This may be the author’s version of a work that was submitted/accepted for publication in the following source: Nierychlo, Marta, McIlroy, Simon J, Kucheryavskiy, Sergey, Jiang, Chen- jing, Ziegler, Anja S, Kondrotaite, Zivile, Stokholm-Bjerregaard, Mikkel, & Nielsen, Per Halkjær (2020) Candidatus Amarolinea and Candidatus Microthrix are mainly responsible for filamentous bulking in Danish municipal wastewater treatment plants. Frontiers in Microbiology, 11, Article number: 1214. This file was downloaded from: https://eprints.qut.edu.au/205774/ c The Author(s) 2020 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. If the docu- ment is available under a Creative Commons License (or other specified license) then refer to the Licence for details of permitted re-use. It is a condition of access that users recog- nise and abide by the legal requirements associated with these rights. If you believe that this work infringes copyright please provide details by email to [email protected] License: Creative Commons: Attribution 4.0 Notice: Please note that this document may not be the Version of Record (i.e. published version) of the work. Author manuscript versions (as Sub- mitted for peer review or as Accepted for publication after peer review) can be identified by an absence of publisher branding and/or typeset appear- ance. If there is any doubt, please refer to the published source. https://doi.org/10.3389/fmicb.2020.01214 fmicb-11-01214 June 5, 2020 Time: 19:43 # 1 ORIGINAL RESEARCH published: 09 June 2020 doi: 10.3389/fmicb.2020.01214 Candidatus Amarolinea and Candidatus Microthrix Are Mainly Responsible for Filamentous Bulking in Danish Municipal Wastewater Treatment Plants Marta Nierychlo1, Simon J. McIlroy1,2, Sergey Kucheryavskiy3, Chenjing Jiang1, Anja S. Ziegler1, Zivile Kondrotaite1, Mikkel Stokholm-Bjerregaard1,4 and Per Halkjær Nielsen1* 1 Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark, 2 Australian Centre for Ecogenomics, The University of Queensland, Brisbane, QLD, Australia, 3 Section of Chemical Edited by: Engineering, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark, 4 Krüger A/S, Aalborg, Shan Yi, Denmark The University of Auckland, New Zealand Filamentous bulking is a common serious operational problem leading to deteriorated Reviewed by: Tomonori Kindaichi, sludge settling that has long been observed in activated sludge biological wastewater Hiroshima University, Japan treatment systems. A number of bacterial genera found therein possess filamentous Feng Ju, Westlake University, China morphology, where some have been shown to be implicated in bulking episodes (e.g., *Correspondence: Ca. Microthrix), the impact of many others is still not clear. In this study we performed Per Halkjær Nielsen a survey of 17 Danish municipal wastewater treatment plants (WWTPs) with nutrient [email protected] removal using 16S rRNA amplicon sequencing over a period of 13 years, where all Specialty section: known filamentous bacteria from 30 genera were analyzed. The filamentous community This article was submitted to constituted on average 13 ± 6%, and up to 43% of total read abundance with the Microbiotechnology, same genera common to all plants. Ca. Microthrix and several genera belonging to a section of the journal Frontiers in Microbiology phylum Chloroflexi were among the most abundant filamentous bacteria. The effect of Received: 11 March 2020 filamentous bacteria on sludge settling properties was analyzed using measurements Accepted: 12 May 2020 of the diluted sludge volume index (DSVI). Strong positive correlations with DSVI were Published: 09 June 2020 observed only for Ca. Microthrix and Ca. Amarolinea, the latter being a novel, recently Citation: Nierychlo M, McIlroy SJ, characterized genus belonging to the phylum Chloroflexi. The bulking potential of other Kucheryavskiy S, Jiang C, Ziegler AS, filamentous bacteria was not significant despite their presence in many plants. Low Kondrotaite Z, phylogenetic diversity was observed for both Ca. Microthrix and Ca. Amarolinea, making Stokholm-Bjerregaard M and Nielsen PH (2020) Candidatus physiological characterization of individual species and potential development of control Amarolinea and Candidatus strategies more feasible. In this study we show that, despite the high diversity of Microthrix Are Mainly Responsible for Filamentous Bulking in Danish filamentous phylotypes in Danish WWTPs, only few of them were responsible for severe Municipal Wastewater Treatment bulking episodes. Plants. Front. Microbiol. 11:1214. doi: 10.3389/fmicb.2020.01214 Keywords: filamentous bulking, activated sludge, 16S rRNA amplicon sequencing, Ca. Microthrix, Ca. Amarolinea Frontiers in Microbiology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 1214 fmicb-11-01214 June 5, 2020 Time: 19:43 # 2 Nierychlo et al. Bulking Filaments in Danish WWTPs INTRODUCTION et al., 2007a; Seviour and Nielsen, 2010), Candidatus Nostocoida (Schade et al., 2002), and several genera belonging to the phylum Filamentous bacteria are commonly observed in activated Chloroflexi (Bjornsson et al., 2002; Kragelund et al., 2007b; sludge in biological wastewater treatment plants (WWTPs) Seviour and Nielsen, 2010; Speirs et al., 2011; McIlroy et al., where they are suggested to importantly provide the structural 2016; Nittami et al., 2017). In more simple activated sludge backbone of well-settling flocs (Seviour and Nielsen, 2010). plants with carbon removal and nitrification (often serving However, overgrowth of some filamentous bacteria is associated specific industries), a number of other genera are commonly with deteriorated sludge settling, leading to sometimes serious encountered including gammaproteobacterial Thiothrix and operational problems known as sludge bulking (Liao et al., 2004; some alphaproteobacterial species, such as Neomegalonema Martins et al., 2004; Lou and de los Reyes, 2005; Vervaeren et al., (formerly Meganema)(Eikelboom and Geurkink, 2002; 2005). Some of those possessing hydrophobic cell surfaces can Kragelund et al., 2005). also cause foam on the surface of process and settling tanks. Studies of activated sludge microbial community composition Filamentous bulking and foaming have long been considered as can be carried out by 16S rRNA gene amplicon sequencing serious problems in biological wastewater treatment systems as but the identification of some abundant bacteria has sometimes they can lead to the carryover of solid particles in the effluent, been hindered by the lack of genus-level taxonomic classification hence reducing treatment efficiency, as well as having a negative and/or the lack of closely related reference sequences in public impact on receiving waters. A large fraction of Danish WWTPs databases (McIlroy et al., 2015, 2017). This is notably an issue for with nutrient removal (BNR) experiences temporal bulking the phylum Chloroflexi, which constitutes a significant fraction problems (Mielczarek et al., 2012) caused by excessive growth of of the filamentous community in Danish WWTPs (Mielczarek certain filamentous phylotypes. Despite the available knowledge et al., 2012), with most of the abundant phylotypes only of filamentous bacteria, it is not possible to predict bulking events classified to the family or order level. Development of ecosystem- and the available control strategies are not always effective. specific MiDAS taxonomy containing exclusively high-quality Identification of filamentous bacteria is a crucial first step for reference sequences from wastewater treatment and anaerobic successful physiological characterization, and selection of proper digesters ecosystem that employs robust taxonomic classification control measures. Traditionally, identification of filamentous (Dueholm et al., 2019; Nierychlo et al., in press) enables species- organisms in activated sludge has been based on microscopic level identification of most activated sludge microbes. The observation of distinct structural features of individual filaments combination of high-throughput sequencing technologies with and subsequent classification into a number of different the activated sludge ecosystem-specific database and taxonomy morphotypes (Eikelboom, 1975, 2000; Jenkins et al., 1993). enables detailed profiling of the total WWTP microbiome, However, it was recently shown that a single morphotype allowing the identification of statistical relationships between the can cover bacteria belonging to phylogenetically distinct abundant phylotypes and process performance parameters. groups that likely vary in their metabolic potential (Nielsen The aim of this study was to perform 16S rRNA gene amplicon et al., 2009a,b; Seviour and Nielsen, 2010). Thus morphotype- sequencing for the survey of the diversity, abundance, and based identification underestimates filament diversity, which distribution of known filamentous microorganisms in Danish may obscure the development and application of suitable full-scale WWTPs with biological nutrient removal (BNR), control strategies. and to investigate the relationship between the abundance of The development of culture-independent molecular methods specific filamentous species and sludge settling properties. The has facilitated more reliable identification and description of the comprehensive survey included more than 600 samples