This may be the author’s version of a work that was submitted/accepted for publication in the following source: McIlroy, SJ, Onetto, CA, McIlroy, B, Herbst, FA, Dueholm, MS, Kirkegaard, RH, Fernando, Warnakulasuriya Eustace Yrosh, Karst, SM, Nierychlo, M, Kristensen, JM, Eales, KL, Grbin, PR, Wimmer, R, & Nielsen, PH (2018) Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems. Frontiers in Microbiology, 9, Article number: 1004. This file was downloaded from: https://eprints.qut.edu.au/204131/ c 2018 The Author(s) This work is covered by copyright. Unless the document is being made available under a Creative Commons Licence, you must assume that re-use is limited to personal use and that permission from the copyright owner must be obtained for all other uses. 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If there is any doubt, please refer to the published source. https://doi.org/10.3389/fmicb.2018.01004 fmicb-09-01004 May 19, 2018 Time: 14:41 # 1 ORIGINAL RESEARCH published: 23 May 2018 doi: 10.3389/fmicb.2018.01004 Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems Simon J. McIlroy1, Cristobal A. Onetto2, Bianca McIlroy1, Florian-Alexander Herbst1, Morten S. Dueholm1, Rasmus H. Kirkegaard1, Eustace Fernando1, Søren M. Karst1, Marta Nierychlo1, Jannie M. Kristensen1, Kathryn L. Eales2, Paul R. Grbin2, Reinhard Wimmer1 and Per Halkjær Nielsen1* 1 Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark, 2 School of Agriculture, Food, and Wine, The University of Adelaide, Adelaide, SA, Australia Edited by: Sabine Kleinsteuber, Helmholtz-Zentrum für Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass Umweltforschung (UFZ), Germany through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the Reviewed by: Christopher Evan Lawson, activity of polyphosphate accumulating organisms (PAOs). However, several alternate University of Wisconsin–Madison, metabolic strategies, without polyphosphate storage, are possessed by other United States organisms, which can compete with the PAO for carbon at the potential expense of Ramesh K. Goel, The University of Utah, United States EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), *Correspondence: which utilize aerobically stored glycogen to energize anaerobic substrate uptake and Per Halkjær Nielsen storage. In full-scale systems the Micropruina spp. are among the most abundant of [email protected] the proposed GAO, yet little is known about their ecophysiology. In the current study, Specialty section: genomic and metabolomic studies were performed on Micropruina glycogenica str. This article was submitted to Lg2T and compared to the in situ physiology of members of the genus in EBPR plants Microbiotechnology, Ecotoxicology and Bioremediation, using state-of-the-art single cell techniques. The Micropruina spp. were observed to a section of the journal take up carbon, including sugars and amino acids, under anaerobic conditions, which Frontiers in Microbiology were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored Received: 05 December 2017 as glycogen for potential aerobic use. Fermentation was not directly demonstrated Accepted: 30 April 2018 Published: 23 May 2018 for the abundant members of the genus in situ, but was strongly supported by the Citation: confirmation of anaerobic uptake of carbon and glycogen storage in the absence McIlroy SJ, Onetto CA, McIlroy B, of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is Herbst F-A, Dueholm MS, Kirkegaard RH, Fernando E, markedly different from the classical GAO model. The amount of carbon stored by Karst SM, Nierychlo M, fermentative organisms has potentially important implications for phosphorus removal – Kristensen JM, Eales KL, Grbin PR, as they compete for substrates with the Tetrasphaera PAO and stored carbon is not Wimmer R and Nielsen PH (2018) Genomic and in Situ Analyses Reveal made available to the “Candidatus Accumulibacter” PAO under anaerobic conditions. the Micropruina spp. as Abundant This study shows that the current models of the competition between PAO and GAO Fermentative Glycogen Accumulating Organisms in Enhanced Biological are too simplistic and may need to be revised to take into account the impact of potential Phosphorus Removal Systems. carbon storage by fermentative organisms. Front. Microbiol. 9:1004. doi: 10.3389/fmicb.2018.01004 Keywords: activated sludge, EPBR, fermentation, Micropruina, GAO, PAO Frontiers in Microbiology| www.frontiersin.org 1 May 2018| Volume 9| Article 1004 fmicb-09-01004 May 19, 2018 Time: 14:41 # 2 McIlroy et al. Characterization of the Micropruina GAO INTRODUCTION anaerobic carbon storage compounds are reported for full-scale activated sludge, including triacylglycerols (TAGs), gamma- Enhanced biological phosphorus removal (EBPR) activated aminobutyric acid (GABA) as well as intracellular pools of sludge systems have been widely implemented for the removal of non-polymerized fermentation by-products, long chain fatty nutrients from wastewaters. Phosphorus (P) removal is achieved acids, amino acids, and trehalose (Satoh et al., 1998; Santos in these systems by cycling of the biomass through carbon-rich et al., 1999; Kristiansen et al., 2013; McIlroy et al., 2013; (feast) anaerobic and carbon-deficient (famine) conditions Nguyen et al., 2015; Marques et al., 2017). Some organisms to encourage the activity of the so-called polyphosphate exhibiting the classical GAO and PAO phenotypes reportedly accumulating organisms (PAOs) [see Oehmen et al.(2007) for utilize sugars and amino acids directly for PHA production review]. (Liu et al., 1996; Burow et al., 2007; Oyserman et al., 2015) Classical models for the PAO phenotype stipulate that and some may also ferment glycogen stores or glucose to aerobically stored polyphosphate provides energy for lactate as an additional anaerobic energy source (McIlroy et al., anaerobic uptake and storage of volatile fatty acids (VFAs) 2014). Most notably, the Tetrasphaera spp. and Microlunatus as polyhydroxyalkanoates (PHAs). Hydrolysis of aerobically phosphovorusT, both within the phylum Actinobacteria, cycle stored glycogen, and activity of the tricarboxylic acid (TCA) polyphosphate without PHA storage with dynamic feast–famine cycle provides required reducing power and additional conditions. Instead, these organisms exhibit a fermentative energy. Stored PHAs are utilized under subsequent aerobic metabolism where polyphosphate supplements anaerobic conditions supporting growth and replenishing glycogen and energy demands (Nakamura et al., 1995; Kong et al., 2005; polyphosphate stores, with wastage of aerobic biomass giving Kristiansen et al., 2013). Importantly, surveys of full-scale net P removal (Comeau et al., 1986; Wentzel et al., 1986: systems revealed that the Tetrasphaera spp. are in much higher Mino et al., 1987). Such a phenotype has been demonstrated abundance than the “Ca. Accumulibacter” PAO, questioning in the widely studied “Ca. Accumulibacter” genus within the the long-held belief that the latter classical-PAO are the most Betaproteobacteria (Hesselmann et al., 1999; He and McMahon, important for EBPR (Nguyen et al., 2011; Mielczarek et al., 2011). 2013a; Saunders et al., 2016) and reinforcing the need to Although EBPR systems are considered as an economical consider diverse physiologies for organisms important for strategy for wastewater treatment, they are subject to periods EBPR. of inefficiency and failure. Deterioration of EBPR has been In addition to the fermentative actinobacterial PAO, it has attributed to a variety of conditions; such as high rainfall, been shown that in dynamic feast–famine systems unidentified nutrient limitation, and high nitrate loading to the anaerobic organisms can store glucose directly as glycogen anaerobically, zone (Oehmen et al., 2007). Another proposed reason is energized by fermentation, without cycling polyphosphate microbial competition, where the proliferation of organisms (Carucci et al., 1999). The role of these “fermentative GAO” competing for anaerobic carbon supply, without excess in EBPR is of interest, given their potential as competitors of polyphosphate storage, is at the theoretical expense of P removal the abundant fermentative Tetrasphaera PAO. A likely candidate efficiency (Satoh et al., 1994). Bacteria with the glycogen for this phenotype is the activated sludge isolate Micropruina accumulating organism (GAO) phenotype have received glycogenicaT – a member of the family Propionibacteriaceae considerable attention as potential