Phenotypic and phylogenetic description of an Italian isolate of "Microthrix parvicella"

" Microthrix parvicella " strain RN1 was isolated from an activated sludge treatment plant in Italy using micromanipulation techniques. The strain grows as thin unbranched filaments which are Gram-positive with Neisser-positive granules. The isolate was characterized by analysis of the 16S rDNA which was amplified directly from cell biomass by the polymerase chain reaction and sequenced. " Microthrix parvicella " strain RN1 presents a very high similarity (100%) with another " M. parvicella " strain recently isolated in Australia, suggesting that this micro-organism, a novel, deep branching member of the actinomycetes subphylum, is the same causing the common events of bulking and foaming phenomena in activated sludge treatment plants throughout the world.     

Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants

Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.     

Quantification of Microthrix parvicella in activated sludge bacterial communities by real-time PCR

AIMS: This study was to develop a simple and reliable method for quantifying Microthrix parvicella 16S rRNA gene copies and its application to activated sludge samples collected from wastewater treatment plants (WWTP) with and without foaming problems. METHODS AND RESULTS: The relative frequency of M. parvicella was determined by combining real-time PCR assays for quantification of total bacterial 16S rRNA gene copies and M. parvicella 16S rRNA gene copies. The developed method was applied to analyse 32 activated sludge samples obtained from German WWTP. The level of M. parvicella 16S rRNA gene copies in the 18 nonfoaming samples was below 3% of the total number of 16S rRNA gene copies and in the range of 0-18% for the 14 foaming samples. CONCLUSIONS: The described method allows reliable monitoring of the amount of M. parvicella in activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method may become an important component of a warning system for forthcoming bulking and foaming episodes.     

Candidatus Monilibacter spp., common bulking filaments in activated sludge, are members of Cluster III Defluviicoccus

Two alphaproteobacterial Neisser negative ‘Nostocoida limicola’ morphotypes differing slightly in their trichome diameter and filament regularity were dominant populations in the Bendigo, Victoria, Australia activated sludge community removing phosphorus (P). Neither responded to the FISH probes available for any of the other alphaproteobacterial ‘N. limicola’ morphotypes. Instead both fluoresced with the DF988 FISH probe designed originally to target alphaproteobacterial cluster II Defluviicoccus tetrad forming organisms. A 16S rRNA based clone library from this biomass revealed that the alphaproteobacterial clones grouped closely with Candidatus ‘Monilibacter batavus’ and Defluviicoccus clones in a cluster separate from the existing cluster I and II Defluviicoccus. When a FISH probe was designed against these, it only hybridized to the thinner and less abundant ‘N. limicola’ morphotype. Micromanipulation–RT-PCR was used to selectively recover the main ‘N. limicola’ morphotype and a FISH probe designed against the 16S rRNA clones generated from it showed only this filament fluoresced. From FISH based surveys, both ‘N. limicola’ variants occurred frequently in phosphorus removal activated sludge systems in Australia treating domestic waste. The data suggest that they represent two new strains of Candidatus ‘Monilibacter’, which on this evidence are filamentous members of the genus Defluviicoccus, a potential competitor for the polyphosphate accumulating organisms in these communities.     

Control of Microthrix parvicella growth in activated sludge

Abstract Effects of aerobic and anaerobic conditions on the growth of Microthrix parvicella in the activated sludge were studied to prevent bulking caused by this filamentous bacterium. The study was conducted on a pilot plant with selector and the data were compared with those observed in a full scale plant subjected to severe bulking due to a massive growth of M. parvicella . Both plants were fed with the same settled waste water. A substantial suppression of the growth of M. parvicella was observed in only the experiments where returned activated sludge was mixed with waste water under aerobic conditions. Both the number of filaments and the sludge volume index (SVI) were lower in the pilot plant than in the full scale plant. Under anerobic conditions, the selector was not able to improve the settleability and avoid the growth of M. parvicella .     

Ultrastructure of Microthrix parvicella from activated sludge

H.M. STRATTON, R. WEBB, E.M. SEVIOUR, L.L. BLACKALL AND R.J. SEVIOUR. 1996. Filaments of Microthrix parvicella grow very poorly in pure culture and have a characteristic uneven appearance containing large numbers of distinctive spherical swollen cells. This feature was only rarely seen with filaments of this organism in activated sludge biomass samples. Ultrastructurally, these spherical swollen cells do not appear to be bacterial endospores or cysts, but do show features that are consistent with them being some form of resting structure. Their production could be a response of M. parvicella to environmental stress, an explanation proposed because of their relative predominance in pure cultures of M. parvicella compared to their infrequent occurrence in filaments of this bacterium observed in activated sludge biomass.     

The in situ physiology of Skermania piniformis in foams in Australian activated sludge plants

The in situ physiology of the filamentous bacterium Skermania piniformis frequently seen in activated sludge foams in Australia was investigated. An oligonucleotide probe, Spin1449, targeting the 16S rRNA of S. piniformis was designed for its identification by fluorescence in situ hybridization (FISH), validated with pure cultures and applied successfully to foam samples from two geographically distant Australian plants. While filaments of this bacterium appeared to be comparatively hydrophobic, the organism had no clear preference for hydrophobic or hydrophilic substrates. In both foams examined using microautoradiography (MAR), filaments selectively took up substrates under aerobic and anoxic (NO(3) (-)) but not anaerobic or anoxic (NO(2) (-)) conditions. Skermania piniformis assimilated oleic acid, palmitic acid, glycerol and glycine. Ectoenzyme activities detected suggest that S. piniformis has an ability to assimilate a greater range of substrates than might be concluded from the MAR data obtained here. Based on the substrate uptake data presented here, an anaerobic selector may work for controlling S. piniformis in activated sludge systems.     

Ecophysiology of mycolic acid-containing Actinobacteria (Mycolata) in activated sludge foams

Increasing incidences of activated sludge foaming have been reported in the last decade in Danish plants treating both municipal and industrial wastewaters. In most cases, foaming is caused by the presence of Actinobacteria; branched mycolic acid-containing filaments (the Mycolata) and the unbranched Candidatus'Microthix parvicella'. Surveys from wastewater treatment plants revealed that the Mycolata were the dominant filamentous bacteria in the foam. Gordonia amarae-like organisms and those with the morphology of Skermania piniformis were frequently observed, and they often coexisted. Their identity was confirmed by FISH, using a new permeabilization procedure. It was not possible to identify all abundant Mycolata using existing FISH probes, which suggests the presence of currently undetectable and potentially undescribed populations. Furthermore, some Mycolata failed to give any FISH signal, although substrate uptake experiments with microautoradiography revealed that they were physiologically active. Ecophysiological studies were performed on the Mycolata identified by their morphology or FISH in both foams and mixed liquors. Large differences were seen among the Mycolata in levels of substrate assimilation and substrate uptake abilities in the presence of different electron acceptors. These differences were ascribed mainly to the presence of currently undescribed Mycolata species and/or differences in foam age.     

Application of quantitative RT-PCR to determine the distribution of <Emphasis Type="Italic">Microthrix parvicella</Emphasis> in full-scale activated sludge treatment systems

Three wastewater treatment plants in South Africa were investigated to understand the phylogeny and distribution of Microthrix parvicella using real-time polymerase chain reaction (RT-PCR). The phylogenetic analysis of the 16S rRNA of M. parvicella revealed 98% to 100% homology of South African clones to M. parvicella reported in Genbank. The standard curves for RT-PCR showed R2 values greater than 0.99, accurate for quantification. The relative occurrence of M. parvicella 16S rRNA gene copies in the three wastewater treatment plants was in the range 0% to 3.97%. M. parvicella copies increased when the environmental temperature (≤20°C) and food/microorganism (F/M) ratio was low. The M. parvicella 16S rRNA copies could be positively correlated to the sludge volume index at low temperature. At higher temperature, there was a rapid reduction in M. parvicella population irrespective of other favorable factors, indicating the strong influence of temperature on filamentous proliferation. RT-PCR has potential applications in wastewater treatment plants to monitor sudden shift in the microbial population and assessing the plants efficacy.     

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_T1038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge.     

Studies on the in situ physiology of Thiothrix spp. present in activated sludge

The in situ physiology of the filamentous sulphur bacterium Thiothrix spp. was investigated in an industrial wastewater treatment plant with severe bulking problems as a result of overgrowth of Thiothrix. Identification and enumeration using fluorescence in situ hybridization (FISH) with species-specific 16S and 23S rRNA probes revealed that 5–10% of the bacteria in the activated sludge were Thiothrix spp. By using a combination of FISH and microautoradiography it was possible to study the in situ physiology of probe-defined Thiothrix filaments under different environmental conditions. The Thiothrix filaments were very versatile and showed incorporation of radiolabelled acetate and/or bicarbonate under heterotrophic, mixotrophic and chemolithoautotrophic conditions. The Thiothrix filaments were active under anaerobic conditions (with or without nitrate) in which intracellular sulphur globules were formed from thiosulphate and acetate was taken up. Thiothrix -specific substrate uptake rates and growth rates in activated sludge samples were determined under different conditions. Doubling times of 6–9 h under mixotrophic conditions and 15–30 h under autotrophic conditions were estimated. The key properties that Thiothrix might be employing to outcompete other microorganisms in activated sludge were probably related to the mixotrophic growth potential with strong stimulation of acetate uptake by thiosulphate, as well as stimulation of bicarbonate incorporation by acetate in the presence of thiosulphate.     

Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation-independent molecular and physical methods. The large, corkscrew-shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group-specific, rRNA-targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga-Flexibacter group of the Cytophaga-Flavobacterium-Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA-targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew-shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named 'Magnospira bakii' and has the taxonomic rank of Candidatus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.     

Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).     

Populations related to Alkanindiges, a novel genus containing obligate alkane degraders, are implicated in biological foaming in activated sludge systems

Activated sludge mixed liquor and biological foam samples were collected from five full-scale municipal wastewater treatment plants in Illinois, all of which were exhibiting biological foaming at the time of sampling. Oligonucleotide probe hybridization consistently measured higher levels of Gammaproteobacteria rRNA in the foam as compared with the mixed liquor for all treatment plants analysed. Cloning and sequencing of 16S rRNA gene amplicons led to the identification of populations which were abundant in each of the treatment plants. These populations were related to the Alkanindiges/Acinetobacter cluster within the Gammaproteobacteria. Further analysis of the 16S rRNA sequences indicated that they clustered in three phylogenetic groups outside the main Alkanindiges/Acinetobacter cluster, suggesting that these groups may represent new taxa. Terminal-restriction fragment length polymorphism analysis showed that these populations were enriched in the foam compared with the underlying mixed liquor similar to the enrichment of the Gammaproteobacteria measured by oligonucleotide probe membrane hybridization. The observed enrichment in foam samples is suggestive of a role for these populations in foam formation or stabilization, and their presence in all treatment plants analysed in this study may be indicative of their widespread abundance in foaming plants.     

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r ²=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19–55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Detection of denitrification genes by in situ rolling circle amplification‐fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells

A target‐primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single‐copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5′‐3′ exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template for in situ RCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal‐to‐noise ratio but low detection frequency (up to 15% for single‐copy genes and up to 43% for the multi‐copy 16S rRNA gene). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA‐FISH was demonstrated on activated sludge by the differential detection of two types of nirS‐defined denitrifiers; one of them was identified as Candidatus Accumulibacter phosphatis by combining in situ RCA‐FISH with 16S rRNA‐targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA‐FISH will allow to link metabolic potential with 16S rRNA (gene)‐based identification of single microbial cells.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.