In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.

Seventeen bacterial strains were isolated from 9 marine mud samples from the inshore environments of the East Sea. Four bacterial strains showed an inhibition against all tested microorganisms Staphylococcus aureus ATCC10832, Escherichia coli JM109, and Fusarium oxysporum. 16S rRNA sequences of four bacterial strains were obtained by PCR using specific primers. PCR products were cloned into E. coli DH5a using pJET1.2 blunt vector. The recombinant plasmids were sequenced and the lengths of these 16S rRNA sequences were ~930bp. The 16S rRNA sequence from the four bacterial DB1.2, DB1.2.3, DB4.2 and DB5.2 strain showed a high identity of 97 to 99% with the 16S rRNA sequence from Photobacterium sp., Oceanisphaera sp., Shigella sp., Stenotrophomonas sp, respectively. / Mười bảy chủng vi khuẩn đã được phân lập từ 9 mẫu bùn biển từ các vùng ven bờ biển Việt Nam. Bốn chủng vi khuẩn được ghi nhận có khả năng ức chế mạnh sự sinh trưởng và phát triển của các chủng vi khuẩn Staphylococcus aureus ATCC10832, Escherichia coli JM109, và thậm chí cả nấm Fusarium oxysporum. Trình tự gene 16S rRNA của bốn chủng vi khuẩn này đã được khuếch đại bằng PCR sử dụng cặp mồi đặc hiệu. Sản phẩm PCR được nối ghép vào vector pJET1.2 blunt sử dụng T4 ligase, hình thành plasmid tái tổ hợp và biến nạp vào E. coli DH5α. Khuẩn lạc có plasmid mang phân đoạn DNA chèn được nuôi cấy và tách plasmid. Trình tự 16S rRNA từ 4 chủng DB1.2, DB1.2.3, DB4.2 and DB5.2 chỉ ra có sự tương đồng 97 ÷ 99% so với trình tự 16S rRNA tương ứng của các chủng vi sinh vật biển trên ngân hàng gene thế giới là Photobacterium sp., Oceanisphaera sp., Shigella sp., và Stenotrophomonas sp.

Purple nonsulfur bacteria are a group of extraordinary metabolic diverse bacteria. They can grow photoautotrophically, photoheterotrophically , chemoheterotrophically or chemoautotrophically. Under various conditions, they can enjoy exceptional flexibility within each of these modes of metabolism. Due to the special physical characteristics properties, they had attracted scientist¡¦s attention in resent years. These bacteria are widely distributed in nature such as lakes, water ponds, coastal lagoons or high concentration organic waste lagoons. Lotus Pond, located in northern Kaohsiung City, is a serious eutrophied artificial lake. Because of receiving sufficient light and having been polluted by significant amounts of soluble organic matter, the ecology of the lake is suitable for the growth of purple nonsulfur bacteria. In the study, the lake water and sediments by using a Winograsdsky column, we successfully isolated 16 strains bacteria from the Lotus Pond. We also amplified the 16S-rDNA fragments of these strains by PCR and sequenced these PCR products, then aligned these sequences with the data of GeneBank. We affirmed that the 16 isolated strains belong to purple nonsulfur bacteria. From phylogenetic analysis, these 16 strains belong to the following three groups of bacteria: Rhodopseudomonas palustris, Rubrivivax gelatinosus, and Rhodobacter sphaeroides. Characteristic studies of these strains, we found that all isolated strains are Gram negative bacteria and contain bacteriochlorophyll a. The strains that belong to R. palustris and R. sphaeroides group can use several different types of short chain organic acid as their carbon source and have denitrification ability. However, only the strains belong to R. palustris group are able to use the aromatic compound benzoate. From salt tolerant studies, we found the strains in R. sphaeroides group can grow well in 3% NaCl, and both R. palustris and R. gelatinosus group can only grow in 1% NaCl.

Family Pinguipedidae belong to the class Actinopterygii, subclass Neopterygii, order Perciformes, suborder Trachinoidei. Currently the interrelationships of the genera within this family and among the families in the Trachinoidei remain unequivocal. Also, whether the Cheimarrichthys should be included in the family Pinguipedidae has also been a controversial issue. This study aimed to reconstruct phylogenetic hypotheses in order to resolve these questions. Species of the Parapercis and Kochichtys in the family Pinguipedidae occur Taiwan. This study used osteological characters, 16S rRNA and Cyt b sequences to conduct phylogenetic analysis such that hypotheses can be proposed. The results revealed the monophyly of Parapercis, a taxonomic view consistent to the prevailary classification. Summarizing all the results, the 17 Parapercis species analysed can be divided into 4 groups. They are (1) Parapercis aurantiaca¡BP. decemfasciata¡BP. mimaseana¡BP. multifasciata¡BP. muronis¡Btwo morphotypes of P. sexfasciata¡F(2) P. cephalopunctata¡BP. clathrata¡BP. hexophthalma¡BP. kamoharai¡BP. tetracantha¡BP. xanthozona¡F(3) P. cylindrica and P. snyderi¡F(4) P. maculata¡BP. ommatura and P. somaliensis. Two color morphotypes have been shown for Parapercis sexfasciata. Data of the present study revealed that the ¡§ autapomorphic¡¨ osteological character known only in Kochichtys also occurred in three Parapercis species. This result supports a close relationship between these species. However, it also challenges the validity of the generic status of Kochichtys. About the dabate of the phylogenetic position of Cheimarrichthys, it should be put into its own family, Cheimarichthyide, rather than placed in the Pinguipedidae. The hypothesis for the sister group of Pinguipedidae to the Cheimarichthyide is not supported by all the data in this study completely. Morphological and molecular evidences are incongruence for closest phylogenetic relationship. Similar results were also obtained when the molecular sequences were analysed using different methods. More data analyses are needed for complete and reliable results. The present study suggests that the Trachinoidei is not a monophyletic group.

In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.

The taxonomic status of the Japanese Wonder Shell, Thatcheria mirabilis is questionable, because it has been classified in the family of Thatcheriidae, Turridae or Conidae (Superfamily: Conoidea). Conoidea is a large and diverse superfamily with more than 10,000 species. Based on shell and radula characters, it is classified into three families, i.e. Conidae, Terebridae and Turridae. However, seven families have been proposed based on foregut structure, shell and radula morphology. In the present study, the molecular phylogeny of Conoidea and the taxonomic status of Thatcheria mirabilis were determined by mitochondria DNA 16S rDNA. The results show that Conoidea includes three clades, presuming Conidae, Terebridae and Turridae. The mean genetic distances within clades were 0.12, 0.10 and 0.10, respectively. And, the distances between clades were 0.14~0.17. Phylogenetic trees reveal that Terebridae and Turridae were within the same group or sister group, Terebridae was closer to Turridae than to Conidae. Although Thatcheria mirabilis and Bathytoma luhdorfi have turrid-form shells, their phylogenetic relationship was close to Conus which was in Conidae`s clade. Some other species, i.e. Oenopota sagamiana¡BPhymorhynchus buccinoides and Raphitoma linearis were also in Conidae`s clade which had been placed in Turridae. In general, the results are consistent with the cladistic classification by Taylor et al (1993), Rosenberg (1998) and Bouchet & Rocroi (2005), but differenrt from the classification by Powell (1966) and Kohn (1998) based on shell characters. Additionally, the hollow, harpoon-like teeth and venom apparatus in Conoidea might independently evolve in each family.

The effect of simple and complex on the composition and diversity of microbial communities and on end-product (biogas and VFAs) synthesis was investigated using an anaerobic batch respirometer at 37 °C and pH 7.2. These experiments, simple substrates were chemically pure and contain a single carbon source (glucose or α-cellulose), while complex substrates were chemically “impure” substrates containing a mixture of two or three carbon sources (biodiesel-derived glycerol or wheat straw) with a substrate/inoculum ratio 6g chemical oxygen demand (COD)/ g volatile solids (VS) seed and 100g of pre-treated dairy manure digestate (DMD), respectively. Concentrations of hydrogen, carbon dioxide, acetate, butyrate, propionate, and ethanol synthesized by different communities selected by growth on the different substrates were measured and confirmed the growth of the microbial communities. 16S rDNA illumina sequencing revealed that DMD without substrates was more diverse than the microbiota cultured by fermentation reactions containing D-glucose, glycerol α-cellulose or wheat straw. The data confirmed that substrates play a crucial role in determining the diversity of species in microbial communities. Dominant operational taxonomic units (OTUs) belonging to families Clostridiaceae, Ruminococcaceae, and Enterobacteriaceae, and the genera Clostridium, Ruminococcus, Sporolactobacillus, and Syntrophomonas were potentially responsible for changes in end-product synthesis patterns in communities cultured with simple and complex substrates. / October 2015

Water sources are susceptible to faecal contamination from animal and human pollution sources. Pollution of our waterways has significant implications on human health, especially from a pathogen perspective. Microbial source tracking (MST) is a promising field which aims to identify the sources of faecal contamination, and thereby allowing for the development of effective management strategies to minimise pollution and the impact on human health. Many of the currently used methods rely on the identification of host-specific markers within the 16S ribosomal RNA (rRNA) gene of bacteria by use of amplification techniques such as polymerase chain reaction (PCR). However, these methods can be limited by sensitivity, quantification, geographical differences and issues of cost which can limit how many markers are evaluated. Developments in DNA sequencing technologies over the past decade have led to a number of next generation sequencing (NGS) platforms which have a rapid, high throughput approach, resulting in an exponential decrease in the cost of sequencing. This has enabled the development of sequence-based metagenomics, where entire communities from environmental samples can be analysed based on their genetic material. The ability to barcode allows for analysis of multiple samples at once, reducing the cost of sequencing environmental samples even further. This is a promising technique for MST, which has had little investigation to date. The primary focus of the studies described in this thesis was to evaluate the use of NGS technology through a metagenomic approach. Roche 454 amplicon sequencing was used to sequence a 16S rRNA gene target, amplified from faecal and water samples from various sources in New Zealand. Barcode strategies were incorporated in the amplification design to allow multiple samples to be sequenced simultaneously. A proof-of-concept study initially utilised a small sequence dataset to evaluate a range of analysis tools available. Taxonomic identification and diversity measures were used to evaluate a selection of currently available tools designed for analysing metagenomic data, with the Quantitative Insights Into Microbial Ecology (QIIME) platform decided upon for further studies. A larger study, including 35 faecal samples from 13 difference sources and 10 water samples, resulted in 522,065 raw sequencing reads. Diversity results suggest three phyla, Bacteroidetes, Firmicutes and Proteobacteria, are strongly represented across all faecal sources analysed. Microbial diversity analysis using clustering techniques provided evidence of host source being the largest influence on bacterial diversity, with samples from each source generally clustering together. This technique could not be used to identify sources of contamination sources in water samples as the water samples all clustered separately from the faecal samples. More successful was the use of taxonomic classifications to determine bacteria genera that were potentially specific to one source. Water samples were screened for these genera, with six out of the ten water samples being indicators of either ruminant or human contamination. Faecal and water samples were also analysed for a selection of published 16S rRNA PCR markers, using a computational motif-based search method. Of the twenty motifs screened for, 14 were found to be relatively source-specific for ruminant, human, dog or pig faecal samples, with some cross-reactivity with chicken and possum samples. Using this method, the contamination source for six of the ten water samples was identified, with the remaining four samples found to not have enough sequences to assess with confidence. Both metagenomic strategies produced comparable results which were consistent with previous MST analysis. This project demonstrates the potential application of next generation sequencing technologies to microbial source tracking, suggesting the possibility this approach to replace existing microbial source tracking methods.

Pinus radiata is the most planted tree in the southern hemisphere. The planted trees are especially susceptible to pathogens, but even the native population, nowadays limited tomerely five locations, are threatened by diseases caused by arthropods, fungi and dehydration. Endophytes are bacteria or fungi that reside inside healthy plant tissue, and often have a beneficial effect on their hosts. Endophytes can help plants adapt to abiotic stress such as drought and protect them against pathogens and insect pests. Given the roles that endophytes play in host stress responses, it is possible that without studying endophytes we may not fully understand a plant’s response to increased temperatures and climate-induced disease.Using Illumina-sequencing of the 16S rRNA-gene the bacterial endophyte community in 15 trees from three of the remaining native populations were studied. By investigating trees from several sites geographical community differences were discovered. The three overall most dominating bacterial taxa can all be connected with genera known to contain members withanti-fungal properties.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T18:17:32Z No. of bitstreams: 1 resumo.pdf: 17263 bytes, checksum: 8e51a65fe7d8eecb448411acb64cf05b (MD5) / Made available in DSpace on 2017-06-13T18:17:32Z (GMT). No. of bitstreams: 1 resumo.pdf: 17263 bytes, checksum: 8e51a65fe7d8eecb448411acb64cf05b (MD5) Previous issue date: 2003-02-20 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / A estirpe probiótica Lactobacillus UFV H2b20, previamente classificada como Lactobacillus acidophilus por suas características de fermentação de açúcares, apresentou-se mais semelhante à espécie Lactobacillus delbrueckii, quanto à seqüência de rDNA 16S, o que levou ao questionamento acerca da identidade da linhagem. Para o esclarecimento da real classificação da linhagem, o método de hibridização DNA-DNA foi empregado. A linhagem apresentou 75,2% e 77,4% de reassociação com L. delbrueckii subsp. lactis (ATCC 12315) e L. delbrueckii subsp. delbrueckii (ATCC 9649), respectivamente. Dado que a homologia de 70% ou mais, por esse método, tem sido usada como padrão para agrupamento de bactérias em uma mesma espécie, sugere-se, aqui, que Lactobacillus UFV H2b20 seja, daqui para frente, denominado L. delbrueckii UFV H2b20. Identificada a linhagem, outro objetivo do trabalho era desenvolver um protocolo para detecção in situ de L. delbrueckii. Uma sonda de 26 nucleotídeos (SA) foi construída e testada com outras espécies de Lactobacillus relacionadas geneticamente entre si. Estes estudos demonstraram que a seqüência de assinatura (SA) estava presente em L. delbrueckii UFV H2b20, L. delbrueckii UFV H2b21, L. delbrueckii subsp. delbrueckii e L. delbrueckii subsp. lactis, o que indica ser ela eficaz para ser usada como sonda para rRNA 16S espécie-específica pelo método de FISH. A hipótese de existência de polimorfismos, levantada em trabalhos prévios no rDNA 16S da linhagem, foi confirmada após as análises dos segmentos de DNA clonados e selecionados do banco genômico construído para a linhagem L. delbrueckii UFV H2b20. As seqüências analisadas demonstraram, também, presença de segmentos correspondentes a quatro genes codificadores de rRNA 16S distintos, e seis segmentos distintos para uma mesma região de rRNA 23S, indicando seis operons putativos. Há evidência de, pelo menos, um operon putativo completo seguido de região codificadora de seis tRNAs. Não se detectou região espaçadora longa entre rDNA 16 e 23S. / Lactobacillus UFV H2b20, a probiotic strain, previously identified as Lactobacillus acidophilus due to its sugar fermentation pattern, was found to be more closely related to Lactobacillus delbrueckii regarding its 16S rDNA sequence. It was demonstrated by DNA-DNA hybridization that this strain presented 75.2% and 77.4% of reassociation with L. delbrueckii subsp. lactis ATCC 12315 and L. delbrueckii subsp. delbrueckii ATCC 9649, respectively. These results place Lactobacillus UFV H2b20 within the L. delbrueckii species, for 70% reassociation as measured by the method used has been a standard to cluster bacteria within the same species. A protocol for in situ detection of L. delbrueckii was developed by means of Fluorescent in situ Hybridization, FISH. A probe consisting of 26 nucleotides labeled with rhodamine was designed based on the signature sequence within the rDNA, and was tested against genetically related Lactobacillus species. A species- specific method was obtained capable of discriminating L. delbrueckii strains from other Lactobacillus species. Previous studies raised the hypothesis of polymorphism among the copies of 16S rDNA in L. delbrueckii UFV H2b20. This was confirmed by sequence analysis of rDNA from a gene library of this strain cloned in phage lambda and subcloned in pBluescript. Sequence analyses of cloned fragments demonstrated the presence of at least four distinct genes encoding 16S rRNAs. Distinct fragments containing 23S rRNA related genes indicated six putative rrn operons. One complete putative rrn operon displays a region encoding 6 different tRNAs. Long spacer regions between 16S and 23S rDNA were not detected.