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This thesis introduced the principle and the evolution of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), described its usage in monitoring the remediation process for oil and other chemical pollution as well as analyzing the nature microbial community, and finally made a comprehensive comment to its future development. PCR- DGGE was initially developed to study DNA mutations because it is a powerful tool to detect DNA base mutations in a PCR-amplified DNA fragment. Its accuracy can be as high as 100%. The principal of DGGE is based on the differences in DNA sequence that affect the melting points of each amplicons, and caused a decrease in the electrophoretic mobility of a partially melted DNA molecule in a polyacrylamide gel containing a linearly increasing gradient of DNA denaturants. PCR-DGGE can effectively detect the community structure of microorganisms in the environments, including the unculturable microorganisms. Nowadays, it has become one of the most frequently applied techniques to study the community structure of microorganisms. Microbiologists can use this technique to understand the microbial ecology, the shift of the microbial community structure during a bioremediation process, such as the oil pollution and other toxic chemical pollution. In addition, PCR-DGGE can also be used for the fermentation studies in food industry and agricultural industries. We can even identify unculturable microorganism by analyzing the DNA fragment sequences. This can help us to design a suitable medium to culture and isolate these ¡¥unculturable organisms¡¦. There are three directions for the further development of PCR-DGGE technique: 1.Improvement of material preparation processes; 2. development of new DGGE-related tools; and 3. combination of other technology with DGGE. The improvement of preparation processes can make DGGE more accuracy to analyze the community structure of microorganisms, improve the sensitivity of DGGE fingerprint detection, and help the DGGE normalization. The development of DGGE-related tools can overcome the limitations imposed on DGGE by co-migration and help us to have a better understanding of the meanings for a DGGE pattern. The combination of other technology with DGGE can help us to have a clear understanding of the microbial ecology, the shift of the microbial community structure, and the function of microorganisms.

This research is focus on the analysis of microbial diversity and existance of methanotrophic microbial strains in Wu-Shan-Ding mud volcano areas. The microbial distribution and diversity in soil surrounding the mud volcano were analyzed by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). The 16S rDNA sequences from PCR-DGGE bands were used to identify the bacterial strains with NCBI gene bank data. The results showed that the microbial diversity in the fresh erupted mud sample varied with other soil samples. The degrees of diversity were propotion to the distance away from the mud volcano. The main bacterial species found in the mud sample are Brevundimonas terrae, Ralstonia solanacearum, Ralstonia taiwanensis, Pseudonocardiaceae bacterium, Ochrobactrum anthropi, Burkholderia phytofirmans, Stenotrophmonas sp., Methylobacterium sp., Cryseobacterium sp., Sphingobacterium sp., Stenotrophomonas sp. MG-3, and Stenotrophomonas sp. EP01. According to documents, all of the above strains are tolerant to high salt and alkaline. The Ochrobactrum anthropi and Burkholderia phytofirmans were found only in the fresh erupted mud sample and the 5-meter soil sample, while Ralstonia mannitolilytica, Ralstonia pickettii, Amycolatopsis rugosa, and Maricaulis sp. were found only in 10-meter soil sample. In other word, all of the soil samples we examed in this study have their own specified bacterial strains. Two methanotrophic microganisms, Methylobacterium organophilum and Ochrobactrum anthropi were also successfully isolated in pure cultures. The biochemical characteristics of these two strains had been studied. These two strains show a potential might be able to use in the bioremediation of petroleum pollution.

Arbuscular mycorrhizal fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Traditional spore morphology-based identification of AMF in biodiversity studies is subjective and requires expertise and time. Researchers have used molecular techniques to investigate community composition of AMF in uncultivated, disturbed, or contaminated soils, but this approach to community analysis of AMF in agricultural soils has not been reported. In this study, a polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) procedure for the detection of fungal 18S ribosomal RNA gene was developed with reference cultures. Five AMF species were procured from the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these INVAM cultures. The methods technical limitations were investigated. The optimized procedures effectiveness was tested by its application to soil samples from 38 farms. Bands from the PCR-DGGE profiles of these samples were excised for sequence analysis. The total number of species recovered was low in comparison to other AMF community surveys of temperate climate locations. The majority of the sequences recovered were Glomus species. Scutellospora calospora, a previously undetected AM fungus in Saskatchewan was found. A trend in AMF distribution in Saskatchewan was observed and it was relatable to their phylogenetic taxonomy. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods.

Arbuscular mycorrhizal fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Traditional spore morphology-based identification of AMF in biodiversity studies is subjective and requires expertise and time. Researchers have used molecular techniques to investigate community composition of AMF in uncultivated, disturbed, or contaminated soils, but this approach to community analysis of AMF in agricultural soils has not been reported. In this study, a polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) procedure for the detection of fungal 18S ribosomal RNA gene was developed with reference cultures. Five AMF species were procured from the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these INVAM cultures. The methods technical limitations were investigated. The optimized procedures effectiveness was tested by its application to soil samples from 38 farms. Bands from the PCR-DGGE profiles of these samples were excised for sequence analysis. The total number of species recovered was low in comparison to other AMF community surveys of temperate climate locations. The majority of the sequences recovered were Glomus species. Scutellospora calospora, a previously undetected AM fungus in Saskatchewan was found. A trend in AMF distribution in Saskatchewan was observed and it was relatable to their phylogenetic taxonomy. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods.

Abstract This research used molecular biological techniques such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to analyze the dynamic microbial community and biodiversity in the groundwater contaminated with petroleum-hydrocarbons. The 16S rDNA sequences from all water samples were compared with the sequences of relative bacteria in the Ribosomal Database Project Bank to construct a phylogenetic tree. The results allowed us to understand the composition of the microbial communities in the petroleum-hydrocarbon contaminated groundwater. In this study, groundwater samples taken from the Chinese Petroleum Corporation Kaohsiung Refinery (CPCKR), Chinese Petroleum Corporation at Ciaotou fuel Tank Farm (CPCCTF) and China Petrochemical Development Corporation at Kaohsiung Factory (CPDCKF) were analyzed. The contaminated sites at CPDCKR and CPCCTF are remeated by natural attenuation. While the CPDCKF site is remeated by an enhanced air sparging bioremediation. In CPDCKR, we found that the low polluted area contained the richest microbial community, followed by the non-polluted area, and the high polluted area. At the CPCCTF site, the microbial community in the non-polluted area was richer than the high-polluted area. Increased microbial populations and variation in microbial community have beenobserved in non-polluted, less polluted, and highly polluted areas. The microbial community showed a dynamic succession of complexity during the bioremediation process at the CPDCKF site. From the 16S rDNA sequence analysis, it is possible that all samples contained petroleum-hydrocarbon degrading bacteria. These petroleum-hydrocarbon degrading bacteria include Methylobacterium, Xanthobacter, Xanthomonas and Pseudomonas at CPCKR site, Flavobacterium at CPCCTF site, Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, and Candida at CPDCKF site. This study also demonstrates that it is more economic and reliable of using molecular techuiques to analyze the groundwater. Thus, groundwater samples can be used to replace soil samples for future work.

In this study, we had established a 16S rDNA-DGGE analys is system to detect the microbial community in petroleum polluted soil and assess the feasibility of using this system to monitor the bioremediation process. Three genomic DNA extracted methods, the KIT, the Bead-beating system, and the Freeze-thaw method were used to evaluate the DNA extraction efficiency and purity. These DNA samples were further tested by DGGE to analysis the microbial community in soil samples. The results showed that KIT method performed advantageous not only in the DNA extraction efficiency and purity, but also expressed the richest bacterial community in its PCR products. From the DGGE analysis, our data indicated that composition of bacterial community were different in the soil samples that were taken from the same site but at different time. This indicated that the species and number of microorganisms in a polluted soil were under a dynamic transition. The combination of DGGE and 16S rDNA gene sequence analysis system were also proven useful in identifying the predominant microbes in a soil sample and monitoring its bacterial community.

Alkylphenol polyethoxylates (APEOs) are commonly present in both industrial and municipal wastewaters. They belong to the nonionic surfactants which have been widely used for years. APEOs themselves are nontoxic to organisms. When released into the environment, the EO chain of APEOs are degraded chemically and biologically. Some of the products, such as nonylphenol and octylphenol, are persistant. According to the partition coefficient constant, the alkylphenols are adsorpted in the sediments and accumulated in the environment. Nonylphenol (NP) is an analog of 17£]-estradiol, a sex hormone. It is one of the environmental hormones which can get into the organisms through the food chain and may interfere with the reproduction function. Previous studies showed the Love River in Kaohsiung was polluted with APEOs considerably. Bacteria capable of using nonylphenol as the sole carbon source were isolated by the enrichment procedures. Some of the bacterial isolates were identified as Serratia marcescens (strain A), Vibrio sp. (strain B) and Aeromonadaceae sp. (strain C) by the 16S rRNA phylogeny. The rates of NP degradation were evaluated by the HPLC-UV. S. marcescens strain A manifested the best degradative. It could degrade almost 25 ppm of NP in 28 days. The degradative capability of Vibrio sp. (strain B), Aeromonadaceae sp. (strain C) and the enriched mix culture were 65%, 25% and 30%, respectively. Additionally, to know the limitation of degrading nonylphenol by strain A, we set the concentration to 100 ppm for the test. Strain A could degrade 72 ppm in this test. Bacterial composition of the enriched consortia was grouped by the DGGE method. The dominants were Ochrobactrum sp. and Alcaligenaceae sp. which may be to applied to environmental bioremediation.

O herbicida ametrina é relativamente persistente no meio ambiente e está entre os cinco mais usados junto à cultura da cana-de-açúcar no Brasil. Técnicas radiométricas e moleculares foram utilizadas neste estudo para avaliar o potencial de sorção/dessorção e a biodegradação da ametrina, além do impacto do tempo de residência deste herbicida sobre a estrutura e a diversidade da comunidade microbiana, respectivamente. Os solos foram coletados em áreas florestais, sem histórico de aplicação de ametrina, sendo que um deles apresentou textura mais argilosa (NVef) e o outro mais arenosa (RQo). A ametrina apresentou baixo a moderado potencial de sorção (Kd = 2,2 e 9,9 L kg-1 nos solos RQo e NVef, respectivamente). Paralelamente, a taxa de biodegradação (t1/2 = 16 e 36 dias nos solos NVef e RQo, respectivamente) e a formação de resíduos ligados de 14C-ametrina ao solo (59 e 38 % da quantidade aplicada nos solos NVef e RQo, respectivamente) foram muito maiores no solo NVef, mostrando sua maior taxa de dissipação. Houve também a formação de um metabólito de importância ambiental, o qual correspondeu a 19 e 26 % da quantidade aplicada após 70 dias da aplicação nos solos RQo e NVef, respectivamente. O DNA total foi extraído dos solos, com posterior uso da técnica de Reação em Cadeia da Polimerase associada à Eletroforese em Gel com Gradiente Desnaturante (PCR-DGGE). A aplicação da ametrina alterou a estrutura da comunidade microbiana em ambos os solos, sendo essa alteração mais evidente no solo NVef, principalmente nos períodos inicias da incubação. Os dados de seqüenciamento evidenciaram o predomínio dos seguintes filos: Proteobacteria, Acidobacteria, Actinobacteria e Bacteroidetes. Houve seleção de alguns filos no solo NVef mesmo com apenas 7 dias de incubação da ametrina; entretanto, não houve redução no número de filos no solo RQo. Isto permitiu concluir que a diversidade da população microbiana pode variar com a aplicação do herbicida ametrina, mas isto dependerá principalmente do tipo de solo / The herbicide ametryne is relatively persistent in the environment and is among the five most used in the sugarcane crop in Brazil. Radiometric and molecular techniques were applied in this study to evaluate sorption/desorption and biodegradation of ametryne and the impact caused its residence time on the structure and diversity of the soil microbial community, respectively. The soils were collected from forested areas, with no history of ametryne application. One of them showed a clayey (NVef) and the other one a sandy (RQo) texture. Ametryne presented low to moderate sorption potential (Kd = 2.2 e 9.9 L kg-1 in the RQo and NVef soils, respectively). In parallel, the biodegradation rate (t1/2 = 16 and 36 days in the NVef and RQo, respectively) and the formation of soil bound residues of 14C-ametryne in soil (59 and 38 % of applied amount in the NVef and RQo, respectively) were much greater in NVef soil, showing its higher dissipation rate. There was also formation of a metabolite of environmental importance, which corresponded to 19 and 26 % of applied amount in the RQo and NVef soils, respectively. The genomic soil DNA was extracted with further application of the Polymerase Chain Reaction associated to the Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technique. Ametryne application changed the structure of microbial community in both soils, but this change was more pronounced in the NVef soil, mainly in the initial periods of incubation. The sequencing data showed predominance of the following phyla: Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes. There was selection of certain phyla in the NVef, even only with 7 days of ametryne application; however, there was no reduction in the phyla number in the RQo. This led to the conclusion that microbial population diversity may vary with ametryne application, but it will depend on the soil type

A microbiota intestinal humana é um ecossistema complexo que abriga centenas de espécies bacterianas e que de um modo geral convive harmonicamente com o hospedeiro. Essa interação promove o desenvolvimento e estimulação do sistema imune. A microbiota tem papel primordial na saúde humana por produzir nutrientes, participar no metabolismo de carboidratos e por competir com bactérias patogênicas na colonização do ambiente intestinal. Ela alcança sua estabilidade em torno do segundo ano de vida. O tipo de parto, de alimentação, as condições sanitárias, sociais e os elementos do hospedeiro, como fatores genéticos, peristaltismo e pH intestinal, influenciam na sua composição. Quando há a instalação de infecções intestinais ou uso de antimicrobianos e de imunossupressores ocorre o desequilíbrio desse sistema. Esse estudo tem como objetivo avaliar o estabelecimento e a diversidade da microbiota intestinal em onze crianças a partir do segundo dia até o décimo segundo mês de vida. As amostras fecais das crianças foram coletadas no segundo e sétimo dias de vida e mensalmente do primeiro ao décimo segundo meses de vida. As análises de \"fingerprinting\" foram realizadas pelo método de eletroforese em gel com gradiente desnaturante (DGGE) usando os iniciadores para a região V3 do gene 16S rRNA. Os perfis de similaridade foram feitos a partir da construção de dendrogramas e para avaliar as relações entre as amostras temporais das crianças e o perfil de bandas obtido com o DGGE foram feitas análises de correspondência. A análise do \"fingerprinting\" mostrou que cada criança apresentou um padrão de colonização distinto. Apesar de compartilharem algumas características como a forma de nascimento, quadro sócio-econômico e terem condições sanitárias semelhantes, observaram-se diferenças no processo de estabelecimento da microbiota de cada uma delas, o que pode ser devido aos fatores individuais e particularidades da alimentação, do uso de medicamentos e das intercorrências infecciosas. As análises de correspondência mostraram agrupamentos temporais, onde as amostras mais tardias (a partir de 10 meses até 12 meses de idade) estão muito relacionadas entre si indicando o início da estabilização da microbiota ao final do 1º ano de vida. O uso da técnica de \"fingerprinting\" por DGGE permitiu uma análise global dos estágios diferentes no estabelecimento da microbiota intestinal. / The human intestinal microbiota is a complex ecosystem that homes hundreds of bacterial species, which generally live in harmony with the host. This interaction promotes the development and stimulation of the immune system. The microbiota plays a major role in human health by producing nutrient involved in carbohydrate metabolism and competes with pathogenic bacteria in the colonization of the intestinal environment. The stability is achieved around the second year of life. The type of delivery, food, sanitation, and social elements of the host, such as genetic factors, peristaltism and intestinal pH, influence their composition. The imbalance of this system happens due the installation of intestinal infections, in the use of antibiotics and immunosuppressants. The aim of this study is to evaluate the establishment and the diversity of the intestinal microbiota of eleven children from the second day of life until the twelfth month of life. The fecal samples were collected from children in the second and seventh days of life and monthly from the first month to the twelfth month of life. Analyses of fingerprinting was performed by the method of denaturing gradient gel electrophoresis (DGGE) using the primers for the V3 region of the 16S rRNA gene. The similarity profiles were made with the construction of dendrograms and to evaluate the relationships between the temporal samples of children and the profile obtained from the DGGE bands were made the correspondence analysis (CA). The fingerprinting analysis showed that each child had a distinct pattern of bands. Although sharing some characteristics such as delivery mode, socio-economic context and similar health conditions were observed differences in the process of establishment of the microbiota of each, which may be due to individual factors, the use of medicine and infectious complications. The correspondence analysis showed temporal clusters, where the later samples (from 10 months to 12 months of age) are closely related to each other indicating the beginning of the stabilization of the microbiota at the end of the first year of life. Using the technique of fingerprintin by DGGE allowed a comprehensive analysis of different stages in the intestinal microbiota establishment.