Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass

2006 ◽  
Vol 54 (1) ◽  
pp. 277-284
Author(s):  
C. Yeates ◽  
L.L. Blackall
Keyword(s):  
Phosphorus Removal ◽  
In Situ Hybridisation ◽  
Metagenomic Library ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Metagenomic Data ◽  
Fluorescence In Situ Hybridisation ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Biological Phosphorus

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, “Candidatus Accumulibacter phosphatis”. DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of non-pure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater.

scholarly journals “Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

2007 ◽  
Vol 73 (18) ◽  
pp. 5865-5874 ◽  
Author(s):  
Shaomei He ◽  
Daniel L. Gall ◽  
Katherine D. McMahon
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
Genetic Marker ◽  
Phosphorus Removal ◽  
Full Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Biological Phosphorus

ABSTRACT We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.

The effect of GAOs (glycogen accumulating organisms) on anaerobic carbon requirements in full-scale Australian EBPR (enhanced biological phosphorus removal) plants

2003 ◽  
Vol 47 (11) ◽  
pp. 37-43 ◽  
Author(s):  
A.M. Saunders ◽  
A. Oehmen ◽  
L.L. Blackall ◽  
Z. Yuan ◽  
J. Keller
Keyword(s):  
Phosphorus Removal ◽  
In Situ Hybridisation ◽  
Full Scale ◽  
Fluorescence In Situ Hybridisation ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Accumulibacter Phosphatis ◽  
Polyphosphate Accumulating Organisms ◽  
Biological Phosphorus

Glycogen-accumulating organisms (GAOs) were present in six full-scale plants investigated and in all but one made a significant contribution to the amount of volatile fatty acid (VFA) taken up anaerobically. While most plants surveyed contain GAOs, it was demonstrated that it is possible for a full-scale plant to operate with an insignificant GAO population.“Candidatus Accumulibacter phosphatis”were the significant polyphosphate-accumulating organisms (PAOs) in all plants surveyed. “Candidatus Competibacter phosphatis” were found in all plants along with other possible GAOs that were observed but not identified. A significant GAO population will increase the carbon requirements by removing VFA that could otherwise have been used by PAOs. Process optimization minimizing GAOs in full-scale plants would lead to a more efficient use of VFA. Enhanced biological phosphorus removal (EBPR), fluorescence in situ hybridisation (FISH), glycogen accumulating organism (GAO); polyphosphate accumulating organism (PAO);

scholarly journals Identity and Ecophysiology of Uncultured Actinobacterial Polyphosphate-Accumulating Organisms in Full-Scale Enhanced Biological Phosphorus Removal Plants

2005 ◽  
Vol 71 (7) ◽  
pp. 4076-4085 ◽  
Author(s):  
Yunhong Kong ◽  
Jeppe Lund Nielsen ◽  
Per Halkjær Nielsen
Keyword(s):  
Phosphorus Removal ◽  
Treatment Plant ◽  
Full Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Fish Analysis ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Polyphosphate Accumulating Organisms ◽  
Biological Phosphorus

ABSTRACT Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up Pi aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pi took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.

scholarly journals Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge, Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

2010 ◽  
Vol 76 (12) ◽  
pp. 3825-3835 ◽  
Author(s):  
Jeong Myeong Kim ◽  
Hyo Jung Lee ◽  
Sun Young Kim ◽  
Jae Jun Song ◽  
Woojun Park ◽  
...  
Keyword(s):  
16S Rrna ◽  
Phosphorus Removal ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Fine Scale ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Accumulibacter Phosphatis ◽  
Biological Phosphorus

ABSTRACT To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.

scholarly journals Bacterial Community and “Candidatus Accumulibacter” Population Dynamics in Laboratory-Scale Enhanced Biological Phosphorus Removal Reactors

2010 ◽  
Vol 76 (16) ◽  
pp. 5479-5487 ◽  
Author(s):  
Shaomei He ◽  
Forrest I. Bishop ◽  
Katherine D. McMahon
Keyword(s):  
Bacterial Community ◽  
Phosphorus Removal ◽  
Community Dynamics ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Reactor Performance ◽  
Total Bacterial Community ◽  
Biological Phosphorus

ABSTRACT “Candidatus Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “Ca. Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “Ca. Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “Ca. Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “Ca. Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “Ca. Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO3-N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “Ca. Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.

Applicability of experience from laboratory reactors with biological phosphorus removal in full-scale plants

2006 ◽  
Vol 54 (1) ◽  
pp. 267-275 ◽  
Author(s):  
E. Tykesson ◽  
L.L. Blackall ◽  
Y. Kong ◽  
P.H. Nielsen ◽  
J. la Cour Jansen
Keyword(s):  
Phosphorus Removal ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Full Scale ◽  
Fluorescence In Situ Hybridisation ◽  
Biological Phosphorus Removal ◽  
Batch Tests ◽  
P Release ◽  
Biological Phosphorus

Enhanced biological phosphorus removal (EBPR) has been used at many wastewater treatment plants all over the world for many years. In this study a full-scale sludge with good EBPR was tested with P-release batch tests and combined FISH/MAR (fluorescence in situ hybridisation and microautoradiography). Proposed models of PAOs and GAOs (polyphosphate- and glycogen-accumulating organisms) and microbial methods suggested from studies of laboratory reactors were found to be applicable also on sludge from full-scale plants. Dependency of pH and the uptake of both acetate and propionate were studied and used for calculations for verifying the models and results from microbial methods. All rates found from the batch tests with acetate were higher than in the batch tests with propionate, which was explained by the finding that only those parts of the bacterial community that were able to take up acetate anaerobically were able to take up propionate anaerobically.

Characterisation of enhanced biological phosphorus removal activated sludges with dissimilar phosphorus removal performances

1998 ◽  
Vol 37 (4-5) ◽  
pp. 567-571 ◽  
Author(s):  
Philip L. Bond ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
Phosphorus Removal ◽  
Batch Reactor ◽  
In Situ Hybridisation ◽  
Chemical Transformations ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Reactor Performance ◽  
Biological Phosphorus ◽  
P Removal

A sequencing batch reactor (SBR) was operated for enhanced biological phosphorus removal (EBPR) and dramatic differences in the P removing capabilities were obtained in different stages of the operation. At one stage extremely poor P removal occurred and it appeared that bacteria inhibiting P removal overwhelmed the reactor performance. Changes were made to the reactor operation and these led to the development of a sludge with high P removing capability. This latter sludge was analysed by fluorescent in situ hybridisation (FISH) using a probe specific for Acinetobacter. Very few cells were detected with this probe indicating that Acinetobacter played an insignificant role in the P removal occurring here. Analysis of the chemical transformations of three sludges supported the biochemical pathways proposed for EBPR and non-EBPR systems in biological models. A change in operation that led to the improved P removal performance included permitting the pH to rise in the anaerobic periods of the SBR cycle.

scholarly journals Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal

2002 ◽  
Vol 68 (10) ◽  
pp. 4971-4978 ◽  
Author(s):  
Katherine D. McMahon ◽  
Michael A. Dojka ◽  
Norman R. Pace ◽  
David Jenkins ◽  
Jay D. Keasling
Keyword(s):  
Activated Sludge ◽  
Phosphorus Removal ◽  
Specific Activity ◽  
Inverse Pcr ◽  
Rrna Genes ◽  
Polyphosphate Kinase ◽  
Type I ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Biological Phosphorus

ABSTRACT A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg2+, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.

Heterotrophic Kinetic Parameter Estimation for Enhanced Biological Phosphorus Removal Processes Operated in Conventional and Membrane-Assisted Modes

2006 ◽  
Vol 41 (1) ◽  
pp. 72-83 ◽  
Author(s):  
Zhe Zhang ◽  
Eric R. Hall
Keyword(s):  
Parameter Estimation ◽  
Phosphorus Removal ◽  
Growth Yield ◽  
Pilot Scale ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Nitrogen And Phosphorus ◽  
Parameter Values ◽  
The Impact ◽  
Biological Phosphorus

Abstract Parameter estimation and wastewater characterization are crucial for modelling of the membrane enhanced biological phosphorus removal (MEBPR) process. Prior to determining the values of a subset of kinetic and stoichiometric parameters used in ASM No. 2 (ASM2), the carbon, nitrogen and phosphorus fractions of influent wastewater at the University of British Columbia (UBC) pilot plant were characterized. It was found that the UBC wastewater contained fractions of volatile acids (SA), readily fermentable biodegradable COD (SF) and slowly biodegradable COD (XS) that fell within the ASM2 default value ranges. The contents of soluble inert COD (SI) and particulate inert COD (XI) were somewhat higher than ASM2 default values. Mixed liquor samples from pilot-scale MEBPR and conventional enhanced biological phosphorus removal (CEBPR) processes operated under parallel conditions, were then analyzed experimentally to assess the impact of operation in a membrane-assisted mode on the growth yield (YH), decay coefficient (bH) and maximum specific growth rate of heterotrophic biomass (µH). The resulting values for YH, bH and µH were slightly lower for the MEBPR train than for the CEBPR train, but the differences were not statistically significant. It is suggested that MEBPR simulation using ASM2 could be accomplished satisfactorily using parameter values determined for a conventional biological phosphorus removal process, if MEBPR parameter values are not available.

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