scholarly journals Microstructure of Anaerobic Granules Bioaugmented with Desulfitobacterium frappieri PCP-1

2002 ◽  
Vol 68 (8) ◽  
pp. 4035-4043 ◽  
Author(s):  
M. Lanthier ◽  
B. Tartakovsky ◽  
R. Villemur ◽  
G. DeLuca ◽  
S. R. Guiot
Keyword(s):  
Growth Rate ◽  
In Situ Hybridization ◽  
Outer Layer ◽  
Specific Growth ◽  
Anaerobic Sludge ◽  
Oligonucleotide Probes ◽  
Reactor Operation ◽  
Mathematical Simulations ◽  
Successful Colonization

ABSTRACT Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day−1 and a low bacterial diffusion of 10−7 dm2 day−1. Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.

scholarly journals Differentiation of Methanosaeta concilii andMethanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy

1999 ◽  
Vol 65 (5) ◽  
pp. 2222-2229 ◽  
Author(s):  
Sylvie Rocheleau ◽  
Charles W. Greer ◽  
John R. Lawrence ◽  
Christiane Cantin ◽  
Louise Laramée ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Outer Layer ◽  
Fluorescent In Situ Hybridization ◽  
Methanosarcina Barkeri ◽  
Confocal Scanning Laser Microscopy ◽  
Oligonucleotide Probes ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

ABSTRACT Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe.Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcinaspecies. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

scholarly journals Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

2006 ◽  
Vol 72 (11) ◽  
pp. 6907-6913 ◽  
Author(s):  
Kohei Nakamura ◽  
Takeshi Terada ◽  
Yuji Sekiguchi ◽  
Naoya Shinzato ◽  
Xian-Ying Meng ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Lytic Enzyme ◽  
Anaerobic Sludge ◽  
Oligonucleotide Probes ◽  
Methanobrevibacter Ruminantium ◽  
The Family ◽  
Methanosphaera Stadtmanae ◽  
Methanothermobacter Thermautotrophicus

ABSTRACT In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H2-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

scholarly journals Fluorescence In Situ Hybridization Using 16S rRNA-Targeted Oligonucleotides Reveals Localization of Methanogens and Selected Uncultured Bacteria in Mesophilic and Thermophilic Sludge Granules

1999 ◽  
Vol 65 (3) ◽  
pp. 1280-1288 ◽  
Author(s):  
Yuji Sekiguchi ◽  
Yoichi Kamagata ◽  
Kazunori Nakamura ◽  
Akiyoshi Ohashi ◽  
Hideki Harada
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Spatial Organization ◽  
Laser Scanning Microscopy ◽  
Upflow Anaerobic Sludge Blanket ◽  
Confocal Laser ◽  
Anaerobic Sludge ◽  
Oligonucleotide Probes ◽  
Thin Sections

ABSTRACT 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35°C) and thermophilic (55°C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-,Methanobacterium-, Methanospirillum-, andMethanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genusMethanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655–2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related toSyntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.

Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH)

2009 ◽  
Vol 55 (4) ◽  
pp. 465-472 ◽  
Author(s):  
Ryohei Ueno
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Rapid Identification ◽  
Oligonucleotide Probes ◽  
Logarithmic Growth Phase ◽  
Prototheca Wickerhamii ◽  
Algal Genus

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

scholarly journals The Polymorphisms of Oligonucleotide Probes in Wheat Cultivars Determined by ND-FISH

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1126 ◽  
Author(s):  
Tianheng Ren ◽  
Maojie He ◽  
Zixin Sun ◽  
Feiquan Tan ◽  
Peigao Luo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Wheat Genome ◽  
Oligonucleotide Probes ◽  
Wheat Cultivars ◽  
Fish Signal

Non-denaturing fluorescence in situ hybridization (ND-FISH) has been used to distinguish wheat chromosomes and to detect alien chromosomes in the wheat genome. In this study, five different oligonucleotide probes were used with ND-FISH to examine 21 wheat cultivars and lines. These oligonucleotide probes distinguished 42 wheat chromosomes and also detected rye chromatin in the wheat genome. Moreover, the signal patterns of the oligonucleotide probes Oligo-pTa535-1 and Oligo-pSc119.2-1 showed high polymorphism in the wheat chromosomes. A total of 17.6% of the A group chromosomes, 25.9% of the B group chromosomes and 8.9% of the D group chromosomes showed obvious mutations when they were compared to the standard ND-FISH signal patterns, and most of them were Oligo-pSc119.2-1 mutants. The results suggested that these polymorphisms could be induced by the crossing of wheat cultivars. The results provided more information for the further application of oligonucleotide probes and ND-FISH.

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