Application of oligonucleotide probes for the detection of Thiothrix spp. in activated sludge plants treating paper and board mill wastes

2002 ◽  
Vol 46 (1-2) ◽  
pp. 559-564 ◽  
Author(s):  
S.B. Kim ◽  
M. Goodfellow ◽  
J. Kelly ◽  
G.S. Saddler ◽  
A.C. Ward
Keyword(s):  
Activated Sludge ◽  
Bacterial Population ◽  
Oligonucleotide Probe ◽  
In Situ Hybridisation ◽  
Filamentous Bacteria ◽  
Phylogenetic Groups ◽  
Investigation Period ◽  
Mixed Populations ◽  
Conventional Microscopy

Filamentous bacteria belonging to the genus Thiothrix were detected in activated sludge samples using the fluorescent in situ hybridisation (FISH) technique. A 16S rRNA-targeted oligonucleotide probe was developed for the detection of members of the T. fructosivorans group, and the performance of probe TNI for the detection of Thiothrix nivea group was enhanced by using an unlabeled competitor. A set of 5 probes covering all phylogenetic groups of Thiothrix were used to examine samples taken from selected activated sludge plants treating paper and board mill wastes. Members of the T. eikelboomii group formed the predominant filamentous bacterial population in plants experiencing poor sludge settleability, whereas members of the T. nivea group were commonly found but not dominantly in the remaining plants. Members of the T. fructosivorans group were not detected at any significant level in any of the samples. The distribution of the main Thiothrix types remained unchanged throughout the investigation period. It was evident that mixed populations of Thiothrix spp. were present in all activated sludge samples investigated, the observed differences were in the relative abundance of the various groups. These findings were supported by the results obtained using conventional microscopy.

scholarly journals Phylogenetic Analysis of and Oligonucleotide Probe Development for Eikelboom Type 021N Filamentous Bacteria Isolated from Bulking Activated Sludge

2000 ◽  
Vol 66 (11) ◽  
pp. 5043-5052 ◽  
Author(s):  
Takahiro Kanagawa ◽  
Yoichi Kamagata ◽  
Shinobu Aruga ◽  
Tetsuro Kohno ◽  
Matthias Horn ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Oligonucleotide Probe ◽  
Sequence Similarity ◽  
Rdna Sequence ◽  
Filamentous Bacteria ◽  
16S Rdna Sequence ◽  
Monophyletic Cluster ◽  
16S Ribosomal Dna ◽  
In Situ Detection

ABSTRACT Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass ofProteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.

Identity, abundance and physiology of Aquaspirillum-related filamentous bacteria in activated sludge

2006 ◽  
Vol 54 (1) ◽  
pp. 237-245 ◽  
Author(s):  
T.R. Thomsen ◽  
C. Kragelund ◽  
P.H. Nielsen
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Nutrient Removal ◽  
Wastewater Treatment Plants ◽  
Oligonucleotide Probe ◽  
Filamentous Bacteria ◽  
Type B ◽  
Anaerobic Conditions ◽  
Almost All

Microcolony-forming bacteria closely related to the genus Aquaspirillum in the Betaproteobacteria were recently observed to be abundant in many nutrient removal wastewater treatment plants. The developed oligonucleotide probe, Aqs997, however, occasionally also targeted some filamentous bacteria in activated sludge samples when fluorescence in situ hybridization was performed. In this study, the identity, abundance, and ecophysiology of these Aqs997-positive filamentous bacteria were studied in detail. Most of the Aqs997-positive filamentous bacteria could morphologically be identified as either Eikelboom Type 1701, Type 0041/0675 or possibly Type 1851, all characterized by epiphytic growth. They were found in almost all 21 wastewater treatment plants investigated. Two morphotypes were found. Type A filaments, which seemed to be the same genotype as the microcolony-forming bacteria targeted by probe Aqs997.Type B filaments also hybridized with probe GNS941, specific for the Chloroflexi phylum, so the true identity remains unclear. Aqs997-positive filaments usually stained Gram-negative, but Gram-positive filaments were also found, stressing the difficulties in identifying bacteria from morphology and simple staining results. Studies on the ecophysiology by microautoradiography showed that Aqs997-positive filamentous bacteria did not consume acetate and glucose, while some took up butyrate, mannose, and certain amino acids. Most likely, some Aqs997-positive filamentous bacteria were able to perform full denitrification such as the Aqs997-positive microcolony-forming bacteria, and some were able to store polyhydroxyalkanoates under anaerobic conditions, potentially being glycogen accumulating organisms.

scholarly journals Simultaneous oligonucleotide probe hybridization and immunostaining for in situ detection of Gordona species in activated sludge

1999 ◽  
Vol 29 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Daniel B Oerther ◽  
Francis L Reyes ◽  
Mark Hernandez ◽  
Lutgarde Raskin
Keyword(s):  
Activated Sludge ◽  
Oligonucleotide Probe ◽  
Probe Hybridization ◽  
In Situ Detection

scholarly journals In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

2011 ◽  
Vol 169 (20) ◽  
pp. 525-525 ◽  
Author(s):  
N. Dinhopl ◽  
M. M. Mostegl ◽  
B. Richter ◽  
N. Nedorost ◽  
A. Maderner ◽  
...  
Keyword(s):  
Oligonucleotide Probe ◽  
In Situ Hybridisation ◽  
Leishmania Species ◽  
Paraffin Wax

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.

Microbial quantification in activated sludge: the hits and misses

2003 ◽  
Vol 48 (3) ◽  
pp. 121-126 ◽  
Author(s):  
S.J. Hall ◽  
J. Keller ◽  
L.L. Blackall
Keyword(s):  
Activated Sludge ◽  
Molecular Genetic ◽  
In Situ Hybridisation ◽  
Biological Nutrient Removal ◽  
Physical Parameters ◽  
Fluorescence In Situ Hybridisation ◽  
Batch Tests ◽  
Polymerase Chain ◽  
Key Microorganisms

Since the implementation of the activated sludge process for treating wastewater, there has been a reliance on chemical and physical parameters to monitor the system. However, in biological nutrient removal (BNR) processes, the microorganisms responsible for some of the transformations should be used to monitor the processes with the overall goal to achieve better treatment performance. The development of in situ identification and rapid quantification techniques for key microorganisms involved in BNR are required to achieve this goal. This study explored the quantification of Nitrospira, a key organism in the oxidation of nitrite to nitrate in BNR. Two molecular genetic microbial quantification techniques were evaluated: real-time polymerase chain reaction (PCR) and fluorescence in situ hybridisation (FISH) followed by digital image analysis. A correlation between the Nitrospira quantitative data and the nitrate production rate, determined in batch tests, was attempted. The disadvantages and advantages of both methods will be discussed.

Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge

1994 ◽  
Vol 17 (3) ◽  
pp. 405-417 ◽  
Author(s):  
Michael Wagner ◽  
Rudolf Amann ◽  
Peter Kämpfer ◽  
Bernhard Assmus ◽  
Anton Hartmann ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Filamentous Bacteria ◽  
Gram Negative ◽  
In Situ Detection

Probing Activated Sludge with Fluorescently Labeled rRNA Targeted Oligonucleotides

1994 ◽  
Vol 29 (7) ◽  
pp. 15-23 ◽  
Author(s):  
M. Wagner ◽  
R. Amann ◽  
H. Lemmer ◽  
W. Manz ◽  
K. H. Schleifer
Keyword(s):  
Activated Sludge ◽  
Ribosomal Rna ◽  
Phosphate Removal ◽  
Municipal Sewage ◽  
Correct Identification ◽  
Rich Medium ◽  
Phylogenetic Groups ◽  
In Situ Investigation ◽  
Nutrient Rich Medium

Activated sludge samples from municipal sewage treatment plants were characterized using 16S and 23S ribosomal RNA targeted oligonucleotide probes specific for defined phylogenetic groups of bacteria. Comparison of in situ community structures as determined by molecular biological methods with the composition of the heterotrophic saprophyte flora isolated on nutrient rich medium revealed large discrepancies. These are caused by the selectivity of media and culture conditions. The most significant effect of cultivation on nutrient rich medium is an underestimation of bacteria belonging to the beta-subclass of Proteobacteria and an overestimation of bacteria belonging to the gamma-subclass of Proteobacteria. Therefore, culture dependent enumerations of the gamma-subclass bacteria of the genus Acinetobacter in plants with enhanced biological phosphate removal (EBPR) resulted in significant overestimations. In situ identification by fluorescent oligonucleotide probing revealed that Acinetobacter numbers were below 8% of the active bacterial cells in the examined EBPR-plants. In situ hybridization techniques also bear the potential for the early and correct identification of filamentous bacteria as indicators for sludge bulking and foaming. A 16S ribosomal RNA targeted oligonucleotide probe specific for Sphaerotilus spec, was developed and successfully applied for in situ investigation of this filamentous bacterium.

Development and application of 16S rRNA-targeted oligonucleotide probe for detection of the phosphate-accumulating bacterium microlunatus phosphovorus in an enhanced biological phosphorus removal process

1998 ◽  
Vol 37 (4-5) ◽  
pp. 481-484 ◽  
Author(s):  
Mamoru Kawaharasaki ◽  
Takahiro Kanagawa ◽  
Hideo Tanaka ◽  
Kazunori Nakamura
Keyword(s):  
16S Rrna ◽  
Activated Sludge ◽  
Phosphorus Removal ◽  
Oligonucleotide Probe ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Phylogenetic Groups ◽  
Removal Process ◽  
Biological Phosphorus ◽  
Total Bacteria

A 16S rRNA-targeted oligonucletide probe (MP2) specific for the phosphate-accumulating bacterium (PAB) M. phosphovorus was designed and applied to a sludge from an enhanced biological phosphorus removal (EBPR) process. Probes specific for defined phylogenetic groups and the polyphosphate staining dye, DAPI (4′, 6-diamidino-2-phenylindol dihydrochloride) were also used to analyze the activated sludge community. M. phosphovorus was about 3% of the total bacteria in the EBPR sludge used. Proteobacteria belonging to the beta subclass were the most abundant. Many coccoid bacteria similar to M. phosphovorus were stained with DAPI. The percentage of PABs detected by DAPI stain was about 9% of the total bacteria.

Phylogenetic analysis and in situ identification of “Nostocoida limicola”-like filamentous bacteria in activated sludge from industrial wastewater treatment plants

2002 ◽  
Vol 46 (1-2) ◽  
pp. 99-104 ◽  
Author(s):  
J. Snaidr ◽  
C. Beimfohr ◽  
C. Levantesi ◽  
S. Rossetti ◽  
J. van der Waarde ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Industrial Wastewater ◽  
Wastewater Treatment Plants ◽  
Molecular Probes ◽  
Special Focus ◽  
Filamentous Bacteria ◽  
Industrial Wastewater Treatment ◽  
Biological Methods

The diversity of filamentous bacteria present in industrial wastewater treatment plants was analysed by a combination of classical and molecular-biological approaches. Many unknown filamentous bacteria were observed in about 80 screened activated sludge samples from different industries with sometimes severe bulking sludge problems. A special focus was paid to filaments which resembled “Nostocoida limicola”, a filamentous bacterium which was found to be present in many WWTPs. These filamentous bacteria are hardly cultivable and only one strain was obtained and maintained in co-culture with a yeast. The 16S rRNA sequences of several other “Nostocoida limicola”-like filamentous bacteria from different sludge samples were obtained by micromanipulation and different molecular-biological methods. The sequences were phylogenetically analyzed and specific molecular probes were developed and applied. The results clearly demonstrate that “Nostocoida limicola”-like filaments from industrial WWTPs are different from all other “Nostocoida limicola” types investigated so far. Our strains are affiliated to the alpha-subclass of Proteobacteria.

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