water Article Low-Temperature Adapted Nitrifying Microbial Communities of Finnish Wastewater Treatment Systems Antonina Kruglova 1,* , Jenni Kesulahti 1, Khoi Minh Le 1, Alejandro Gonzalez-Martinez 2, Anna Mikola 1 and Riku Vahala 1 1 Department of Built Environment, Aalto University, FI-00076 AALTO, Tietotie 1E, P. O. Box 15200, 02150 Espoo, Finland; [email protected] (J.K.); [email protected] (K.M.L.); anna.mikola@aalto.fi (A.M.); riku.vahala@aalto.fi (R.V.) 2 Institute of Water Research, University of Granada, 18071 Granada, Spain; [email protected] * Correspondence: antonina.kruglova@aalto.fi Received: 30 June 2020; Accepted: 26 August 2020; Published: 31 August 2020 Abstract: In this study, the microbial community of nitrifying activated sludge adapted to Finnish climate conditions was studied to clarify the microbial populations involved in low-temperature nitrification. Microbial community analysis of five full-scale wastewater treatment plants (WWTPs) showed several differences compared to WWTPs from other countries with a similar climate. In particular, very low abundance of ammonium oxidizing bacteria (AOBs) (altogether < 0.25% of total community) as well as typical NOBs (<0.35%) and a high abundance of orders Cytophagales and Micrococcales was observed in all Finnish WWTPs. To shed light on the importance of autotrophic and heterotrophic nitrifying processes, laboratory studies of activated sludge were carried out with a presence of and a lack of organic carbon in wastewater at 10 1 C. Two different sludge retention ± ◦ times (SRTs) were compared to determine the effect of this operational parameter on low-temperature nitrogen removal. The important role of previously reported Candidatus Nitrotogaarctica for nitrite oxidizing in cold climate conditions was confirmed in both full-scale and laboratory scale results. Additionally, potential participation of Dokdonella sp. and Flexibacter sp. in nitrogen removal at low-temperatures is proposed. Operation at SRT of 100 days demonstrated more stable and efficient nitrogen removal after a sharp temperature decrease compared to 14 days SRT. Keywords: nitrogen removal; activated sludge; ammonium oxidizing bacteria; nitrogen oxidizing bacteria; low temperature 1. Introduction Today, the main responsibility of conventional municipal wastewater treatment systems is to remove organic nutrients such as nitrogen and phosphorus from wastewaters in order to prevent environmental pollution due to human activities. Conventional biological nitrogen removal, based on nitrification and denitrification, is an underlying process in the majority of wastewater treatment plants (WWTPs). The stable and high efficiency of these WWTPs depends on the microbial composition and diversity of functional microbial groups in activated sludge [1]. Consequently, design and operational control of existing WWTPs are based on the gained knowledge of the optimal parameters for the functional microbial communities. A successful nitrification (the oxidation of ammonia to nitrite, and further to nitrate) is traditionally related to sequential activity of ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB, respectively) [2]. Temperature is considered to be one of the key factors affecting growth of the most-described AOBs and NOBs with an optimum value being around 28 ◦C, however cold-adapted AOB and NOB species have been reported in several studies [3,4]. Water 2020, 12, 2450; doi:10.3390/w12092450 www.mdpi.com/journal/water Water 2020, 12, x FOR PEER REVIEW 2 of 17 value being around 28 °C, however cold-adapted AOB and NOB species have been reported in several studies [3,4]. Water 2020, 12, 2450 2 of 16 In Finland, average the temperature of wastewater is in the range of 5–12 °C during most of the year and 14–21 °C in the shorter and warmest period, whilst stable and efficient nitrification can be reachedIn Finland, at down average to 8 °C [5,6]. the temperature There are only of wastewatera few studies is on in activated the range sludge of 5–12 microbial◦C during communities most of the yearoperated and 14–21at this◦ Ctemperature in the shorter range. and warmestAccording period, to Gonzalez-Martinez whilst stable and eetffi ciental. [7], nitrification the diversity can beof reachedbacteria atand down archaea to 8 ◦inC[ WWTPs5,6]. There decreases are only with a few the studies decrease on of activated operational sludge temperature. microbial communities Decrease of operatedAOB and atammonium this temperature oxidizing range. archaea According (AOA) diversities to Gonzalez-Martinez due to a low temperature et al. [7], the effect diversity was also of bacteriareported and by Urakawa archaea in et WWTPs al. [8]. Accordingly, decreases with low the conc decreaseentrations of operational of conventional temperature. nitrifiers Decrease have been of AOBdetected and in ammonium activated sludge oxidizing of WWT archaeaPs in (AOA) northern diversities Finland dueas well to a as low in pilot temperature WWTPs eoperatedffect was at also 10 reported± 2 °C [7,9]. by Urakawa Unknown et al.temperature-tolerant [8]. Accordingly, low AOA concentrations and heterotrophic of conventional nitrifiers nitrifierswere proposed have been as detectedpossible contributors in activated sludge[9,10]. However, of WWTPs at inthe northern moment Finland there is asno well available as in pilotdata WWTPson the key operated nitrifying at 10bacteria2 C[ and7,9 archaea]. Unknown of low-temperature temperature-tolerant adapted AOA municipal-activated and heterotrophic sludge nitrifiers and were thus proposed operational as ± ◦ possibleparameters contributors of WWTPs [9 ,are10]. not However, optimized at the for moment cold regions. there is At no the available same time, data onKruglova the key et nitrifying al. [5,6] bacteriareportedand high archaea sensitivity of low-temperature of nitrifying processes adapted municipal-activatedto temperature fluctuation sludge andat typical thus operational operation parametersconditions ofwhile WWTPs operation are not at optimized longer sludge for cold retention regions. Attime the same(SRT) time, increased Kruglova the etresistance al. [5,6] reported of the highnitrifying sensitivity community of nitrifying to low-temperature processes to temperature stress accompanied fluctuation by at microbial typical operation population conditions shifts. while operationThe objectives at longer sludgeof this retention research timewere (SRT) to stud increasedy the microbial the resistance community of the nitrifying structure community of activated to low-temperaturesludge adapted to stress Finnish accompanied climate conditions. by microbial Clar populationifying the shifts.groups of microorganisms, which are importantThe objectives for low-temperature of this research werenitrification, to study thecan microbial help to community improve structureday-to-day of activatedoperation sludge and adaptedtroubleshooting to Finnish of climatethe activated conditions. sludge Clarifying processes the at groups WWTPs. of microorganisms, To shed light on which the importance are important of forautotrophic low-temperature and heterotrophic nitrification, nitrifying can help processes, to improve laboratory day-to-day studies operation were carried and troubleshooting out with the ofpresence the activated and lack sludge of organic processes carbon at WWTPs. in wastewat To sheder. In light parallel, on the experiments importance with of autotrophic long SRT were and heterotrophicperformed in nitrifyingorder to processes,evaluate whether laboratory optimizi studiesng were this carried operational out with parameter the presence could and stabilize lack of organicefficient carbonnitrogen in removal wastewater. in WWTPs In parallel, in cold experiments regions. with long SRT were performed in order to evaluate whether optimizing this operational parameter could stabilize efficient nitrogen removal in WWTPs2. Material in coldand regions.Methods 2.2.1. Material Sampling and Methods 2.1. SamplingThe full-scale activated sludge samples were obtained from five large highly nitrifying WWTPs of Finland. The laboratory low-temperature-adapted activated sludge was obtained from four pilot reactorsThe full-scaleoperated activatedin water sludge laboratory samples of wereAalto obtained University, from fiveEspoo. large The highly sampling nitrifying locations WWTPs are of Finland.presented The in laboratoryFigure 1. low-temperature-adapted activated sludge was obtained from four pilot reactors operatedThe inmain water characteristics laboratory of of Aalto WWT University,Ps are presented Espoo. The in Table sampling 1. locations are presented in Figure1. Figure 1. Sampling locations. 1–5–full-scale wastewater treatment plants (WWTPs), 6–9 pilot reactors. Water 2020, 12, 2450 3 of 16 The main characteristics of WWTPs are presented in Table1. Table 1. General characteristics of studied wastewater treatment plants (WWTP1-5). Name WWTP1 WWTP2 WWTP3 WWTP4 WWTP5 Sample Location Helsinki Porvoo Hyvinkää Turku Tampere Spring 11.5 1 6.9 0.5 8.6 0.5 11 0.5 15 0.5 T ( C) ± ± ± ± ± month.ave ◦ Summer 17.1 0.5 17 0.5 14.5 0.5 17.9 0.5 20.5 0.5 ± ± ± ± ± Q ave Spring 280,000 16,000 112,000 180,000 85,000 (m3/day) Summer 280,000 10,000 9500 60,000 75,000 Spring 6.1 7.1 7.3 7.4 7.5 pH Summer 6.2 7.1 7.4 7.3 7.4 BOD7ATU Spring 350 370 190 220 100 (mg/L) Summer 320 270 200 360 310 Influent Spring 570 780 450 550 810 COD (mg/L) characteristics Cr Summer 660 580 650 750 540 Spring 51 46 48 43 61 N (mg/L) tot Summer 60 55 52 76 54 Spring 35 27 45 26 28 NH -N