Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2017-02-16T18:12:51Z No. of bitstreams: 1 PDF - Bruna Regina dos Santos Silva.pdf: 4083927 bytes, checksum: f31770fedd1b2c616664828556da3d87 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2017-03-07T16:47:15Z (GMT) No. of bitstreams: 1 PDF - Bruna Regina dos Santos Silva.pdf: 4083927 bytes, checksum: f31770fedd1b2c616664828556da3d87 (MD5) / Made available in DSpace on 2017-03-07T16:47:15Z (GMT). No. of bitstreams: 1 PDF - Bruna Regina dos Santos Silva.pdf: 4083927 bytes, checksum: f31770fedd1b2c616664828556da3d87 (MD5) Previous issue date: 2017-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Red rice is practically unknown by most Brazilians, but in Paraíba as in other Northeastern states, it is highly cultivated and has great socioeconomic importance, cultivated mainly by small farmers. In agricultural activity, large quantities of cellulolytic materials are generated. This material is degraded by cellulolytic microorganisms, which hydrolyze and metabolise cellulose efficiently. There is a great commitment to the development of renewable alternatives to fossil fuels and one of these alternatives is second generation ethanol production, using enzymatic hydrolysis in vegetable biomass derivatives and subsequent fermentation. Microorganisms exhibit immense genetic diversity and play a variety of roles in maintaining ecosystems. One of these functions is the production of extracellular enzymes, which help in the degradation of organic matter. Cellulase is an enzyme that hydrolyzes cellulose by breaking the β-1,4 linkage. In the search for new non- cultured microorganisms, metagenomics appears as a tool that isolates DNA from environmental sources to identify enzymes with biotechnological potential. In this work, a gene encoding an endoglucanase was cloned from red rice culture residues using the metagenomic strategy. The amino acid identity between this gene and its nearest published congeners is less than 70%. The gene found has potential for use in the production of ethanol from cellulosic biomass (second generation ethanol). / O arroz vermelho é praticamente desconhecido pela maioria dos brasileiros, mas na Paraíba assim como em outros estados do Nordeste, é bastante cultivado por pequenos agricultores, apresentando uma grande importância socioeconômica. Na atividade agrícola, grandes quantidades de materiais celulolíticos são gerados. Esse material é degradado por microrganismos celulolíticos, que hidrolisam e metabolizam a celulose de forma eficiente. Existe um grande empenho para o desenvolvimento de alternativas renováveis aos combustíveis fósseis e uma dessas alternativas é a produção etanol de segunda geração, utilizando a hidrólise enzimática em derivados de biomassa vegetal e sua posterior fermentação. Os microrganismos apresentam uma imensa diversidade genética e desempenham vária funções na manutenção de ecossistemas. Uma dessas funções é a produção de enzimas extracelulares, que ajudam na degradação da matéria orgânica. A celulase é uma enzima que hidrolisa a celulose por meio da quebra da ligação β-1,4. Na busca por novos microrganismos não-cultivados, a metagenômica surge como uma ferramenta que isola o DNA a partir de fontes ambientais para identificar enzimas com potencial biotecnológico. Neste trabalho, um gene que codifica uma endoglucanase foi clonado a partir de resíduos de cultura do arroz vermelho utilizando a estratégia metagenômica. A identidade de aminoácidos entre este gene e os seus congêneres mais próximos publicado é inferior a 70%. O gene encontrado possui potencial para uso na produção de etanol a partir de biomassa celulósica (segunda etanol geração).

Submitted by isabel silva (isabel_manaus@yahoo.com.br) on 2017-06-22T15:28:22Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-06-23T13:18:22Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-06-23T13:18:48Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) / Made available in DSpace on 2017-06-23T13:18:48Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) Previous issue date: 2017-04-05 / CAPES / The advancement of molecular technologies in the scientific field has enabled the development of various forms of access to the genetic material of organisms in order to locate, map, isolate, characterize and decode the target genes of a particular individual or a set of individuals coexisting in a specific environment. Metagenomic studies are very promising in several areas of biotechnology, such as the discovery of new molecules of industrial biotechnological interest, the investigation of new antibiotics and drugs, the bioremediation of environments impacted with toxic metals and the prospection of various enzymes. The objective of this work was characterize enzymatically a previously screened lipase enzyme from Metagenomic Library of Terra Preta de Índio. Lipase gene sequence was isolated from the metagenomic library and expressed in Pichia pastoris under control of PGK promoter. Recombinant lipase was characterized by hydrolysis activity of lipid substrates and synthesis ability in organic solvent. In silico analyzes infer the identification of extracellular lipase belonging to the lipase family, superfamily -hydrolase and lipase activity molecular function. Enzyme was efficiently produced in P. pastoris and recombinant lipase showed activity of 374.59 U/mL hydrolyzing p-nitrophenyl palmitate, Vmax (ap.) 143,4 U/mL.min-1, Km (ap.) 1,4 mM and Kcat (ap.) 103,7 S-1. Enzyme had maximum activity at pH 8.0, temperature 90 ºC and remained stable at high temperatures. Synthesis ability was evaluated by the formation of ethyl laurate by esterification reaction of lauric acid with ethanol, yielding 70% conversion in 45 minutes. Recombinant protein is characterized as an alkaline, thermotolerant, activated by calcium, EDTA e detergents. / O avanço das tecnologias moleculares no campo científico, possibilitou o desenvolvimento de diversas formas de acesso ao material genético dos organismos, de forma a ser possível localizar, mapear, isolar, caracterizar e decodificar os genes alvo de um indivíduo particular ou de um conjunto de indivíduos coexistentes em um ambiente específico. Estudos metagenômicos são bastante promissores em diversas áreas da biotecnologia, como nas descobertas de novas moléculas de interesse biotecnológico industrial, na investigação de novos antibióticos e fármacos, na biorremediação de ambientes impactados com metais tóxicos e na prospecção de enzimas diversas. O objetivo deste trabalho foi caracterizar enzimaticamente uma enzima lipase previamente rastreada de uma Biblioteca Metagenômica de Terra Preta de Índio. A sequência correspondente ao gene de lipase foi isolada da biblioteca metagenômica, clonada e expressada em Pichia pastoris sob controle do promotor PGK. A lipase recombinante foi caracterizada pela atividade de hidrólise de substratos lipídicos e capacidade de síntese em solvente orgânico. Análises in silico da sequência proteica inferem a identificação de uma lipase extracelular pertencente à / - hidrolase e com função molecular para atividade lipásica. A enzima foi produzida eficientemente em P. pastoris e a lipase recombinante apresentou atividade de 374,59 U/mL hidrolisando p-nitrofenil palmitato, Vmax(ap.) 143,4 U/mL.min-1, Km(ap.) 1,4 mM e Kcat(ap.) 103,7 S-1. A enzima possuiu atividade máxima em pH 8,0, temperatura 90 ºC e se manteve estável em altas temperaturas. A capacidade de síntese foi avaliada pela formação de laurato de etila pela reação de esterificação do ácido láurico com etanol apresentando rendimento de 70% em 45 minutos de reação. A proteína recombinante se caracteriza como uma enzima alcalina, termotolerante, ativada por cálcio, EDTA e detergentes.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Lipases have been one of the important biocatalysts that catalyse the transformation of lipids to yield very important products that can be of beneficial in food, agriculture, pharmaceutical medicine and for the biodiesel production. In the search for novel biocatalysts, notably lipases, the conventional culture-based techniques were used but it can only address sourcing the biomolecule from 1-10% of the microbial population leaving the wealth of the biomolecules packed in 90-99% of the microbial community unaccounted for. Metagenomic technique, which is culture-independent, was developed as a comprehensive approach to address literally 100% of the microbial population thereby maximizing the chances of obtaining novel biocatalysts with superior physico-chemical and catalytic traits. In principle, any biomolecule including lipase could be sourced from any biologically-active environment, of which animal rumen is one. However, among the rumenant animals goat has diverse feeding habit, thereby laying ground for increased microbial diversity in its gastro-intestinal tract. It was thus, postulated that goat rumen could be potential source of novel lipase isoforms. Therefore, the aim of the study was to extract metagenomic DNA from goat rumen and construct a metagenomic fosmid library and screen the library for lipase isoforms. The fosmid clones were functionally screened using 1% tributyrin as a substrate and five positive clones were selected. From the five clones, two fosmids were selected for further study. Following nucleotide sequencing and in-silico analysis of the insert of the two selected clones, one lipase encoding open reading frame (Lip-VUT3 and Lip-VUT5) from each fosmid clones of approximately 212 and 248 amino acids, respectively, was identified. The amino acid sequences of the Lip-VUT3 ORF contained a classical conserved lipase GSDL sequence motif while the amino acid sequences of the Lip-VUT5 ORF contained a classical G-L-S-L-G conserved lipase/esterase motif sequence. The two genes (Lip-VUT3 and Lip-VUT5) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 60 °C and 70 °C, and respective pH optima of 6.0 and 10.0. Further biochemical characterisation indicated that Lip-VUT3 and Lip-VUT5 lipases showed tolerance towards a wide concentration (50%-100%) of methanol. Lip-VUT3 had a Km value of 0.287 mM while Lip-VUT5 had a Km value of 0.556 Mm. This shows that Lip-VUT3 lipase has a higher affinity for olive oil than Lip-VUT5. Lip-VUT3 and Lip-VUT5 were characterised to be true lipases that have been recovered from the rumen environment through metagenomic approach. Therefore, the study proved that metagenomic approach helps in recovering novel lipase isoforms with potential down stream industrial and therapeautic applications from goat rumen metagenome, a rich but untapped source.

Water reuse has become a promising alternative to alleviate stress on conventional freshwater resources in the face of population growth, sea level rise, source water depletion, eutrophication of water bodies, and climate change. Potable water reuse intentionally looks to purify wastewater effluent to drinking water quality or better through the development and implementation of advanced treatment trains. While membrane-based treatment has become a widely-adopted treatment step to meet this purpose, there is growing interest in implementing treatment trains that harness microorganisms as a more sustainable and less energy-intensive means of removing contaminants of emerging concern (CECs), through biological degradation or transformation. In this dissertation, various aspects of the operation of a microbially-active carbon-based advanced treatment train producing water intended for potable reuse are examined, including fate of dissolved organic carbon, underlying microbial populations, and functional genes are explored. Further, dynamics associated with antibiotic resistance genes (ARGs), identified as a microbially-relevant CECs, are also assessed. Overall, this dissertation advances understanding associated with the interplay between and within treatment processes as they relate to removal of various organic carbon fractions, microbially community dynamics, functional genes, and ARGs. Further, when relevant, these insights are contextualized to operational conditions, process upsets, water quality parameters, and other intended water uses within the water industry with the goal of broadening the application of advanced molecular tools beyond the scope of academic research. Specifically, this dissertation illuminates relationships among organic carbon fractions and molecular markers within an advanced treatment train employing flocculation, coagulation, and sedimentation (FlocSed), ozonation, biologically active carbon (BAC) filtration, granular active carbon (GAC) contacting, and UV disinfection. Biodegradable dissolved organic carbon (BDOC) analysis was adapted specifically as an assay relevant to assessing dissolved organic carbon biodegradability by BAC/GAC-biofilms and applied to profile biodegradable/non-biodegradable organic carbon as wastewater effluent passed through each of these treatment stages. Of particular interest was the role of ozonation in producing bioavailable organic carbon that can be effectively removed by BAC filtration. In addition to understanding the removal of fractionalized organic carbon, next generation DNA sequencing technologies (NGS) were utilized to better understand the microbial dynamics characteristic of complex microbial communities during disinfection and biological treatment. Specifically, this analysis was focused on succession and colonization of taxa, genes related to a wide range of functional interests (e.g. metabolic processes, horizontal gene transfer, DNA repair, and nitrogen cycling), and microbial CECs. Finally, NGS technologies were employed to assess the differences between a wide range of water use categories, including conventional drinking water, potable reuse, and non-potable reuse effluent's microbiomes to identify core and discriminatory taxa associated with intended water usage. The outcomes of this dissertation provide valuable information for optimizing carbon-based treatment trains as an alternative to membrane-based treatment for sustainable water reuse and also advance the application of NGS as a diagnostic tool for assessing the efficacy of various water treatment technologies for achieving treatment goals. / Doctor of Philosophy / Several factors have led to increased stress on conventional drinking water sources and widespread global water scarcity. Projections indicate that continued population growth, increased water demand, and degradation of current freshwater resources will negatively contribute to water needs and underscore the need to secure new potable (i.e. fit for human consumption) sources. Water reuse is a promising alternative to offset the growing demands on traditional potable sources and ameliorate negative consequences associated with water scarcity. Discharge of treated wastewater to marine environments is especially a lost opportunity, as the water will no longer be of value to freshwater habitats or as a drinking water source. Water reuse challenges the conventional wastewater treatment paradigm by providing advanced treatment of wastewater effluent to produce a valuable resource that can be safely used directly for either non-potable (e.g., irrigation, firefighting) or potable (i.e., drinking water) applications. The means of achieving advanced treatment of wastewater effluents can take many forms, commonly relying on the utilization of membrane filtration. However, membrane filtration is an intensive process and suffers from high initial costs, high operational costs, membrane fouling with time, and the production of a salty and difficult to dispose of waste stream. These drawbacks have motivated the water reuse industry to explore more sustainable approaches to achieving high quality effluents. One such alternative relies on the utilization of microorganisms to provide biological degradation and transformation of contaminants through a process known as biologically active filtration (BAF). Comparatively to membrane systems, BAF is more cost effective and produces significantly fewer byproducts while still producing high quality treated water for reuse. However, the range in quality of the resulting treated water has not yet been fully established, in part due to the lack of understanding of the complex microbial communities responsible for biological treatment. As water and wastewater treatment technologies have evolved over the past century, many biological treatments have remained largely 'black box' due to the lack of effective tools to identify the tens of thousands of species of microbes that inhabit a typical system and to track their dynamics with time. Instead, analysis has largely focused on basic water quality indicators. This dissertation takes important steps in advancing the implementation of the study of DNA and biodegradable organic carbon (BDOC) analysis to improve understanding of the mechanisms that drive different water reuse treatment technologies and to identify potential vulnerabilities. Insights gained through application of these tools are contextualized to observed operational conditions, process upsets, and water quality measurements. This helped to advance the use of DNA-based tools to better inform water treatment engineering practice. Specifically, this dissertation dives into the relationships between organic carbon and DNA-based markers within an advanced treatment train employing flocculation, coagulation, and sedimentation (FlocSed), ozonation, biologically active carbon (BAC) filtration, granular active carbon (GAC) contacting, and UV disinfection. Development and application of the BDOC test revealed that the bulk of organic carbon entering the treatment train is dissolved. Further, BDOC analysis served to characterize the impact of specific treatment processes and changes in operational conditions on both biodegradable and non-biodegradable organic carbon fractions. Such information can help to inform continued process optimization. Utilization of DNA-based technologies shed light on the functional capacity of microbial communities present within each stage of treatment and the fate of antibiotic resistance genes (ARGs). ARGs are of concern because, when present in human pathogens, they can result in the failure of antibiotics to cure deadly infections. Other functional genes of interest were also examined using the DNA-based analysis, including genes driving metabolic processes and nitrogen cycling that are critical to water purification during BAF treatment. Also, the DNA-based analyses made it possible to better understand the effects of disinfectants on microbes. Interestingly, some ARG types increased in relative abundance (a measure analogous to percent composition) response to treatments, such as disinfection, and others decreased. Characterization of the microbial communities and their dynamic response to changing operation conditions were also observed. For example, it was possible to characterize how the profiles of microbes changed with time, an ecological process called succession, during BAC filtration and GAC contacting. Generally, this analysis, coupled with the functional analysis, shed light on the important, divergent roles of bacterial communities on organic degradation during both BAC and GAC treatment. Finally, a study was conducted that compared the microbiome (i.e. entire microbial community) between a wide range of conventional drinking water, potable reuse water, and non-potable reuse waters. Here it was found that significant differences existed between the microbial communities of water intended for potable or non-potable usage. This work also looked to expand the application of NGS technologies beyond strictly academic research by developing the application of more advanced DNA-based tools for treatment train assessment and monitoring.