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.

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.

Structure and function of nitrifying biofilms as determined by molecular techniques and the use of microelectrodes

2002 ◽  
Vol 46 (1-2) ◽  
pp. 233-241 ◽  
Author(s):  
S. Okabe ◽  
H. Naitoh ◽  
H. Satoh ◽  
Y. Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Bacterial Community ◽  
Spatial Organization ◽  
Domestic Wastewater ◽  
Rotating Disk ◽  
Molecular Techniques ◽  
Bacterial Populations ◽  
Steady State Condition ◽  
Synthetic Media

The phylogenetic diversity of a nitrifying bacterial community of two types of nitrifying biofilms, a domestic wastewater biofilm and an autotrophic nitrifying biofilm grown on rotating disk reactors (RDR), was characterized by 16S ribosomal DNA (rDNA)-cloning analysis. Thereafter, successional development of nitrifying the bacterial community within both biofilms was visualized in situ by fluorescent in situ hybridization (FISH) with a set of fluorescently labeled 16S rRNA-targeted DNA probes. In situ hybridization revealed that Nitrosomonas ureae was the numerically dominant species of the ammonia-oxidizing population in the domestic wastewater biofilm and that a population shift from N. urea to N. europaea and N. eutropha occurred when the culture medium was switched to the synthetic media from the domestic wastewater. After reaching the steady-state condition, microprofiles of NH4+, NO2−, NO3−, and O2 in the biofilms were measured by use of microsensors, and the spatial distributions of in situ nitrifying activities were determined. The relationship between the spatial organization of nitrifying bacterial populations and the in situ activity of these populations within the biofilms was discussed. Microelectrode measurements revealed that the active ammonia-oxidizing zone was vertically separated from the active nitrite-oxidizing zone. This vertical separation became more evident with increase of the substrate C/N ratio, leading to deterioration of nitrification efficiency. The combined use of these techniques made it possible to relate in situ nitrifying activity directly to the occurrence of nitrifying bacterial populations.

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.

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.

Effect of dissolved oxygen concentration on the biofilm and in situ analysis by fluorescence in situ hybridization (FISH) and microelectrodes

2003 ◽  
Vol 47 (1) ◽  
pp. 49-57 ◽  
Author(s):  
A. Jang ◽  
P.L. Bishop ◽  
S. Okabe ◽  
S.G. Lee ◽  
I.S. Kim
Keyword(s):  
In Situ Hybridization ◽  
Dissolved Oxygen ◽  
Nitrifying Bacteria ◽  
Dry Density ◽  
Ammonia Oxidizing Bacteria ◽  
Biofilm Thickness ◽  
Experimental Findings ◽  
And Control ◽  
Microelectrode Measurements

A better understanding of microbiology and ecology of nitrifying bacteria in inner biofilms is an important part of improving process performance and control. Microelectrodes and fluorescent in situ hybridization (FISH) in biofilm research have been used to investigate the spatial distributions of various microbial activities in biofilms and have led to new experimental findings as well as modifications of the homogeneous assumptions in the biofilm kinetic models. The objective of this study is to try the combination of two methods, both FISH and microelectrode measurements, and to provide reliable and in situ information on nitrifying bacterial activity in biofilms. The characteristics of biofilm developed on tygon slides were different according to the change of dissolved oxygen (DO). When the DO increased from 2 to 10 μg DO/L, the rate of the biofilm thickness increased and its dry density changed from 50-70 to 25-90 mg/cm3. Ammonia oxidizing bacteria were not uniformly distributed in biofilm, and were found at the deeper layer where oxygen is depleted, they were detected primarily in the upper and middle layers of the biofilm.

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.

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.

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.

Bio-Augmentation of Nitrifying Microorganisms to Increase the Efficiency of Oxidation of Nitrogen Compounds during Wastewater Biofiltration

2020 ◽  
Vol 36 (2) ◽  
pp. 99-107
Author(s):  
A.S. Sirotkin ◽  
J.V. Kobeleva ◽  
E.S Gorshkova
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Municipal Wastewater ◽  
Ammonium Nitrogen ◽  
Nitrifying Bacteria ◽  
Nitrogen Compounds ◽  
Laboratory Setup ◽  
Start Up ◽  
Nitrite Oxidizing Bacteria

The efficiency of nitrifying bacteria bio-augmentation into biofilm microbiocenosis during 30-day continuous biofiltration of municipal wastewater model solution has been assessed. The laboratory setup consisted of two parallel operating biofilters, in one of which, after start-up period, cultures of ammonium-oxidizing and nitrite-oxidizing bacteria of the Nitrobacter genus were sequentially introduced. It was established that the bio-augmentation of ammonium-oxidizing bacteria into the biofilm microbiocenosis led to an increase in the efficiency of ammonium nitrogen removal by an average of 1.6 times compared to the control biofilter. The subsequent bio-augmentation of nitrite-oxidizing bacteria caused an increase in the amount of nitrates in purified water by 2 times on average. As a result of bio-augmentation of nitrifying bacteria into the biofilm microbiocenosis, the nitrification process was intensified. Quantitative and qualitative identification of microorganisms via fluorescence in situ hybridization showed an increase in the number of nitrifying microorganisms in the biofilm of experimental biofilter, which confirms the efficiency of introduction of microorganisms and correlates with the data on biotransformation of nitrogen compounds. nitrifying microorganisms, wastewater biofiltration, biofilms, bio-augmentation, fluorescence in situ hybridization.

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