Correlation of Seasonal Nitrification Failure and Ammonia-Oxidizing Community Dynamics in a Wastewater Treatment Plant Treating Water from a Saline Thermal Spa

Ann Microbiol (2014) 64:1671–1682 DOI 10.1007/s13213-014-0811-5

ORIGINAL ARTICLE

Correlation of seasonal nitrification failure and ammonia-oxidizing community dynamics in a wastewater treatment plant treating water from a saline thermal spa

Luciano Beneduce & Giuseppe Spano & Francesco Lamacchia & Micol Bellucci & Francesco Consiglio & Ian M. Head

Received: 21 August 2013 /Accepted: 9 January 2014 /Published online: 24 January 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract In this work we evaluated the effect of a perturba- sampling period. This study reports a clear association of tion on nitrification performance in a wastewater treatment microbial community dynamics (strongly correlated to salin- plant (WWTP) treating urban and saline thermal bath waste- ity, temperature, and dissolved oxygen) to nitrification perfor- water, which regularly occurred during summer months. We mance. Particularly, ammonia-oxidizing bacteria are severely wanted to find out if this related to changes in the ammonia affected by drastic changes in operational conditions, with oxidizing communities. The bacterial and ammonia-oxidizing direct consequences on WWTP performance. bacterial (AOB) community from three different basins of the WWTP were evaluated using PCR-DGGE and cloning and Keywords Activated sludge . DGGE (denaturing gradient gel sequencing of 16S rRNA gene fragments, over a six month electrophoresis) . Diversity . Nitrification . AOB . Wastewater survey. Both eubacterial and AOB communities underwent treatment continuous change over time, with a particularly prominent shift between the third and fourth month of monitoring for eubacteria and the fourth month and fifth month for AOB. At Introduction the same time, reduction of nitrification performance was observed in the WWTP. The AOB community in the activated Nitrification in wastewater treatment plants (WWTP) is main- sludge was dominated by clones with high 16S rRNA se- ly driven by the activity of aerobic, chemolithoautotrophic quence identity to halophilic-halotolerant organisms from the ammonia oxidizing bacteria (AOB) and nitrite-oxidizing bac- Nitrosomonas oligotrohpa and Nitrosomonas marina clusters. teria (NOB). The former are typically considered rate limiting A significant correlation (R=0.97) was detected between the for nitrification, since AOB are slow growing and more sen- structure of the AOB community and environmental parame- sitive to environmental factors such as dissolved oxygen (DO) ters, indicating that the AOB community structure changed in content, salinity, light and substrate concentration (Wagner line with the environmental changes that took place over the et al. 1995; Koops et al. 2003; Geets et al. 2006). Failure of nitrogen removal from sewage in WWTP can dramatically : : L. Beneduce (*) G. Spano F. Lamacchia affect surface water environments receiving final effluent, Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, leading to eutrophication and other ecological risks (Painter Università di Foggia, via Napoli 25, 71122 Foggia, Italy 1986; Philips et al. 2002;Jensen2003). e-mail: [email protected] AOB form two evolutionary distinct groups. The first falls : L. Beneduce M. Bellucci within the Gammaproteobacteria and comprises the STAR* Agroenergy Research Group, Università di Foggia, Foggia, Nitrosococcus species, and the second forms a monophyletic Italy group within the Betaproteobacteria, with the genera F. Consiglio Nitrosomonas and Nitrosospira (Purkhold et al. 2000). More Acquedotto Pugliese S.p.A. U.C.I.S. compartimento di Foggia, recently, ammonia-oxidizing Archaea have been detected in Foggia, Italy WWTP (Park et al. 2006); however, their significance in nitrogen removal in wastewater treatment remains open to I. M. Head School of Civil Engineering and Geosciences, Newcastle University, debate (Wells et al. 2009;YeandZhang2011). In addition, Drummond Building, Newcastle upon Tyne, NE1 7RU England, UK a further group belonging to the order Planctomycetales is 1672 Ann Microbiol (2014) 64:1671–1682 associated with the anaerobic ammonia oxidation (Anammox) conducted a survey of AOB communities and nitrification process (Jetten et al. 2001). performance over a 6-month period in which the salinity of The phylogeny of AOB has been described on the basis of the wastewater increased by 30 %. comparative 16S rRNA sequence analysis (Head et al. 1993; The aim of this study was to establish the effect of this Teske et al. 1994;Purkholdetal.2000). To date, all AOB perturbation on nitrification performance and whether this isolated from soil and freshwater habitats belongs to the related to changes in the AOB communities. During the 6- Betaproteobacteria, whereas AOB belonging to month survey the nitrogen removal performance was severely Gammaproteobacteria have only been isolated from marine compromised by environmental conditions. and other saline environments (Ward and O’Mullan 2002). Culture based studies of the diversity and ecology of AOB in natural and engineered systems are severely hampered by Materials and methods the slow growth rates and autotrophic metabolism of AOB, which are easily outcompeted by heterotrophic organisms Sampling (Watson et al. 1989; Wagner et al. 1993). Analysis of 16S rRNA genes and genes encoding the alpha Sludge samples were collected from an activated sludge subunit of ammonia monooxygenase (amoA), Fluorescence wastewater treatment plant, located in Margherita di Savoia In-Situ Hybridization (FISH) and denaturing gradient gel (Apulia, Italy), treating urban and saline spa wastewater. The electrophoresis (DGGE) have been used widely to analyse WWTP consisted of a primary settler, a pre-denitrification AOB populations and communities in many environments basin with recirculation from oxidation/nitrification, an acti- and have led to many aspects of the ammonia oxidizing vated sludge oxidation/nitrification basin and a secondary bacterial community ecology being unravelled in detail. settler. The average hydraulic retention time was 12.5 h and AOB community composition and diversity in natural and the average solid retention time was 8.4 days. engineered systems have been related to variables such as Sampling was conducted at intervals of 30 days, over a ammonia content (Lydmark et al. 2007), salinity (Freitag period of six months, from March to August. A total of six et al. 2006), type of substratum (Gorra et al. 2007), DO time-weighted (12 h) average samples were taken in triplicate concentrations (Bellucci et al. 2011), reactor design (Rowan from the pre-denitrification basin (DEN), oxidation basin et al. 2003) and the presence of heavy metals (Principi et al. (OXI) and recirculating sludge from the secondary settler 2009). Moreover, characterization of the AOB community (RIC), respectively. After sampling, sludge samples were composition and diversity, distribution patterns and ecophys- frozen at −20 °C and transported to the laboratory for DNA iology has been reviewed (Koops et al. 2003) while 16S extraction. The pH, conductivity and DO concentration were rRNA and amoA based phylogeny has been extended and determined directly at the time of sampling. Total suspended improved (Purkhold et al. 2003). solids (TSS), nitrite, nitrate and ammonium content were Correlations among the structure and functional stability of assayed on unfrozen aliquots of the same samples used for AOB communities in WWTPs, physicochemical parameters microbial community analysis. and efficiency of bioreactors have been postulated and investigated by many authors (Wittebolle et al. 2005, 2008; DNA extraction and PCR amplification Siripong and Rittman 2007;Belluccietal.2013). However, many aspects still remain to be ascertained. For instance, the Ten milliliters of mixed liquor samples were centrifuged at role of functional redundancy, which may allow communities 10,000 g for 2 min and the supernatant was discarded. Nucleic to maintain physiological capabilities while their composition acids extraction from 0.25 g of sludge samples was carried out changes in response to environmental conditions, is not yet with a Power Soil DNA extraction kit (MoBio, Carlsbad, CA well understood. – USA) according to the manufacturer’s protocol. Each DNA In this paper we monitored bacterial and AOB community extraction was performed in duplicate, the amount of DNA composition and diversity in a wastewater plant treating a was estimated by visualization on a 1 % agarose gel stained mixture of urban wastewater and wastewater from a thermal with ethidium bromide, and the molecular weight was esti- bath (spa). mated by comparison with Gelpilot 100 bp plus ladder This WWTP is regularly subject to an increase in salinity, (Qiagen, Milan, Italy) used as a DNA molecular weight mark- temperature and reduced dissolved oxygen content every year er. Extracted DNA concentration was adjusted to 10 ng μl−1 during the summer months. The changes in salinity in the with sterile deionised water and stored at −20 °C until wastewater resulting from seasonal changes in the contribu- analysis. tion from the spa bath waters provide an excellent opportunity To study total Bacteria and AOB communities, a separate to assess the effects of short term salinity changes on AOB primer set and PCR protocols were used. PCR amplification communities and nitrification performance. We, therefore, of eubacterial 16S rRNA gene fragments was performed using Ann Microbiol (2014) 64:1671–1682 1673

Primer 357f-GC (5′-CGC CCG CCG CGC GCG GCG GGC acrylamide solution (final volume ca. 15.0 ml). Gels were run GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG at 60 °C in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA, CAG CAG-3′) and 518r (5′-ATTACC GCG GCT GCT GG- pH 8.3) for 900 V hour. A gradient of 30 % to 55 % denaturant 3′) (Muyzer et al. 1993). PCR reaction was carried out in a was used to obtain the best resolution of DNA fragments on total volume of 50.0 μl, containing 1.0 μl of DNA, 40 pmol the gel. Gels were stained with 1× SYBR green solution each of forward and reverse primers, 2.0 units of Taq DNA (Flucka Biockemika, Buchs, Switzerland) in 1× TAE for polymerase, (Qiagen), 1.5 mM MgCl2, 1× buffer (supplied by 40 min. Thereafter, gels were visualised on a Versadoc imag- the enzyme manufacturer), 10 nmol each of desoxynucleoside ing system (Bio-Rad), and images were acquired and convert- triphosphate and molecular biology grade water up to the final ed to TIFF format for subsequent analyses. volume. Ammonia-oxidizing Betaproteobacteria were analysed using primers CTO189f (5′- GGA GRA AAG YAG GGG Cloning of PCR amplified 16S rRNA gene fragments ATC G-3′) and CTO654r (5′-CTA GCY TTG TAG TTT CAA ACG C-3′). A nested approach was used for DGGE analysis Aliquots of PCR-amplified AOB 16S rRNA gene fragments of AOB PCR products, using primers 357f-GC and 518r in a from samples OXI2 and OXI5 were cloned with the p-GEM-T second amplification step (Kowalchuk et al. 1997). easy vector cloning kit (Promega, France) according to man- ’ Ammonia-oxidizing Arcahea were checked by PCR using ufacturer s instructions. Competent DH10B Escherichia coli primers Arch-amoAF (5′-STAATGGTCTGGCTTAGACG- cells were transformed with ligated PCR products. ′ ′ Transformed cells were screened on Luria-Bertani (LB) 3 ) and Arch-amoAR (5 -GCGGCCATCCATCTGTATGT- −1 3′) targeting the archaeal AmoA gene and using the PCR Agar plates containing 0.1 mM IPTG, 25 mg ml X-Gal μ −1 protocol previously reported by Francis et al. (2005). and 100 gml ampicillin. White colonies were spread on Primers were purchased from Sigma Proligo (Paris, France). LB agar plates containing ampicillin, and subsequently a Each PCR reaction was carried out in a 50.0 μl volume, single colony from each plate was used for plasmid extraction containing 3.0 μl of DNA, 20 pmol each of forward and with a Qiaprep spin miniprep kit (Qiagen). In order to screen reverse primers, 1.5 units of Taq DNA polymerase, for the presence of inserts of the expected size, aliquots of extracted plasmids were used as a DNA template for PCR (Qiagen), 1.5 mM MgCl2, 1× buffer, 10 nmol each of desoxynucleoside triphosphate and molecular biology grade reaction, following the same protocol as reported for AOB water up to final volume. An iCycler thermal cycler (Bio-Rad, PCR. Then, 30 clones were randomly selected for each sample Hercules, CA, USA) was used for all PCR reactions. PCR and screened by DGGE to ascertain the presence of different conditions for bacterial 16S rRNA were as follows: denatur- clone types by comparing migration distances. Thirty clones ationat95°Cfor2min,then24cyclesofdenaturationat per library with the correct fragment size insert were subjected – 95 °C for 1 min, annealing at 65 °C for 1 min (temperature to PCR with primers 357fGC 518r, and PCR products were was reduced by 1 °C at each cycle), and extension at 72 °C for screened by DGGE. After evaluation of a different migration 1 min. Finally, 15 cycles of 95 °C for 1 min, 53 °C for 1 min, pattern of the single band generated by each clone from the 72 °C for 1 min, and final extension at 72 °C for 10 min were environmental samples, 30 clones were selected for done. The PCR condition for beta-proteobacterial AOB were: sequencing. denaturation at 95 °C for 2 min, then 30 cycles of denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min and extension Sequence analyses at 72 °C for 1 min, then final extension at 72 °C for 10 min. Size and quality of all PCR products were analysed by Cloned 16S rRNA gene fragments (n=30) were sequenced agarose gel electrophoresis (2 % agarose). All PCR analyses with an ABI 310 DNA sequencer (Applied Biosystems, were conducted in triplicate in order to assess the reproduc- Foster City, CA, USA). The most closely matching sequences ibility of the results. in the EMBL-EBI nucleotide database (European Bioinformatics Institute, http://www.ebi.ac.uk)were DGGE determined using the FASTA algorithm, specifically the FASTA33 program. Phylogenetic analyses were conducted Denaturing gradient gel electrophoresis analyses were con- using MEGA version 4.1 (Tamura et al. 2007). ducted using a D-code universal mutation detection system (Bio-Rad). Ten percent polyacrylamide gels (10 % polyacrylamide, 1.0 mm thick, 16×16 cm) were cast using a Bio-Rad Nucleotide sequence accession number gradient maker and a peristaltic pump, with a flow rate of 2.5 ml min−1. For gel polymerization, 15.0 μl of TEMED and Nucleotide sequences are available at the following GenBank 150.0 μl of 10 % ammonium persulfate were added to each accession number: from FN552767 to FN552796. 1674 Ann Microbiol (2014) 64:1671–1682

Statistical analysis 5.6 mg l−1) with a reduction of ammonia removal from 72.5–79.7 % to 11.6–13.8 % in July and August (Fig. 1b). Rarefaction curves were calculated using the matrices from No significant reduction in performance with respect to BOD the AOB and total bacteria DGGE profiles considering one removal was observed, since both BOD and COD in the final band as OTU by PAST3 (Hammer et al. 2001). DGGE pat- effluent were below the legislation limits for the plant (40.0 terns from triplicate PCR samples were processed and and 160.0 mg l−1 , respectively) during all six months. analysed with Bionumerics 6.0 software (Applied Maths, Saint-Martens-Latem, Belgium). Similarity matrices, based PCR-DGGE analyses of bacterial, AOA and AOB upon Pearson product–moment correlation coefficients, were communities calculated from the densitometric curves of DGGE patterns and subsequently used for cluster analysis. Statistical analyses No PCR product was obtained by archaeal AmoA primers, were conducted using Minitab 14.0 software. even when amplification conditions were changed by reduc- Multivariate Canonical Correlation Analysis (CCA) was ing annealing temperature to 40 °C. DGGE profiles of both used to test and quantify the relationship between the AOB bacterial and AOB communities were checked for reproduc- community structure and the environmental conditions (tem- ibility by running triplicate PCR reaction each for duplicate perature, DO content and conductivity, respectively). CCA is DNA samples on the same DGGE gel (data not shown) and a method of correlating two sets of multidimensional vari- evaluating the banding pattern by Pearson correlation. The ables; more specifically, CCA can be seen as the problem of DGGE profiles from replicate PCR showed 98.9±3.25 mean finding latent vectors for two sets of variables such that their similarity values and duplicate DNA reactions showed 95.7± correlation is mutually maximized. In other words, a pair of 3.8 mean similarity values, calculated by the Pearson correla- linear transformations (canonical axis), one for each of the sets tion. Thus, in both cases there was significant similarity be- of variables, is obtained such that they are maximally corre- tween replicates and duplicates assessed, (P<0.005, Mann– lated. Analogous with Principal Component Analysis, the first Whitney U test) confirming the reproducibility of the method. canonical axis within the same set of variables is independent Moreover, the bacterial community in the WWTP showed from the others and accounts for the highest amount of vari- clear temporal variations with DGGE profiles clustering with ance; like ordinary correlation, the squared canonical correla- respect to sampling date (Fig. 2a). Indeed, three broad groups tion is the percentage of the total variance accounted for in the were recovered in cluster analysis (month 1–4, month 5 and analysis. month 6). The bacterial communities at all sampling times were significantly different (P<0.005, Mann–Whitney U test) with the exception of the communities analysed in month 1 Results and 2 which were not significantly different (P>0.5, Mann– Whitney U test). The mean similarity of AOB DGGE patterns Physical and chemical changes in the WWTP from the different sections of the WWTP at months 1, 2, 3 and 6 ranged between 87.6±4.1 to 92.7±3.1. Also, in the case of The wastewater treatment plant, which receives a mixture of AOB, a lower mean similarity between independent samples urban and thermal-bath wastewater, serves a population of ca from different parts of the WWTP was found in month 5 (70.1 13,000 inhabitants. In the activated sludge basin, the temper- ±12.2). Moreover, the sample RIC 4 had a very low similarity ature constantly increased over time from 10 °C (March) to value with respect to DEN4 and OXI4 (39.9±0.9). Like 26.5 °C (August) (Fig. 1a), while pH was constant in the range bacterial communities, a clear temporal variation was found 7.2–7.4 (data not shown). DO content decreased from 7.8 to for AOB communities, which clustered according to sampling 3.5 mg l−1. Conductivity (used as an indirect index of salinity) time in two separated groups (months 1–3 and months 4–6; was high during the whole sampling period, ranging from Fig. 2b). All AOB communities analysed at different sampling 4.200 μScm−1 in March to 6.110 μScm−1 in August; the times were found to be significantly different (P<0.005 month in which the WWTP receives the highest input from Mann–Whitney U test). Rarefaction curves of the AOB and the thermal bath and spa (Fig. 1a). Ammonia nitrogen in the total bacteria richness as a function of the sampling number influent ranged between 23.0 and 28.0 mg l−1 (data not were calculated and reported in Fig. 3. The AOB curve has an shown). Ammonia nitrogen in the activated sludge mixed asymptote, while the total bacterial one did not reach a liquor increased from an average of 17.7±2.5 mg l−1 in the plateau. first three months to 27.2±1.9 mg l−1 in the last 3 months, The variation of DGGE profiles with time was further − − whereas NO2 -N and NO3 -N dropped below the detection analysed by comparing the AOB and bacterial DGGE profiles level (Fig. 1b). Failure of nitrification was confirmed by the over the six month sampling period with the profiles obtained high level of ammonia nitrogen found in the final effluent in at the first sampling time (Fig. 4a) and relative to the samples the last two months of sampling (21.0±3.2 and 24.6± taken in consecutive months (i.e., month n vs. month n-1, Ann Microbiol (2014) 64:1671–1682 1675

Fig. 1 Chemical data of WWTP. a Conductivity (black diamond), Temperature (black square)and dissolved oxygen (black triangle) content determined in the oxidation basin. b ammonia (black square), nitrite (black triangle)and nitrate (black diamond) -nitrogen determined in the oxidation basin

Fig. 4b). This analysis confirmed that both bacterial and AOB conductivity). The first canonical axis (with respect to both communities were dynamic over the sampling period, with the AOB community and environmental conditions) bacterial community similarity dropping from 85 % to 50 % accounted for a very large part of the total variance (approx- and AOB community similarity dropping from 70 % to 20 %. imately 87 %). The CCA analysis shows a strict linear The most pronounced shift occurred from month 4 to month 5 correlation but also a progressive change of the AOB com- for bacteria (reduction in similarity from 65 % to 31 %) and munity over time, considering the quite clear separation of from month 3 to month 4 for AOB (reduction in similarity T1, T2 and T3 from T4, T5 and T6 (Fig. 5). from 67 % to 14 %; Fig. 4b). Cloned sequences analysis Canonical correlation analysis For a detailed evaluation of the nature of the shift in AOB In order to relate the shift of the AOB community over time communities in the WWTP, we investigated the phylogeny of to the environmental conditions within the WWTP, AOB species present in the main clusters identified by DGGE Canonical Correlation Analysis (CCA) was used. A strong analysis by building two clone libraries of AOB 16S rRNA and significant correlation (R=0.97) was detected between gene fragments, consisting of 30 clones from sample OXI2 the structure of the AOB community and the set of environ- and OXI5, respectively. All 60 clones were checked by DGGE mental parameters (temperature, DO content and and grouped on the basis of their co-migration with the most 1676 Ann Microbiol (2014) 64:1671–1682

Fig. 2 PCR-DGGE profiles of bacterial (a)andAOB(b)16S rRNA gene fragments in the WWTP over a 6-month sampling period. Dendrograms were constructed from the Pearson product–moment correlation using UPGMA clustering. OXI active sludge, DEN pre- denitrification, RIC secondary settler. Terminal number corresponds to the month of sampling (1=March, 6=August). In Fig. 2b a DGGE profile from pooled DNA is presented, with labelled bands (from I to VI) related to grouped cloned sequences (see Fig. 6)

dominant bands seen in DGGE profiles (data not shown). rRNA gene sequences with high sequence identity (96.5– Even though 18–29 DGGE bands were detected in the PCR- 99.7 %). Among them, 86 % of OXI2 and 63 % of OXI5, DGGE analyses, most of these bands were quite weak with matched with ammonia-oxidizing betaproteobacteria (group only a few bands dominating the profile. The 16S rRNA gene III and IV) whereas other clone sequences matched with fragments from all of the 60 clones co-migrated with six of the Azovibrio sp. (e.g., clone B14, group I), Dechloromonas sp. most intense bands found in the DGGE profiles. Thus, by (clone B10 and B12, group II) and denitrifying Fe-oxidizing cross referencing the clone library data with the DGGE pro- bacteria belonging to unclassified Betaproteobacteria (clone files, we were able to group all clones, from both libraries, into B5 and A69, group V and VI). Interestingly, in both samples six groups (Fig. 6). all of the clone AOB 16S rRNA genes were recovered from Nucleotide sequences of 30 cloned 16S rRNA gene frag- the genus Nitrosomonas, particularly within the ments, representative of each of the six groups previously N. oligotropha/N. marina lineages. This phylogenetic cluster determined, were compared with sequences from the EMBL comprises halophilic and halotolerant species isolated from database using FASTA 3 (Table 1). All recovered sequences freshwater and marine water, soil and WWTP (Cébron et al. matched with previously identified Betaproteobacteria 16S 2004;McCaigetal.1999; Purkhold et al. 2000). Ann Microbiol (2014) 64:1671–1682 1677

Fig. 3 Rarefaction curves of the total bacterial (continuous line) and AOB (dashed line)OTUs detected in the WWTP as a function of the sample numbers

Fig. 4 Variation of similarity a values between DGGE profiles of the AOB (black square)and bacterial (black diamond)DGGE data over the six months (a)and comparison relative to the similarity values of consecutive months (b)

b 1678 Ann Microbiol (2014) 64:1671–1682

and abundance of ammonia-oxidizing Archaea with respect to AOB in WWTP was also reported (Zhang et al. 2009; Limpiyakorn et al. 2011; Ye and Zhang 2011). In our study, AOAwas not detected by PCR, probably the number of AOA in the WWTP falls below the detection limit of the method. Other studies (Wells et al. 2009)reportedthatarchaealAmoA was detectable by PCR only in less than 15 % of samples from a 1 year weekly survey in a WWTP. This is not surprising because the role of AOA in WWTP is still unclear (Mußmann et al. 2011). Several studies have focused on the relationship between the shifts in AOB communities and the WWTP performance with the aim of establishing a correlation between distribution patterns and activities of AOB and nitrification efficiency Fig. 5 Canonical Correlation between AOB DGGE patterns and envi- (Layton et al. 2005; Wittebolle et al. 2005, 2008; Otawa ronmental conditions. The labels are referred to subsequent sampling- et al. 2006; Lydmark et al. 2007). Salinity was found to be a times (T1-T6=month 1–6). In the figure only axis 1 is portrayed, since this accounted for a large fraction of the total species-environment significant driving factor affecting AOB community compo- variance sition in natural and artificial environments (Pommerening- Röser et al. 1996; Freitag et al. 2006). Here, we evaluated the Discussion structure and dynamics of the AOB community in a WWTP treating saline water from thermal-bath facilities which were Composition and diversity of ammonia-oxidizing bacterial severely affected by seasonal malfunctioning. The aim of our communities in wastewater treatment plants have been inves- work was to monitor changes in AOB diversity and compo- tigated in recent years (Ballinger et al. 1998; Rowan et al. sition in relation to WWTP performance during a six month 2003; Limpiyakorn et al. 2005). More recently, occurrence survey.

Fig. 6 Clone frequencies from samples OXI2 and OXI5. Clones were grouped by roman numbers (I to VI) based upon their co- migration with bands in DGGE profiles from the original samples Ann Microbiol (2014) 64:1671–1682 1679

Table 1 Clones grouped by closest sequence matches via the EMBL database

Group Clone Reference seq. Accession n. Identity (%) Source Literature reference

IB1Azovibrio sp. R-25062 AM084040 98.3 Activated sludge, Ghent Belgium Heylen et al. 2006 B14 98.1 B22 98.2 B24 98.2 B20 96.6 II B10 Dechloromonas sp. CL24 AF288775 96.5 Enrichment culture from sediment Achenbach et al. 2001 B12 96.8 III B59 Estuarine Nitrosomonas sp. AF338207 97.3 Estuarine water Chesapeake Bay Voytek 1996 B64TA 921-i 97.8 B65 97.7 B69 97.7 A21 96.9 A29 97.9 A39 98.5 A44 98.5 A67 97.8 A68 97.7 A75 97.6 A91 99.3 A98 97.4 IV B30 Nitrosomonas sp. Is343 AJ621032 98.0 Brakish water Netherlands: River Schelde B33 97.8 B55 97.8 B62 97.6 A4 97.8 A38 98.0 A45 97.8 A99 97.8 V A69 Denitrifying Fe-oxidizing U51102 99.7 Feshwater mud samples, town ditches of Straub et al. 1996 bacteria Bremen VI B5 Aquabacterium parvum NR024874 99.0 Water distribution system biofilm, Berlin Kalmbach et al. 1999 strain B6

The AOB and total bacteria diversity was measured and The temporal variation of the bacterial community evi- compared using rarefaction curves based on the DGGE pro- denced by the clustering of the DGGE profiles, and the files. As expected, the diversity of the total bacteria was high relative stability in the first two months, could be explained and not fully discovered, while the AOB diversity was by the first period of survey being characterized by relative completely sampled as the rarefaction curve reached a plateau stability of physical and chemical parameters of the WWTP. (Fig. 3). By examining DGGE patterns of sludge bacterial Similarly to the total bacteria, the changes in the AOB communities among the three examined sections of the community observed at months 4 and 5 could be linked to WWTP, we observed high similarity values (Fig. 2a), suggest- the beginning of the perturbing effect generated by increased ing that the dominant bacterial communities were stable salinity and temperature and reduction of DO content that throughout the WWTP. The exception observed in month 5, began in the fourth month of the survey (see Fig. 1a). where the mean similarity among sampling locations was The observed dynamics of AOB communities at different lower, suggests that this deviation of bacterial community locations in the same WWTP was in accordance with Gorra stability in the whole WWTP could be the first indication that et al. (2007), who reported similar patterns in a constructed the WWTP microbial community was responding to the wetland. Wittebolle et al. (2005) also observed a clear AOB perturbing effect of increased salinity and reduced DO con- community shift, both by DGGE profile clustering and mov- centration that occurred at this time (see Fig. 1a). ing window analysis, correlated with changing nitrification 1680 Ann Microbiol (2014) 64:1671–1682 parameters in pharmaceutical wastewater treatment plants. species was inhibited by the changes in effluent salinity, DO Particularly, the authors assessed that failure of nitrification and environmental parameters (T), as confirmed by canonical was reflected in community shifts of the AOBs. The same correlation analysis that indicated a strong correlation between authors (Wittebolle et al. 2008), when evaluating the dynam- the shift of AOB-DGGE profiles over time and environmental ics of AOB communities on a short term basis in two func- parameters. It should be noted that the AOB-DGGE profiles tionally stable reactors, found that the AOB community was comprised bands that were found to be related to other non- not stable in either reactor over a period of more than AOB Betaproteobacteria, as confirmed by clone libraries one month. By contrast, in another study seasonal variations analysis. Particular care must be taken when dealing with in the composition of AOB communities, which were found in PCR-DGGE profiles analysis in order to avoid misinterpreta- a survey of 12 different WWTPs, were not correlated with the tion of population dynamics by weighing non-AOB se- ammonia removal performances (Limpiyakorn et al. 2005). In quences that could affect the data output. In our case, we our study a continuous shift in DGGE patterns over time, with stated that 25 % of clones in our libraries derived from the more drastic shift in the fifth month of sampling for bacteria same sludge samples were other Betaproteobacteria. and fourth month of sampling for AOB, was observed. Siripong and Rittman 2007, by evaluating AOB and NOB The high correlation of the structure of the AOB commu- communities in seven WWTPs, found that stable and similar nity with environmental parameters and the progressive nitrifier communities are shared among different reactors and change of the same community over time, evidenced by that functional redundancy (coexistence of various nitrifiers CCA analysis, clearly indicates that the AOB community having different growth and survival characters) may be valu- structure changed in line with the changes in the environmen- able for maintaining the stability and performance of nitrifying tal parameters that took place over the sampling period where bioreactors. Moreover, Wittebolle et al. (2008)demonstrated electrical conductivity and temperature increased and DO that a small group of AOB species can play a dominant role in decreased. a nitrifying reactor and speculated that less dominant species As reported in the results, some of the recovered sequences could constitute a reserve of AOB potentially able to replace of cloned 16S rRNA gene fragments belonged to dominant species as conditions change. Thus, functional sta- Betaproteobacteria other from AOB. Several authors reported bility can arise from having a diverse group of organisms that that CTO primers also amplify 16S rRNA gene sequences can carry out the same primary function under different con- from Betaproteobacteria other than AOB (Purkhold et al. ditions. When environmental factors change, the dominant 2000; Rowan et al. 2003;Cébronetal.2004;Belluccietal. organisms are selected on the basis of their ability to function 2011). Indeed, some of our Betaproteobacteria sequences optimally under the new conditions. In the case of the slow- were already reported in other studies using the same primer growing AOB, rapid changes in environmental conditions can set. For example, clones B10 and B12 identified in this work take the dominant organisms outside their optimum operation- were highly similar to clone AF527026 -43Ft- reported by al conditions; then, lower abundance organisms that may be Rowan et al. (2003). It has to be noted that the closest relatives optimally active under the new conditions do not have time to of the most represented AOB sequences come from estuarine grow sufficiently, hence, the process fails. samples (e.g., group IV with Estuarine Nitrosomonas sp. TA When assessing the AOB community dynamics, quantita- 921i). Accordingly to previous studies (Freitag et al. 2006), tive analysis is a powerful tool for evaluating the relationships we hypothesize that the presence of a dominant AOB popu- between microbial populations and stability of reactor perfor- lation of halophilic/halotolerant species is likely to be influ- mances. In our case we found that the percentage of AOB enced by the selective pressure of salinity and may vary in its clones clearly decreased between OXI2 and OXI5 (group III adaptation to other chemical and physical conditions and IV in Fig. 5). More specifically, clones belonging to group (Bollmann and Laanbroek 2002; Lydmark et al. 2007). The III are the most affected, with a reduction from 36.6 to 13.3 % influent wastewater for this WWTP comprises a mix of urban of the whole clone libraries. Acknowledging that this is not the wastewater and water from a saline thermal bath. It is charac- most robust way to quantitatively assess changes in the AOB terized by a constantly high level of salinity as assessed by community, and that FISH (fluorescence in situ hybridization) conductivity measurements (around 4.000 μScm−1,witha or Q-PCR (quantitative real-time PCR) would have provided seasonal increase up to 6.000 μScm−1) related with the more more definitive quantitative information, we can argue that the intense activities of spa facilities during the summer months decrease in the relative abundance of AOB sequences in 16S (Fig. 1b). Both the intensity of DGGE bands and relative rRNA gene clone libraries may relate to the reduction in abundance of clones belonging to groups III and IV suggest ammonia removal performance. In a recent study (Wang that AOB closely related with N. oligotropha/N. marina line- et al. 2012), it was evidenced by the Q-PCR approach that age are the representative AOB in the WWTP. The low the stable function of nitrification was correlated with a stable diversity of the key AOB could be the cause of nitrification AOB number in the pilot-scale WWTP and suggested a sig- failures. Indeed, the functionality of the few dominant AOB nificantly positive correlation between ammonia removal Ann Microbiol (2014) 64:1671–1682 1681 efficiency and total AOB population number. More recently community structure in the lower Seine River: impact of Paris – Terada et al. (2013), by applying quantitative FISH analyses on wastewater effluents. Appl Environ Microbiol 70:6726 6737 Francis CA, Roberts KJ, Beman MJ, Santoro AE, Oakley BB (2005) two different bioreactors, were able to assess different dominance Ubiquity and diversity of ammonia-oxidizing Archaea in water of fast growing alophilic and halotollerant AOB Nitrosomonas columns and sediments of the ocean. PNAS 102:14683–14688 spp. versus slower growing AOB Nitrosospira spp. Freitag TE, Chang L, Prosser JI (2006) Changes in the community In our study, we found that a WWTP treating wastewater structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater–marine gradient. Environ with high salinity and with uncertain nitrification performance Microbiol 8:684–696 is characterized by an AOB community restricted to a few key Geets J, Boon N, Verstraete W (2006) Strategies of aerobic ammonia- species. 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Electron 4(1):9pp Therefore, we suggest that the reduction of nitrification per- Head IM, Hiorns WD, Embley TM, McCarthy AJ, Saunders JR (1993) formance was due to reduction of activity and abundance of The phylogeny of autotrophic ammonia-oxidizing bacteria as deter- the dominant AOB populations without substitution by new mined by analysis of 16S ribosomal RNA gene sequences. J Gen Microbiol 139:1147–1153 AOB better adapted to the changed operational conditions. Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P Indeed, the observed reduction of the principal AOB popula- (2006) Cultivation of denitrifying bacteria: optimization of isolation tion (clone from group III and IV) could be due to a lower conditions and diversity study. Appl Environ Microbiol 72:2637– tolerance to multiple stresses (reduction of oxygen, increase of 2643 Jensen FB (2003) Nitrite disrupts multiple physiological functions in temperature and salinity). 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