High Diversity and Functional Potential of Undescribed “Acidobacteriota” in Danish Wastewater Treatment Plants

Microbial communities in water resource recovery facilities encompass a large diversity of poorly characterized lineages that could have undescribed process-critical functions. Recently, it was shown that taxa belonging to “Acidobacteriota” are abundant in Danish full-scale activated sludge wastewater treatment plants (WWTP), and here we investigated their diversity, distribution, and functional potential. “Acidobacteriota” taxa were identified using a comprehensive full-length 16S rRNA gene reference dataset and amplicon sequencing surveys across 37 WWTPs. Members of this phylum were diverse, belonging to 14 families, eight of which are completely uncharacterized and lack type strains. Several lineages were abundant, with relative abundances of up to 5% of the microbial community. Genome annotation and metabolic reconstruction of 50 high-quality “Acidobacteriota” metagenome-assembled genomes (MAGs) from 19 WWTPs showed high metabolic diversity and potential involvement in nitrogen and phosphorus removal and iron reduction. Fluorescence in situ hybridization (FISH) using newly-designed probes revealed cells with diverse morphologies, predominantly located inside activated sludge flocs. FISH in combination with Raman microspectroscopy revealed ecophysiological traits in probe-defined cells from the families Holophagaceae, Thermoanaerobaculaceae, and Vicinamibacteraceae, and families with the placeholder name of midas_f_502, midas_f_973, and midas_f_1548. Members of these lineages had the potential to be polyphosphate-accumulating organisms (PAOs) as intracellular storage was observed for the key compounds polyphosphate and glycogen.

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The capacity of wastewater treatment plants drives bacterial community structure and its assembly

Scientific Reports ◽

10.1038/s41598-019-50952-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Young Kyung Kim ◽

Keunje Yoo ◽

Min Sung Kim ◽

Il Han ◽

Minjoo Lee ◽

...

Keyword(s):

Wastewater Treatment ◽

Community Structure ◽

Bacterial Community ◽

Activated Sludge ◽

Community Assembly ◽

Bacterial Communities ◽

Wastewater Treatment Plants ◽

High Capacity ◽

Operating Conditions ◽

Rrna Gene

Abstract Bacterial communities in wastewater treatment plants (WWTPs) affect plant functionality through their role in the removal of pollutants from wastewater. Bacterial communities vary extensively based on plant operating conditions and influent characteristics. The capacity of WWTPs can also affect the bacterial community via variations in the organic or nutrient composition of the influent. Despite the importance considering capacity, the characteristics that control bacterial community assembly are largely unknown. In this study, we discovered that bacterial communities in WWTPs in Korea and Vietnam, which differ remarkably in capacity, exhibit unique structures and interactions that are governed mainly by the capacity of WWTPs. Bacterial communities were analysed using 16S rRNA gene sequencing and exhibited clear differences between the two regions, with these differences being most pronounced in activated sludge. We found that capacity contributed the most to bacterial interactions and community structure, whereas other factors had less impact. Co-occurrence network analysis showed that microorganisms from high-capacity WWTPs are more interrelated than those from low-capacity WWTPs, which corresponds to the tighter clustering of bacterial communities in Korea. These results will contribute to the understanding of bacterial community assembly in activated sludge processing.

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“Candidatus Dechloromonas phosphoritropha” and “Ca. D. phosphorivorans”, novel polyphosphate accumulating organisms abundant in wastewater treatment systems

The ISME Journal ◽

10.1038/s41396-021-01029-2 ◽

2021 ◽

Author(s):

Francesca Petriglieri ◽

Caitlin Singleton ◽

Miriam Peces ◽

Jette F. Petersen ◽

Marta Nierychlo ◽

...

Keyword(s):

Wastewater Treatment ◽

Wastewater Treatment Plants ◽

Niche Partitioning ◽

16S Rrna Gene Sequencing ◽

Abundant Species ◽

Phosphate Removal ◽

Rrna Gene ◽

Biological Phosphorus Removal ◽

Polyphosphate Accumulating Organisms

AbstractMembers of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.

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Do initial concentration and activated sludge seasonality affect pharmaceutical biotransformation rate constants?

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11475-9 ◽

2021 ◽

Author(s):

Tamara J. H. M. van Bergen ◽

Ana B. Rios-Miguel ◽

Tom M. Nolte ◽

Ad M. J. Ragas ◽

Rosalie van Zelm ◽

...

Keyword(s):

Microbial Community ◽

Activated Sludge ◽

Community Composition ◽

Rate Constants ◽

Wastewater Treatment Plants ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Initial Concentration ◽

Different Seasons

Abstract Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (kb) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC–MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and kb values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different kb for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different kb values were measured, depending on initial concentration. In general, biodegradable compounds had a higher kb when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the kb in order to reliably predict the fate of pharmaceuticals in the WWTP. Key points • Biotransformation and sorption of pharmaceuticals were assessed in activated sludge. • Higher initial concentrations resulted in higher biotransformation rate constants for biodegradable pharmaceuticals. • Summer and winter inocula produced slightly different biotransformation rate constants although microbial community composition did not significantly change. Graphical abstract

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Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants

Applied and Environmental Microbiology ◽

10.1128/aem.03044-19 ◽

2020 ◽

Vol 86 (9) ◽

Cited By ~ 5

Author(s):

Jannie Munk Kristensen ◽

Marta Nierychlo ◽

Mads Albertsen ◽

Per Halkjær Nielsen

Keyword(s):

Wastewater Treatment ◽

Pathogenic Bacteria ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Content Type ◽

Effluent Quality ◽

Culture Independent ◽

Influent Wastewater

ABSTRACT Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the “clean” effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock. IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri. Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

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The Microbial Database for Danish wastewater treatment plants with nutrient removal (MiDas-DK) – a tool for understanding activated sludge population dynamics and community stability

Water Science & Technology ◽

10.2166/wst.2013.151 ◽

2013 ◽

Vol 67 (11) ◽

pp. 2519-2526 ◽

Cited By ~ 18

Author(s):

A. T. Mielczarek ◽

A. M. Saunders ◽

P. Larsen ◽

M. Albertsen ◽

M. Stevenson ◽

...

Keyword(s):

Wastewater Treatment ◽

Activated Sludge ◽

Nutrient Removal ◽

Wastewater Treatment Plants ◽

Amplicon Sequencing ◽

Temporal Variations ◽

Full Scale ◽

16S Rrna Amplicon Sequencing ◽

Specific Factors ◽

Operational Data

Since 2006 more than 50 Danish full-scale wastewater treatment plants with nutrient removal have been investigated in a project called ‘The Microbial Database for Danish Activated Sludge Wastewater Treatment Plants with Nutrient Removal (MiDas-DK)’. Comprehensive sets of samples have been collected, analyzed and associated with extensive operational data from the plants. The community composition was analyzed by quantitative fluorescence in situ hybridization (FISH) supported by 16S rRNA amplicon sequencing and deep metagenomics. MiDas-DK has been a powerful tool to study the complex activated sludge ecosystems, and, besides many scientific articles on fundamental issues on mixed communities encompassing nitrifiers, denitrifiers, bacteria involved in P-removal, hydrolysis, fermentation, and foaming, the project has provided results that can be used to optimize the operation of full-scale plants and carry out trouble-shooting. A core microbial community has been defined comprising the majority of microorganisms present in the plants. Time series have been established, providing an overview of temporal variations in the different plants. Interestingly, although most microorganisms were present in all plants, there seemed to be plant-specific factors that controlled the population composition thereby keeping it unique in each plant over time. Statistical analyses of FISH and operational data revealed some correlations, but less than expected. MiDas-DK (www.midasdk.dk) will continue over the next years and we hope the approach can inspire others to make similar projects in other parts of the world to get a more comprehensive understanding of microbial communities in wastewater engineering.

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Activated Sludge Microbial Community and Treatment Performance of Wastewater Treatment Plants in Industrial and Municipal Zones

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17020436 ◽

2020 ◽

Vol 17 (2) ◽

pp. 436 ◽

Cited By ~ 6

Author(s):

Yongkui Yang ◽

Longfei Wang ◽

Feng Xiang ◽

Lin Zhao ◽

Zhi Qiao

Keyword(s):

Wastewater Treatment ◽

Microbial Community ◽

Activated Sludge ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Treatment Plant ◽

Wastewater Sludge ◽

Community Diversity ◽

Nitrogen And Phosphorus ◽

Metabolic Functions

Controlling wastewater pollution from centralized industrial zones is important for reducing overall water pollution. Microbial community structure and diversity can adversely affect wastewater treatment plant (WWTP) performance and stability. Therefore, we studied microbial structure, diversity, and metabolic functions in WWTPs that treat industrial or municipal wastewater. Sludge microbial community diversity and richness were the lowest for the industrial WWTPs, indicating that industrial influents inhibited bacterial growth. The sludge of industrial WWTP had low Nitrospira populations, indicating that influent composition affected nitrification and denitrification. The sludge of industrial WWTPs had high metabolic functions associated with xenobiotic and amino acid metabolism. Furthermore, bacterial richness was positively correlated with conventional pollutants (e.g., carbon, nitrogen, and phosphorus), but negatively correlated with total dissolved solids. This study was expected to provide a more comprehensive understanding of activated sludge microbial communities in full-scale industrial and municipal WWTPs.

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Anaerobic co-digestion of sludge with other organic wastes and phosphorus reclamation in wastewater treatment plants for biological nutrients removal

Water Science & Technology ◽

10.2166/wst.2006.420 ◽

2006 ◽

Vol 53 (12) ◽

pp. 177-186 ◽

Cited By ~ 18

Author(s):

D. Bolzonella ◽

P. Pavan ◽

P. Battistoni ◽

F. Cecchi

Keyword(s):

Wastewater Treatment ◽

Activated Sludge ◽

Wastewater Treatment Plants ◽

Biogas Production ◽

Organic Wastes ◽

Organic Loading Rate ◽

Waste Activated Sludge ◽

Nutrients Removal ◽

Nitrogen And Phosphorus ◽

Biological Nutrients Removal

This paper deals with the performances obtained in full scale anaerobic digesters co-digesting waste activated sludge from biological nutrients removal wastewater treatment plants, together with different types of organic wastes (solid and liquid). Results showed that the biogas production can be increased from 4,000 to some 18,000 m3 per month when treating some 3–5 tons per day of organic municipal solid waste together with waste activated sludge. On the other hand, the specific biogas production was improved, passing from 0.3 to 0.5 m3 per kgVS fed the reactor, when treating liquid effluents from cheese factories. The addition of the co-substrates gave minimal increases in the organic loading rate while the hydraulic retention time remained constant. Further, the potentiality of the struvite crystallisation process for treating anaerobic supernatant rich in nitrogen and phosphorus was studied: 80% removal of phosphorus was observed in all the tested conditions. In conclusion, a possible layout is proposed for designing or up-grading wastewater treatment plants for biological nutrients removal process.

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The development and use of real-time PCR for the quantification of nitrifiers in activated sludge

Water Science & Technology ◽

10.2166/wst.2002.0488 ◽

2002 ◽

Vol 46 (1-2) ◽

pp. 267-272 ◽

Cited By ~ 14

Author(s):

S.J. Hall ◽

P. Hugenholtz ◽

N. Siyambalapitiya ◽

J. Keller ◽

L.L. Blackall

Keyword(s):

Wastewater Treatment ◽

Activated Sludge ◽

Real Time ◽

Real Time Pcr ◽

Wastewater Treatment Plants ◽

Quantitative Polymerase Chain Reaction ◽

Analytical Data ◽

Full Scale ◽

Rrna Gene ◽

Dna Templates

Chemical analytical data has long been used to monitor the performance of activated sludge plants even though the process relies on the performance of microorganisms. It is now evident that a rapid and reliable quantitative method is required, to be able to monitor the organisms responsible for nutrient transformation and their activities, allowing avenues for more efficient nutrient removal. The development of real-time or quantitative polymerase chain reaction (PCR) also known as TaqMan® or 5′-nuclease assay has allowed the rapid, quantitative analysis of DNA templates, eliminating some of the variability traditionally associated with other quantitative techniques. In this study analysis of Nitrospira spp., one of the key organisms in nitrite oxidation in wastewater treatment, was used to validate real-time PCR for the their quantification in activated sludge. A probe and primer set, targeting the 16S rRNA gene of Nitrospira spp. was designed according to the constraints of the TaqMan® specifications. Samples used to evaluate the method included DNA from the sludge from full-scale wastewater treatment plants and laboratory scale systems. The reproducibility, quantitative efficiency and specificity were assessed in the evaluation. It was concluded that the method is sensitive and reproducible but has some constraints on the quantitative efficiency. A survey of full-scale systems for Nitrospira spp. was carried out and the results are presented here.

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The limits and ultimate possibilities of technology of the activated sludge process

Water Science & Technology ◽

10.2166/wst.2008.545 ◽

2008 ◽

Vol 58 (8) ◽

pp. 1671-1677 ◽

Cited By ~ 3

Author(s):

A. F. van Nieuwenhuijzen ◽

A. G. N. van Bentem ◽

A. Buunnen ◽

B. A. Reitsma ◽

C. A. Uijterlinde

Keyword(s):

Wastewater Treatment ◽

The Netherlands ◽

Activated Sludge ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Treatment Method ◽

Activated Sludge Process ◽

Municipal Wastewater Treatment ◽

Nitrogen And Phosphorus ◽

Activated Sludge Processes

The (low loaded) biological nutrient removing activated sludge process is the generally accepted and applied municipal wastewater treatment method in the Netherlands. The hydraulical and biological flexibility, robustness and cost efficiency of the process for advanced removal of nutrients like nitrogen and phosphorus without (too much) chemicals results in a wide application of the activated sludge process within Dutch waterboards. Presumably, wastewater treatment plants will have to contribute to the improvement of the quality of the receiving surface waters by producing cleaner effluent. In this perspective, the Dutch research organisation STOWA initiated a research project entitled “The Boundaries of the Activated Sludge Process” to investigate the possibilities and limitations of activated sludge processes to improve the effluent quality. It is concluded that the activated sludge process as applied and operated at WWTP's in the Netherlands has the potential to perform even better than the current effluent discharge standards (10 mg Ntotal/l and 1 mg Ptotal/l). Reaching the B-quality effluent (<5mg Ntotal/l and <0.3 mg Ptotal/l) will be possible at almost all WWTPs without major adjustments under the conditions that: the sludge load is below 0.06 kg BOD/kg TSS.d the internal recirculation is above 20 the BOD/N ratio of the influent is above 3. Complying with the A-quality effluent (<2.2 Ntotal/l and <0.15 mg Ptotal/l) seems to be difficult (but not impossible) and requires more attention and insight into the activated sludge process. Optimisation measures to reach the A-quality effluent are more thorough and are mostly only achievable by additional construction works (addition of activated sludge volume, increasing recirculation capacity, etc.). It is furthermore concluded that the static HSA-results are comparable to the dynamic ASM-results. So, for fast determinations of the limits of technology of different activated sludge processes static modelling seems to by sufficient.

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16S rRNA gene-based primer pair showed high specificity and quantification accuracy in detecting freshwater Brocadiales anammox bacteria

FEMS Microbiology Ecology ◽

10.1093/femsec/fiaa013 ◽

2020 ◽

Vol 96 (3) ◽

Cited By ~ 4

Author(s):

Chang Ding ◽

Lorenz Adrian ◽

Yongzhen Peng ◽

Jianzhong He

Keyword(s):

Wastewater Treatment ◽

16S Rrna ◽

16S Rrna Gene ◽

Denaturing Gradient Gel Electrophoresis ◽

Wastewater Treatment Plants ◽

High Specificity ◽

Amplicon Sequencing ◽

Anammox Bacteria ◽

Rrna Gene ◽

Detection And Quantification

ABSTRACT Anaerobic ammonium oxidizing (anammox) bacteria are widely distributed and contribute significantly to the global nitrogen cycle. Traditionally, identification and quantification based on the 16S rRNA gene were considered not reliable because of low 16S rRNA gene sequence identity within Brocadiales. Here we hypothesize that by using appropriate primers and methodology, 16S-based detection and quantification of anammox bacteria can be accurate. We modified an existing 16S rRNA gene-based primer pair (Amx694F–Amx960R) by changing one nucleotide (Amx694F position 18, G→C) (Amx694PF–Amx960R) so that they match the sequences of most Brocadiales anammox bacteria, and evaluated the modified primer pair with 29 freshwater samples from microcosms, anammox reactors and wastewater treatment plants of various geographical origins. The primer pair showed high specificity in detection and quantification of anammox populations in samples that contained >0.1% anammox bacteria. Quantification of anammox abundance by quantitative real-time PCR and delineation of anammox species by denaturing gradient gel electrophoresis agreed well with amplicon sequencing results. A clear shift of anammox population towards ‘Candidatus Kuenenia’ was observed under laboratory cultivation conditions. With the help of amplicon sequencing, we demonstrated that 16S rRNA gene-based anammox-specific primers are able to achieve qualitative and quantitative monitoring of anammox communities in wastewater treatment plants and natural freshwater environments.2007;73:5261–7.

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