradation pathway, aerobic and mediated by monooxygenases, and isolated bacterial strains that may be used as consortia which immobilized in HFBRs as an efficient alternative in the remediation of PCE contaminated areas.

O tetracloroeteno (PCE), um dos principais contaminantes de águas subterrâneas, é uma molécula recalcitrante, com toxicidade elevada. Processos de biorremediação de água ou solo contaminados com PCE são normalmente limitados pela baixa eficiência de microrganismos sabidamente envolvidos em sua degradação. No entanto, a prospecção de novos microrganismos, mais eficientes na degradação do PCE é uma alternativa para otimizar esses processos. Os objetivos deste estudo foram desenvolver uma técnica de biorremediação utilizando um reator horizontal de leito fixo (RHLF) contendo consórcios de microrganismos eficientes na degradação do PCE, e caracterizar a via de degradação do PCE utilizada pelo consórcio selecionado. Para tanto, amostras de sedimento de dois poços de monitoramento de água foram coletadas de uma metalúrgica com histórico de contaminação com PCE. Os sedimentos foram imobilizados, acondicionados em RHLFs específicos e submetidos a cultivo de enriquecimento em meio mínimo suplementado com PCE. A estrutura das comunidades e a diversidade bacteriana dos RHLFs foram avaliadas e comparadas com as amostras do PM1 e PM2, por PCR-DGGE e sequenciamento de bibliotecas de clones do gene rRNA 16S. Os resultados evidenciaram a seleção de populações bacterianas no RHLF contendo o inóculo do PM1 (In1) após o cultivo de enriquecimento, enquanto no RHLF contendo o inóculo do PM2 (In2), a estrutura da comunidade bacteriana não diferiu daquela observada no PM2. Ensaios de degradação do PCE nos RHLFs, usando cromatografia gasosa associada à espectrometria de massas (CG/EM), mostraram, após 12 horas, uma eficiência de 87 % na degradação do PCE no reator com In1 e 96 % no reator com In2. Foi feito o isolamento e identificação, por sequenciamento do gene rRNA 16S, das bactérias dos RHLFs, sendo identificados 4 isolados do In1, similares a Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans e Stenotrophomonas maltophilia e 7 isolados do In2, similares a Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii e Comamonas sp. Compostos orgânicos voláteis nos reatores com In1 e In2 foram analisados por CG/EM, identificando a produção de clorofórmio (TCM) e 1,1,1-tricloroetano (TCA) como produtos da degradação do PCE. Consórcios formados por bactérias isoladas dos reatores In1 (IIn1) e In2 (IIn2) foram imobilizados e acondicionados em RHLFs distintos para avaliar o potencial dos mesmos na degradação do PCE. Após 12 horas, 92 % do PCE foi degradado nos reatores com IIn1 e IIn2, com produção de TCM e TCA. Testes de degradação usando células em suspensão foram conduzidos para avaliar a eficiência de cada isolado na degradação do PCE. O isolado I8 do In2 (I8In2), identificado como Comamonas sp., teve 68 % de eficiência na degradação do PCE. Ensaios com inibidor de monoxigenases do citocromo P-450 (1-aminobenzotriazole) mostraram que a degradação do PCE nos RHLFs, contendo IIn1, IIn2 e I8In2, foram dependentes dessa enzima. Como conclusão, nós identificamos uma nova via de degradação do PCE altamente eficiente, aeróbia e mediada por monoxigenases e isolamos cepas bacterianas que podem ser usadas como consórcios imobilizados nos RHLFs como uma alternativa eficiente na remediação de áreas contaminadas com PCE. / Tetrachloroethene (PCE), one of the main contaminants of groundwater, is a recalcitrant molecule with high toxicity. Bioremediation processes of water or soil contaminated with PCE are usually limited by the low efficiency of microorganisms known to be involved in its degradation. However, the exploration of new and more efficient microorganisms in the degradation of PCE is an alternative to optimize these processes. The objectives of these studies were to develop a bioremediation technique using horizontal fixed bed reactor (HFBR) containing microbial consortia effective in the PCE degradation, and to characterize the PCE degradation pathway used by the selected consortium. For that, sediment samples of two groundwater monitoring wells were collected from a metallurgical plant with historical of PCE contamination. The sediments were immobilized, packed in specific HFBRs and subjected to enrichment in minimal medium supplemented with PCE. The bacterial community structure and diversity in the HFBRs were evaluated and compared to samples from the MW1 and MW2, by PCR-DGGE and sequencing of 16S rRNA gene clone libraries. The results revealed the selection of bacterial populations in the HFBR containing inoculum from MW1 (In1) after enrichment, while in the HFBRs containing inoculum from MW2 (In2), the bacterial community structure did not differ from that observed in MW2. Tests of PCE degradation in HFBRs using gas chromatography-mass spectrometry (GC/MS) showed, after 12 hours, an efficiency of 87 % in the PCE degradation in the In1 reactor and 96 % in the In2 reactor. Bacteria from HFBR were isolated and identified by sequencing of 16S rRNA gene, and 4 isolates from In1, similar to Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans and Stenotrophomonas maltophilia, and 7 isolates from In2, similar to Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii and Comamonas sp. were identified. Volatile organic compounds in the reactors with In1 and In2 were analyzed by GC/MS, showing the production of chloroform (TCM) and 1,1,1-trichloroethane (TCA) as PCE degradation products. Consortia composed of bacteria isolated from the In1 (IIn1) and In2 (IIn2) reactors were immobilized and packed in distinct HFBRs to evaluate the potential of specific consortia in PCE degradation. After 12 hours, 92 % of PCE was degraded in reactors with IIn1 and IIn2, with the production of TCM and TCA. Degradation tests using cells suspension were conducted to evaluate the efficiency of each isolate in PCE degradation. Isolate I8 from In2 (I8In2), identified as Comamonas sp., showed 68 % efficiency in the PCE degradation. Assays using inhibitor of monooxygenases cytochrome P-450 (1-aminobenzotriazole) showed that the PCE degradation in HFBRs containing IIn1, IIn2 and I8In2 were dependent of this enzyme. In conclusion, we have identified a new highly efficient PCE degradation pathway, aerobic and mediated by monooxygenases, and isolated bacterial strains that may be used as consortia which immobilized in HFBRs as an efficient alternative in the remediation of PCE contaminated areas.