scholarly journals Evaluating heterotrophic growth in a nitrifying biofilm reactor using fluorescence in situ hybridization and mathematical modeling

2005 ◽  
Vol 52 (7) ◽  
pp. 135-141 ◽  
Author(s):  
R. Nogueira ◽  
D. Elenter ◽  
A. Brito ◽  
L.F. Melo ◽  
M. Wagner ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Retention Time ◽  
Biofilm Reactor ◽  
Laser Scanning Microscopy ◽  
Nitrifying Bacteria ◽  
Heterotrophic Growth ◽  
Biofilm Reactors

The objective of this study was to evaluate the significance of heterotrophic growth in nitrifying biofilm reactors fed only with ammonium as an energy source. The diversity, abundance and spatial distribution of nitrifying bacteria were studied using a combination of molecular tools and mathematical modeling, in two biofilm reactors operated with different hydraulic retention times. The composition and distribution of nitrifying consortia in biofilms were quantified by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes combined with confocal laser scanning microscopy (CLSM) and digital image analysis. Autotrophic and heterotrophic biofilm fractions determined by FISH were compared to the output from a multispecies model that incorporates soluble microbial products (SMP) production/consumption. In reactor R1 (short retention time) nearly 100% of the total bacteria could be identified as either ammonia- or nitrite-oxidizing bacteria by quantitative FISH analyses, while in reactor R2 (long retention time) the identification rate was only 73%, with the rest probably consisting of heterotrophs. Mathematical simulations were performed to evaluate the influence of the hydraulic retention time (HRT), biofilm thickness, and substrate utilization associated SMP production on the growth of heterotrophic bacteria. The model predicts that low HRTs resulted in a lower availability of SMPs leading to purely autotrophic biofilms. These model predictions are consistent with experimental observations. At HRTs that are about an order of magnitude larger than the reciprocal of the net maximum growth rate the majority of the active biomass will grow suspended in the bulk phase rather than in the biofilm.

Fluorescence in situ hybridization analysis of nitrifiers in piggery wastewater treatment reactors

2004 ◽  
Vol 49 (5-6) ◽  
pp. 333-340 ◽  
Author(s):  
D.J. Kim ◽  
T.K. Kim ◽  
E.J. Choi ◽  
W.C. Park ◽  
T.H. Kim ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Ammonia Concentration ◽  
Biofilm Reactor ◽  
Nitrifying Bacteria ◽  
Fish Analysis ◽  
Ammonia Oxidizing Bacteria ◽  
Piggery Wastewater

Fluorescence in situ hybridization (FISH) was performed to analyze the nitrifying microbial communities in an activated sludge reactor (ASR) and a fixed biofilm reactor (FBR) for piggery wastewater treatment. Heterotrophic oxidation and nitrification were occurring simultaneously in the ASR and the COD and nitrification efficiencies depend on the loads. In the FBR nitrification efficiency also depends on ammonium load to the reactor and nitrite was accumulated when free ammonia concentration was higher than 0.2 mg NH3-N/L. FISH analysis showed that ammonia-oxidizing bacteria (NSO1225) and denitrifying bacteria (RRP1088) were less abundant than other bacteria (EUB338) in ASR. Further analysis on nitrifying bacteria in the FBR showed that Nitrosomonas species (NSM156) and Nitrospira species (NSR1156) were the dominant ammonia-oxidizing and nitrite-oxidizing bacteria, respectively, in the piggery wastewater nitrification system.

Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

2003 ◽  
Vol 47 (1) ◽  
pp. 97-104 ◽  
Author(s):  
D.W. Han ◽  
J.S. Chang ◽  
D.J. Kim
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Selective Inhibition ◽  
Biofilm Reactor ◽  
Microbial Community Analysis ◽  
Nitrifying Bacteria ◽  
Nitrite Accumulation ◽  
Fish Analysis ◽  
Oxidation Activity

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Comparison of detection specificity of nitrifying bacteria in biofilm using fluorescence in situ hybridization and in situ fluorescent antibody methods

2003 ◽  
Vol 47 (5) ◽  
pp. 129-132
Author(s):  
N. Noda ◽  
Y. Ebie ◽  
M. Matsumura ◽  
S. Tsuneda ◽  
A. Hirata ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Specific Fluorescence ◽  
Antibody Method ◽  
In Situ Detection ◽  
Detection Specificity ◽  
Fluorescent Antibody Method

The in situ fluorescent antibody and fluorescence in situ hybridization (FISH) methods are very useful in the in situ detection of specific bacteria like nitrifiers in a biofilm. In this study, simultaneous staining using the FISH and in situ fluorescent antibody methods was examined. As a result, no specific fluorescence was observed with either method when FISH was performed followed by the in situ fluorescent antibody method; however, when the in situ fluorescent antibody method was performed first followed by FISH, specific fluorescence was observed in both cases. Moreover, it was suggested that the detection specificities of FISH and the in situ fluorescent antibody method are almost identical.

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

1998 ◽  
Vol 37 (4-5) ◽  
pp. 441-449 ◽  
Author(s):  
Michael Wagner ◽  
Daniel R. Noguera ◽  
Stefan Juretschko ◽  
Gabriele Rath ◽  
Hans-Peter Koops ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
In Situ Hybridization ◽  
Activated Sludge ◽  
Heterotrophic Bacteria ◽  
Sewage Treatment Plant ◽  
Laser Scanning Microscopy ◽  
Industrial Plant ◽  
Ammonia Oxidizing Bacteria ◽  
Ammonia Oxidizers

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O2 within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters are not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 16S rDNA sequence analysis confirmed the affiliation suggested by FISH.

scholarly journals Numerical Dominance and Phylotype Diversity of Marine Rhodobacter Species during Early Colonization of Submerged Surfaces in Coastal Marine Waters as Determined by 16S Ribosomal DNA Sequence Analysis and Fluorescence In Situ Hybridization

2002 ◽  
Vol 68 (2) ◽  
pp. 496-504 ◽  
Author(s):  
Hongyue Dang ◽  
Charles R. Lovell
Keyword(s):  
In Situ Hybridization ◽  
Salt Marsh ◽  
Fluorescence In Situ Hybridization ◽  
Laser Scanning Microscopy ◽  
23S Rrna ◽  
Marine Waters ◽  
Surface Colonization ◽  
Early Stages ◽  
Coastal Marine

ABSTRACT Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the α subdivision of the division Proteobacteria (α-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the α-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.

Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions

2015 ◽  
Vol 61 (6) ◽  
pp. 417-428 ◽  
Author(s):  
Edith R. Valle ◽  
Gemma Henderson ◽  
Peter H. Janssen ◽  
Faith Cox ◽  
Trevor W. Alexander ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser

In this study, methanogen-specific coenzyme F420autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

scholarly journals Groping for Quantitative Digital 3-D Image Analysis: An Approach to Quantitative Fluorescence In Situ Hybridization in Thick Tissue Sections of Prostate Carcinoma

1997 ◽  
Vol 15 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Karsten Rodenacker ◽  
Michaela Aubele ◽  
Peter Hutzler ◽  
P. S. Umesh Adiga
Keyword(s):  
Image Analysis ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Cell Nucleus ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Necessary Condition ◽  
Confocal Laser ◽  
Thin Sections

In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH). The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA) can be estimated by counting the number of FISH signalsper cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI) to a software package for display, inspection, count and (semi‐)automatic analysis of 3‐D images for pathologists is outlined including the underlying methods of 3‐D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer‐aided analysis of large 3‐D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3‐D data is not in sight. A semi‐automatic segmentation method is thus presented here.

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