Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 122-137). Also available on the World Wide Web.

Submitted by RUTH HELENA GIANSANTE null (ruthhgster@gmail.com) on 2018-04-03T13:04:19Z No. of bitstreams: 1 RUTH_GIANSANTE_DISS_versão final (20DEZ2017) (5)6.pdf: 858614 bytes, checksum: 2838c4b704bfcc265b052ef9b29dcb67 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-04-03T18:23:48Z (GMT) No. of bitstreams: 1 giansante_rh_me_jabo.pdf: 858614 bytes, checksum: 2838c4b704bfcc265b052ef9b29dcb67 (MD5) / Made available in DSpace on 2018-04-03T18:23:48Z (GMT). No. of bitstreams: 1 giansante_rh_me_jabo.pdf: 858614 bytes, checksum: 2838c4b704bfcc265b052ef9b29dcb67 (MD5) Previous issue date: 2017-11-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Rizobactérias são excelentes candidatas à aplicação em processos de bioacumulação de elementos potencialmente tóxicos, pois desenvolveram mecanismos para a incorporação intracelular de uma ampla gama de íons. A sensibilidade e a capacidade de remoção de cádmio (Cd2+) de duas espécies de rizobactérias: Rizobium tropici (LBMP-C01) e Ensifer meliloti (LBMPC02), foram estudadas. A concentração mínima inibitória (CMI) das bactérias foi determinada pelo cultivo em meio contendo CdCl2.2H2O (0,025 a 4 mmol L-1). Foram realizados testes de viabilidade das células das duas estirpes na CMI e ensaios de bioacumulação com suspensões de células bacterianas nas doses de 10, 20 e 30 %(v/v) em solução contendo 100 mg L-1 de Cd2+. As estirpes LBMP-C01 e LBMP-C02 foram sensíveis a concentrações de Cd2+ superiores a 1,0 e 0,05 mmol L-1, respectivamente. As células de LBMP-C01 e LBMP-C02 apresentaram-se viáveis nas CMI 1,0 e 0,05 mmol L-1 Cd2+, respectivamente. A estirpe LBMP-C01 não removeu Cd2+ nos ensaios de bioacumulação e a estirpe LBMP-C02 foi capaz de remover 80 % deste íon em solução contendo 100 mg L-1 Cd2+, após 72 h de contato e 30 %(v/v) do bioacumulador. Os espectros de absorção molecular na região do infravermelho, de ambas as espécies estudadas praticamente não indicaram diferenças nos grupos funcionais presentes nas moléculas da biomassa celular. A observação por microscopia eletrônica de transmissão mostrou a presença de maior número de grânulos eletrodensos no citoplasma da estirpe de LBMP-C02 em relação à LBMP-C01 quando estas foram cultivadas com Cd2+. A estirpe LBMP-C02 foi a mais eficiente na remoção de Cd2+. A resistência a metais dessas duas bactérias envolve mecanismos diferentes. / Rhizobacteria are excellent candidates for use in the processes of bioaccumulation of potentially toxic elements because they have developed mechanisms for the intracellular uptake of a wide range of ions. Here, the sensitivity and capacity to remove cadmium (Cd2+) of two species of rhizobacteria, Rhizobium tropici (LBMP-C01) and Ensifer meliloti (LBMP-C02), were studied. The minimum inhibitory concentration (MIC) of the bacteria was determined by culturing them in medium containing CdCl2·2H2O (0.025 to 4 mmol L-1 ). Cell viability tests of the two strains were performed at MIC, and bioaccumulation assays were performed with 10, 20, and 30 %(v/v) bacterial cell suspensions in a Cd2+ solution 100 mg L-1 . Strains LBMP-C01 and LBMP-C02 were sensitive to Cd2+ concentrations above 1.0 and 0.05 mmol L-1 , respectively. LBMP-C01 and LBMP-C02 cells were viable at the MICs of Cd2+ solution 1.0 and 0.05 mmol L-1 , respectively. LBMP-C01 did not remove Cd2+ in the bioaccumulation assays, whereas LBMP-C02 removed 80 % of this ion in Cd2+ solution 100 mg L-1 , after 72 h of contact and 30 %(v/v) of the bioaccumulator. The infrared absorption spectra of both species did not indicate differences in the functional groups present in the molecules of the cell biomass. Transmission electron microscopy showed the presence of a larger number of electron-dense granules in the cytoplasm of LBMP-C02 compared to LBMP-C01 when they were cultured with Cd2+. The LBMP-C02 strain was the most efficient in the Cd2+ removal. The metal resistance of these two bacteria involves different mechanisms.

Humic substances (HS) are the humified portions of totally decomposed soil organic matter that are ubiquitous in nature. Although these substances have been studied for more than 200 years, neither their metabolic capabilities nor a specific chemical structure has yet to be determined. HS have been studied as a carbon source in many environments where they are degraded; however, previous studies have shown that some microorganisms are capable of utilizing humic substances as electron acceptors and electron donors in anaerobic respiration. Even though there have been humic-reducing and humic-oxidizing microorganisms isolated and studied in recent years, the mechanism of humics metabolism and its interaction in the natural environment are not well understood. However, it is known that the continuous change in the redox state of HS is important to the cycling of iron, stability of nitrogen and carbon, and the mobility and bioavailability of inorganic and organic environmental pollutants. In this study, microbial communities were examined to evaluate the community dynamics of nitrate-dependent HS-oxidizing populations and to provide a snapshot of the phylogenetic diversity of these microorganisms. Column studies were performed using nitrate as the sole electron acceptor and the following as the electron donors in different columns: reduced humic acids, oxidized humic acids, and acetate as the control. Liquid buffered media was added to a separate column to serve as an additional control. Polymerase chain reactions of the 16S rRNA genes using DNA from the column studies were performed and analyzed by constructing 16S rDNA clone libraries and by performing denaturing gradient gel electrophoresis (DGGE). Clones from the library have been sequenced and analyzed to paint a phylogenetic picture of the microbial community under the various conditions. Results indicate that the majority of the clones were assigned to four well-characterized divisions, the Acidobacteria, the Bacteroidetes, the Firmicutes, and the Proteobacteria. Additional findings suggest that members related to Bacteroidetes are involved in some sort of HS cycling in the environment or may be excellent electron scavengers enabling them to outcompete other microorganisms and that members of Proteobacteria may be the dominant HS-oxidizing microorganisms in natural environments. An additional aspect of this project hypothesizes that specific genes are differentially expressed when HS-oxidizing bacteria are growing on reduced HS as compared to acetate or in the presence of oxidized HS. To test this hypothesis, the global gene expression profile of Acidovorax ebreus strain TPSY was assessed using microarray analysis. This method led to the identification of several genes potentially involved in nitrate-dependent HS oxidization and a proposed model for this respiratory process in strain TPSY. The final section in this project was designed to assess in-service teachers' perceived levels of familiarity with and interest in learning more about selected microbiology concepts and their actual understanding of the selected microbiology concepts. Sixty-two in-service elementary, middle, and high school teachers from several school districts across southern Illinois completed a three-part instrument that included additional open-ended questions to gain more information about the teachers' conceptual understanding. The results of this study suggest that teachers who hold a teaching certification specific for teaching life science have taken more life science courses and scored significantly higher on the familiarity and conceptual knowledge sections of this study. The current research explores what is currently known about humic substances, specifically humics as an electron donor, analyzes the community structure in a humics oxidizing environment, identifies genes that are induced under nitrate-reducing, HS-oxidizing conditions, and evaluates the importance of microbiology to biological scientific literacy in today's society.

Metal pollution has been a great challenge in most industrialized countries as a result of waste generated from industrial activities being introduced into the environment. Unicellular green algae have been considered a potential biological tool for bioremediation of metal pollutants due to its metal sequestration properties. However, methods for further improving unicellular green algae metal sequestration by manipulating metal uptake and tolerance in unicellular green algae have not been studied in detail. In this study, a family metal transport protein named MTP1 - MTP4 from C. reinhardtii were screened by yeast heterologous expression for metal transport activity. MTP1 was able to strongly rescue the Zn and Co sensitivity of the zrc1cot1 strain, MTP3 could weakly mediate Zn and Co growth, but MTP2 and MTP4 appeared to have no Zn or Co tolerance activity. MTP2, MTP3 and MTP4 but not MTP1 could strongly rescue the Mn sensitivity of the pmr1 strain. When MTP4 was over-expressed in C. reinhardtii the strain showed a significant increase in Cd tolerance compared to the wild type, but no significant difference in Mn tolerance and uptake. AtHMA4 a Zn2+ and Cd2+ transporter from the plant Arabidopsis thaliana, which is a member of the Heavy Metal ATPase family, was also expressed in C. reinhardtii. HMA4 full length and C-terminal tail expression strains were screened for Zn and Cd tolerance and uptake. Both sets of strains showed a significant increase in Cd and Zn tolerance and uptake compared to the wild type. Metal tolerance and uptake was compared between the genetically engineered C. reinhardtii strains and unicellular green algal strains that are naturally adapted to metal tolerance which were P. hussi, P. kessleri, and C. luteoviridis. Results showed significant increase in Zn and Cd tolerance and uptake in the natural strains compared to the engineered strains. Therefore in addition to genetically engineered strains, naturally adapted strains could also be used as tools for effective metal bioremediation and pollutant treatment.

Acid mine drainage (AMD) is an environmental problem of a global scale. Passive remediation strategies utilizing the metabolism of sulfate-reducing bacteria have emerged as promising options for the mitigation of impacted AMD sites. In order to test the effect of varying complex and simple carbon sources on AMD remediation efficiency, pilot-scale bioreactors were constructed and exposed to AMD in situ over a ten-month period. Geochemical analyses suggested that the efficiency of AMD remediation depended more on the seasonal weather patterns of Southern Illinois, USA than the substrate composition of each bioreactor. Enrichment cultures targeting sulfate-reducing organisms yielded several isolates most closely related to members of the genera Desulfovibrio and Clostridium. Microbial community analysis was performed using fluorescent in situ hybridization, 16S rRNA gene targeted pyrosequencing, and quantitative polymerase chain reaction (qPCR). Results suggested that the depth from which samples were taken as well as the substrate composition impacted the microbial communities within each bioreactor. Over the course of the experiment the community changed from one similar to that of a bovine rumen to one more adapted to the acidic nature and high metal content of AMD. Community abundance based on 16S rRNA gene and dsrB gene copy number suggested an overall decrease in the bacterial population over the course of the study.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-02Bitstream added on 2014-06-13T19:55:59Z : No. of bitstreams: 1 paixao_daa_me_jabo.pdf: 1354562 bytes, checksum: 375f9e3806bd3d094111e5d1f95f8ddd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A estratégia de clonagem e sequenciamento do gene 16S rRNA é uma das técnicas moleculares que permite estimar e comparar a diversidade microbiana de diferentes amostras ambientais. O objetivo deste trabalho foi estimar a diversidade de microrganismos pertencentes ao Domínio Bactéria em um consórcio degradador de óleo diesel, por meio do sequenciamento parcial do gene 16S rRNA, assim como desenvolver uma nova metodologia de rastreamento em bibliotecas metagenômicas. O consórcio bacteriano foi obtido através de solo enriquecido com óleo diesel. O DNA metagenômico foi extraído com o auxílio do kit Fast DNA spin Kit for soil (Bio101- Quantum Biotechnologies) e amplificado por uma reação de PCR (Reação em Cadeia da Polimerase) com os oligonucleotídeos iniciadores FD1 e RD1 específicos para a o gene 16S rRNA. Os produtos de PCR foram clonados em vetor pGEM T Easy (Promega) e transformados em células competentes de Escherichia Coli DH5 . O sequenciamento parcial dos clones foi feito com oligonucleotideos universais do vetor. Para a prospecção gênica foi utilizado membranas de nylon com “pools” de DNA de todas as placas. A biblioteca obtida gerou 431 clones. Os clones obtidos apresentaram similaridade com o filo Proteobacteria, com representantes das classes Gammaproteobacteria, Alphaproteobacteira e Betaproteobacteria. O gênero Pseudomonas apresentou-se com maior frequência de clones na biblioteca. O “software” DOTUR foi usado para determinar o número de unidades taxonômicas operacionais (OTUs). A curva de extinção indicou que os 431 clones sequenciado foram suficientes para estimar a diversidade bacteriana do consórcio. A metodologia testada baseado em “pools” de DNA foi eficiente na detecção e isolamento do gene Alkb na bilbioteca metagenomica. / Cloning and sequencing of 16S rRNA gene it is one of the molecular techniques that permits estimate and compare the microbial diversity of different environmental samples. The aim of this work was estimate the diversity of microorganisms that belong to Bacteria domain in a consortium specialized in diesel oil degradation, through partial sequencing of 16S rRNA gene, as well as develop a new methodology for screening libraries in metagenomics. This consortium was obtained through enrichments achieved using diesel oil in soil samples. The metagenomic DNA was obtained using Fast DNA spin Kit for soil (Bio 101-Quantum Biotechnologies) and amplified by PCR (Polymerase chain reaction) with FD1 and RD1 oligonucleotides, which are specific for 16S rRNA gene. The PCR products were cloned into pGEM-TEasy vector (Promega) and Escherichia coli DH5 was used as the host cell for recombinant DNAs. The partial clones sequencing was obtained using universal primers of the vector. For the exploration of gene were used nylon membranes with Pools of DNA from all plates. The library generated from 431 clones. All clones sequenced showed similarity with the phylum Proteobacteria, distributed in Gammaproteobacteria, Alphaproteobacteira and Betaproteobacteria classes. The Pseudomonas genus was the most abundant genus found in the metagenomic library. The DOTUR software was used to assigns sequences to operational taxonomic units (OTUs). Using the OTUs composition data, rarefaction curves were made to show that 431 sequences were enough to obtain a satisfactory coverage of diversity of the microbial consortium. The test methodology based on Pools of DNA isolation was effective in detecting the gene Alkb Library metagenomics.

Thesis (MTech (Chemical Engineering))--Peninsula Technikon, 2001 / Certain fungi have been shown to excrete extracellular enzymes, including peroxidases, laccases, etc. These enzymes are useful for bioremediation of aromatic pollutants present in industrial effluents (Leukes, 1999; Navotny et aI, 1999). Leukes (1999) made recent significant development in the form of a capillary membrane gradostat (fungal) bioreactor that offers optimal conditions for the production of these enzymes in high concentrations. This system also offers the possibility for the polluted effluent to be treated directly in the bioreactor. Some operating problems relating to continuous production of the enzymes and scale-up of the capillary modules, were, however, indentified. In an attempt to solve the above-mentioned identified problems the research group at Peninsula Technikon considered a number of alternative bioreactor configurations. A pulsed packed bed bioreactor concept suggested by Moreira et at. (1997) was selected for further study. Their reactor used polyurethane pellets as the support medium for the fungal biofilm and relied upon pulsing of the oxygen supply and recycle of nutrient solution in order to control biomass accumulation. These authors reported accumulation due to the recycle of proteases that were believed to destroy the desired ligninases. We experimented with a similar concept without recycle to avoid backrnixing and thereby overcome protease accumulation. In our work, a maximum enzyme productivity of 456 Units.L1day·1 was attained. Since this was significantly greater than the maximum reported by Moreira et aI, 1997 (202 Units.L-1day-I) it appeared that the elimination of recycle had significant benefits. In addition to eliminating recycle we also used a length / diameter (L / D) ratio of 14: 1 (compared with 2.5: 1 used by Moreira et aI, 1997) in order to further reduce backrnixing. Residence time distributions were investigated to gain insight into mechanisms of dispersion in the reactor. It was found that the pulsed packed bed concept presented problems with regard to blockage by excess biomass. This led us to consider the advantages of a fluidized bed using resin beads. Accordingly, growth of fungi on resin beads in shake flasks was investigated with favorable results. An experimental program is proposed to further investigate the fluidized bed concept with a view to extending the operation time of the bioreactor. From our literature survey to date, packed bed fungal bioreactors are still the best reactor configuration for continuous production ofligninolytic enzymes. An interesting study of the application of laccases to the degradation of naphthalene and MTBE is described in an addendum to this thesis.

Thesis submitted in fulfilment of the requirements for the degree Magister Technologiae: Chemical Engineering in the Faculty of Engineering at the Cape Peninsula University of Technology 2014 / The legislative requirements for handling cyanide containing wastewater have become stringent internationally. Cyanide properties make it indispensable in the mining industry especially for gold recovery. The resultant wastewater generated is discarded to tailing ponds. Any leakages or total collapse of tailing ponds can result in the contamination of surface water bodies; endangering aquatic organisms’ and humans’ alike. The over reliance on physical and/or chemical treatment methods for cyanide wastewater treatment is not sustainable due to high input costs and the generation of by-products. A feasible alternative treatment method for cyanide contaminated wastewater is the biodegradation method, as a wide range of microorganisms can degrade cyanide. In this study, the cyanide biodegradation ability of Fusarium oxysporum was assessed in two stages. Firstly, optimal operating conditions for maximum cyanide biodegradation were determined using a central composite design (CCD) at an elevated cyanide concentration, i.e. 500 mg F-CN/L. Thereafter, using the optimum conditions obtained, (i.e temperature 22°C and pH 11), cyanide biodegradation kinetics and microbial growth kinetics in the cultures at lower cyanide concentrations of 100, 200 and 300 mg F-CN/L were assessed. This was followed by the assessment of cyanide biodegradation at a temperature of 5°C, which was used to simulate winter conditions. In general, lower cyanide concentrations are used in the extraction of gold, therefore, the resultant wastewater will contain free cyanide concentration less than 300 mg F-CN/L. For the first stage of experiments, an isolate, Fusarium oxysporum from cyanide containing pesticides was cultured on potato dextrose agar (PDA) plates, followed by incubation at 25°C for 5 days. A response surface methodology (RSM) was used to evaluate design parameters for the biodegradation of cyanide by this fungus. The temperature evaluated at this stage ranged from 9°C to 30°C and pH range of 6 to 11 in cultures solely supplemented with agrowaste, i.e Beta vulgaris waste. Beta vulgaris is commonly known as Beetroot. The Fusarium oxysporum inoculum (2% v/v) was grown on a Beta vulgaris waste solution (20% v/v), as the sole carbon source in a synthetic gold mine wastewater (39% v/v) containing heavy metals; arsenic (7.1 mg/L), iron (4.5 mg/L), copper (8 mg/L), lead (0.2 mg/L) and zinc (0.2 mg/L), for 48 hours using a rotary shaker at 70 rpm. Thereafter, free cyanide as a potassium cyanide solution (39% v/v), was added to the cultures to make a final cyanide concentration of 500 mg F-CN/L in the culture medium which was incubated for a further 72 hours at 70 rpm. Optimal operating conditions for the biodegradation of cyanide were then determined using a numerical option in the Design-Expert® software version 6.0.8 (Stat-Ease Inc., USA). Subsequently, using the optimal pH obtained (pH =11) and a preselected temperature of 5°C (to represent winter conditions), cyanide biodegradation rates and microbial growth kinetic studies were carried out using Beta vulgaris waste containing a Fusarium oxysporum (0.7% v/v; grown overnight) inoculum in wastewater (32.7% v/v) and potassium cyanide in phosphate buffer (53.7% v/v). The cultures contained 100, 200 and 300 mg F-CN/L. The cultures were incubated in an orbital shaker at 70 rpm for 144 hours and samples taking every 24 hours. An Ordinary Differential Equation (ODE) solver (Polymath) was used for modelling cyanide degradation kinetics while the Monod’s growth kinetic model was used to monitor the microbial growth parameters of the cultures. For the first stage, the optimum operating conditions were determined as a temperature of 22°C and a pH of 11 for maximum cyanide biodegradation of 277 mg F-CN/L from an initial cyanide concentration of 500 mg F-CN/L over a 72 hour period, with residual ammonium-nitrogen and nitrate-nitrogen of 150 mg NH4+-N/L and 37 mg NO3--N/L, respectively. Although, the residual ammonium-nitrogen inhibited cyanide biodegradation, it was consumed as a nitrogen source for microbial growth. The Beta vulgaris waste was determined to be a suitable substrate for cyanide degradation. From the biodegradation response quadratic model, temperature was determined to influence cyanide biodegradation. For the cyanide degradation kinetics, at an optimum temperature of 22°C, the biodegradation efficiency was 77%, 58% and 62% with the corresponding maximum microbial population of 1.56 x 107, 1.55 x 107 and 1.57 x 107 CFU/mL for 100, 200 and 300 mg F-CN/L, being achieved. An indication that the F. oxysporum cultures were efficient at lower cyanide concentration. Furthermore, at a temperature of 5°C, the biodegradation efficiency, although slightly lower, was 51%, 43% and 44% with the corresponding maximum microbial population of 1.21 x107, 1.11 x 107 and 1.12 x 107 CFU/mL for 100, 200 and 300 mg F-CN/L cultures, respectively, with minimal differences observed for cultures with 200 and 300 mg F-CN/L. The cyanide biodegradation rates increased with temperature increases and varied with different cyanide concentrations below 500 mg F-CN/L. The estimated energy of activation for cyanide degradation for a change in temperature from 5°C to 22°C using the Arrhenius model was 19.6, 12.7 and 14.9 kJ/mol for 100, 200 and 300 mg F-CN/L, respectively. The means and standard deviations for rate of degradation of cyanide at 5°C and 22°C for the ODE models was 0.0052 (± 0.0011) h-1 and 0.0084 (± 0.0027) h-1, respectively. The inhibitory effect of the cyanide was quantitatively pronounced under cold temperature as the heavy metals, residual ammonium-nitrogen and nitrate-nitrogen hindered the cyanide degradation. Similarly, microbial growth rates increased with a temperature rise (from 5°C to 22°C), resulting with a reduction in the microbial populations’ doubling time. When compared with the simulated winter conditions, the specific population growth rate increased 4-fold, 5-fold and 6-fold in 100, 200 and 300 mg F-CN/L, respectively, for higher temperatures; an indication that the Fusarium oxysporum isolate prefers higher temperature. The estimated energy of activation for cellular respiration was 44.9, 54 and 63.5 kJ/mol for 100, 200 and 300 mg F-CN/L cultures, respectively, for the change in temperature from 5°C to 22°C. The means and standard deviations of microbial growth rate at 5°C and 22°C were: 0.0033 (± 0.0013) h-1 and 0.0151 (± 0.0027) h-1, respectively. The difference in error (standard deviation) of the cyanide biodegradation rate and microbial growth rate was insignificant (0.02% at 5°C) especially at temperature 22°C where there were minimal differences, indicating the reliability and reproducibility of this biodegradation system in batch operated bioreactors.

Orientador: Eliana Gertrudes Macedo Lemos / Banca: Alessandro Minillo / Banca: Janete Apparecida Desidério Sena / Resumo: A estratégia de clonagem e sequenciamento do gene 16S rRNA é uma das técnicas moleculares que permite estimar e comparar a diversidade microbiana de diferentes amostras ambientais. O objetivo deste trabalho foi estimar a diversidade de microrganismos pertencentes ao Domínio Bactéria em um consórcio degradador de óleo diesel, por meio do sequenciamento parcial do gene 16S rRNA, assim como desenvolver uma nova metodologia de rastreamento em bibliotecas metagenômicas. O consórcio bacteriano foi obtido através de solo enriquecido com óleo diesel. O DNA metagenômico foi extraído com o auxílio do kit Fast DNA spin Kit for soil (Bio101- Quantum Biotechnologies) e amplificado por uma reação de PCR (Reação em Cadeia da Polimerase) com os oligonucleotídeos iniciadores FD1 e RD1 específicos para a o gene 16S rRNA. Os produtos de PCR foram clonados em vetor pGEM T Easy (Promega) e transformados em células competentes de Escherichia Coli DH5 . O sequenciamento parcial dos clones foi feito com oligonucleotideos universais do vetor. Para a prospecção gênica foi utilizado membranas de nylon com "pools" de DNA de todas as placas. A biblioteca obtida gerou 431 clones. Os clones obtidos apresentaram similaridade com o filo Proteobacteria, com representantes das classes Gammaproteobacteria, Alphaproteobacteira e Betaproteobacteria. O gênero Pseudomonas apresentou-se com maior frequência de clones na biblioteca. O "software" DOTUR foi usado para determinar o número de unidades taxonômicas operacionais (OTUs). A curva de extinção indicou que os 431 clones sequenciado foram suficientes para estimar a diversidade bacteriana do consórcio. A metodologia testada baseado em "pools" de DNA foi eficiente na detecção e isolamento do gene Alkb na bilbioteca metagenomica. / Abstract: Cloning and sequencing of 16S rRNA gene it is one of the molecular techniques that permits estimate and compare the microbial diversity of different environmental samples. The aim of this work was estimate the diversity of microorganisms that belong to Bacteria domain in a consortium specialized in diesel oil degradation, through partial sequencing of 16S rRNA gene, as well as develop a new methodology for screening libraries in metagenomics. This consortium was obtained through enrichments achieved using diesel oil in soil samples. The metagenomic DNA was obtained using Fast DNA spin Kit for soil (Bio 101-Quantum Biotechnologies) and amplified by PCR (Polymerase chain reaction) with FD1 and RD1 oligonucleotides, which are specific for 16S rRNA gene. The PCR products were cloned into pGEM-TEasy vector (Promega) and Escherichia coli DH5 was used as the host cell for recombinant DNAs. The partial clones sequencing was obtained using universal primers of the vector. For the exploration of gene were used nylon membranes with Pools of DNA from all plates. The library generated from 431 clones. All clones sequenced showed similarity with the phylum Proteobacteria, distributed in Gammaproteobacteria, Alphaproteobacteira and Betaproteobacteria classes. The Pseudomonas genus was the most abundant genus found in the metagenomic library. The DOTUR software was used to assigns sequences to operational taxonomic units (OTUs). Using the OTUs composition data, rarefaction curves were made to show that 431 sequences were enough to obtain a satisfactory coverage of diversity of the microbial consortium. The test methodology based on Pools of DNA isolation was effective in detecting the gene Alkb Library metagenomics. / Mestre

This research has investigated the biodegradation of hydrocarbons particularly n-alkanes using open mixed culture which is relevant for both microbial enhanced oil recovery (MEOR) and the bioremediation of hydrocarbon contaminated soils. Biodegradation of n-C12, C14, C16, C18, C20 and some readily biodegradable substrates (glucose, acetic acid and ethanol) was studied using a respirometric method developed to assess the biodegradability of these compounds. Laboratory batch and semi-continuous experiments were performed in small-scale bioreactors at room temperature and 40oC under various conditions i.e. aerobic, anoxic with nitrate, sulfate reducing and completely anaerobic conditions using two different sources of open mixed microbial cultures obtained from an agricultural site and anaerobic digestion plant. Glucose, acetic acid, ethanol, C12, C14 and C16 were degraded microbially under aerobic batch conditions to nondetectable levels at room temperature and 40oC using the two sources of inoculum whereas C18 and C20 were degraded partially under room temperature and to nondetectable levels at 40oC with the two inocula sources. Under aerobic semi-continuous, glucose and the n-alkane substrate were biodegraded even at low hydraulic retention time (HRT). Under anaerobic conditions, the n-alkanes were utilized by the soil inoculum at room temperature and at 40oC with nitrate as the electron acceptor but no microbial activity was observed under sulfate reducing and completely anaerobic conditions. The open mixed cultures require an initial acclimation period before utilizing the substrates. The acclimation period was significantly shorter under aerobic conditions than anaerobic conditions for the n-alkanes. Acclimation periods of approximately 1-2 days under aerobic conditions was observed for the readily biodegradable substrates and 2 days for glucose under anoxic conditions. The acclimation periods for the nalkanes was between 3-5 days under aerobic conditions and approximately 2 weeks under anoxic conditions. The acclimation period was not affected by the substrate concentration and inoculum type however, for the n-alkanes, the acclimation period was reduced by 1-2days under aerobic conditions at 40oC. The biodegradation of the liquid hydrocarbons was more significant than the solids at room temperature but in general higher temperature increased the degree of biodegradation. The electron acceptor consumption data i.e. dissolved oxygen and nitrate consumption data obtained was mathematically modelled using Monod kinetics to obtain biokinetic parameters. Good fittings between the model solution and the experimental data was obtained. The biokinetic parameters obtained were within the range of values reported in literature. The use of respirometric data for the estimation of biodegradation kinetic parameters can be very reliable. The consistency of the data obtained show that the approach is very reproducible and quality information can be obtained. The results of this study showed that the open mixed microbial cultures from soil and AD inocula contained diverse microorganisms capable of utilizing both liquid and solid n-alkanes at room temperature and 40oC under aerobic and anoxic conditions.