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

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.

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The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6875-6887.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6875-6887 ◽

Cited By ~ 157

Author(s):

Justyna Adamczyk ◽

Martin Hesselsoe ◽

Niels Iversen ◽

Matthias Horn ◽

Angelika Lehner ◽

...

Keyword(s):

Community Structure ◽

Activated Sludge ◽

Fluorescence Labeling ◽

Specific Substrate ◽

Ammonia Oxidizing Bacteria ◽

Microarray Hybridization ◽

Independent Manner ◽

And Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

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Molecular and modeling analyses of the structure and function of nitrifying activated sludge

Water Science & Technology ◽

10.2166/wst.1999.0012 ◽

1999 ◽

Vol 39 (1) ◽

pp. 51-59 ◽

Cited By ~ 17

Author(s):

Bruce E. Rittmann ◽

Chrysi S. Laspidou ◽

Jodi Flax ◽

David A. Stahl ◽

Vincent Urbain ◽

...

Keyword(s):

Activated Sludge ◽

Structure And Function ◽

And Function

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Effects of hydroxylamine on microbial community structure and function of autotrophic nitrifying biofilms determined by in situ hybridization and the use of microelectrodes

Water Science & Technology ◽

10.2166/wst.2004.0805 ◽

2004 ◽

Vol 49 (11-12) ◽

pp. 61-68 ◽

Cited By ~ 19

Author(s):

T. Kindaichi ◽

S. Okabe ◽

H. Satoh ◽

Y. Watanabe

Keyword(s):

In Situ Hybridization ◽

Community Structure ◽

Microbial Community ◽

Microbial Community Structure ◽

Structure And Function ◽

Ammonia Oxidizing Bacteria ◽

Low Concentrations ◽

Nitrite Oxidizing Bacteria ◽

And Function

Effects of hydroxylamine (NH2OH), an intermediate of NH4+ oxidation, on microbial community structure and function of two autotrophic nitrifying biofilms fed with and without NH2OH were analyzed by a 16S rRNA approach and the use of microelectrodes. In the NH2OH-added biofilm, partial oxidation of NH4+ to NO2- was observed, whereas complete oxidation of NH4+ to NO3- was achieved in the control biofilm. In situ hybridization results revealed that no nitrite-oxidizing bacteria (NOB) hybridized with any specific probes were detected in the NH2OH-added biofilm. Thus, the addition of low concentrations of NH2OH (250 mM) completely inhibited the growth of NOB. Phylogenetic analysis of 16S rDNA indicated that the ammonia-oxidizing bacteria (AOB) detected in both biofilms were closely related to Nitrosomonas europaea, and that the clone sequences from both biofilm libraries have more than 99% similarity to each other. However, in situ hybridization results revealed that the addition of NH2OH changed the form of growth pattern of the dominant Nitrosomonas spp. from dense clusters mode to single scattered cells mode. Microelectrode measurements revealed that the average NH4+ consumption rate calculated in the NH2OH-added biofilm was two times higher than that in the control biofilm. This clearly demonstrated that the oxidation of NH4+ was stimulated by NH2OH addition.

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Deammonification in biofilm systems: population structure and function

Water Science & Technology ◽

10.2166/wst.2002.0481 ◽

2002 ◽

Vol 46 (1-2) ◽

pp. 223-231 ◽

Cited By ~ 18

Author(s):

C. Helmer-Madhok ◽

M. Schmid ◽

E. Filipov ◽

T. Gaul ◽

A. Hippen ◽

...

Keyword(s):

Population Structure ◽

Stable Process ◽

Cost Effective ◽

Structure And Function ◽

Combined Application ◽

Cost Effective Treatment ◽

And Function ◽

The Cost ◽

Micro Organisms ◽

Nitrogen Elimination

For the development of alternative concepts for the cost effective treatment of wastewaters with high ammonium content and low C/N-ratio, autotrophic consortia of micro-organisms with the ability to convert ammonium directly into N2 are of particular interest. Several full-scale industrial biofilm plants eliminating nitrogen without carbon source for years in a stable process, are suspected for some time to harbor active anaerobic ammonium oxidizers in deeper, oxygen-limited biofilm layers. In order to identify the processes of the single-stage nitrogen elimination (deammonification) in biofilm systems and to allocate them to the responsible micro-organisms, a deammonifying moving-bed pilot plant was investigated in detail. 15N-labelled tracer compounds were used as well as 16S rDNA libraries and in situ identification of dominant organisms. The usage of rRNA-targeted oligonucleotide probes (FISH) was particularly emphasized on the ammonium oxidizers of the β-subclass of Proteobacteria and on the members of the order Planctomycetales. The combined application of these methods led to a deeper insight into the population structure and function of a deammonifying biofilm.

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