Variability of type 021N in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes

1998 ◽  
Vol 37 (4-5) ◽  
pp. 423-430 ◽  
Author(s):  
P. H. Nielsen ◽  
K. Andreasen ◽  
M. Wagner ◽  
L. L. Blackall ◽  
H. Lemmer ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Treatment Plant ◽  
Strain Differences ◽  
Molecular Probes ◽  
Clear Correlation ◽  
Substrate Uptake ◽  
Organic Substrates ◽  
Uptake Pattern

Bulking sludges were investigated in seven industrial or municipal activated sludge treatment plants from Denmark, Germany and Australia. The dominating filaments were all identified as type 021N according to the Eikelboom key. The extent of variability in the filament taxonomy was assessed using fluorescence in situ hybridization (FISH) with rRNA-targeted nucleic acid probes specific for type 021N, Thiothrix and Leucothrix. Not all of the filaments morphologically identified as type 021N hybridized with the 021N probe. In one treatment plant the predominant filament hybridized with the probe for Thiothrix and in one treatment plant the predominant filament did not hybridize with any of these probes. In none of the plants did filaments hybridize with the probe for Leucothrix. A study of the in situ uptake of different organic substrates by the various filaments was also conducted using microautogradiography. The uptake of 6 different organic substrates under aerobic conditions was studied by providing C-14 or H-3 labeled substrates (acetate, glucose, ethanol, glycine, leucine and oleic acid) in incubations of a period of 3 hours. No filaments took up all the tested substrates, and type 021N from the various treatment plants varied in their uptake abilities. The study demonstrated that strain differences with regard to substrate utilization are likely to occur among bacteria within the same genera and designated types which are indistinguishable on the basis of morphological observations alone and by the molecular probes used in this study for identification. Whether there is a clear correlation between type of wastewater and the capability of taking up the various organic substrates for the filaments remains to be elucidated.

In situ detection of cell surface hydrophobicity of probe-defined bacteria in activated sludge

2001 ◽  
Vol 43 (6) ◽  
pp. 97-103 ◽  
Author(s):  
J. L. Nielsen ◽  
L. H. Mikkelsen ◽  
P. H. Nielsen
Keyword(s):  
Activated Sludge ◽  
Surface Properties ◽  
Treatment Plant ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Substrate Uptake ◽  
Filamentous Bacteria ◽  
In Situ Conditions ◽  
Bacterial Surfaces

The surface hydrophobicity of different types of bacteria in activated sludge were investigated under in situ conditions by following the adhesion of fluorescent microspheres with defined surface properties to bacterial surfaces (the MAC-method). This technique was combined with identification of the bacteria with fluorescence in situ hybridization with rRNA-targeted oligonucleotides (FISH) and could thus be used for characterization of surface properties of probe-defined bacteria directly in a complex system without prior enrichment or isolation. This MAC-FISH technique could be used for single bacteria as well as filamentous bacteria. In the investigated activated sludge from an industrial wastewater treatment plant, two types of filamentous bacteria dominated. One morphotype consistently attracted only very few hydrophobic microspheres, indicating that the thin sheath of exopolymers around the cells had a hydrophilic surface. Use of a hierarchical set of gene probes revealed that these filaments were sulphide oxidising Thiothrix spp. The other predominating filamentous morphotype had a thick, very hydrophobic exopolymeric sheath. This filamentous bacterium was found to belong to the alpha-Proteobacteria. The relevance of the significant differences in surface hydrophobicity for the two morphotypes in respect to substrate uptake and floc formation is discussed.

In situ characterization of substrate uptake by Microthrix parvicella using microautoradiography

1998 ◽  
Vol 37 (4-5) ◽  
pp. 19-26 ◽  
Author(s):  
Kjær Andreasen ◽  
Per Halkjær Nielsen
Keyword(s):  
Oleic Acid ◽  
Activated Sludge ◽  
Control Strategy ◽  
Nutrient Removal ◽  
Substrate Uptake ◽  
Organic Substrates ◽  
Aerobic Conditions ◽  
Anoxic Conditions ◽  
Microthrix Parvicella

Microthrix parvicella is a filamentous microorganism responsible for bulking and foaming problems in many activated sludge treatment plants. The problems have increased with the introduction of nutrient removal in many countries, and presently, there is no reliable control strategy for M. parvicella. Little is known about the physiology of M. parvicella, and conflicting data exist about its preferred organic substrates, and whether it is able to be physiologically active under anaerobic and anoxic conditions. In this study, the ability of M. parvicella to take up various radioactively labeled organic compounds was investigated in situ at three nutrient removal plants using a microautoradiographic technique. Of 12 compounds tested under aerobic conditions only the long chain fatty acids (LCFA), oleic acid and palmitic acid, and to some extent a lipid, trioleic acid, were assimilated. None of the simple substrates such as acetate, propionate, butyrate, glucose, ethanol, glycine and leucine were taken up. Furthermore, the uptake of oleic acid was compared under anaerobic, anoxic and aerobic conditions, and it was demonstrated that in addition to aerobic conditions M. parvicella was also able to take up oleic acid under anaerobic and anoxic conditions. No difference in substrate uptake pattern for M. parvicella was found among the tested activated sludge plants. The results strongly indicate that a better control strategy against M. parvicella must rely on a better understanding of presence and availability of triglycerides and LCFA, and an improved knowledge of the physiology of M. parvicella under anaerobic and anoxic conditions.

scholarly journals Application of the FISH Technique to Visualize Sex Chromosomes in Domestic Cat Spermatozoa

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2106
Author(s):  
Barbara Kij-Mitka ◽  
Halina Cernohorska ◽  
Svatava Kubickova ◽  
Sylwia Prochowska ◽  
Wojciech Niżański ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Sex Chromosomes ◽  
Chromosomal Abnormalities ◽  
Molecular Cytogenetics ◽  
Molecular Probes ◽  
Domestic Cat ◽  
Fish Technique ◽  
Y Chromosomes

Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear chromatin decondensation, this technique has already been used to visualize chromosomes in human, mouse, cattle, swine, horse, and dog spermatozoa. Until now, FISH has not been performed on domestic cat sperm; therefore, the aim of this study was to visualize sex chromosomes in domestic cat sperm. The results showed the presence of X and Y chromosomes in feline spermatozoa. The procedure used for sperm decondensation and fluorescence in situ hybridization was adequate to visualize chromosomes in domestic cat spermatozoa and, in the future, it may be used to determine the degree of chromosomal abnormalities in these gametes.

scholarly journals Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

1999 ◽  
Vol 65 (4) ◽  
pp. 1746-1752 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Amino Acid Uptake ◽  
Cell Types ◽  
Black Box ◽  
Acid Uptake ◽  
Substrate Uptake

ABSTRACT We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.

scholarly journals Time-series genome-centric analysis unveils bacterial response to operational disturbance in activated sludge

2019 ◽  
Author(s):  
María Victoria Pérez ◽  
Leandro D. Guerrero ◽  
Esteban Orellana ◽  
Eva L. Figuerola ◽  
Leonardo Erijman
Keyword(s):  
Growth Rate ◽  
Time Series ◽  
Wastewater Treatment ◽  
Activated Sludge ◽  
Microbial Communities ◽  
Wastewater Treatment Plant ◽  
Treatment Plant ◽  
Operating Conditions ◽  
The Face

ABSTRACTUnderstanding ecosystem response to disturbances and identifying the most critical traits for the maintenance of ecosystem functioning are important goals for microbial community ecology. In this study, we used 16S rRNA amplicon sequencing and metagenomics to investigate the assembly of bacterial populations in a full-scale municipal activated sludge wastewater treatment plant over a period of three years, including a period of nine month of disturbance, characterized by short-term plant shutdowns. Following the reconstruction of 173 metagenome-assembled genomes, we assessed the functional potential, the number of rRNA gene operons and thein situgrowth rate of microorganisms present throughout the time series. Operational disturbances caused a significant decrease in bacteria with a single copy of the ribosomal RNA (rrn) operon. Despite only moderate differences in resource availability, replication rates were distributed uniformly throughout time, with no differences between disturbed and stable periods. We suggest that the length of the growth lag phase, rather than the growth rate, as the primary driver of selection under disturbed conditions. Thus, the system could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions.IMPORTANCEIn this work we investigated the response of microbial communities to disturbances in a full-scale activated sludge wastewater treatment plant over a time-scale that included periods of stability and disturbance. We performed a genome-wide analysis, which allowed us the direct estimation of specific cellular traits, including the rRNA operon copy number and the in situ growth rate of bacteria. This work builds upon recent efforts to incorporate growth efficiency for the understanding of the physiological and ecological processes shaping microbial communities in nature. We found evidence that would suggest that activated sludge could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions. This paper provides relevant insights into wastewater treatment process, and may also reveal a key role for growth traits in the adaptive response of bacteria to unsteady environmental conditions.

scholarly journals Molecular probes for the identification of avian Haemoproteus and Leucocytozoon parasites in tissue sections by chromogenic in situ hybridization

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Tanja Himmel ◽  
Josef Harl ◽  
Anna Kübber-Heiss ◽  
Cornelia Konicek ◽  
Nuhacet Fernández ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Molecular Probes ◽  
Tissue Sections ◽  
Chromogenic In Situ Hybridization

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.

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