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 Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology

1999 ◽  
Vol 65 (3) ◽  
pp. 1289-1297 ◽  
Author(s):  
Natuscka Lee ◽  
Per Halkjær Nielsen ◽  
Kjær Holm Andreasen ◽  
Stefan Juretschko ◽  
Jeppe Lund Nielsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Aerobic Incubation

ABSTRACT A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli andHerpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.

Significance of substrate C/N ratio on structure and activity of nitrifying biofilms determined by in situ hybridization and the use of microelectrodes

2000 ◽  
Vol 41 (4-5) ◽  
pp. 317-321 ◽  
Author(s):  
H. Satoh ◽  
S. Okabe ◽  
N. Norimatsu ◽  
Y. Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Heterotrophic Bacteria ◽  
Outer Part ◽  
Experimental Result ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Spatial Distributions ◽  
Interspecies Competition ◽  
Oxidation Rates

The effect of substrate C/N ratio on the spatial distributions of ammonia-oxidizing bacteria and their activity was investigated by using microelectrodes with high spatial resolution and fluorescent in situ hybridization (FISH) technique. In this study, an interspecies competition for O2 between ammonia-oxidizing bacteria and heterotrophic bacteria was experimentally evaluated. An autotrophic nitrifying biofilm originally cultured at C/N=0 was used as a model biofilm to study changes in specific NH4+ oxidation rate profiles in the biofilm when the substrate C/N ratio was varied. As C/N ratio increased, specific NH4+ oxidation rates decreased in the outer part of the biofilm due to interspecies competition, while they were unchanged in the inner part. The increase in substrate C/N ratio (i.e., addition of acetate) immediately induced the interspecies competition for O2 between ammonia-oxidizing bacteria and heterotrophic bacteria at the outer part of the biofilm. As a result of the interspecies competition, NH4plus; oxidation was restrained, resulting in a decrease in the ammonia-oxidizing bacterial populations. This experimental result clearly explains the stratified spatial distributions of ammonia-oxidizing bacteria within the biofilms at higher substrate C/N ratios. The combined application of microelectrodes and FISH techniques provides new insights into microbial ecology and population dynamics of nitrifying bacteria within multi-species biofilms.

Membrane bioreactor versus conventional activated sludge system: population dynamics of nitrifiers

2005 ◽  
Vol 52 (10-11) ◽  
pp. 417-425 ◽  
Author(s):  
R. Manser ◽  
W. Gujer ◽  
H. Siegrist
Keyword(s):  
Activated Sludge ◽  
Community Composition ◽  
Membrane Bioreactor ◽  
Membrane Separation ◽  
Heterotrophic Bacteria ◽  
Microbial Community Composition ◽  
Laser Scanning Microscopy ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Conventional Activated Sludge

Although membrane bioreactors have attracted increasing attention in recent years, little research has been undertaken on the influence of the membrane separation on the microbial community composition. This paper compares the startup behaviour and the performance of the subsequent eight months of a membrane bioreactor with a conventional activated sludge pilot plant. Both plants were operated in parallel at the same sludge age and treated the same domestic wastewater. The identification of the nitrifying community composition using fluorescent in situ hybridization revealed only minor differences between the two reactors for both ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. Accordingly, both systems exhibited the same maximum nitrification rates. Confocal laser scanning microscopy showed that the aggregates formed by nitrifying bacteria were located mostly in the inner part of the flocs and were overgrown by heterotrophic bacteria. It is concluded that the membrane separation itself does affect neither the nitrifying community composition nor the nitrification performance. However, impacts on kinetic parameters are emphasized.

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.

scholarly journals Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization

2001 ◽  
Vol 67 (12) ◽  
pp. 5810-5818 ◽  
Author(s):  
Holger Daims ◽  
Niels B. Ramsing ◽  
Karl-Heinz Schleifer ◽  
Michael Wagner
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Environmental Samples ◽  
Internal Standard ◽  
Laser Scanning Microscopy ◽  
Ammonia Oxidizer ◽  
Confocal Laser ◽  
Ammonia Oxidizing Bacteria ◽  
Widespread Application

ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 × 107 ± 1.9 × 107 cells ml−1. Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

Monitoring the development of anaerobic biofilms using fluorescent in situ hybridization and confocal laser scanning microscopy

2000 ◽  
Vol 41 (12) ◽  
pp. 69-77 ◽  
Author(s):  
J. C. Araujo ◽  
G. Brucha ◽  
J. R. Campos ◽  
R. F. Vazoller
Keyword(s):  
In Situ Hybridization ◽  
Confocal Laser Scanning Microscopy ◽  
Fluorescent In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Anaerobic Consortium

In this study we investigated the development of anaerobic biofilm using a laboratory reactor. We were especially interested in comparing the organization of anaerobic cells (particularly those that are very common in domestic sewage sludge) in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescent in situ hybridization (FISH) with domain and group specific probes directed against 16S ribosomal RNA were used to quantify microbial composition in the biofilm. FISH and confocal laser scanning microscopy (CLSM) were used to elucidate spatial distribution of microbes in the biofilms. Two experiments were carried out, one with pure methanogenic organisms and the other with a microbial anaerobic consortium. The pure methanogen cultures, Methanobacterium formicicum (DSM 1535); Methanosaeta concilli (DSM 3671) and Methanosarcina barkeri (DSM 800) were used to seed the modified Robbins Device (MRD) to allow the development of biofilms on polypropylene and glass surfaces during the 9-days experiment. The results showed that all the three species were colonizing both surfaces after two and nine days of experimental period. In another experiment, with polypropylene coupons only, MRD was seeded with a microbial anaerobic consortium and biofilm formation was studied during 11 days. At the end of this period, the biofilms generated were of uneven thickness with areas of minimal or no surface coverage and areas where the biofilm attained a thickness of 7.0 to 9.0 μm as revealed by CLSM. The results showed that the modified Robbins Device together with the fluorescent in situ hybridization and confocal laser scanning microscopy are suitable tools to study anaerobic biofilm development in different kinds of support materials.

scholarly journals Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa

2004 ◽  
Vol 70 (8) ◽  
pp. 4648-4657 ◽  
Author(s):  
Maria Vila ◽  
Rafel Simó ◽  
Ronald P. Kiene ◽  
Jarone Pinhassi ◽  
José M. González ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gulf Of Mexico ◽  
Fluorescence In Situ Hybridization ◽  
Marine Bacteria ◽  
Heterotrophic Bacteria ◽  
Phytoplankton Bloom ◽  
Mediterranean Coast ◽  
Marine Bacterioplankton ◽  
Significant Uptake

ABSTRACT The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [35S]DMSP ranged between 27 and 51% of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [3H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [35S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [3H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (α-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [35S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the γ-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and γ-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating 35S from DMSP (around 50%). Altogether, the application of AU with [35S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.

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