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If there is any doubt, please refer to the published source. https://doi.org/10.1016/j.copbio.2019.03.008 Available online at www.sciencedirect.com ScienceDirect Re-evaluating the microbiology of the enhanced biological phosphorus removal process Per Halkjær Nielsen, Simon J McIlroy, Mads Albertsen and Marta Nierychlo We have critically assessed some of the dogmas in the agriculture or the P can be released as orthophosphate, often microbiology of enhanced biological phosphorus removal in an anaerobic digester, and precipitated, for example, as (EBPR) and argue that the genus Tetrasphaera can be as struvite, and applied as fertilizer [4]. important as Ca. Accumulibacter for phosphorus removal; and that proliferation of their competitors, the glycogen Ever since the EBPR process was first described by James accumulating organisms, does not appear to be a practical Barnard in South Africa in the 70s [6], the PAOs and their problem for EBPR efficiency even under tropical conditions. An proposed competitors, the glycogen accumulating organisms increasing number of EBPR-related genomes are changing our (GAOs),havebeenintensivelystudiedandtheir microbiology understanding of their physiology, for example, their potential considered reasonably well described. Studies on organisms to participate in denitrification. Rather than trying to identify thought tobe importantforEBPR have historicallyfocusedon organisms that adhere to strict phenotype metabolic models, populations that appear to adhere to the canonical PAO and we advocate for broader analyses of the whole microbial GAO phenotypes that are enriched in volatile fatty acids communities in EBPR plants by iterative studies with isolates, (VFA) fed lab-scale systems where the community composi- lab enrichments, and full-scale systems. tion is often not determined. However, full-scale systems Address provide substantially more complex substrate compositions Center for Microbial Communities, Department of Chemistry and and growth environments, providing diverse niches for organ- Bioscience, Aalborg University, Aalborg, Denmark isms. Many of the proposed PAO and GAO are found to be absent or present only in low abundance in full-scale systems Corresponding author: Nielsen, Per Halkjær ([email protected]) [7 ]. Moreover, several variations of the classical phenotypes have been observed that question our common understanding Current Opinion in Biotechnology 2019, 57:111–118 of the microbiology and ecology of the EBPR process. The This review comes from a themed issue on Environmental aim of this review is to outline the new studies and critically biotechnology assess some common dogmas in EBPR microbiology related Edited by Lutgarde Raskin and Per Halkjær Nielsen to the role of PAOs and GAOs in full-scale EBPR plants. For a complete overview see the Issue and the Editorial Available online 5th April 2019 Which PAOs are important to full-scale EBPR? https://doi.org/10.1016/j.copbio.2019.03.008 Most studies in the field of EBPR microbiology have con- 0958-1669/ã 2019 The Authors. Published by Elsevier Ltd. This is an centrated on the canonical model PAO Candidatus Accumu- open access article under the CC BY-NC-ND license (http://creative- libacter. This genus is commonly found in full-scale plants commons.org/licenses/by-nc-nd/4.0/). and is relatively easy to enrich in VFA fed lab-scale reactors. Assuch,Ca.Accumulibacterhaslongbeenassumedtobethe most important of the known PAOs, yet very few studies have concurrently assessed the abundance of the several populations proposed to possess the phenotype in full-scale plants [8 ,9,10]. Our comprehensive surveys of more than Introduction 20 full-scale Danish wastewater treatment plants (WWTP) Wastewater treatment using the enhanced biological phos- over several years with both Fluorescence in situ Hybridiza- phorus removal (EBPR) process is becoming increasingly tion (FISH) and 16S rRNA gene amplicon sequencing popular because the demand for achieving low effluent [7 ,11] clearly show that although Ca. Accumulibacter is phosphorus (P) concentrations without addition of chemical common (mean abundance 0.5%), members of the actino- precipitants is increasing across the world. The EBPR bacterial Tetrasphaera PAO are often observed in higher process relies on polyphosphate accumulating organisms abundances. Our subsequent survey of 32 full-scale EBPR (PAOs) which take up and store excessive amounts of P, plants in 12 countries (covering 5 continents) (Figure 1) and compared to chemical precipitation, EBPR is a sustain- supports a higher abundance of Tetrasphaera in most plants able, effective, and economical process [1–4]. Furthermore, globally. There are examples of plants where Tetrasphaera it is a suitable process for recovery of P, which is a limited are absent or present in low abundance, primarily pilot-scale non-renewable resource, particularly important as fertilizer experiments [8 ], but this could partly be due to extraction [5]. The P-rich biomass can be used directly as a fertilizer in bias as this genus is underestimated by standard DNA www.sciencedirect.com Current Opinion in Biotechnology 2019, 57:111–118 112 Environmental biotechnology Figure 1 Actinobacteria; Tetrasphaera Proteobacteria; Dechloromonas Proteobacteria; Candidatus Accumulibacter %Read Actinobacteria; Tessaracoccus Abundance 10.0 Actinobacteria; Microlunatus Cyanobacteria; Candidatus Obscuribacter 1.0 Gemmatimonadetes; Gemmatimonas 0.1 Proteobacteria; Pseudomonas Proteobacteria; Candidatus Accumulimonas Actinobacteria; Friedmanniella m Asia ChinaAsia Japan EuropeEurope Poland Austria nited Kingdo Europe Portugal Denmark MiDAS Asia South Korea Australia Australia Africa SouthNorth AmericaAfrica USA urope U EuropeE The Netherlands Current Opinion in Biotechnology Relative read abundance of all putative PAOs [7 ] in full-scale activated sludge WWTPs across 12 countries from 5 continents. Average values for each country (except Denmark) are based on 3 to 9 samples from 2 to 4 EBPR plants collected in years 2011-2014. PAO abundance in Denmark is an average of 18 full-scale WWTPs; data sourced from MiDAS survey [64]. extraction procedures [12]. Interestingly, the dominant quantification of the intracellular content of poly-P, OTUs (97% identity) from both Tetrasphaera and Ca. Accu- polyhydroxyalkanoates (PHA), and glycogen [16 ]. mulibacter were the same in most plants across the world. Interestingly, the cellular content of poly-P in Ca. Accu- However, there may be significant microdiversity within the mulibacter was approx. 3 times higher than in Tetra- dominant OTUs as use of the ppk1 gene as phylogenetic sphaera, when considered per cell, but very similar per À13 À3 marker for Ca. Accumulibacter generally reveals large diver- biovolume (0.7 Â 10 gP mm ), as the cell volume of sity in studies around the globe [10,13–15]. Other putative Tetrasphaera is 3–5 times smaller. A P-mass balance on PAOs consistently found in a relatively high abundance in biomass from the aeration tank, which contained the some plants included the genera Dechloromonas and Tessar- highest amount of intracellular P, showed that both acoccus, although their abundance appears to be overesti- Tetrasphaera and Ca. Accumulibacter made a significant mated by sequencing based methods (possibly due to known contribution to P-removal. Also, an enriched lab-scale biases with extraction efficiency, primer choice, and copy culture of Tetrasphaera was able to carry out at least 80% number [12]), and they likely play a minimal role in P of the P-removal [19], supporting the suggestion that removal [7 ]. To date, the genera Tetrasphaera and Ca. both Ca. Accumulibacter and Tetrasphaera are important Accumulibacter appear to be the only known PAOs to be for P-removal in full-scale EBPR plants. consistently found in abundances where they could be considered critical to P removal performance. However, Are GAOs really problematic in full-scale several unknown bacteria are observed to contain excess EBPR plants? polyphosphate (poly-P) reserves and future work should GAOs are thought to be a cause of EBPR failure in focus