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 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.

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.

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.

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.

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.

scholarly journals Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization

2004 ◽  
Vol 70 (3) ◽  
pp. 1641-1650 ◽  
Author(s):  
Tomonori Kindaichi ◽  
Tsukasa Ito ◽  
Satoshi Okabe
Keyword(s):  
Acetic Acid ◽  
In Situ Hybridization ◽  
Bacterial Community ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Heterotrophic Bacteria ◽  
Nitrifying Bacteria ◽  
Community Members ◽  
Biofilm Community

ABSTRACT Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4 + as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (α-Proteobacteria), 23%; γ-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. α- and γ-Proteobacteria dominated the utilization of [14C]acetic acid and 14C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-d-[1-14C]glucosamine (NAG). The GNSB accounted for 9% of the 14C-amino acid-consuming bacteria and 27% of the [14C]NAG-consuming bacteria but did not utilize [14C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.

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.

scholarly journals Measurements of nitrite production in and around the primary nitrite maximum in the central California Current

2013 ◽  
Vol 10 (11) ◽  
pp. 7395-7410 ◽  
Author(s):  
A. E. Santoro ◽  
C. M. Sakamoto ◽  
J. M. Smith ◽  
J. N. Plant ◽  
A. L. Gehman ◽  
...  
Keyword(s):  
Ammonia Oxidation ◽  
Heterotrophic Bacteria ◽  
California Current ◽  
Euphotic Zone ◽  
Ammonia Oxidizing Bacteria ◽  
Central California ◽  
Oxidation Rates ◽  
Ammonia Oxidizing Archaea ◽  
Nitrite Production ◽  
Nh3 Oxidation

Abstract. Nitrite (NO2−) is a substrate for both oxidative and reductive microbial metabolism. NO2− accumulates at the base of the euphotic zone in oxygenated, stratified open-ocean water columns, forming a feature known as the primary nitrite maximum (PNM). Potential pathways of NO2− production include the oxidation of ammonia (NH3) by ammonia-oxidizing bacteria and archaea as well as assimilatory nitrate (NO3−) reduction by phytoplankton and heterotrophic bacteria. Measurements of NH3 oxidation and NO3− reduction to NO2− were conducted at two stations in the central California Current in the eastern North Pacific to determine the relative contributions of these processes to NO2− production in the PNM. Sensitive (< 10 nmol L−1), precise measurements of [NH4+] and [NO2−] indicated a persistent NH4+ maximum overlying the PNM at every station, with concentrations as high as 1.5 μmol L−1. Within and just below the PNM, NH3 oxidation was the dominant NO2− producing process, with rates of NH3 oxidation to NO2− of up to 31 nmol L−1 d−1, coinciding with high abundances of ammonia-oxidizing archaea. Though little NO2− production from NO3− was detected, potentially nitrate-reducing phytoplankton (photosynthetic picoeukaryotes, Synechococcus, and Prochlorococcus) were present at the depth of the PNM. Rates of NO2− production from NO3− were highest within the upper mixed layer (4.6 nmol L−1 d−1) but were either below detection limits or 10 times lower than NH3 oxidation rates around the PNM. One-dimensional modeling of water column NO2− production agreed with production determined from 15N bottle incubations within the PNM, but a modeled net biological sink for NO2− just below the PNM was not captured in the incubations. Residence time estimates of NO2− within the PNM ranged from 18 to 470 days at the mesotrophic station and was 40 days at the oligotrophic station. Our results suggest the PNM is a dynamic, rather than relict, feature with a source term dominated by ammonia oxidation.

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