Transition of bacterial spatial organization in a biofilm monitored by FISH and subsequent image analysis

2004 ◽  
Vol 49 (11-12) ◽  
pp. 365-370 ◽  
Author(s):  
Y. Aoi ◽  
S. Tsuneda ◽  
A. Hirata
Keyword(s):  
Image Analysis ◽  
Outer Layer ◽  
Spatial Organization ◽  
Heterotrophic Bacteria ◽  
Great Influence ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrite Oxidizing Bacteria ◽  
Growth Activity ◽  
Do Concentration

The dynamic transition of bacterial community structure in a biofilm was monitored by the fluorescence in situ hybridization (FISH) technique and subsequent image analysis. Heterotrophic bacteria that had occupied the outer layer were gradually decreased whereas ammonia-oxidizing bacteria (AOB) gradually increased their growth activity and extended their existence area to the outer layer of the biofilm through the gradual reduction of the C/N ratio. The spatial organization of AOB in the biofilm dynamically changed responding to the environmental conditions such as pH fluctuation and lack of dissolved oxygen (DO) and had great influence on the nitrification activity. The accumulation of nitrite was observed at lower DO concentration, which might be due to the property that nitrite-oxidizing bacteria (NOB) possess of higher Km values for oxygen than AOB.

Structure and function of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes

2000 ◽  
Vol 42 (12) ◽  
pp. 21-32 ◽  
Author(s):  
S. Okabe ◽  
Y. Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Spatial Organization ◽  
Settling Tank ◽  
Outer Part ◽  
Domestic Wastewater ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Bacterial Populations ◽  
Nitrite Oxidizing Bacteria

Time dependent development of the spatial organization of NH4+- and NO2−-oxidizing bacterial populations in a domestic wastewater biofilm and in an autotrophic nitrifying biofilm were investigated by fluorescent in situ hybridization (FISH) with a set of 16S rRNA-targeted oligonucleotide probes. Population dynamics of nitrifying bacteria in the biofilms were correlated with the biofilm performance. In situ hybridization indicated that Nitrosomonas spp. (excluding probe NEU stained NH4+-oxidizing bacteria: i.e., N. marina-lineage, N. europaea-lineage, N. eutropha, and N. halophila) and Nitrospira-like bacteria were the numerically dominant nitrifying species in the domestic wastewater biofilm. However, probe NEU stained NH4+-inoxidizing bacteria became dominant populations in the autotrophic nitrifying biofilm (which were initially cultured with the primary settling tank effluent) after switching to the synthetic media. This population shift might be attributed to the effect of NO2−-–N accumulation and higher growth rates of N. europaea-lineage and N. eutropha, outcompeting other Nitrosomonas spp. in the synthetic medium. This evidence indirectly supports that N. europhaea has been most commonly isolated and studied in most of the previous researches. For the spatial organization of NH4+- and NO2−-oxidizing bacterial populations, bacteria of the genus Nitrobacter could not be detected, instead Nitrospira-like bacteria were found as the main nitrite-oxidizing bacteria in both biofilms. Whereas most of the ammonia-oxidizing bacteria were found throughout the biofilms, the location of nitrite-oxidizing bacteria was restricted to the active nitrite-oxidizing zone, which was detected in the inner part of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of NO2−-oxidizing bacteria, as determined with FISH. These observations have considerable significance to our understanding of microbial nitrification occurring in wastewater treatment processes and in the natural environment.

scholarly journals In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

1999 ◽  
Vol 65 (7) ◽  
pp. 3182-3191 ◽  
Author(s):  
Satoshi Okabe ◽  
Hisashi Satoh ◽  
Yoshimasa Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Spatial Organization ◽  
Outer Part ◽  
Domestic Wastewater ◽  
Ammonia Oxidizing Bacteria ◽  
Bacterial Populations ◽  
Nitrite Oxidizing Bacteria ◽  
Microelectrode Measurements ◽  
Inner Parts

ABSTRACT We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.

Nitrogen loss in a nitrifying biofilm system

1999 ◽  
Vol 39 (7) ◽  
pp. 13-21 ◽  
Author(s):  
C. Helmer ◽  
S. Kunst ◽  
S. Juretschko ◽  
M.C. Schmid ◽  
K.-H. Schleifer ◽  
...  
Keyword(s):  
Nitrogen Loss ◽  
Comparative Sequence Analysis ◽  
Organic Substrate ◽  
Ammonia Oxidizing Bacteria ◽  
Autotrophic Nitrification ◽  
Batch Tests ◽  
Causative Agents ◽  
Do Concentration ◽  
Nitrogen Elimination

In a biological contactor that is part of the biological pretreatment of landfill leachate in Mechernich (Germany) nitrogen elimination of 60% or more was observed under low dissolved oxygen (DO) conditions. Ammonia was converted without accumulation of nitrite and with only little nitrate production. Interestingly, due to limited supply with organic substrate in the system, this observation cannot simply be explained by a combination of conventional autotrophic nitrification and heterotrophic denitrification. In situ hybridization with 16S rRNA-targeted probes revealed the presence of large microcolonies of at least three different types of ammonia-oxidizing bacteria in those biofilm regions where extremely high nitrogen losses occurred. These results were confirmed by comparative sequence analysis of biofilm-derived amoA (encoding the active-site polypeptide of ammonia-monooxygenase) clones for molecular fine-scale analysis of the ammonia-oxidizing population. In batch tests inoculated with biofilm material nitrogen loss occurred without dosage of organic substrate at a DO concentration of 1 mg/l. The simultaneous presence of ammonia and nitrite in the reactor induced the process of complete nitrogen elimination. N2 was identified to be the gaseous end product of the reaction. These results indicate that under low DO concentrations autotrophic ammonia-oxidizers might be the causative agents of the observed nitrogen loss by performing aerobic/anoxic denitrification with nitrite as electron acceptor and ammonia (or perhaps hydroxylamin) as electron donor.

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 In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

2007 ◽  
Vol 73 (15) ◽  
pp. 4931-4939 ◽  
Author(s):  
Tomonori Kindaichi ◽  
Ikuo Tsushima ◽  
Yuji Ogasawara ◽  
Masaki Shimokawa ◽  
Noriatsu Ozaki ◽  
...  
Keyword(s):  
Organic Compounds ◽  
Spatial Organization ◽  
Flow Direction ◽  
Anammox Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Reactor Performance ◽  
Inlet Port ◽  
Anammox Activity ◽  
Total Bacteria

ABSTRACT We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH4 + and NO2 − consumption rates decreased from 0.68 and 0.64 μmol cm−2 h−1 at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm−2 h−1 at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH4 + and NO2 − and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O2 or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

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

2004 ◽  
Vol 49 (11-12) ◽  
pp. 61-68 ◽  
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.

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.

In situ analysis of nitrifying bacteria in sewage treatment plants

1996 ◽  
Vol 34 (1-2) ◽  
pp. 237-244 ◽  
Author(s):  
Michael Wagner ◽  
Gabriele Rath ◽  
Hans-Peter Koops ◽  
Janine Flood ◽  
Rudolf Amann
Keyword(s):  
Activated Sludge ◽  
Sewage Treatment ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Microbiological Methods ◽  
Nitrite Oxidizing Bacteria ◽  
Identification Technique ◽  
Engineered Systems ◽  
Simultaneous Identification

Autotrophic microbial nitrification is the key process in the removal of ammonia from wastewater. To avoid the limitations of traditional microbiological methods an in situ identification technique for ammonia- and nitrite-oxidizing bacteria was developed. Based on comparative sequence analyses we designed a collection of 16S ribosomal RNA-targeted oligonucleotide probes for all validly described members of the genusNitrobacter . Whole cell hybridizations of target and reference cells with fluorescent probe derivatives were used to determine the optimal hybridization stringency for each of the probes. These probes were applied together with a recently developed probe for important members of the genus Nitrosomonas for simultaneous identification of ammonia- and nitrite-oxidizing bacteria in natural and engineered systems. Ammonia-oxidizing bacteria were identified in situ in river water, epiphytic biofilms from eutrophic wetlands, oligotrophic biofilms, a nitrifying trickling filter biofilm as well as in all analyzed nitrifying activated sludge samples. In none of these samples could Nitrobacter cells be detected in situ. However, all hitherto describedNitrobacter species and a strain of Nitrobacter sp. isolated from one of the analyzed nitrifying activated sludge samples showed bright hybridization signals with all Nitrobacter specific probes. Possible reasons for the absence of in situ detectable Nitrobacter cells are discussed.

scholarly journals Distribution and Rate of Microbial Processes in an Ammonia-Loaded Air Filter Biofilm

2009 ◽  
Vol 75 (11) ◽  
pp. 3705-3713 ◽  
Author(s):  
Susanne Juhler ◽  
Niels Peter Revsbech ◽  
Andreas Schramm ◽  
Martina Herrmann ◽  
Lars D. M. Ottosen ◽  
...  
Keyword(s):  
Heterotrophic Bacteria ◽  
Experimental Period ◽  
Protonated Form ◽  
Product Inhibition ◽  
Nitrifying Bacteria ◽  
Outlet Section ◽  
Ammonia Oxidizing Bacteria ◽  
Biological Degradation ◽  
Activity Measurements

ABSTRACT The in situ activity and distribution of heterotrophic and nitrifying bacteria and their potential interactions were investigated in a full-scale, two-section, trickling filter designed for biological degradation of volatile organics and NH3 in ventilation air from pig farms. The filter biofilm was investigated by microsensor analysis, fluorescence in situ hybridization, quantitative PCR, and batch incubation activity measurements. In situ aerobic activity showed a significant decrease through the filter, while the distribution of ammonia-oxidizing bacteria (AOB) was highly skewed toward the filter outlet. Nitrite oxidation was not detected during most of the experimental period, and the AOB activity therefore resulted in NO2 −, accumulation, with concentrations often exceeding 100 mM at the filter inlet. The restriction of AOB to the outlet section of the filter was explained by both competition with heterotrophic bacteria for O2 and inhibition by the protonated form of NO2 −, HNO2. Product inhibition of AOB growth could explain why this type of filter tends to emit air with a rather constant NH3 concentration irrespective of variations in inlet concentration and airflow.

scholarly journals Microscale Distribution of Populations and Activities ofNitrosospira and Nitrospira spp. along a Macroscale Gradient in a Nitrifying Bioreactor: Quantification by In Situ Hybridization and the Use of Microsensors

1999 ◽  
Vol 65 (8) ◽  
pp. 3690-3696 ◽  
Author(s):  
Andreas Schramm ◽  
Dirk de Beer ◽  
Johan C. van den Heuvel ◽  
Simon Ottengraf ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Specific Activity ◽  
Reaction Rates ◽  
Bulk Water ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrite Oxidizing Bacteria ◽  
Water Gradient ◽  
Bacterial Aggregates

ABSTRACT The change of activity and abundance of Nitrosospiraand Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 μm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.

Sign in / Sign up

Export Citation Format

Share Document